analyses of the function of the palmitoyl …
TRANSCRIPT
The Pennsylvania State University
The Graduate School
Intercollege Graduate Program in Neuroscience
ANALYSES OF THE FUNCTION OF THE PALMITOYL TRANSFERASES
GODZ AND SERZ‐BETA IN KNOCK‐OUT MICE
A Dissertation in
Neuroscience
by
Shoko Murakami
© 2008 Shoko Murakami
Submitted in Partial Fulfillment
of the Requirements
for the Degree of
Doctor of Philosophy
December 2008
ii
The dissertation of Shoko Murakami was reviewed and approved* by the following:
Bernhard Lüscher Professor of Biology, Biochemistry and Molecular Biology, and Psychiatry Dissertation Advisor Chair of Committee Richard Ordway Associate professor of Biology Gong Chen Associate Professor of Biology Robert F. Paulson Associate professor of Veterinary and Biomedical Sciences Erin D. Sheets Assistant professor of Chemistry Robert L. Sainburg Associate professor of Kinesiology and Neurology Chair of Neuroscience Graduate Program *Signatures are on file in the Graduate School.
iii
ABSTRACT Type A GABA receptors (GABAARs) are the major sites of fast synaptic inhibition in the central nervous system (CNS) and mediate the therapeutic effects of many clinically important drugs. The efficacy of synaptic inhibition is critically dependent on the postsynaptic accumulation of GABAARs. Golgi-specific DHHC zinc finger protein (GODZ; aka, DHHC3) and SERZ-β (aka, DHHC7) have been identified as palmitoyl acyl-transferases (PATs) of the γ2 subunit of GABAARs. In addition, the cell adhesion molecule neuroligin 2 (NL2) is localized at GABAergic synapses and implicated in GABAergic synapse formation. Previous analyses of GODZ by RNAi in cultured neurons had suggested that they are important for postsynaptic accumulation of GABAARs at inhibitory synapses. In addition these studies pointed to a novel possible role of GABAARs in the assembly of GABAergic inhibitory synapses.
A first objective of this doctoral thesis was to elucidate the function of GODZ and SERZ-β in vivo. In order to achieve this purpose, global and conditional knock-out (KO) mice were generated for both GODZ and SERZ-β. Genetic deletion of GODZ and SERZ-β was confirmed by genomic Southern blotting, RT-PCR and immunofluorescence analyses. Global GODZ and SERZ-β KO mice were found to be viable, whereas GODZ and SERZ-β double knock-out (DKO) mice showed a partially penetrant perinatally lethal phenotype. Male, but not female, GODZ KO mice exhibited a small but significant reduction in body weight compared to wild type (WT) littermate controls. A more dramatic reduction in body weight was evident in DKO mice, as quantified in females but also overtly evident in males. Compared to WT littermate controls, GODZ KO mice showed complex behavioral alterations characterized by excessive jumping in response to handling, hindleg clenching upon suspension by their tail, hyperlocomotion in an Open Field test, as well as enhanced Prepulse Inhibition of acoustic startle responses. However, GODZ KOs showed normal anxiety related behavior in Free Choice and Elevated Plus Maze tests. Immunofluorescence analyses of cortical neuron cultures prepared from GODZ KO or GODZ/SERZ-β DKO mice revealed unaltered numbers of postsynaptic clusters of γ2 subunit containing GABAARs and the inhibitory postsynaptic scaffold protein gephyrin. Similarly, the number of clusters of the corresponding presynaptic marker glutamate decarboxylase (GAD) and vesicular inhibitory amino acid transporter (VIATT) were also unchanged. Consistent with unaltered clustering in pure DKO cultures, the surface expression levels of the γ2 subunit and NL2 assessed by biotinylation was unaltered in DKO compared to WT cultures. However, when mutant neurons were co-cultured with an excess of WT neurons, punctate staining for postsynaptic GABAAR clusters and presynaptic inhibitory synaptic markers of GODZ KO and DKO neurons was dramatically reduced compared to neurons of pure WT
iv
cultures. Similarly, γ2 subunit heterozygous neurons co-cultured with an excess of WT neurons showed a significant reduction in inhibitory synapse formation, suggesting that γ2 subunit-containing GABAARs contribute to normal GABAergic innervation. In addition, electrophysiological analyses of DKO neurons co-cultured with WT neurons revealed unaltered GABA-induced whole cell current but significant deficits in the frequency and amplitude of miniature inhibitory postsynaptic currents (mIPSCs) compared to WT neurons. These results suggest that GODZ and/or SERZ-β are dispensable for normal surface expression of GABAARs in pure cultures but necessary for postsynaptic clustering of GABAARs and normal formation and function of inhibitory synapses under competitive conditions.
As a second objective I further analyzed the role of GABAARs in GABAergic synapse formation. Preliminary immunofluorescence analyses of GABAAR γ2 subunit KO neurons had revealed a dramatic loss of postsynaptic NL2 at inhibitory synapses, indicating that perhaps the γ2 subunit was required for normal expression of a synaptic adhesion molecule, which in turn was required for GABAergic inhibitory synapse formation. To test this idea and to analyze the surface expression of GABAARs in γ2 subunit KO neurons I performed a series of biotinylation assays, comparing the surface expression of NL2, α and β subunits of GABAARs in WT and γ2 subunit KO cultures. Although the total amount of NL2 and β2/3 subunit expressed in γ2 KO cultures was unaltered, the surface expression level of NL2, α1 and β2/3 subunits was greatly reduced in γ2 KO compared to WT neurons. This indicated that the γ2 subunit is necessary for the surface expression of both NL2 and GABAARs. Previously, co-aggregation assay in transfected HEK293T cells provided evidence that NL2 interacts with α and/or β subunits, but not the γ2 subunit, in the plasma membrane. These results together suggest a mechanism in which GABAAR-dependent presentation of NL2 at the cell surface promotes interaction of the postsynaptic apparatus with GABAergic presynaptic terminals, thereby ensuring apposition of functionally matching pre- and postsynaptic elements.
v
TABLE OF CONTENTS
List if Figures ................................................................................................................................ viii
List of Tables .................................................................................................................................. ix
List of Abbreviations ....................................................................................................................... x Acknowledgement ........................................................................................................................ xiii
Chapter 1 INTRODUCTION ........................................................................................................... 1 1.1. GABAergic synapses ............................................................................................................... 1 1.1.1. GABAA receptors ................................................................................................................. 1
1.1.1.1. Physiological and therapeutic relevance of GABAARs .............................................. 1 1.1.1.2. Structure of GABAARs ............................................................................................... 1 1.1.1.3. Intracellular trafficking of GABAARs ......................................................................... 4 1.1.1.4. Postsynaptic clustering of GABAARs ......................................................................... 7 1.1.2. Neuroligins ........................................................................................................................... 9
1.1.2.1. Cell adhesion molecules in nervous system ................................................................ 9 1.1.2.2. Structure, localization and function of neurexin and neuroligin ............................... 10
1.2. Protein palmitoylation ............................................................................................................ 12 1.2.1. Palmitoylation in general ................................................................................................... 12 1.2.2. Enzymes involved in palmitoylation .................................................................................. 14 1.2.3. Palmitoylation of neural proteins ....................................................................................... 17 1.2.4. Palmitoylation of γ2 subunit of GABAARs and NL2 ......................................................... 20
1.3. Aim of study .......................................................................................................................... 21 Chapter 2 Materials and methods ................................................................................................. 23 2.1. Gene Targeting....................................................................................................................... 23 2.1.1. Overall strategies of generation of conditional KO mice ................................................... 23 2.1.2. Construction of targeting vectors ....................................................................................... 24
2.1.2.1. GODZ targeting vector ............................................................................................. 24 2.1.2.2. SERZ-β targeting vector .......................................................................................... 25 2.1.2.3. Partial digestion ....................................................................................................... 26 2.1.2.4. Three way/partial ligation ......................................................................................... 26
2.1.3. Production of targeted embryonic stem cell clones ........................................................... 27 2.1.3.1. General growth conditions ....................................................................................... 27 2.1.3.2. Preparation of primary mouse embryonic fibroblasts (PMEFs) ............................... 27 2.1.3.3. Linearization of targeting vector DNA .................................................................... 28 2.1.3.4. Transfection of ES cells by electroporation ............................................................. 28 2.1.3.5. Screening for targeted ES cell colonies .................................................................... 29 2.1.3.6. Expansion of positive clones ..................................................................................... 31 2.1.4. Blastocyst injection and generation of chimeric mice ....................................................... 32 2.1.5. Generation of floxed and knockout allele of GODZ and SERZ-β ..................................... 32
2.2. Genotyping ............................................................................................................................. 33
vi
2.2.1. Genomic DNA isolation for genotyping ........................................................................... 33 2.2.2. Primers and PCR ................................................................................................................ 33
2.3. RTPCR ................................................................................................................................... 35
2.3.1. RNA isolation .................................................................................................................... 35
2.3.2. First-Strand cDNA Synthesis ............................................................................................. 36 2.3.3. PCR using synthesized cDNA .......................................................................................... 36 2.3.3.1. RTPCR ...................................................................................................................... 36 2.3.3.2. Quantitative RTPCR ................................................................................................. 38
2.4. Behavioral tests ..................................................................................................................... 39 2.4.1. Mouse husbandry .............................................................................................................. 39 2.4.2. Open Field test .................................................................................................................. 39 2.4.3. Elevated plus maze ............................................................................................................ 39 2.4.4. Free choice exploration ..................................................................................................... 39 2.4.5. Hot plate test ..................................................................................................................... 40 2.4.6. Prepulse Inhibition ............................................................................................................ 40
2.5. Tissue culture ........................................................................................................................ 41 2.5.1. Neuron culture .................................................................................................................. 41 2.5.2. Preparation of glial cells ................................................................................................... 42
2.6. Immunofluorescent analyses ................................................................................................. 43 2.6.1. Immunofluorescent staining of cultured cortical neurons ................................................. 43 2.6.2. Microscopic analysis .......................................................................................................... 43 2.6.3. Quantification of immunofluorescent staining .................................................................. 44
2.7. Western blot analyses ........................................................................................................... 44 2.8. Metabolic labeling of cortical neurons with 3H-palmitic acid .............................................. 45 2.8.1. Concentration of 3H-palmitic acid .................................................................................... 46 2.8.2. Metabolic labeling ............................................................................................................ 46 2.8.3. Protein extraction and Immunoprecipitation for NL2 ........................................................ 46 2.8.4. Protein extraction and Immunoprecipitation for γ2 subunit of GABAARs ....................... 47 2.8.5. Fluorography ..................................................................................................................... 48
2.9. Surface biotinylation assay ................................................................................................... 49 2.9.1. Surface biotinylation assay ............................................................................................... 49 2.9.2. Quantification of surface biotinylation assay .................................................................... 50
2.10. Electrophysiology ............................................................................................................... 50 Chapter 3 Results .......................................................................................................................... 52 Part I: Analyses of the Functions of the Palmitoyl Transferases GODZ & SERZ-β in Knock-out Mice .............................................................................................................................................. 52 3.1.1. Introduction and aim of study ........................................................................................... 52 3.1.2. Generation of fGODZ and fSERZ-β mutant and global KO mice ................................... 52
3.1.2.1. Design and generation of targeting vector ............................................................... 52 3.1.2.2. Screening of positive clones .................................................................................... 54
vii
3.1.2.3. Establishing the fGODZ, fSERZ-β GODZ-/-, SERZ-β-/- and DKO mouse lines ... 54 3.1.2.4. Confirmation of deletion of exon 3/4 of GODZ and SERZ-β by Southern hybridization and RT-PCR........................................................................................ 58 3.1.2.5. Confirmation of loss of GODZ and SERZ-β protein in KO neurons ............................................................................................................... 61 3.1.3. Normal clustering of pre- and postsynaptic markers of GABAergic synapses in GODZ KO and GODZ/SERZ-β DKO cultures ................................................................ 61 3.1.4. Deficits in GABAAR postsynaptic clustering of GODZ KO and DKO cortical neurons co-cultured with WT neurons ................................................................................ 63 3.1.5. γ2 subunit containing GABAARs are required for efficient GABAergic innervation ............................................................................................................................ 64 3.1.6. Surface expression of γ2 subunit and NL2 in DKO ........................................................... 68 3.1.7. Functional deficits of DKO neurons in inhibitory and excitatory neurotransmission ....... 71 3.1.8. Overt phenotype of GODZ-/-, SERZ-β-/- and DKO mice: Body weight, lethality, fertility and posture ........................................................................................................... 74 3.1.9. Behavioral characterization of GODZ KO mice ................................................................ 78
3.1.9.1. GODZ KO mice show hyperlocomotion in the Open Field test .............................. 78 3.1.9.2. GODZ KO mice show normal level of anxiety ....................................................... 80 3.1.9.3. GODZ KO show enhanced Prepulse Inhibition ....................................................... 80 3.1.9.4. Other behavioral phenotypes ................................................................................... 82
3.1.10. Upregulation of ZDHHC15 mRNA in brains of GODZ KO and DKO neurons ............ 82 Part II: GABAA receptor-dependent presentation of NL2 at inhibitory synapses ....................... 87 3.2.1. Introduction and aim of study ........................................................................................... 87 3.2.2. The γ2 subunit of GABAARs is required for normal GABAAR and NL2 surface expression ....................................................................................................................................................... 87
Chapter 4 Discussion .................................................................................................................... 90 4.1. Overview of findings ............................................................................................................. 90 4.2. GODZ and SERZ-β are required for γ2 subunit postsynaptic clustering, but dispensable for normal surface expression ............................................................................................................. 91 4.3. DKO neuron exhibit deficit not only in inhibitory but also in excitatory neurotransmissions 92 4.4. GODZ might be the major PAT for the γ2 subunit of GABAARs ......................................... 94 4.5. Compensation of loss of GODZ/SERZ-β function by other DHHC family proteins ............. 94 4.6. Behavioral phenotypes of GODZ KO mice ............................................................................ 95 4.6.1. GODZ KO mice demonstrate reverse phenotype with NL2 transgenic mice .................... 95
4.6.2. Hyperekplexia-like phenotype in GODZ KO mice ~ Possible link between GODZ/SERZ-β with glycine receptors ................................................................................................................ 96 4.7. Technical limitation in the palmitoylation assay in neurons .................................................. 98 4.8. Palmitoylation and lipid raft ................................................................................................ 100 4.9. Palmitoylation and ubiquitination ........................................................................................ 102 4.10. Possible mechanism of cooperative function of NL2 and γ2 to induce inhibitory synapse 104 4.11. Outlook .............................................................................................................................. 105 Bibliography ............................................................................................................................... 109
viii
LIST OF FIGURES
Figure 1.1. GABAA receptor and an individual subunit .................................................................. 2 Figure 1.2. GABAergic synapse and trafficking and membrane targeting of GABAA receptors .. 15 Figure 1.3. Phylogenic tree of 25 DHHC proteins ......................................................................... 16 Figure 3.1. Targeting strategy for GODZ and SERZ-β ................................................................. 53 Figure 3.2. Screening of G418-resistant ES cells .......................................................................... 55 Figure 3.3. Chimeric mice ............................................................................................................. 56 Figure 3.4. Detection and verification of GODZ and SERZ-β deletion mutations ....................... 59 Figure 3.5. Normal clustering of pre- and postsynaptic markers of GABAergic synapses in GODZ KO cortical neurons ....................................................................................................................... 62 Figure 3.6. Deficits in GABAAR postsynaptic clustering of GODZ KO and DKO cortical neurons co-cultured with WT neurons ........................................................................................................ 65 Figure 3.7. Subtle deficits in γ2 subunit-containing GABAARs of γ2+/- neurons result in deficits in GABAergic innervation ............................................................................................................. 69 Figure 3.8. Analyses of surface expression of γ2 subunit of GABAARs and NL2 by surface biotinylation of cultured cortical neurons ...................................................................................... 70 Figure 3.9. Functional deficits of DKO neurons in inhibitory and excitatory neurotransmission . 72 Figure 3.10. Weight loss observed in GODZ KO and DKO mice ................................................. 75 Figure 3.11. GODZ KO mice show an increased muscle tension ................................................. 77 Figure 3.12. GODZ KO mice show hyperlocomotion in the Open Field test ............................... 79 Figure 3.13 GODZ KO mice show normal level of anxiety .......................................................... 81 Figure 3.14. GODZ KO show enhanced Prepulse Inhibition ........................................................ 83 Figure 3.15. GODZ KO mice exhibit nociceptive sensory motor deficits ..................................... 84 Figure 3.16. mRNA of ZDHHC15 is upregulated in brain of GODZ KO and DKO mice ........... 86 Figure 3.17. Reduced surface expression of NL2, α1 and β2, 3 subunits of GABAARs in γ2 subunit KO neurons ....................................................................................................................... 88
ix
LIST OF TABLES
Table 2.1: Wash solutions/500 ml ................................................................................................ 30 Table 2.2: Primers for genotyping ................................................................................................. 34 Table 2.3: Parameters of genotyping PCR reactions ..................................................................... 35 Table 2.4: Primers for RT-PCR ..................................................................................................... 37 Table 2.5: Parameters for RT-PCR reactions ................................................................................. 37 Table 2.6: Primers for qRT-PCR ................................................................................................... 38 Table 2.7: Parameters for qRT-PCR .............................................................................................. 38 Table 2.8: Primary Antibodies for Western blot analyses ............................................................. 45 Table 3.1: Loci contained in each mosaicism ................................................................................ 57 Table 3.2: Number of offspring of Chimera x EIIaCre and GODZ/SERZ-β alleles present ......... 57 Table 3.3: Genotype of offspring of Mosaic D x 129 SvJ ............................................................. 58 Table 3.4: Clenching phenotype of GODZ and SERZ-β KO mice ............................................... 76
x
LIST OF ABBREVIATIONS
4-OH-TM 4-hydroxy tamoxifen
AChE Acetylcholinesterase
AMPAR α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor
APT1 Acylprotein thioesterase-1
BAC Bacterial artificial chromosome
BIG2 Brefeldin-A-inhibited GDP/GTP exchange factor 2
bpA Bovine growth hormone polyadenylation site
BZ Benzodiazepine
CAML Calcium-Modulating cyclophilin Ligand
CNS Central nervous system
DHHC-CRD Aspartate-histidine-histidine-cysteine-cyteine rich domain
DKO Double knock-out
DMP Dimethyl pimelimidate
DMSO Dimethyl sulfoxide
DRM Detergent-resistant membrane
ER Endoplasmic reticulum
ES cell Embryonic stem cell
FBS Fetal bovine serum
FRAP Florescence Recovery After Photobleaching
G418 Geneticin
GABA Gamma-aminobutyric acid
xi
GABAAR GABAA receptor
GABARAP GABAAR-associated protein
GAD Glutamate decarboxylase
GAP43 Growth associated protein 43
GAPDH Glyceraldehyde-3-phosphate dehydrogenase
GluR Glutamate receptor
GlyT Glycine transporter
GODZ Golgi-specific DHHC zinc finger protein
GPI Glycosylphosphatidylinositol
HAP1 Huntingtin-associated protein 1
HEK cell Human embryonic kidney cell
HFY His-FLAG-YFP
INCL Infantile neuronal ceroid lipofuscinosis
KO Knock-out
LNS domain Laminin, neurexin, sex-hormone binding protein domain
lox P Locus of cross-over in P1
LRAT Lecithin retinol acyl transferase
MBOAT Membrane-bound O-acyltransferase
MEM Modified Eagle’s medium
mEPSC Miniature excitatory postsynaptic current
Meu Mosaic, early embryonic stage and ubiquitously
mIPSC Miniature inhibitory postsynaptic current
NBA Neurobasal-A Media
NCAM Neuronal cell adhesion molecule
xii
neo Neomycin
NL Neuroligin
NSF N-ethylmaleimide-sensitive factor
PAT Protein acyltransferase
PBS Phosphate buffered saline
PGK Phosphoglycerate kinase I
PMEF Primary mouse embryonic fibroblast
Porc Porcupine
PPI Pre-pulse inhibition
PPT Protein palmitoyl thioesterase
PRIP Phospholipase-C-related catalytically inactive protein
PSD Postsynaptic density
RPE65 Retinal pigment epithelium-specific protein 65kDa
RT Room temperature
SERZ Sertoli cell gene with a zinc finger domain
shRNA Small-hairpin RNA
SNP Single-nucleotide polymorphism
TM Transmembrane
UPS Ubiquitin proteasome system
Wg Wingless
WT Wild type
Yck2 Yeast casein kinase 2
xiii
ACKNOWLEDGMENTS This thesis is a sum of a long, long journey from 12 years ago when I first attempted to study towards to PhD degree. All people whom I encountered during these years gave me their thoughts, shared the challenges and difficulties and supported me. These were the all nourishment for this journey. Therefore, I would like to express my gratitude to the following people:
First, I would like offer my great thanks to Dr. Bernhard Lüscher, my thesis advisor, for his guidance and generosity. Without your vast scientific knowledge and thoughtfulness, it would be impossible to complete this work. My appreciation goes to collaborators Dr. Gong Chen and Xia Wu for the electrophysiological analyses.
Additionally, I thank Rachnanjali Lal for the imaging and quantification of GODZ KO cortical neurons.
I would like to thank to my committee members: Drs. Bernhard Lüscher, Richard Ordway, Gong Chen, Robert Paulson, and Erin D Sheets for their advice and support.
I would like to thank to Drs. Xu Yuan, John Clinton Earnheart, Cheng Fang and Nadia Sahir for sharing scientific knowledge, great support and encouragement.
I also thank to Yao Guo and Sue Lingenfelter for technical support and encouragement.
I would like to thank to Qiuying Shen and Zhen Ren for providing a cheerful environment.
I would like to thank to the rest of the Luscher Lab members both present and past: Dr. Melissa Alldred, Shuai Shi, Dr. Beth Luellen, Ashley Stull, Sebnem Nur Tuncdemir, Scott DiLoreto and Anjuli Datta for the technical help and support all along the way.
I thank to Dr. Susumu Hirose who was the supervisor of my first graduate program and supported me when I decided to go back to school.
I would like to thank to my father, mother, brother, sister and friends for their love.
Finally my husband, Dr. Katsuhiko Murakami, my son, Taiki Murakami, and daughters Sakura and Momo, deserve my gratitude from bottom of my heart. You were always by me and gave me a warm, happy school life. I am also thankful for Katsu’s critical scientific advice. I would have never made it to the end without you.
1
CHAPTER ONE
INTRODUCTION 1.1. GABAergic synapses
1.1.1. GABAA receptors
1.1.1.1. Physiological and therapeutic relevance of GABAARs
Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter and mediates
most fast neural inhibition in mammalian central nervous system (CNS) by acting on
GABAA receptors (GABAARs). Fine-tuning of GABAAR function is known to be critical
for modulation of neural excitability in CNS. The importance of its role is evident based
on GABAergic deficits associated with a wide range of neurological and mental disorders
including epilepsy (Baulac et al., 2001, Wallace et al., 2001, Scheffer and Berkovic,
2003), anxiety disorders (Malizia, 2002, Lydiard, 2003), autism (Schmitz et al., 2005)
and schizophrenia (Lewis et al., 2005, Petryshen et al., 2005). In addition, GABAARs are
also clinically relevant drug targets for some general anesthetics and sedative and
anxiolytic agents including benzodiazepines (BZs) and barbiturates (Korpi et al., 2002,
Rudolph and Antkowiak, 2004). Very interestingly, a recent study showed that the
specific GABAAR subtypes containing α2 and α3 subunit were critical components of
spinal pain control (Knabl et al., 2008). These facts have been facilitating the
investigation on the mechanisms that regulate intracellular trafficking and accumulation
of GABAARs on the neural plasma membrane.
1.1.1.2. Structure of GABAARs
GABAARs belong to a heteropentameric ligand-gated ion-channel superfamily which
also includes the nicotinic acetylcholine receptors, glycine receptors and 5-
hydroxytryptamine 3 receptors (Barnard et al., 1998, Chen and Olsen, 2007) (Fig. 1.1a).
GABAARs are assembled into pentamers from subunits encoded by 19 separate but
homologous genes (α1−6, β1−3, γ1−3, δ, ρ1−3, ε, π and θ) resulting in considerable
receptor heterogeneity (reviewed by (Sieghart et al., 1999, Sieghart and Sperk, 2002)).
2
Figure 1.1. GABAA receptor and an individual subunit. a, GABAA receptors are heteropentameric chloride ion channels and commonly consist of two α, two β and one γ2 subunit. Upon binding of GABA at the cleft between α and β subunits, channel opens and allows the influx of Cl- ions into the cell, resulting in the hyperpolarization. b, Each subunit of GABAA receptors contains same domain organization with large extracellular N-terminus followed by four transmembrane (TM) domains and short extracellular C-terminus, as well as a large intracellular loop between TM3 and TM4.
3
The structural diversity of GABAARs is further increased by alternative splicing of these
subunits. All GABAAR subunits share the same domain organization with a large
extracellular N-terminus followed by four transmembrane (TM) domains and a short
extracellular C-terminus (Fig. 1.1b). They also contain a large intracellular loop between
TM3 and TM4. This cytoplasmic loop serves as an interacting domain with many other
proteins and includes phosphorylation sites that regulate these interactions and thus the
localization and function of GABAARs (see below for detail).
The structural heterogeneity of different GABAAR subtypes results in functional
heterogeneity as illustrated by differences in agonist affinity, channel opening probability
and pharmacology, as well as differences in cellular and brain regional localization of
different GABAAR subtypes. Despite the number of possible subunit combinations, these
GABAAR subtypes are assembled in defined neuronal populations (Fritschy and Mohler,
1995) and at different developmental stages with surprising selectivity (Laurie et al.,
1992, Fritschy et al., 1994, Paysan et al., 1994). For example, the α1/β2,3/γ2 combination
is detected in numerous cell types, whereas α2/β2,3/γ2, α3/β2,3/γ2 and α5/β2,3/γ2 is
expressed in discrete cell populations of neurons in rat adult brain (Fritschy and Mohler,
1995). In addition, the α2 subunit is expressed throughout brain at birth and disappears as
α1 expression is increased during the first postnatal week (Fritschy et al., 1994). The
assembly of these subunits is carefully regulated in the endoplasmic reticulum (ER). In
heterologous expression systems, coexpression of α, β and γ subunit is required for
formation of receptors that mimic the electrophysiological and pharmacological
parameters of native receptors (reviewed in (Whiting, 1999)). Recent studies also indicate
that δ subunit-containing GABAARs are highly sensitive to GABA and ethanol
(Sundstrom-Poromaa et al., 2002, Wallner et al., 2003) and responsible for tonic currents
that are enhanced by a low doses of ethanol (Glykys et al., 2007). However, the majority
of GABAA receptors consist of α and β subunits together with the γ2 subunit in a ratio of
2:2:1. While γ1 and γ3 subunit are functionally similar to the γ2 subunit, they are part of
minor populations of GABAARs only (Laurie et al., 1992, Wisden et al., 1992). Similarly,
the expression patterns of the δ, ε and θ subunits is also highly restricted to specific
neural cell types (Laurie et al., 1992, Wisden et al., 1992, Sieghart et al., 1999).
4
1.1.1.3. Intracellular trafficking of GABAARs
After the assembly in the ER, GABAARs are trafficked to the Golgi apparatus and
transported to cell surface by transport vesicles (Fig. 1.2) (Leil et al., 2004). Several
proteins have been identified as GABAAR-associated proteins that regulate GABAAR
intracellular trafficking. For instance, GABAAR-associated protein (GABARAP)
interacts with the γ1−3 subunit major intracellular loop domains (Wang et al., 1999,
Nymann-Andersen et al., 2002) and is known to increase surface expression of
GABAARs upon overexpression (Leil et al., 2004, Chen et al., 2005). GABARAP also
interacts with microtubules (Wang and Olsen, 2000) and N-ethylmaleimide-sensitive
factor (NSF) (Kittler et al., 2001). Using siRNA to knock-down GABARAP, a recent
study showed that GABARAP was dispensable for maintenance of basal surface
expression of GABAARs, but necessary for an NMDA-induced increase in surface
expression of GABAARs (Marsden et al., 2007). Phospholipase-C-related catalytically
inactive proteins (PRIP1 and PRIP2) bind to GABARAP and the intracellular domains of
β and γ2 subunits of GABAARs (Kanematsu et al., 2002, Uji et al., 2002). A recent study
showed that the association between GABAARs and GABARAP was significantly
reduced in PRIP1/2 DKO neurons (Mizokami et al., 2007). In addition, the surface
number of diazepam binding sites in PRIP1/2 DKO mice was reduced, suggesting that
PRIP1/2 are required for normal expression of γ2 subunit containing GABAARs
(Mizokami et al., 2007). The surface expression of GABAARs was also reduced by
disruption of the direct interaction between the β3 subunit and PRIP using a peptide
competitor corresponding to the PRIP1 binding site (Mizokami et al., 2007). Taken
together, PRIP is thought to serve as a bridging protein between GABARAP and
GABAARs (Luscher and Keller, 2004, Mizokami et al., 2007, Jacob et al., 2008).
Brefeldin-A-inhibited GDP/GTP exchange factor 2 (BIG2) is also known to regulate
GABAAR trafficking. BIG2 is localized to the trans-Golgi network and vesicle-like
structures in the dendritic cytoplasm and at the synapse. In yeast two-hybrid assay, the C-
terminus of BIG2 interacted with the intracellular loop of the β3 subunit of GABAAR.
Upon transfection in human embryonic kidney (HEK) 293 cells, BIG2 promoted the exit
of GABAARs from the ER (Charych et al., 2004). Phosphorylation is also known to
5
Figure 1.2. GABAergic synapse and trafficking and membrane targeting of GABAA receptors. In presynaptic terminals, GABA is converted from glutamate by glutamate decarboxylase (GAD) and transported into neurotransmitter vesicles. Presynaptic neurexin interacts extracellularly with postsynaptic NL2. GABAA receptors are assembled from subunits in the ER. Correctly assembled receptors exit the ER via Golgi, a mechanism that may be regulated by GODZ/SERZ-β dependent palmitoylation. Other receptors and subunits maybe subject to ubiquitination and degraded by the proteasome (Kittler et al., 2002). GODZ/SERZ-β and other proteins such as GABARAP, NSF, BIG2 and PRIP are known to regulate the intracellular trafficking of GABAA receptors. GABAA receptors that reach the cell surface distribute between extrasynaptic and synaptic sites by lateral diffusion, a mechanism that is regulated by interaction with the postsynaptic cytoskeleton (Jacob et al., 2005, Tretter et al., 2008). Endocytosis is mediated by AP2 and clathrin-coated pits. The endocytosed receptors can be recycled back to the cell surface or degraded by targeting to lysosomes.
6
regulate GABAARs trafficking. Akt, a serine/threonine kinase, phosphorylates Ser410 in
the intracellular loop of the β2 subunit of GABAARs in response to insulin and this
phosphorylation leads to an increase in the number of GABAARs at the cell surface
(Wang et al., 2003). Phosphorylation also regulates the number of GABAARs on the cell
surface by endocytosis (Fig. 1.2). Clathrin adaptor AP2 binds to β and γ subunit of
GABAARs and, thus facilitates constitutive GABAAR endocytosis in a clathrin-dependent
manner (Kittler et al., 2000). Blockade of clathrin-dependent endocytosis causes reduced
GABAAR internalization, resulting in a large increase in the amplitude of miniature
inhibitory postsynaptic currents (mIPSCs) (Kittler et al., 2000). Interestingly, the AP2
binding motifs in the β3 (Kittler et al., 2005) and γ2 subunits (Kittler et al., 2008) contain
phosphorylation sites for protein kinase A/C and Src kinase, respectively. The
phosphorylation of these sites dramatically decreases AP2 interaction with GABAARs.
Consistent with negative regulation of endocytosis of GABAARs by phosphorylation of
these sites, GABAAR subunit-derived phosphomimetic peptides containing these AP2
binding sites interfered with protein-protein interaction between AP-2 and GABAARs in
vitro and, upon infusion of the peptides into neurons via electrophysiological recording
pipette, enhanced the mIPSCs amplitude (Kittler et al., 2005, Kittler et al., 2008). Taken
together, phosphorylation of GABAARs at distinct AP2 binding sites promotes the cell
surface stability of GABAARs and thereby increases the strength of inhibitory synapses
(Kittler et al., 2005, Jacob et al., 2008, Kittler et al., 2008).
Endocytosed GABAARs have two fates; they can be rapidly recycled back to the
cell surface, or on a slower time scale, targeted for lysosomal degradation (Kittler et al.,
2004) (Fig. 1.2). Huntingtin-associated protein 1 (HAP1) is known to regulate the sorting
decision through a direct interaction with the intracellular loop of the β subunit of
GABAARs. HAP1 binding inhibits receptor degradation and facilitates receptor recycling
(Kittler et al., 2004), thereby enhancing GABAergic mIPSCs. A recent study in our
laboratory revealed that Calcium-Modulating cyclophilin Ligand (CAML) also regulates
recycling of endocytosed GABAARs (Yuan et al., 2008). CAML binds to the C-terminal
portion of the γ2 subunit including the C-terminal half of the major cytoplasmic domain
and the fourth transmembrane domain (Yuan et al., 2008), which has previously been
7
shown to be essential for normal accumulation of GABAARs at synapses (Alldred et al.,
2005). In CAML knock-out (KO) cortical neurons the recycling of endocytosed
GABAARs was significantly reduced as evidenced by surface biotinylation of cultured
neurons (Yuan et al., 2008). Consistent with this observation, knock-down of CAML
expression by a CAML-specific small-hairpin RNA (shRNA) in cortical neurons resulted
in reduced expression of postsynaptic GABAARs, reduced GABA-evoked whole cell
currents and deficits in GABAergic synaptic function (Yuan et al., 2008).
1.1.1.4. Postsynaptic clustering of GABAARs
GABAARs inserted into the plasma membrane can be subdivided into two groups: one is
localized in the extrasynaptic membrane and the other at synapses and these two receptor
pools mediate tonic and phasic inhibition, respectively (Fig. 1.2). Most receptors are first
delivered to extrasynaptic sites from where they are believed to reach synaptic sites by
lateral diffusion within or close to the plasma membrane (Bogdanov et al., 2006) (Fig.
1.2).
The localization at extrasynaptic or synaptic is at least in part determined by the
subunit composition of GABAARs. Most γ2 subunit containing GABAARs are synaptic
(Essrich et al., 1998), whereas receptors containing α5, β2, β3 and δ subunit
preferentially localize at extrasynaptic sites (Nusser et al., 1996, Fritschy et al., 1998,
Brickley et al., 1999, Brunig et al., 2002, Herd et al., 2008). In mutant mice lacking α5
subunit gene due to a chromosomal deletion, the γ2 subunit punctuate immunoreactivity
representing postsynaptic clustering were unaltered in the axon initial segment of
pyramidal cells assessed in brain sections. However, diffusive γ2 subunit-specific
staining was greatly reduced in these mutant mice, suggesting that the selective loss of
extrasynaptic receptors containing α5 and γ2 subunits (Fritschy et al., 1998).
The postsynaptic clustering of GABAARs is important for efficient inhibitory
synaptic transmission. The mechanisms that regulate postsynaptic clustering of
GABAARs are, however, still poorly understood. One protein, gephyrin, is a scaffold
protein localized at both glycinergic and GABAergic postsynaptic sites and has been
8
implicated in the regulation of postsynaptic clustering of GABAARs. Knock-out of
gephyrin genetically or by shRNA in neurons showed a reduced postsynaptic
accumulation of GABAARs containing α2 and γ2 subunits, whereas total expression of
these subunits was unchanged (Kneussel et al., 1999, Kneussel et al., 2001, Jacob et al.,
2005). However, GABAergic inhibitory postsynaptic current still exist in gephyrin KO
neurons (Levi et al., 2004). Interestingly, the receptor clusters on the cell surface were
significantly more mobile in the absence of gephyrin compared with those in control
neurons, suggesting a role of gephyrin in stabilization of GABAARs at synapse (Jacob et
al., 2005). On the other hand, γ2 subunit deficient mice showed significant reductions in
both synaptic GABAARs and gephyrin clusters. These studies indicated that γ2 subunit
and gephyrin are interdependent component that is critical for the postsynaptic clustering
of abundant subtypes of GABAARs (Essrich et al., 1998). In agreement with these early
findings, recent evidence indicates that GABAARs bound to gephyrin through a 10 amino
acid hydrophobic motif in the intracellular domain of the α2 subunit (Tretter et al., 2008).
Furthermore, the interaction between this motif and gephyrin was critical for the
postsynaptic clustering of GABAARs containing α2 subunit. The intracellular loops of
the γ2 and β3 subunits showed only minimal binding to gephyrin under the same
condition (Tretter et al., 2008).
Some studies indicated that there is a gephyrin-independent mechanism that
regulates the postsynaptic clustering of GABAARs (Kneussel et al., 2001, Levi et al.,
2004). In hippocampal cultures from gephyrin KO animals, α2 and γ2 subunit do cluster
at pyramidal synapses with a reduced level compared with control neurons (Levi et al.,
2004). In spinal cord from gephyrin KO mice, the punctate staining of α2, α3, β2/3 and
γ2 subunit was strikingly reduced, whereas clustering of the α1 and α5 subunit was
unaltered (Kneussel et al., 2001). These results are consistent with unaltered mIPSCs in
gephyrin KO cultures (Levi et al., 2004), and the notion that gephyrin interacts with the
α2 subunit but not or much less with other subunits (Tretter et al., 2008), suggesting the
existence of gephyrin-independent postsynaptic clustering mechanisms of GABAARs.
This gephyrin-independent mechanism might include a factor that bridges GABAARs and
cytoskeleton or other synapse-resident protein. To reveal this mechanism, further work is
9
needed. Taken together, currently available data indicate that the GABAAR subunit
composition and receptor-specific interactions with cytoskeletal proteins determine the
synaptic versus extrasynaptic distribution of different GABAAR subtypes.
1.1.2. Neuroligins
1.1.2.1. Cell adhesion molecules in nervous system
The nervous system is highly complex yet remarkably well organized. In the human brain,
each of the 1011 nerve cells establishes average of 1000-10000 contacts with other cells.
The resulting 1015 contacts are essential for all brain functions from motor control to
emotional and cognitive behavior. The building of this sophisticated network is mediated
by the spatially and temporally controlled expression of selective cell adhesion molecules
that are differentially expressed on different neural cell surfaces. Although the proteins
that govern the selective adhesion in nervous system are not yet fully determined, many
adhesion protein families have been identified and studied in some detail (reviewed in
(Benson et al., 2001, Missler, 2003, Yamagata et al., 2003, Shapiro et al., 2007)). In
addition to the differences between adhesion family proteins, the diversification of
adhesion molecule is generated by a alternative splicing (Missler and Sudhof, 1998,
Schmucker et al., 2000). This diversity is believed to enable to establish the extraordinary
network of nervous system. To date, four families of adhesion protein have been
identified in nervous system: (a) cadherins, (b) neuronal cell adhesion molecule (NCAM),
(c) integrins, and (d) the neurexin/neuroligin system.
It has been suggested that synapse formation between specific types of neurons
involves heterophilic transsynaptic signaling (Brose, 1999). In presynaptic terminal, a
complex membrane trafficking machinery serves to regulate neurotransmitter secretion.
On the other hand, postsynaptic structures are specialized for modulation of signal
transduction, containing scaffold proteins and ion channels. Although the homophilic cell
adhesion molecule may play a critical role in synaptic recognition and adhesion,
heterophilic cell adhesion has been postulated to explain the high specificity of
synaptogenesis (Brose, 1999). To date, the neurexin/neuroligin (NL) system represents
the only known heterophilic cell adhesion system present at synapses. Rapidly emerging
10
data suggest that these two protein families are critically important for organizing
molecules for excitatory glutamatergic and inhibitory GABAergic synapses in
mammalian brain as described below.
1.1.2.2. Structure, localization and function of neurexin and neuroligin
There are three neurexin genes in mammals. Each of these genes has two different
promoters, generating a larger α-neurexin and a shorter β-neurexin (Tabuchi and Sudhof,
2002). The diversity of neurexins is also multiplied by an alternative splicing at five sites
(Tabuchi and Sudhof, 2002). In contrast to β-neurexins containing only one extracellular
LNS domain (laminin, neurexin, sex-hormone binding protein domain), α-neurexins
contain six extracellular LNS domains with three EGF-like domains (Missler and Sudhof,
1998). At the carboxy terminus, α- and β- neurexin contain the same transmembrane
domain and short intracellular tail containing PDZ domain binding site (Missler and
Sudhof, 1998). With in situ hybridization, neurexins of six major forms showed fairly
broad overlapping expression patterns in brain (Ullrich et al., 1995). Neurexin was
originally found as a binding protein of α-latroroxin, a component of black widow spider
venom, which triggers a massive neurotransmitter release from presynaptic terminals.
The presynaptic localization of neurexin was also confirmed by antibody labeling
(Ushkaryov et al., 1992, Dean et al., 2003). A recent study using immunoelectron
microscopy, however, revealed the pre- and postsynaptic localization of neurexin
(Taniguchi et al., 2007). This cis-expressed neurexin inactivated neuroligin in the
postsynapse and promoted destabilization of synapses, suggesting an additional role of
neurexin in silencing the activity of synaptic adhesion molecules (Taniguchi et al., 2007).
NLs are encoded by four genes in mice and widely expressed in brains and in
some other tissues (Ichtchenko et al., 1996, Philibert et al., 2000). NLs are type I
transmembrane proteins that include a large extracellular domain, a single
transmembrane domain and a C-terminal cytoplasmic domain containing a PDZ
recognition sequence (Missler and Sudhof, 1998). The major extracellular domain is
homologous to acetylcholinesterase (AChE) and mediates neurexin binding in Ca2+-
dependent manner, but lacks cholinesterase activity. This AChE domain is also required
11
for NLs homo-multimerization and its activity in synaptogenesis (Dean et al., 2003). NL1
contains two alternative splice sites (A and B) in the extracellular domain. NL1AB, the
most abundant splice variant of NL1 contains insertions at both splice sites A and B and
is localized selectively at the postsynaptic membranes of glutamatergic excitatory
synapses (Song et al., 1999). The splice site A but not site B of NL1 is present also in
NL2. The major isoform of NL2 with an insertion at site A, NL2A is almost exclusively
found at GABAergic synapses (Graf et al., 2004, Varoqueaux et al., 2004, Chih et al.,
2005, Chih et al., 2006) (Fig. 1.2). Hereafter, NL1AB and NL2A are referred to as NL1
and NL2, respectively. The intense studies on neurexin/NLs system in synaptogenesis
were triggered by the findings that neuroligins presented on the surface of fibroblast cells
induced the formation of presynaptic terminals in contacting axons (Scheiffele et al.,
2000, Graf et al., 2004). In addition, the Ca2+-dependent adhesion between β-neurexin
and NLs is required for this synaptogenesis activity (Dean et al., 2003). Strikingly, study
using these hemisynapses showed a miniature excitatory postsynaptic current (mEPSC)-
like activity in HEK293 cells that were expressing NLs and glutamate receptors (GluRs)
and cocultured with neurons (Fu et al., 2003, Sara et al., 2005). In neurons, NL1
overexpression enhanced excitatory synapse numbers which is consistent with the
increased amplitude and frequency of mEPSCs, as well as that of mIPSC with lesser
extent (Prange et al., 2004, Chih et al., 2005, Levinson and El-Husseini, 2005a, Nam and
Chen, 2005). In addition, complement phenotypes were shown in acute hippocampal
slices from NL1 KO mice (Chubykin et al., 2007), suggesting that observation in the
overexpression experiments indicate physiological roles of NL1. In contrast, current
studies strongly suggested that NL2 plays a critical role at GABAergic synapses,
consistent with that NL2 is exclusively localized at inhibitory synapses in the CNS
(Varoqueaux et al., 2004). Although NL2 overexpression induced the formation of both
glutamatergic and GABAergic terminals (Chih et al., 2005, Levinson and El-Husseini,
2005a), at functional level it selectively increased inhibitory, but excitatory, synaptic
responses (Chubykin et al., 2007). Accordingly, in acute cortical slices from NL2 KO
mice a deficit of neurotransmission was observed selectively at GABAergic but
glutamatergic neurons (Chubykin et al., 2007). Parallel morphological experiments
12
showed that the number of inhibitory synapses was selectively decreased in NL2 KO
mice. These studies strongly support an important function of NL2 specifically at
GABAergic synapses (Chubykin et al., 2007).
Structural and biochemical analyses of the β-neurexin LNS domain revealed that
the presence or absence of alternatively spliced residues at splice site 4 (S4) in the LNS
domain decide the ability of β-neurexin to cluster different types of NLs (Graf et al.,
2004, Graf et al., 2006). Addition of the S4 insert into neurexin 1β selectively reduced
the clustering of NL1, 3, 4 and the glutamatergic postsynaptic protein postsynaptic
density 95 (PSD-95), whereas the clustering of NL2 and GABAergic postsynaptic protein
gephyrin was unaffected and remained strong (Graf et al., 2006). Furthermore, the
insertion of S4 reduced the affinity of β-neurexin with NL1 and NL4, but maintained
high affinity binding to NL2 and NL3 (Graf et al., 2006). In addition to preferential
induction of GABAergic synapses by presynaptic neurexin 1β containing the S4 insert, α
neurexins also preferentially interact with NL2, rather than NL1 and contribute
exclusively to GABAergic and not glutamatergic synapses (Kang et al., 2008). Thus,
different binding affinities of alternatively spliced isoforms of neurexins and neuroligins
play an instructive role in establishment of different types of synapses. However, it
apparently not only be a mechanism as observed normal synaptic contact density in triple
KO mice of NL1, 2 and 3 (Varoqueaux et al., 2006). A recent study showed that the
effects of overexpression of either NL1 or NL2 require synaptic signaling verified by
using pharmacological inhibition (Chubykin et al., 2007). Therefore, the neuronal activity
might also a key component to specify the types of synapses in response to distinct NLs.
1.2. Protein palmitoylation
1.2.1. Palmitoylation in general
Protein lipid modifications have been thought to facilitate protein association with
membrane by exerting hydrophobicity. Four types of lipid modification have been
described to date: myristoylation, isoprenylation, palmitoylation and
glycosylphosphatidylinositol (GPI) modification. N-myristoylation refers to covalent
13
attachment of the 14-carbon saturated fatty acid myristoylate to an N-terminal glycine
residue via amid linkage (Resh, 1999). Isoprenylation is an attachment of either 15-
carbon farnesyl or 20-carbon geranylgeranyl isoprenoids to a C-terminal cysteine residue
(Zhang and Casey, 1996). Palmitoylation exists in several variations and includes
mechanisms of co-translational acylation or transamidation of N-terminal Gly or Cys
residues or posttranslational acylation of Cys residues to thioesters of palmitic acid
virtually anywhere in the polypeptide chain (Smotrys and Linder, 2004, Greaves and
Chamberlain, 2007). All three lipid modifications occur in the cytoplasm or on the
cytoplasmic face of vesicular membranes (Resh, 1999, Nadolski and Linder, 2007).
In comparison to myristoylation and N-palmitoylation, which occur co-
translationally and appear to be permanent, S-palmitoylation (hereafter referred to simply
as palmitoylation) occurs post-translationally and is reversible; thereby allowing a protein
to undergo repeated palmitoylation-depalmitoylation cycles (Smotrys and Linder, 2004,
Resh, 2006). The palmitoylation status of a protein may be changed repeatedly and in
response to extracellular signals (Nadolski and Linder, 2007). Importantly, specific
physiological stimuli have been suggested to dynamically alter protein-palmitoylation
levels, a mechanism that is thought to contribute to synaptic plasticity (el-Husseini Ael
and Bredt, 2002, Hayashi et al., 2005).
Recent studies revealed several distinct functions of palmitoylation. First,
palmitoylation can increase the affinity of a protein for membranes. For example, small
GTPases N-Ras and H-Ras can be palmitoylated at one or two cysteines, respectively, a
mechanism that determines their differential cellular localization. Dually palmitoylated
H-Ras accumulates to a greater extent at the plasma membrane than the
monopalmitoylated N-Ras, which is localized at the Golgi. The distinct distribution of
these two proteins is critical for their different functions (Mor and Philips, 2006). Second,
palmitoylation can prevent ubiquitination of proteins that are subject to both types of
posttranslational modification. For example, the yeast DHHC protein Swf1 palmitoylates
the yeast SNARE protein Tlg1. The palmitoylated Tlg1 is retained on trans-Golgi
network/endosome membranes and thereby protected from ubiquitination and
degradation by the ubiquitin proteasome system (UPS) (Valdez-Taubas and Pelham,
14
2005). Third, palmitoylation interferes with protein-protein interactions, as discussed
further below. At last, palmitoylation may affect the conformation of proteins. For
example, palmitoylation of the Wnt effector signaling protein LRP6 at a
juxtamembraneous cysteine has been proposed to result in extension and tilting of the
transmembrane domain (Abrami et al., 2008). Deficit in the palmitoylation of these
cysteines appears to lead to a hydrophobic mismatch, followed by ER retention by the ER
quality control system (Abrami et al., 2008). Similarly, it has also been hypothesized that
palmitoylation might relieve hydrophobic mismatch by leading the protein into the
membrane domain with appropriate thickness for the TM domains of proteins (de
Planque and Killian, 2003, Kandasamy and Larson, 2006, Greaves and Chamberlain,
2007). Cholesterol composition is a key player in controlling bilayer thickness (Nezil and
Bloom, 1992). Palmitoylation might facilitate to translocate proteins harboring thicker
TM domain to membrane microdomains such as cholesterol-rich lipid rafts by lateral
movement (Greaves and Chamberlain, 2007). Thus, palmitoylation is thought to regulate
protein localization, trafficking and stability through diverse mechanisms.
1.2.2. Enzymes involved in palmitoylation
To date, proteins that belong to three different protein families have been reported as
palmitoyl acyltransferases: the membrane-bound O-acyltransferase (MBOAT) family
(Hofmann, 2000), the lecithin retinol acyl transferase (LRAT) family and the DHHC
family (Bartels et al., 1999, Lobo et al., 2002).
The MBOAT family of proteins possess a short but significant conserved
sequence including histidine residue that is a presumptive active site (Hofmann, 2000).
Distinct MBOAT proteins catalyze fatty acid transfer not only to proteins, but also to
cholesterol, alcohols or diacylglycerol (Hofmann, 2000). Only one protein, Hhat/Rasp
(aka, skinny hedgehog or sightless), has been identified and confirmed biochemically as a
palmitoyl acyltransferase that mediates N-palmitoylation of proteins (Chamoun et al.,
2001, Lee and Treisman, 2001, Micchelli et al., 2002, Tanaka et al., 2002). Porcupine
(Porc) is also an MBOAT member and has been speculated as a palmitoyl acyltransferase
of Wingless (Wg) due to the fact that Porc binds to the N-terminus of Wg containing
15
putative palmitoylation sites (Tanaka et al., 2002). However, further analyses are required
to establish this mechanism as fact. LRAT palmitoylates a retinoid isomerase, retinal
pigment epithelium-specific protein 65kDa (RPE65), required for the synthesis of 11-cis-
retinal (Xue et al., 2004). The so-called palmitoylation switch hypothesis suggests that
palmitoylation of RPE65 increase its affinity to membrane, and hence to its substrate all-
trans retinyl ester (Xue et al., 2004). However, a recent study showed that palmitoylation
of RPE65 by LRAT is not required for its association with membrane (Jin et al., 2007).
Thus, the function of palmitoylation by LRAT remains to be elucidated.
Although palmitoylation was first described three decades ago (Schmidt et al., 1979,
Magee and Courtneidge, 1985), the molecular machinery that carries out palmitoylation
was only recently identified, using yeast genetics (Bartels et al., 1999, Lobo et al., 2002,
Roth et al., 2002). Erf2 and Akr1 were isolated as the first protein acyltransferases
(PATs) for Ras and yeast casein kinase 2 (Yck2), respectively (Bartels et al., 1999, Lobo
et al., 2002, Roth et al., 2002). Both proteins contain the Asp-His-His-Cys (DHHC) motif
embedded in a cysteine-rich domain (CRD) that is critical for PAT activity (Lobo et al.,
2002). These findings shed light on the DHHC-CRD family of proteins and indicated that
they might function as palmitoyl acyl-transferases (PATs). By palmitoyl-proteome
analyses of yeast cells whose six genes encoding DHHC-CRD family proteins had been
mutated, it was shown that DHHC family proteins are responsible for most of the
palmitoylation in these cells (Roth et al., 2006). So far, 25 different DHHC genes have
been identified in the mammalian genome (Mitchell et al., 2006) (our unpublished
results) (Fig. 1.3). Heterologous expression of these genes in HEK 293T cells was used to
test their ability to palmitoylate various putative substrates. These experiments suggested
that some of these enzymes (DHHC3, 7) have relatively broad substrate specificity, while
others were able to palmitoylate only one substrate (DHHC9, 17, 18) or none at all
(Fukata et al., 2004, Fang et al., 2006, Fernandez-Hernando et al., 2006, Greaves et al.,
2008, Tsutsumi et al., 2008).
16
Figure 1.3. Phylogenic tree of 25 DHHC proteins. The phylogenic tree shows relative relationship of DHHC proteins. zDHHC3 is GODZ and its most close homologue is zDHHC7, SERZ-β.
17
In contrast to the large number of palmitoyl acyltransferases, only three
acylprotein thioesterases have been identified. Protein palmitoyl thioesterases I (PPT1)
and its homolog PPT2 are soluble lysosomal lipases that cleave fatty acids from cysteine
residues during protein degradation (Linder and Deschenes, 2003). PPT1 deacylates
cysteine thioesters from palmitoylated proteins, peptides and palmitoyl-cysteine, whereas
PPT2 has different substrate preference (Calero et al., 2003, Linder and Deschenes, 2003).
The importance of PPT1 is underscored by mutations in the PPT1 gene that cause
infantile neuronal ceroid lipofuscinosis (INCL), a severe neurodegenerative disorder
(Vesa et al., 1995). The phenotype of this disorder includes accumulation of fatty acid
modified proteins as granular osmiophilic deposits in CNS cells (Haltia et al., 1973).
While most cell types remain unaffected despite the presence of deposits, cortical
neurons are lost during the disease process. PPT2 mutant mice show a neurodegenerative
phenotype that is however distinct of that of PPT1 mutant mice or that found in INCL
patients (Gupta et al., 2003). The third enzyme, acylprotein thioesterase-1 (APT1),
removes palmitate from protein on the cytosolic surface of membrane (Duncan and
Gilman, 1998). Three palmitoylated proteins, α subunit of heterotrimeric G protein, H-
Ras and endothelial nitricoxide synthase, have been identified as substrates of APT1 in
vitro (Duncan and Gilman, 1998, Yeh et al., 1999). However, the role of APT1 in vivo is
still poorly understood.
1.2.3. Palmitoylation of neural proteins
Many proteins in CNS are known to be palmitoylated and recent studies found that
neuronal protein palmitoylation plays an important role to regulate neural development
and synaptic function.
Palmitoylation of the growth associated protein (GAP43), which stimulates GTP
binding of the small G-protein G0, has been reported to be crucial for regulation of axon
growth (Strittmatter et al., 1990, Benowitz and Routtenberg, 1997). In particular,
palmitoylation of two cysteines in GAP43 is required for its plasma membrane
localization and thereby for its interaction with G0 (Sudo et al., 1992). A recent study
found that expression of a specific palmitoylated motif in GAP43 was sufficient to
18
increase the number of filopodia and the branching of dendrites and axons in neurons
(Gauthier-Campbell et al., 2004). These data suggest that palmitoylation of this motif
could create local changes in membrane tension and thereby trigger changes in axon
structure (Gauthier-Campbell et al., 2004). The presynaptic SNARE protein SNAP25,
which is required for neurotransmitter release, is also palmitoylated. However, in this
case palmitoylation is dispensable for membrane localization, but necessary for SNARE
complex disassembly (Washbourne et al., 2001). In contrast, palmitoylation of
synaptotagmin I, another protein component of the membrane fusion machinery,
contributes to its proper sorting to presynaptic terminals (Kang et al., 2004).
Palmitoylation is also thought to cooperate with other protein trafficking signals.
Glutamate decarboxylase 65 kDa (GAD65), one of two isoforms of the enzyme that
mediates the last step in the synthesis of GABA, has three different targeting signals
located in tandem at the N-terminus of GAD65: a Golgi targeting signal, a membrane
anchoring signal and a palmitoylation signal. The first two of these are required for Golgi
targeting of GAD65, whereas palmitoylation of GAD65 is required for trafficking from
the Golgi to GABAergic terminals (Kanaani et al., 2002, Kanaani et al., 2004, Kanaani et
al., 2008).
Several postsynaptic proteins are also known to be palmitoylated. One of the
interesting molecules is PSD95, a scaffold protein that is known to be required for
assembly of postsynaptic complexes containing cell adhesion molecules and glutamate
receptors at excitatory synapses. It binds directly to NMDA receptors (Cho et al., 1992,
Kornau et al., 1997) and indirectly with AMPA receptors (AMPARs) through stargazin
that binds to both AMPARs and PSD95 (Chen et al., 2000). Palmitoylation at the N-
terminus of PSD95 is critical for its postsynaptic targeting and multimerization (Craven
et al., 1999, El-Husseini et al., 2000, Christopherson et al., 2003, Xu et al., 2008).
AMPARs are palmitoylated at two cysteines in the TM2 domain and the intracellular C-
terminal region, respectively. Palmitoylation in TM2 domain is required for Golgi
targeting, which results in the reduction of surface expression level of AMPARs (Hayashi
et al., 2005). C-terminal palmitoylation interferes with binding of AMPARs to the
cytoskeleton-associated protein 4.1N which leads to enhancement of AMPAR
19
internalization (Hayashi et al., 2005). These studies suggested that depalmitoylated
AMPARs are stabilized at the cell surface, whereas palmitoylated receptors are subject to
internalization. Thus, trafficking of AMPARs is regulatd by two independent
mechanisms that involve palmitoylation of the receptor itself and a scaffold protein,
respectively.
Interestingly, the palmitoylation status of proteins can be dynamically regulated
by cellular stimuli. For example, the palmitoylation level of GAP43 is largely and
specifically reduced in adult compared to neonatal rat brain (Patterson and Skene, 1999).
Neuronal activity is also known to regulate the palmitoylation status of SNAP25 and
synaptotagmin I (Kang et al., 2004), PSD95 (El-Husseini Ael et al., 2002), indicating a
role of palmitoylation in activity-dependent synaptic plasticity (El-Husseini Ael et al.,
2002, Huang and El-Husseini, 2005). Thus, palmitoylation may be one of the critical
mechanisms to respond to environmental stimuli and modifies cellular response
accordingly.
The physiological significance of palmitoylation is underscored by the findings of
the association between schizophrenia and genetic variation in the 22q11 gene containing
ZDHHC8 (Murphy et al., 1999, Badner and Gershon, 2002, Chakravarti, 2002, Liu et al.,
2002, Mukai et al., 2004). Single-nucleotide polymorphisms (SNPs) found in intron 4 of
ZDHHC8 has been shown to associate with a familial form of this disease (Mukai et al.,
2004). However, the same mutation does not appear to be broadly associated with
schizophrenia (Otani et al., 2005, Liu et al., 2007). DHHC8 -/- mice show abnormal
fearfulness and rearing behavior, are resistant to the locomotor activating effect of a
psychomimetic NMDA blocker, and thus considered to constitute an animal model of
schizophrenia. In addition, both schizophrenia patients and DHHC8 KO mice show a
deficit in pre-pulse inhibition (PPI) (Mukai et al., 2004) (reviewed in (Braff et al., 2001)).
Besides DHHC8, a mutation affecting alternative splicing of the DHHC15 was found to
be associated with X-linked mental retardation (Mansouri et al., 2005). DHHC15 is a
close paralog of GODZ and SERZ-β and therefore of special interest in the context of
this thesis.
20
1.2.4. Palmitoylation of γ2 subunit of GABAARs and NL2
Through efforts to identify players that regulate the intracellular and postsynaptic
trafficking of GABAARs, our lab has identified Golgi-specific DHHC zinc finger protein
(GODZ; aka, DHHC3) and SERZ-β (aka, DHHC7) as interacting proteins of the γ2
subunit of GABAARs (Keller et al., 2004, Fang et al., 2006). Coexpression of the γ2
subunit with GODZ or SEZ-β but none of the other DHHC family proteins resulted in
palmitoylation of the γ2 subunit in heterologous cells. Furthermore, the native γ2 subunit
was palmitoylated in neurons (Keller et al., 2004). Two groups including our own
independently showed that palmitoylation was necessary for accumulation of GABAARs
at inhibitory synapses (Rathenberg et al., 2004, Fang et al., 2006). Inhibition of
palmitoylation with the non-specific inhibitor 2-bromopalmitate, or mutation of cysteine
residues representing putative palmitoylation sites of the γ2 subunit abolished
postsynaptic accumulation of γ2 subunit-containing GABAARs (Rathenberg et al., 2004).
Knockdown of GODZ by dominant negative GODZ or shRNA also suggested that
GODZ plays a critical role in normal postsynaptic trafficking of γ2 subunit containing
GABAARs and normal GABAergic inhibitory transmission (Fang et al., 2006).
Surprisingly, In addition to these postsynaptic effects, postsynaptic GODZ deficits also
resulted in indirect and selective deficits in GABAergic innervation (Fang et al., 2006).
Heterologous expression assays indicate that, in addition to the γ2 subunit,
GODZ also palmitoylates diverse other proteins including the Gs-protein α subunit,
SNAP25, PSD-95 (Fukata et al., 2004), the GluR1-4 subunits of AMPARs (Hayashi et al.,
2005) and the DnaJ-family chaperon, cysteine-string protein (Greaves et al., 2008). PSD-
95 and AMPARs are of interest because the palmitoylation status of these proteins has
been implicated in functional plasticity of excitatory synapses (el-Husseini Ael and Bredt,
2002, Fukata et al., 2004, Hayashi et al., 2005). However, amplitude and frequency of
mEPSCs in GODZ deficit neurons were unaffected, suggesting that GODZ is dispensable
for glutamatergic transmission (Fang et al., 2006). Moreover, recent evidence indicates
that palmitoylation of PSD-95 is dispensable for LTP and LTD of excitatory synapses
analyzed in brain slices (Xu et al., 2008). Finally, recent experiments in our laboratory
have identified NL2 as a selective substrate of GODZ and SERZ-β in heterologous cells
21
(Shi et al, unpublished results). GODZ shRNA transfected neurons showed deficits in
postsynaptic accumulation of NL2 (Cheng Fang et al, unpublished results), suggesting
that deficits in innervation observed in GODZ deficient neurons may reflect reduced
palmitoylation and deficits in trafficking of NL2, rather than or in addition to GABAARs.
Interestingly, immunofluorescent analysis of NL2 in γ2 subunit KO cultures revealed a
striking loss of NL2 from GABAAR deficient synapse, and indicated that GABAARs are
part of a stable complex that includes NL2 and perhaps other proteins (Melissa Alldred,
unpublished results). These preliminary data suggest that the γ2 subunit is required for
postsynaptic clustering of NL2-GABAAR complexes and that normal trafficking of this
complex requires GODZ.
1.3. Aim of study
Part I:
As a first aim of this PhD thesis, I generated global and conditional KO mice of GODZ
and SERZ-β towards functional analysis of these two enzymes in vivo. To explore the
overall deficits, several behavioral paradigms were utilized to test on GODZ global KO
mice. The effects of GODZ/SERZ-β KO at the cellular level were also assessed. In
particular, this thesis study focused on the effects of GODZ/SERZ-β KO on the
intracellular trafficking and postsynaptic clustering of γ2 subunit of GABAARs. In
addition, the functional effects on the GABAergic synapse was analyzed by patch
clumping electrophysiology. In particular this included:
(1) Generation of global and conditional mouse KO alleles of GODZ and SERZ-β in
mice
(2) Confirmation of gene KOs using three independent methods
(3) Analyses of the effects of GODZ and SERZ-β KO on the surface expression of
GABAARs and NL2
(4) Analyses of the effects of GODZ and SERZ-β KO on GABAergic synapse
formation and function
(5) Characterization of behavioral phenotypes of GODZ KO mice
22
Part II:
As a second aim of my thesis I further assessed the role of the γ2 subunit for surface
expression of other GABAAR subunits, as well as NL2.
23
CHAPTER TWO
MATERIALS AND METHODS 2.1. Gene Targeting
2.1.1. Overall strategies of generation of conditional KO mice
Gene targeting has been widely used to manipulate genomic modification to mutate,
delete or insert genes in many different loci to assess phenotypes of these specific
genetic modification in the organisms. Global KO technique allows generation and
analyses of animals that carry deletions in genomic allele of interest in all cells. However,
such gene deletion in regionally/temporally unrestricted way might cause to severe
developmental defects or lethal phenotype or to the unspecific effects on the other tissues.
To overcome these problems, cell type- or temporal-restricted (conditional) gene KO
technique is well established with Cre-loxP system that was originally from
bacteriophage P1 (Lakso et al., 1992, Orban et al., 1992, Gu et al., 1994). Cre is a
member of λ integrase superfamily that cleave DNA at specific recognition sequence,
locus of cross-over in P1 (loxP), and ligate it to the other cleaved end at the second
recognition site, resulting in a deletion of the DNA fragment in between these two
recognition sites (Argos et al., 1986). The genomic DNA modified with loxP sequences
at the region to be knocked out (called a “floxed gene”) is excised upon the Cre
expression that is controlled by a cell type- or temporal-specific transcriptional promoter.
Three different Cre transgenic lines have been used in our lab: (1) CaMKIICre2834
(Schweizer et al., 2003), (2) Emx1-Cre (Iwasato et al., 2000) and (3) Esr1-Cre (Hayashi
and McMahon, 2002). Usage of these different Cre transgenic mice makes it possible to
investigate the role of gene product in different cell type and developmental point selectively.
(1) CaMKIICre2834 mouse harbors Cre transgene under the control of CaMKIIα promoter.
CaMKIICre2834-induced recombination is first detected in the hippocampus at postnatal
day (P) 17 and gradually increases to near adult levels by P34. In adult mouse,
CaMKIICre2834-induced recombination is limited to the forebrain with highest levels in
the CA1 region of the hippocampus and cerebral cortex (Schweizer et al., 2003).
24
(2) Emx1-Cre mouse harbors Cre transgene that is inserted in the Emx1 locus, thereby
expresses Cre in the developing brain as well as adult brain, especially in cerebral cortex,
olfactory bulb and hippocampus. The loxP recombination is detectable as early as
embryonic day (E) 11.5 (Iwasato et al., 2000).
(3) Esr1-Cre mouse harbors a transgene encoding 4-hydroxy tamoxifen (4-OH-TM)-
inducible ERTM-Cre (Hayashi and McMahon, 2002). Widespread expression of Cre-
ERTM has been shown in embryonic and adult mouse by injecting 4-OH-TM
intraperitoneally into pregnant mouse or adult mouse itself, respectively, and in cell
culture by treating them with 4-OH-TM (Hayashi and McMahon, 2002).
2.1.2. Construction of targeting vectors
2.1.2.1. GODZ targeting vector
A bacterial artificial chromosome (BAC) clone (RPCI-23-338G21) that contained the
entire GODZ gene was obtained from BACPAC Resources Center, Children’s hospital
Oakland Research Institute, California (Luscher lab plasmid number p666). A 4.9 kb Eco
RI fragment of p666 containing exons 3-5 of GODZ was subcloned into p681 to yield
p684. Plasmid 674 (generously provided by Dr. Cavener, Penn State University, Dept of
Biology) contains PGK-neo cassette (Soriano et al., 1991), which consists of neomycin
(neo) gene that is under the control of phosphoglycerate kinase I (PGK) promoter and
bovine growth hormone polyadenylation site, flanked by loxP sites. The Eco RI fragment
containing the floxed PGK-neo cassette was filled in by Klenow and cloned into the filled
in Nhe I site of p684, thereby restoring the Eco RI sites of the PGK neo cassette (named
as GRAMP1_5 kb/PGK-neo).
A 1.1 kb Nco I – Eco RI GODZ genomic fragment that is located just upstream of
the 4.9 kb Eco RI fragment containing exons 3-5 was amplified by PCR using p666 as a
template and NEco upper (5’-GACAG CGGCC GCCCT ACAGA ACCAG AGG-3’)
and NEco lower (5’-CTGGA ATTCT GGAAT G-3’) primers. Note that the Nco I site
was changed to a Not I site (underlined) in NEco upper primer. This Not I site later
served as a unique restriction site for linearization of the targeting vector. This PCR
product was cloned into the Not I/Eco RI sites of pBluescript II KS (Stratagene, La Jolla,
CA) to yield GRAMP1_1.1 kb/pBS. This plasmid was modified at the Eco RI site by
25
insertion of a double stranded oligonucleotide containing Bam HI site and a loxP site
flanked by Eco RI cohesive ends using the following oligonucleotides: Upper oligo: 5’-
AATTG GATCC GTCAT CTGTA TCATT GAAGC GTTCG GGAAC TTCGT ATAAT
GTATG CTATA CGAAG TTATG-3’, Lower oligo: 5’-AATTC ATAAC TTCGT
ATAGC ATACA TTATA CGAAG TTCCC GAACG CTTCA ATGAT ACAGA
TGACG GATCC-3’ (named as GRAMP1_1.1 kb/loxP/pBS).
The GRAMP1_5 kb/PGK-neo plasmid was linearized with Sal I in the vector
back bone and partially digested with Eco RI. The resulting 6.5 kb Eco RI fragment was
inserted into Eco RI site of GRAMP1_1.1 kb/loxP/pBS, generating GODZ targeting
vector (given the plasmid number p729). The direction of the insert was verified by
diagnostic restriction enzyme digestion and the sequence of the three loxP sites verified
by sequencing.
2.1.2.2. SERZ-β targeting vector
A BAC clone containing the entire SERZ-β gene BAC RP23-87F1 (aka p667) was
obtained from BACPAC Resources Center. A 2.9 kb Bgl II fragment of p667 containing
exon 2-4 was subcloned into the Bgl II site of a variant of pEGFP-N2 (aka p493,
Clontech, Mountain View, CA) in which the Bam HI site had been destroyed to produce
p688-BamHI. Analogously, a 6.6 kb Bgl II fragment of p667 containing exon 5 and 6 was
inserted into the Bam HI site of pBluescript (SK-) (named as GR2_6.6/pBS).
A double stranded oligonucleotide containing a Kpn I site and a loxP site flanked
by Bam HI cohesive sites were prepared by annealing the following oligonucleotides:
Upper oligo: 5’-GATCG GTACC GGACT CGTCA CTTGT TAGAT AGCAG GAACT
TCGTA TAATG TATGC TATAC GAAGT TATG-3’, Lower oligo: 5’-GATCC
ATAAC TTCGT ATAGC ATACA TTATAC GAAGT TCCTG CTATC TAACA
AGTGA CGAGT CCGGT ACC-3’. This double stranded oligonucleotide was inserted
into Bam HI site of p688-BamHI, resulting in the plasmid Bgl-loxP-Bgl_2.9/pEGFP-N2.
PCR was performed using GR2 ex3upp primer (5’-CAGAG GTTCT TCGTG
CTTTT CACC-3’) and GRAMP2ex6 lower primer (5’-GGCCT CCTTT CCTGG
TCCTC A-3’) and p667 as template, followed by Nhe I digestion to purify 737 bp Nhe I
26
fragment that was cloned into the Nhe I site of p493, resulting in NN/pEGFP-N2. The
direction of the insert was confirmed by digestion with Bgl II and Sac I, respectively. The
plasmid NN/pEGFP-N2 was linearized with Eco RI in the vector backbone and partially
digested by Nhe I to produce a 5.4 kb Eco RI-Nhe I fragment that was ligated with the 4.4
kb Nhe I–Eco RI fragment of GR2_6.6/pBS (named as NNE/pEGFP-N2). The Eco RI
fragment containing PGK-neo-loxP from p674 was filled in by Klenow and cloned into
filled in Bgl II site of NNE/pEGFP-N2, The resulting construct (N-PGKneo-NE/pEGFP-
N2) was confirmed by restriction digests and partial sequencing.
The SERZ-β targeting vector (p730) was generated by three way/partial ligation
of a 7.6 kb Eco RI-Nhe I fragment of Bgl-loxP-Bgl_2.9/ pEGFP-N2 (obtained by partial
digestion with Nhe I) with 2.4 and 4.4 kb Eco RI-Nhe I fragments of N-PGKneo-
NE/pEGFP-N2. Proper assembly of the fragments was verified by restriction enzyme
digestions and the three loxP sites of p730 confirmed by sequencing.
2.1.2.3. Partial digestion
A 2 µg of linearized plasmid was digested by 2 units of restriction enzyme in a total
volume of 20 µl. The reaction was stopped by adding 10 mM EDTA (pH 8.0) at 0, 1, 3, 5,
15, and 30 min and the DNA fragments analyzed by agarose gel.
2.1.2.4. Three way/partial ligation
Three DNA fragments to be ligated were purified from agarose gel. Two of the fragments
were first ligated using 1 µl of T4 Ligase in a total volume of 10 µl at room temperature
(RT) for 10 min and the ligated product corresponding to the proper size was gel purified.
The purified fragment was then ligated with the third DNA fragment using 1 µl of T4
ligase in a total volume of 10 µl at 16 °C overnight. Ligations were transformed into E.
Coli and screened by standard procedures.
27
2.1.3. Production of targeted embryonic stem cell clones
2.1.3.1. General growth conditions
Embryonic stem (ES) cell and feeder cells were grown in a 5% CO2 incubator at 37 °C.
To avoid undetected contamination of cells with mycoplasma no antibiotics were used.
Flasks and plates were treated with 0.1% gelatin for 2 hours and the gelatin aspirated
immediately before use. Unless used for DNA purification, ES cells were grown on the
feeder cells previously grown on gelatinized flasks and plates (2.0 x 104 cells/cm2).
2.1.3.2. Preparation of primary mouse embryonic fibroblasts (PMEFs)
To avoid cross contamination, each embryo was processed separately in all step to
prepare PMEFs. Embryonic day 14 (E14) mouse embryos were harvested aseptically and
collected in a Petri dish containing cold phosphate buffered saline (PBS) containing 5
mM glucose. Embryo’s head and liver were removed and the remaining tissue washed
with 70% ethanol and transferred into a fresh Petri dish containing 1 ml cold PBS per
embryo. The tissue was minced with sharp sterile tweezers (size 5), followed by passing
twice through a sterile 18 gauge needle. The tissue was transferred into a 15 ml tube and
let sediment for 2 min, and the excess liquid removed. To the tissue sediment, 1 ml of
trypsin-EDTA (Invitrogen, cat # 25300-054, stored at -20 °C) was added and the slurry
incubated in a bacterial shaker at 37 °C, 200 rpm for 20 min. The cell suspension was let
sit on the bench for 4 min and the supernatant transferred to a new tube containing one
volume of PMEF culture media with 10% fetal bovine serum (FBS) to inactivate the
trypsin. The remaining tissue sediment was once more trypsinized as above and pooled
with the first supernatant. The cells were collected by centrifugation at 160 x g for 5 min,
resuspended in PMEF culture media, and seeded in 75 cm2 tissue culture flasks. The
culture media was changed on the next day. After two days, the flasks were nearly
confluent and each T75 was passed into one T175 flask. The media was changed on the
next day and the cells in each T175 flask were passed again into three T175 flask. After
they were confluent, all cells were trypsinized, collected for irradiation with 4000 RAD
in a cobalt x-ray source in culture media at 3 x 106 cells/ml and kept on ice in the mean
time. One volume of ice-cold media containing 20% dimethyl sulfoxide (DMSO) was
28
added slowly while swelling the cell suspension. Cells were aliquoted into NUNC
CryoTubes (1 ml, 1.5 x 106 cells/ml) and aliquots wrapped with babble wrap and placed
in a styropore box in –80 °C overnight, then transferred into liquid nitrogen.
2.1.3.3. Linearization of targeting vector DNA
To transfect into ES cells, the GODZ and SERZ-β targeting vectors (p729 and 730) were
linearized with Not I and Bgl II, respectively, purified by phenol extraction, precipitated
with isopropanol and washed with 70% ethanol. The air dried vector DNA was dissolved
in sterile Tris-EDTA (TE) buffer (10 mM Tris-Cl pH8.0, 0.5 mM EDTA pH 8.0).
2.1.3.4. Transformation of ES cells by electroporation
The ES cell line BK4 (Ito et al., 1995) was provided by the Transgenic Mouse Facility,
University Park, at passage #8. The ES cells were grown in two T75 flasks to 50-80%
confluency and the complete media was changed 3 h prior to harvesting for
electroporation. Cells of two T75 flasks (4.0-10.0 x 107 cells) were trypsinized,
centrifuged (160 x g, 5 min) and resuspended in PBS. The cells were collected by
centrifugation again, resuspended in 1.6 ml of PBS and divided into two electroporation
cuvettes (BIORAD) with 4 mm electrode gap. Each sample was mixed with 10 µl of
linearized targeting vector DNA (1 µg/µl in TE) and incubated at RT for 5 min. The
suspension was then electroporated at RT using a BIORAD Gene pulser set at 500 µF,
240 V, resulting in a time constants of 6.8-8.7 msec. After recovering at RT for 5 min,
the cell suspensions were diluted with 20 ml complete media that was pre-warmed at
37 °C and distributed over six 6 cm Petri dishes using three different ES cell densities (2,
3, and 5 ml cell suspension/dish). After 24 h the culture media was replaced with fresh
media containing 350 µg/ml Geneticin (G418). The selection media was refreshed every
day. Colony screening was started on day 8-10 after electroporation.
2.1.3.5. Screening for targeted ES cell colonies
G418-resistant ES cell cultures grown in a 6 cm Petri dish were washed with PBS once
and the dish overlaid with 3 ml of fresh PBS. Under a dissecting microscope, ES cell
29
colonies were selected using a P20 micropipette adjusted to 2 µl and transferred into
individual wells of a 96-well round bottom plate containing 30 µl of PBS/well, with
leaving every other column empty to avoid cross contamination. After picking 48
colonies, 50 µl of trypsin-EDTA were added to each well and the plate incubated at 37 °C
for 5 min. The reaction was stopped with 200 µl/well of ES cell media containing 300
µg/ml of G418 and the colonies disrupted by pipetting up and down about 10 times. The
cell suspension was divided into three equal volumes (~90 µl) and transferred into
individual wells of two flat bottom 96-well plates that were previously seeded with
irradiated feeder cells (6.4 x 103 cells/well) and one 48-well plate that was only
gelatinized. The plates were incubated in the 5% CO2 incubator at 37 °C and 50% of the
media replaced every 24-48 hrs. Once the ES cells in the 96-well plates reached 50-80%
confluency, the wells were washed with PBS and the cells trypsinized (20 µl of trypsin-
EDTA/well, 3 min, 37 °C), followed by addition of 30 µl/well of media containing 300
µg/ml of G418 and trituration to disrupt the colonies. The cells were diluted with one
volume (50 µl/well) of media containing 20% DMSO and the plates wrapped with bubble
wrap and placed in a styropore box that was transferred to –80 °C for storage and further
expansion of colonies of interest at a later time.
The ES cells seeded in 48-well plates were grown to complete confluency and used to
purify genomic DNA for screening. The ES cells were washed with PBS, digested with
150 µl of trypsin-EDTA/well (37 °C, 5 min) and transferred into 1.5 ml tubes. The cells
were collected by centrifugation (160 x g, 5 min, 4 °C), washed by resuspension in PBS,
repelleted and lysed by the addition of 500 µl of lysis buffer (100 mM Tris-HCl pH 8.5, 5
mM EDTA, 0.2% SDS, 200 mM NaCl, 100 µg/ml proteinase K) and incubation at 55 °C
for 16 h. Genomic DNA was purified by sequential extraction with phenol and CHCl3,
and precipitation with isopropanol. The DNA pellet was washed with 70% ethanol, air-
dried and dissolved in 30 µl TE. To remove RNA, the DNA was treated with 1 µl RNase
A (1 mg/ml, 50 °C, 5 min) and stored at -20 °C.
30
For screening of positive clones by Southern hybridization, the purified genomic DNAs
were subjected to restriction digest overnight, followed by phenol extraction and
precipitation with ethanol. The DNA pellets were washed with 70% ethanol, air dried,
dissolved in 10 µl TE and separated on a 0.75% agarose gel (15 x 25 cm). The agarose
gel was then soaked in denaturing/transfer solution (0.5 M NaOH, 1.5 M NaCl) for 15
min and the DNA transferred to N+ membrane (Hybond-N+, GE Healthcare, Piscataway,
NJ) using capillary transfer. The membrane was laid on 3 M NaOAc pH5.2 poured on a
piece of Saran wrap with the DNA side facing up for 20 s, removed and air-dried on
3MM paper for 10 min. The DNA was crosslinked to the membrane using a Stratalinker
(standard conditions, Stratagene) that was baked at 80 °C for 1 h, re-hydrated with
distilled water, soaked in 2 x SSC and prehybridized at 65 °C for 4 h in hybridization
buffer (0.5 M sodium phosphate pH 7.2, 7% SDS, 1 mM EDTA) containing 1% BSA
(Sigma-Aldrich, cat # A7030) and 50 µg/ml herring sperm DNA (Invitrogen, denatured
at 95 °C for 2 min). The [32P]-labeled hybridization probe (Fig. 3.1) (approx. 2 x 106
cpm/ml final hybridization solution, random hexamer primed with Ready-To-GoTM DNA
labeling beads and [α32P]dCTP, 3000 Ci/mMol, GE Healthcare) was mixed with one
volume of formamide, denatured at 80 °C for 2 min and added to the pre-warmed
hybridization solution at 65 °C. The membrane was hybridized for 16 h at 65 °C, washed
with solution II (see below) four times for 5 min each at 55 °C, twice at 60-65 °C, twice
at 65 °C, and finally with solution III twice for 5 min each at 65 °C. The membrane was
placed in Saran wrap, sealed and exposed to a Phosphor Screen (Packard, Meriden, CT)
overnight and the signal detected using a Cyclone Phosphor imager (Packard, Meriden,
CT).
Solution II Solution III Solution IV
1 M NaPi pH 7.2 62.5 ml 25 ml 12.5 ml
20% SDS 25 ml 25 ml 25 ml
0.5 M EDTA pH 8.0 500 µl 500 µl 500 µl
Table 2.1: Wash solutions/500 ml
31
2.1.3.6. Expansion of positive clones
The positive ES cells identified using 5’ probe were expanded from stock ES cell library
in 96-well plates kept in –80 °C. The cells were thawed by placing the 96-well plates in
37 °C tissue culture incubator and transferred to a 15 ml tube containing 1 ml of media
with 300 µg/ml G418. The cells were collected by a centrifugation at 160 x g for 5 min,
resuspended in 1 ml of cell media containing 300 µg/ml G418, and seeded in two wells of
48-well plate at different cell densities. When ES cell colonies started appearing, media
was replaced to that without G418 (here after, media contained no G418). The cells that
had reached 60-70% confluency were passed into 3 wells of 24-well plate at different cell
densities. Once the ES cells reached 70% confluence, they were passed to T25 flasks and
subsequently to T75 flasks. At 70% confluence, cells were collected by trypsinization and
centrifugation and resuspended in media. A 90% of cells were diluted to 5 x 106 cells/ml
using media containing 20% DMSO and 15% FBS (DMSO final concentration should be
more than 10%), aliquoted in NUNC CryoTubes, frozen as described above overnight
and transferred into liquid nitrogen (Passage # 14). The 10% cells were seeded in a
gelatinized T25 flask and passed into a T75 flask for genomic DNA purification as
described above.
Media for PMSFs
1 x DMEM (Invitrogen, cat # 11965-092)
10% FBS
1% 200 mM L-Glu (100x) (Invitrogen, cat # 25030-081)
1% 10 mM MEM Non-Essential Amino Acids Solution (100x) (Invitrogen, cat # 11140-
050)
Media for ES cells
500 ml DMEM (Invitrogen, cat # 11965-092)
75 ml ES cell Qualified FBS (Invitrogen, cat # 16141)
6 ml 200 mM L-Glu (100 x) (Invitrogen, cat # 25030-081)
4 µl 2-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, cat # M7522)
1000 units/ml ESGRO (LIF) (Millipore, cat # ESG1107)
32
Other substances needed
Geneticin (G418) (Invitrogen, cat # 11811)
Trypsin-EDTA (Invitrogen, cat # 25300-047)
2.1.4. Blastocyst injection and generation of chimeric mice
One tube of the positive clone stock was thawed and seeded into a T75 flask. The media
was changed the next morning and two days later the cells were trypsinized and ¾ of the
cells frozen in three aliquots (passage #15). The remaining cells were seeded in three T25
flasks at three different densities. Flasks that had reached 70-80% confluency were used
for blastocyst injection by the Penn State transgenic facility.
2.1.5. Generation of floxed and knockout allele of GODZ and SERZ-β
The chimeric male mice with high chimerism were used to mate with female B6.FVB-
Tg(EIIa-cre)C5379Lmgd/J (here after EIIa-Cre) (The Jackson laboratory, Bar Harbor,
ME). The offspring was PCR genotyped using primer GZ1-GZ2, GZ1-g2.20t, and GZ1-
GZ4 for GODZ and primer SZ1-SZ6, SZ1-g2.20t and SZ5-SZ4 for SERZ-β. The mice
harboring KO allele (GODZ+/- and SERZ-β+/-, Fig. 3.1) were used to gain global knock-
out mice of GODZ and SERZ-β (GODZ-/- and SERZ-β-/-), assessed by PCR using
primer GZ1-4 and GZ3-GZ4 for GODZ and primer SZ5-SZ4 and SZ1-SZ6 for SERZ-β,
as well as Cre primers. Mice with mosaicism of fGODZ/+ and GODZ+/- or fSERZ-β/+
and SERZ-β+/- were mated with 129SvJ to segregate discrete recombinant allele. The
offspring was PCR genotyped using primer GZ1-GZ2 and GZ1-GZ4 for GODZ and
primer SZ1-SZ6 and SZ5-SZ4 for SERZ-β, as well as Cre primers. After the segregation,
homozygotic mice were obtained for fGODZ and fSERZ-β.
To gain double knock-out of GODZ and SERZ-β (DKO), GODZ-/-, cre-/- was
mated with SERZ-β-/-, cre-/-, followed by the mating of GODZ+/-, SERZ-β +/- x
GODZ+/-, SERZ-β +/-. The offspring was genotyped by PCR using primer GZ1-GZ4,
GZ3-GZ4, SZ5-SZ4 and SZ1-SZ6. These genotypings were further confirmed by
Southern blot analyses.
33
2.2. Genotyping
2.2.1. Genomic DNA isolation for genotyping
Genomic DNA was isolated from tail biopsies that was clipped 5 mm in length with a
framed scissor to ensure that there is no cross contamination of DNA between each
animal. The tail biopsies were digested in 500 µl of lysis buffer by incubating end-to-end
at 55 °C for 3 h. Undigested materials were removed by centrifugation at 15,000 x g for
10 min at RT. Genomic DNA was precipitated with isopropanol and dissolved in 500 µl
of TE by mixing end-to-end at 55 °C for 1 h. The genomic DNA was stored at -20 °C
until to use.
2.2.2. Primers and PCR
Optimal PCR primers were designed using OLIGO 4.0 program (Medprobe, Norway).
Sequences of all primers used for genotyping and the parameter of all PCR programs are
shown in Table 2.2 and 2.3, respectively. All genotyping PCRs were performed in 1 x
PCR buffer (50 mM KCl, 1.5 mM MgCl2, 10 mM Tris-HCl pH 9.0 at RT) in the presence
of 200 µM of each deoxynucleotide, 0.4 µM of each primer, 0.6 units of Taq DNA
polymerase and 1 µl of genomic DNA per 15 µl total volume of reaction. The PCR
products were analyzed on agarose gel.
34
Table 2.2: Primers for genotyping
Name Sequence
GZ1 primer 5’-GAGGCTTCAGAATAGTCTCTTAC-3’
GZ2 primer 5’-GCACCGCTTACAAACACTGAAAT-3’
GZ3 primer 5’-TGCCAGCCCAGCCTCATTTTATT-3’
GZ4 primer 5’-GCTCCCCAACTCTTACTTGAATG-3’
SZ1 primer 5’-TGAGCCAGGATGGATTTCAGACA-3’
SZ3 primer 5’-GAGGGGCTGAACTTGTTTTGTTG-3’
SZ4 primer 5’-TCCCCTGATGTATGCGAATGTCC-3’
SZ5 primer 5’-AACAGGTGCCTTTTGAATGTCAG-3’
SZ6 primer 5’-TGCCCTCGGACGCAGGAGATGAA-3’
g2.20t primer 5’-ATGCTCCAGACTGCCTTGGGAAAAGT-3’
Cre up 5’-AGATGTTCGCGATTATC-3’
Cre low 5’-AGCTACACCAGAGACGG-3’
35
Allele Locus detected Primer used
Annealing temp. and duration
Elongation temp. and duration
Number of cycles
GODZ Eco RI site where loxP site 1 was inserted (upstream of ex.3)
GZ1, GZ2
57 °C, 30 sec
65 °C, 120 sec
40
GODZ Eco RI – Nhe I fragment containing ex. 3 and ex. 4
GZ1, GZ4
55 °C, 30 sec
65 °C, 120 sec
40
GODZ Nhe I site where PGK-neo cassette was inserted (downstream of ex. 4)
GZ3, GZ4
53 °C, 30 sec
65 °C, 90 sec
40
GODZ Neo gene in GODZ allele GZ1, g2.20t
70 °C, 30 sec
70 °C, 120 sec
40
GODZ Neo gene in GODZ allele GZ3, g2.20t
60 °C, 30 sec
65 °C, 60 sec
40
SERZ-β Bam HI site where lop site 1 was inserted (upstream of ex. 3)
SZ1, SZ6
65 °C, 30 sec
65 °C, 90 sec
40
SERZ-β Bam HI –Bgl II fragment containing ex. 3 and ex. 4
SZ4, SZ5
56 °C, 30 sec
65 °C, 150 sec
40
SERZ-β Bgl II site where PGK-neo cassette was inserted (downstream of ex. 4)
SZ3, SZ4
55 °C, 30 sec
65 °C, 30 sec
40
SERZ-β Neo gene in SERZ-β allele SZ1, g2.20t
67 °C, 30 sec
65 °C, 140 sec
40
SERZ-β Neo gene in SERZ-β allele SZ3, g2.20t
60 °C, 30 sec
65 °C, 60 sec
40
Cre Cre gene Cre up, Cre low
55 °C, 40 sec
65 °C, 120 sec
36
Table 2.3: Parameters of genotyping PCR reactions
2.3. RT-PCR
All reagents used for RNA were made with DEPC-treated ddH2O.
2.3.1. RNA isolation
RNA was isolated from brain and liver of adult mice. A 1/8 size of brain and the same
size of liver were quickly frozen in 1.5 ml tubes on dry ice, homogenized in 500 µl
TRIZOL® Plus RNA Purification System (Invitrogen), followed by addition of another
500 µl TRIZOL® and incubation for 5 min at RT. CHCl3 (0.2 ml) was added and the
mixture shaken by hand for 15 s, then incubated for 2-3 min at RT. The samples were
36
centrifuged at 12,000 x g for 15 min at 4 °C and the upper phase that contained the RNA
was transferred to new 1.5 ml tubes. RNA was precipitated by incubation with
isopropanol at RT for 10 min and centrifugation at 12,000 x g for 10 min at 4 °C. The
RNA pellet was washed by vortexing with 1 ml of 70% ethanol and recollected by
centrifugation at 7,500 x g for 5 min at 4 °C. The RNA pellet was air-dried for 10 min at
RT and dissolved in 20 µl of RNase free ddH2O. The DNA was removed by incubation
with DNase I (Amplification Grade, Invitrogen) using 1 µg of RNA in a total volume of
10 µl (15 min, RT). The DNase I was inactivated by incubating with 1 µl of 25 mM
EDTA (65 °C, 10 min). Samples were analyzed by agarose gel electrophoresis to confirm
that the 18S and 28S rRNA were not degraded. The purified RNA was kept at -80 °C.
2.3.2. First-Strand cDNA Synthesis
The first-strand cDNA synthesis was carried out using SuperScript® III First-Strand
Synthesis System with Oligo(dT) as a primer (Invitrogen). All reagents and protocols
provided by the manufacture were used. Briefly, RNA/primer mixture was prepared with
0.1 µg of DNase I treated RNA, 1 µl of 50 µM Oligo(dT)20, 1 µl of dNTP mix in a total
volume of 10 µl. The RNA/primer mixture was incubated at 65 °C for 5 min and on ice
for 1 min. cDNA synthesis mixture was prepared with 2 µl 10xRT buffer, 4 µl of 25 mM
MgCl2, 2 µl of 0.1 M DTT, 1 µl of RNaseOUT (40 U/µl) and 1 µl of SuperScriptTM III
RT (200 U/µl) on ice. The cDNA synthesis mixture was added to the chilled RNA/primer
mixture and incubated at 50 °C for 50 min and the reaction terminated by incubating at
85 °C for 5 min. After chilled on ice, the samples were briefly span down and incubated
with 1 µl RNase H (37 °C, 20 min). The synthesized cDNA was kept at -20 °C.
2.3.3. PCR using synthesized cDNA
2.3.3.1. RTPCR
The same PCR protocol with 1 µl of cDNA solution was utilized as described above.
Primers and PCR conditions were listed in Table 2.4 and 2.5.
37
Name Annealing locus
(Forward/Reverse)
Sequence
GP1 GODZ exon1
(F)
5-’CTAGAATTCGCCACCATGCTTATCCCCACCCATC-3’
GP2 GODZ exon 4
(R)
5’-GCAATGCAGGAAGTGGAATC-3’
GP3 GODZ exon 6
(R)
5’-TCGATAAGCTTGCGACCACATACTGGTACGGGTC-3’
SP1 SERZ-β exon1
(F)
5’-CTAGAATTCGCCACCATGCAGCCGTCGGGAC-3’
SP2 SERZ-β exon4
(R)
5’-TGCCCTCGGACGCAGGAGATGAA-3’
SP3 SERZ-β exon6
(R)
5’-AGTCTCGAGCTCAGACAGAGAACTCGG-3’
Table 2.4: Primers for RTPCR
allele Locus
detected
Primers
used
Annealing
temp. and time
Elongation
temp. and time
Number of cycles
GODZ ex. 1 – ex. 4 GP1,
GP2
53 °C, 30 sec 65 °C, 1 min 40
GODZ ex. 1 – ex. 6 GP1,
GP6
50 °C, 30 sec 65 °C, 1 min 40
SERZ-β ex. 1 – ex. 4 SP1, SP2 55 °C, 30 sec 72 °C, 2 min 40
SERZ-β ex. 1 – ex. 6 SP1, SP6 55 °C, 30 sec 72 °C, 1 min 40
Table 2.5: Parameters for RTPCR reactions
38
2.3.3.2. Quantitive RTPCR (qRT-PCR)
The same PCR protocol with 1 µl of cDNA solution was utilized as described above,
except using VentR® DNA polymerase for ZDHHC21 as the bacterial genomic DNA
contamination in Taq DNA polymerase gives false-positive PCR products in ZDHHC21
PCR. Primers and PCR conditions were listed below (Table 2.6, 2.7). For normalization,
house keeper gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used (Abu
et al., 2008).
Name Sequence
dhhc15upper 5’-CTCGTCGTTCTCTGGTCCTACTA-3’
dhhc15lower 5’-GCCACAAAGAGGAGAAAGAGGAC-3’
dhhc21upper 5’-TGGGCTTGATTGTCTTTGTCTGG-3’
dhhc21lower 5’-AGTAAGCAGCTCAGTGTAGAAAC-3’
dhhc25upper 5’-AAAACCACGGCACATCTGAGCAC-3’
dhhc25lower 5’-CACCAGGAGGAGAAAGAGGATAG-3’
GAPDH F 5’- GTGGCAAAGTGGAGATTGTTGCC-3’
GAPDH R 5’- GATGATGACCCGTTTGGCTCC-3’
Table 2.6: Primers for qRT-PCR
allele Primers used Annealing temp. and
duration
Elongation temp. and
duraiton
Number of
cycles
Zdhhc15 dhhc15 upper,
dhhc15 lower
63 °C, 30 sec 65 °C, 1 min 30
Zdhhc21 dhhc21 upper,
dhhc21 lower
63 °C, 30 sec 65 °C, 1 min 35
Zdhhc25 dhhc25 upper,
dhhc25 lower
63 °C, 30 sec 65 °C, 1 min 40
GAPDH GAPDH F,
GAPDH R
57 °C, 30 sec 68°C, 1 min 30
Table 2.7: Parameters for qRT-PCR
39
2.4. Behavioral tests
Behavioral tests were performed at the age of 8-12 weeks, except the Prepulse inhibition
paradigm, which was performed at 22 weeks of age, during the first 6 h of the dark phase,
unless indicated otherwise. The behavior in all paradigms except an open field test and a
prepulse inhibition test were video recorded for subsequent off-line quantification.
2.4.1. Mouse husbandry
The cages were not changed until being weaned to avoid any stress. After being weaned,
males and females were separated and kept in Gang cages in separate room until tests.
2.4.2. Open Field test
Open field test was performed using the Omnitech Degiscananimal activity monitor
(Omnitech electronics, INC.), which provides 21 different parameters on mouse behavior
including horizontal activity, total travel distance, number of movements, vertical activity,
stereotypy, margin distance and center distance. To acclimatize to the testing room, mice
(in their home cages) were transferred to the testing room 3 days before the test. To
initiate the test, each mouse was placed into the left front corner of the empty ‘Open Field’
square arena (16.5” x 16.5” x 12”). The animal’s behavior was recorded over three
consecutive 5 min test intervals for a total of 15 min. After the test, the weight of each
mouse was recorded.
2.4.3. Elevated plus maze
Mice were placed on the center square on an elevated crossbar (30 cm per arm x 5 cm
wide x 40 cm tall) with two walled (20 cm, transparent) and two open arms. The behavior
was video recorded for 5 min. The number of entries and the total time spent on closed
and open arms were recorded (Crestani et al., 1999).
2.4.4. Free choice exploration
In a compartment containing 6 units (10 x 10 x 20 cm), mice were placed individually
into one segment comprising three interconnected units contained bedding and moist
40
foods. After 24 h of familiarization, the remaining three novel units were made accessible
and the retraction from entering the novel segment, the number of visits and total time
spent in familiar and novel units were recorded over the duration of 5 min (Crestani et al.,
1999).
2.4.5. Hot plate test
The test was performed in the last 3 h of the light phase. Pain reflexes in response to a
thermal stimulus were measured. A two litter glass beaker was placed in a crysterization
dish (190 x 10 cm) filled with 55 °C water. For the even temperature distribution, a
distance holder of 2 cm elevation made from a plastic 150 mm bacterial Petri plate was
inserted between the beaker and the dish. Mice were placed in the beaker for 30 s. The
latency to respond with a hindpaw lick was measured and the number of licking of
hindpaw was counted. The mouse was immediately removed from the hot plate and
returned to its home cage. Animals were not habituated to the apparatus prior to testing.
2.4.6. Prepulse Inhibition
Prepulse inhibition (PPI) was examined using an SR-LAB acoustic startle response
accelerometer box (San Diego Instruments, San Diego, CA) and SR-LAB software. The
sound level was calibrated against an external dB meter and the output from the
pizioelectric platform in millivolts was checked by oscilloscope. Animal enclosure (6”
(L) x 2 ¼” (ID)) within the test cabinet was appropriate size to reduce restraint stress
during startle sessions. The mice were first placed in the chamber for a 5 min acclimation
period with a 70 dB background noise. This was followed by a 15 min testing session in
which three trial types were randomly presented: 1) 120 dB 40 ms pulse (P-ALONE), 2)
the same 120 dB 40 ms pulse preceded 100 ms by one of three 20 ms prepulse that was 4,
8 or 12 dB above background (actual 74, 78 and 82 db), 3) no stimulus (NOSTIM). Each
test session consisted of four experimental blocks. The 1st and 4th blocks provided data
only on the magnitude of the normal startle response generated by four P-ALONE trials.
The change in startle magnitude between the 1st and 4th block served as the main
measure of startle habituation. The 2nd and 3rd blocks consisted of eight P-ALONE and
41
five prepulse trials of each type as described above, presented in pseudorandom order
with variable inter-trial intervals with average 15 s (range 7-23 s). NOSTIM
measurements were made randomly throughout all blocks. Mean NOSTIM values were
subtracted from all startle values by trial block to eliminate any contribution of non-
startle motor artifact to measures of startle magnitude. PPI was calculated as the percent
reduction in startle amplitude in the presence of the prepulse stimulus compared to the
amplitude in the absence of the prepulse stimulus according to the following formula:
[100-(100 x amplitude on prepulse trial/amplitude on P-ALONE trial)]. Thus, a large
"percent score" indicates a high degree of PPI, while a smaller "percent score" indicates
less PPI.
2.5. Tissue culture
2.5.1. Neuron culture
Cortical neuron cultures were prepared from E14 mouse embryos. On the first day,
embryos were beheaded and the heads kept in Hibernate E (BrainBitsTM, Springfield, IL)
supplemented with 2% B27 (Invitrogen) at 4 °C while the genotyping was performed. On
the second day, the cortices of appropriate genotype were dissected under a dissecting
microscope and collected in cold PBS containing 5 mM glucose. Tissue was incubated
with papain solution (PBS containing 10 mM glucose, 0.1% BSA, 0.5 mg/ml papain
(Sigma-Aldrich), and 10 µg/ml DNase I) for 15 min at RT and dissociated by trituration
with a fire-polished Pasteur pipette. The papain reaction was stopped with one volume of
DMEM/2% FBS/10 mM HEPES (Invitrogen) and the cell suspension span down at 160 x
g for 5 min at RT. For immunocytochemistry, palmitoylation assay and surface
biotinylation assay, the cells were re-suspended in MEM/2% FBS (MEM with 2 % FBS,
2 mM Glutamax I, 1 mM sodium pyruvate, 5.2 mg/ml glucose and 100 units/ml
penicillin/streptomycin) and diluted to 2.3 x 105 cells/ml. For immunocytochemistry,
cells were seeded on poly-L-lysine (Sigma-Aldrich, cat#1524) coated coverslips at 3.6 x
104 cells/cm2. For palmitoylation assay and surface biotinylation cells were seeded in
plates with poly-L-lysine (Sigma-Aldrich, cat #6282) coated 100 and 60 mm dishes,
respectively, at 4.1 x 104 cells/cm2. After 1 h, the media was replaced with fresh
42
MEM/10% FBS (MEM with 10 % FBS, 2 mM Glutamax I, 1 mM sodium pyruvate, 5.2
mg/ml glucose and 100 units/ml penicillin/streptomycin). After 24 h, the neuron cultures
were washed three times with PBS and the coverslips was flipped over onto glial feeder
cells grown in 35 mm plate with Neuron culture media (Neurobasal-A Media (NBA), 2%
B27, 2 mM Glutamax I, and 100 units/ml penicillin/streptomycin). The neuron cultures
grown on dishes were maintained in Neuron culture media without any glial feeder cells.
For electrophysiological analyses, the dissected neurons after papain treatment
were resuspended in MEM/5% FBS (MEM with 5 % FBS, 2% B27, 200 µg/ml of
NaHCO3, 20 mM D-glucose, 0.5 mM L-glutamine, and 25 units/ml of
penicillin/streptomycin) and seeded at 1.7 x 105 cells/cm2 onto poly-D-lysine (BD
Biosciences, cat#354210) coated colverslips covered by a monolayer of glial cells. The
neuron cultures were maintained in a 10% CO2/95% air incubator except the one for
electrophysiology that was maintained in a 5% CO2/95% air incubator.
2.5.2. Preparation of glial cells
Feeder cells were prepared from cortices of newborn rat pups as described previously
(Banker, 1998). Briefly, 2-3 day old postnatal rat pups were euthanized and their cortices
placed into cold Hanks’s Buffered Salt Solution (1X HBSS (Invitrogen), 1 mM HEPES,
100 units/ml penicillin/streptomycin). Cortices were minced and centrifuged and
supernatant removed, then the tissue was incubated in 1.5 ml each of 2.5% trypsin and
1% DNase for 15 min at 37 ºC. Tissue was triturated with a fire-polished Pasteur pipette
and passed through a 72 µm nylon filter. Cells were centrifuged, resuspended in glial
media (MEM, 0.6% glucose, 100 units/ml penicillin/streptomycin, 10% FBS, 2 mM
Glutamax I) and maintained at 37 ºC in 5% CO2/95% air incubator. After cells reached
confluency, they were split 1:3 and then grown to confluency again, after which they
were plated on 35-mm dishes. At 70-80% confluency, their growth was stopped with
poly-uridine (35 µg uridine, 15 µg fluoro-deoxyuridine per dish, Sigma-Aldrich) and
media changed to neuronal media.
43
2.6. Immunofluorescent analyses
2.6.1. Immunofluorescent staining of cultured cortical neurons
Cortical neurons at DIV18 were washed three times with PBS (136 mM NaCl, 1.75 mM
NaH2PO4, 8.25 mM Na2HPO4), fixed with 4% (w/v) paraformaldehyde in 0.15 M
phosphate buffer (pH 7.4) at RT for 15 min on a shaker and washed again three times.
Neurons were permeabilized for 5 min at RT with PBS containing 0.2% Triton X-100
and 10% normal donkey serum (Jackson ImmunoResearch, West Grove, PA), except for
GODZ immunostaining in which 0.15% saponin was used instead of Triton X-100 and
contained in all solutions hereafter. The neurons were washed three times with PBS and
incubated with affinity purified rabbit anti GODZ antibody (1: 200) (Keller et al., 2004),
rabbit anti SERZ-β antiserum (1: 200, gift of M.K. Skinner, Washington State University,
Pullman, WA)(Chaudhary and Skinner, 2002), mouse anti-gephyrin mAb 7a (gift of H.
Betz, Max Plank Institute, Frankfurt, Germany, 1: 2000), guinea pig anti-γ2 subunit (gift
of J.M. Fritschy, University of Zurich, Switzerland, 1:1500), rabbit anti-γ2 subunit
(generated in our lab, 1:200), rabbit anti-vesicular inhibitory amino acid transporter
(VIATT) (gift of B. Gasnier, Center Nationale de la Recherche Scientific, Paris, France,
1:5000) (Dumoulin et al., 1999) or mAb GAD-6 (0.5 µg/ml; Developmental Studies
Hybridoma Bank, University of Iowa, IA) in PBS containing 10% normal donkey serum
overnight at 4 °C. After washing with PBS (3 washes, 15 min/each, RT), AlexaFluor
488-conjugated goat anti-rabbit, AlexaFluor 647-conjugated donkey anti-mouse
(Molecular Probes, Eugene, OR), or Cy3 donkey anti-mouse or guinea pig (Jackson
ImmunoResearch) were applied (1:500) as appropriate in PBS containing 10% normal
donkey serum for 45 min at RT followed by repetition of wash steps. The coverslips were
mounted on slides with mounting solution (50% v/v glycerol, 50% 0.1 M NaHCO3
pH7.4) and stored at 4 °C until imaging.
2.6.2. Microspopic analysis
Fluorescent images were captured using a Zeiss Axiophot2 microscope equipped with a
40 x 1.3 NA objective and an ORCA-100 video camera linked to an OpenLab imaging
system (Improvision, Lexington, MA). Digital gray scale images were pseudo-colored
44
using green, red and blue for the sequentially recorded fluorescences. Images were
adjusted for contrast using OpenLab and assembled into figure palettes using Adobe
Photoshop.
2.6.3. Quantification of immunofluorescent staining
Immunofluorescent staining for synaptically clustered proteins were quantified as
described (Alldred et al., 2005). Cells innervated by GABAergic axons were selected
based on immunofluorescent staining for GAD or VIATT. Two properly innervated
dendritic segments of 40 µm in length were chosen from 8-59 cells in three to four
independent experiments. Immunoreactive puncta representing pre- or postsynaptic
clusters of proteins were automatically selected using OpenLab imaging software
(Improvision, Lexington, MA). Protein clusters were defined as immunofluorescent
puncta within the region of interest that exceeded a fluorescence intensity threshold that
was 2-fold greater than the diffuse fluorescence measured on the shaft of the same
dendrite and fit a target size range of 0.2 to 2 µm in diameter. To determine the
percentage of colocalization, the fraction of immunofluorescent puncta specific for one
protein that were maximally one pixel apart from the puncta of the other protein puncta
was counted in dendritic segments of 40 µm in length and their average size (area) was
recorded using Openlab and was then computed. Statistical comparisons were performed
using student’s T-test.
2.7. Western blot analyses
Protein samples were heated at 95 °C for 2 min and separated by 10% polyacrylamide gel
containing 0.1% SDS. After equilibration in the transfer buffer (39 mM glycine, 48 mM
Tris base, 0.04% SDS, 20% methanol) for 30 min at RT, protein was transferred to PVDF
membrane (activated by immersion in 100% methanol for 20 s and equilibrated with the
transfer buffer for 5 min) using a semidry blotter at 15 volts (Bio-Rad, Hercules, CA).
The membrane was then blocked with 5% nonfat dry milk (Carnation) in tris-buffered
saline with tween-20 (TBST) buffer (10 mM Tris-Cl pH 7.5, 150 mM NaCl, 0.5% Tween
20) for 2 h at RT and incubated with primary antibody in 5% nonfat dry milk in TBST
45
buffer for 2 h at RT or overnight at 4 °C. The membrane was washed with RIPEA buffer
(10 mM Tris-Cl pH7.5, 60 mM NaCl, 2 mM EDTA, 0.4% Triton X-100, 0.4% SDS,
0.4% deoxycholate) for 15 min, followed with TBST buffer (4 times, 10 min each, RT).
The membrane was re-blocked for 30 min at RT and incubated with appropriate
secondary antibody conjugated to horseradish peroxidase (HRP) (1:5000, Jackson
ImmunoResearch) for 2 h at RT. The membrane was washed as above with RIPEA and
TBST buffer and the signals were detected using ECL Plus Kit (GE Healthcare).
Primary antibody Source Dilution
Rabbit anti-NL2
antiserum
Given by N. Brose (Max-Planck-Institute for
Experimental Medicine, Göttingen, Germany)
(Varoqueaux et al., 2004)
1:2000
Rabbit
anti-GluR2/3
antibody
Millipore (cat # AB1506, Billerica, MA) 1:300-500
Mouse anti-
β-tubulin Isotype
I+II antibody
Sigma-Aldrich (cat # T8535, St. Louis, MO) 1:1000
BD17 Given by D. Benke (University of Zurich, Switzerland) 1:5000
Rabbit
anti-α1 antibody
Given by D. Benke (University of Zurich, Switzerland) 1:500
Rabbit
anti-γ2 antibody
Sigma-Aldrich (cat # G9919, St. Louis, MO) 1:500
Rabbit
anti-γ2 antibody
Generated in our lab by GenScript Corporation
(Piscataway, NJ)
1:200
Table 2.8: Primary Antibodies for Western blot analyses
2.8. Metabolic labeling of cortical neurons with 3H-palmitic acid
In all procedure using 3H-palmitic acid, pipette tips, polypropylene centrifuge tubes and
15 ml conical tubes were coated with SIGMACOAT® (Sigma-Aldrich), dried and rinsed
with ddH2O.
46
2.8.1. Concentration of 3H-palmitic acid
Palmitic acid, [9,10-3H(N)] (NEN-Perkin-Elmer, Wellesley, MA) was concentrated in the
Speed Vac® at low heat to approximately 300 mCi/ml. The concentrated 3H-palmitic
acid was stored in -20 °C.
2.8.2. Metabolic labeling
The media in cortical neuron culture was reduced to 5 ml and concentrated 3H-palmitic
acid diluted in a portion of media and added to neurons as such the final concentration
was 2 mCi/ml. The neurons were incubated with 3H-palmitic acid for 4 h under the
standard condition.
2.8.1. Protein extraction and immunoprecipitation of NL2
The culture media containing 3H-palmitic acid was removed and neurons were washed
with PBS three times. The neurons were scraped in a 1.3 ml SDS buffer (10mM Tris-HCl
pH 8.0 at 4 °C, 150 mM NaCl, 1% SDS, 1 mM EDTA pH 8.0, 1 mM PMSF, 1 µg/ml of
each leupeptin, aprotinin, pepstatin A and antipain) at RT, transferred to 15 ml conical
tubes and mixed with 2 ml of cold Triton buffer (the same buffer with SDS buffer except
containing 1% Triton X-100 instead of SDS). The neurons were sonicated by Sonifier
450 (Branson, Danbury, CT) approximately 2-3 s at 2.5 output control on constant pulse.
The protein extract was mixed with 7.1 ml of cold Triton buffer and divided into two 15
ml conical tubes: one was mixed with 3.5 µl of rabbit anti-NL2 antibody (rabbit #799,
gift of N. Brose, Max-Planck-Institute for Experimental Medicine, Göttingen, Germany)
and the other with 3.5 µg of control rabbit-IgG (Jackson ImmunoResearch). The sample
was mixed end-to-end for 16 h followed by 4 h with Protein A (Sigma-Aldrich, see below
for preparation) at 4 °C. The resins were precipitated by a centrifuge at 2,500 x g for 2
min at 4 °C and washed by mixing end-to-end with 3 ml NL2 IP wash buffer (50 mM
Tris-HCl pH 7.4 at 4 °C, 150 mM NaCl, 2 mM EDTA pH 8.0, 0.125% SDS, 0.875%
Triton X-100, 1 mM PMSF, 1 µg/ml of each leupeptin, aprotinin, pepstatin A and
antipain) (6 times, 15 min each, 4 °C) and 2 ml cold PBS contaning1 mM PMSF, 1
µg/ml of each leupeptin, aprotinin, pepstatin A and antipain (10 min, 4 °C). The resins
47
were transferred to 1.5 ml tubes and stored at this stage in – 80 °C or preceded to
immunoblotting and fluorography. The proteins were eluted from the beads by incubation
with 20 µl of 2 x SDS sample buffer (125 mM Tris–HCl pH 6.8, 20% glycerol, 4% SDS,
10 mM DTT and 0.002% bromophenol blue) for 2 min at 95°C. Note that SDS loading
buffer contains 5-10 mM of DTT of final concentration was used in this study due to the
fact the thioester bond is sensitive to reducing agents (Bizzozero, 1995).The samples
were then split into two aliquots and analyzed by SDS-PAGE on duplicate gels, with the
first gel containing 25% of each sample used for immunoblotting and the second gel
containing the rest of the samples processed for fluorography.
To prepare Protein A agarose resins for NL2 Immunoprecipitation, Protein A agarose
resins (60 µl of resin slurry/sample) were washed with 1 ml of cold PBS and spun down
at 2,500 x g for 2 min to remove supernatant. After washing 3 times, the resins were
mixed with BSA (30 µg/60 µl of Protein A resin) accompanied with restriction enzyme
(New England Biolabs, Ipswich, MA), and then with 1 ml of cold PBS. The resins were
precipitate by centrifugation as above and the supernatant removed.
2.8.2. Protein extraction and immunoprecipitation of the γ2 subunit of
GABAARs
Immunoprecipitation of the γ2 subunit from cultured cortical culture was described
previously (Keller et al., 2004). Briefly, cultured cortical neurons were scraped into PBS
and washed with PBS twice by centrifugation. The neurons were then resuspended in 10
mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.5% deoxycholate, 0.05% phosphatidylcholine, 1
mM benzamidine, 100 µg/ml bacitracin, 1 mM PMSF, 1 µg/ml of each leupeptin,
aprotinin, pepstatin A and antipain, sonicated briefly, and extracted on ice for 15 min.
Extracts were cleared by centrifugation at 10,000 x g for 5 min. For
immunoprecipitations, anti-α1 affinity resins and anti-γ2 affinity resins were generated
with rabbit anti-α1 antiserum (gift of Dr. D. Benke, University of Zurich, Switzerland) or
rabbit anti-γ2-antibody (gift of Dr. A. De Blas, University of Connecticut, CT),
respectively, via cross-linking to Protein A agarose beads as described below. Protein
48
extract was incubated with antibody-coated beads corresponding to 2–3 µg anti-α1
antiserum, or equivalent amounts of control rabbit-IgG, for 2 h at 4 °C. After incubation,
the beads were collected by centrifugation at 2,500 x g for 2 min and washed three times
for 15 min each at 4 °C in wash buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.2%
Triton X-100, 0.1% deoxycholate, 2 mM EDTA, 0.02% NaN3, 1 mM PMSF, 1 µg/ml of
each leupeptin, aprotinin, pepstatin A and antipain), followed by a 10 min wash at 4 °C in
PBS. The proteins were eluted from the beads and separated on SDS-PAGE as described
in NL2 immunoprecipitation.
For affinity resin preparation, all tubes and pipette tips were coated with SIGMACOAT®.
Protein A beads (100 µl slurry) were washed three times with 1 ml equilibration buffer
(0.02 M NaH2PO4, 0.15 M NaCl, pH8.0). Antiserum containing 200 µg of IgG or rabbit-
IgG as control was diluted in a total volume of 500 µl PBS and added to the equilibrated
beads. The sample was mixed end-to-end for 1 h at RT, washed twice with 1 ml 0.2 M
NaBO4 (pH 9.0) and resuspended in a final volume of l ml of 0.2 M NaBO4 (pH 9.0). A
crosslinker agent dimethyl pimelimidate (DMP) was added to a final concentration of 20
mM and mixed end-to-end for 30 min at RT. The reaction was terminated by washing the
resin with 1 ml 0.2 M ethanolamine (pH 8.0) and mixing end-to-end with fresh 0.2 M
ethanolamine (pH 8.0) for 2 h at RT. The resins were washed with PBS twice,
resuspended in a final total volume of 1 ml PBS containing 0.01% thimerosal and kept at
4 °C. To use the generated affinity resins, appropriate amount of resins were washed with
PBS three times and mixed with BSA. The resins were washed with PBS again and
precipitate by centrifugation as above to remove the supernatant.
2.8.3. Fluorography
The 3H-labeled sample was separated by SDS-PAGE and the SDS-PAGE gel fixed for 1
h in 10% glacial acetic acid/30% methanol followed by incubation in EN3HANCE
Autoradiography Enhancer (NEN-Perkin-Elmer, Wellesley, MA) for 1 h with gentle
agitation. After remove the EN3HANCE solution, an excess amount of cold ddH2O was
added to precipitate the fluorescent material inside the gel and incubated for only 30 min
49
for optimum results. The gel was placed onto a piece of blotting paper, dried under
vacuum for 2 h at 80 °C, and exposed directly to Kodak XAR film for 2 months at –
80 °C.
2.9. Surface Biotinylation assay
2.9.1. Surface biotinylation assay
A 2-3 6 cm dishes/genotype DIV14 (NL2 in γ2KO, γ2 subunit in DKO) or DIV20 (α1
and β2/3 subunits in γ2KO, NL2 in DKO) cortical neurons were processed at the same
time and analyzed on the same SDS-PAGE gel for later quantification analyses. Neurons
were incubated for 45 min with 100 µg/ml of media leupeptin, washed twice with 3 ml
cold PBS containing 2.5 mM CaCl2 and 1 mM MgCl2 (PBS/CaCl2/MgCl2) and incubated
with 1 mg/ml NHS-SS-biotin (EZ-LinkTM Sulfo-NHS-SS-biotin, Pierce, Rockford, IL) in
PBS/CaCl2/MgCl2 (freshly made immediately before use) for 20 min on ice in cold room.
The unreacted biotin was quenched by washing briefly once with cold 50 mM glycine in
PBS/CaCl2/MgCl2 followed by incubation with the same but fresh solution. After further
washes with 0.1% BSA (Sigma, cat# 7030) in PBS/CaCl2/MgCl2 three times, the neurons
were collected and lysed in cold 800 µl IP buffer (1% Triton X-100, 150 mM NaCl, 10
mM Tris–HCl pH 7.5, 1 mM EDTA pH 8.0, 1 mM phenylmethylsulfonyl fluoride
(PMSF), 1 µg/ml of each leupeptin, pepstatin A, antipain and aprotinin). Protein was
extracted for 15 min on ice and the extract cleared by centrifugation at 10,000 x g for 10
min at 4 °C. Protein concentration was determined using Bradford assay. A 200 µg of
protein extract was mixed end-to-end with 50 µl Immobilized NeutrAvidinTM protein
(Pierce) for 2 h in cold room. Samples were collected by centrifugation at 2,000 x g for 2
min at 4 °C, added 1 ml cold IP buffer to wash resins (repeat 3 times). The protein was
eluted in 10 µl 3 x SDS/β-mercaptoethanol loading buffer and processed for Western blot
analyses. The signals were detected by ECL Plus Kit (GE Healthcare) and exposed to
HyperfilmTM ECL film (GE Healthcare).
50
2.9.2. Quantification of surface biotinylation assay
The signals acquired from immunoblotting in surface biotinylation assay were scanned
by EPSON EXPRESSION 1600 (Epson, Long Beach, CA) and analyzed by ImageJ
1.38x software (http://rsb.info.nih.gov/ij), which gives an area x intensity value for each
signal. For the quantification of surface protein in mutant neuron cultures, the value of
each biotinylated protein was first normalized to the value determined for the same
protein or beta-tubulin in the corresponding unpurified sample. The average of these
values from two or three samples/genotype processed on the same SDS-gel was then
normalized to that the corresponding value of a WT sample processed in parallel on the
same gel. The final normalized values were averaged across three or more experiments
performed on different days. Statistical comparisons were performed using student’s T-
test.
2.10. Electrophysiology (collaboration with Xia Wu and Dr. Gong Chen)
Cortical neurons at DIV9-11 were used for electrophysiological analyses. Whole-cell
recording were performed by using Multiclamp 700A (Molecular Devices Corporation
(MDC), Sunnyvale, CA). Patch pipettes were pulled from borosilicate glass and fire-
polished to the final resistance of 4-6 MΩ. The recording chamber was continuously
perfused with a bath solution containing 128 mM NaCl, 30 mM D-glucose, 25 mM
HEPES, 5 mM KCl, 2 mM CaCl2, and 1mM MgCl2 (pH 7.3, adjusted using NaOH). Patch
pipettes were filled with 147 mM KCl, 5 mM disodium phosphocreatine, 2 mM EGTA,
10 mM HEPES, 2 mM MgATP, and 0.3 mM Na2GTP (pH 7.3, adjusted with KOH).
Series resistances were typically 10-20 MΩ. Data were acquired using PCLAMP 8
software, and sampled at 10 kHz and filtered at 1-2 kHz. To assess whole-cell currents,
pulses (20 s) of GABA (100 µM) or glutamate (500 µM) were applied through a glass
pipette with the pipette tip close to the cell soma and evoked currents were recorded with
the membrane potential clamped at -70 mV. Current peak amplitudes were measured
using Clampfit 9 software (MDC) to obtain GABAA or AMPA receptor-mediated whole
cell current (pA). Miniature IPSCs and mEPSCs were recorded in the presence of 0.5 µM
tetrodotoxin (TTX) and either 10 µM 6-cyano-7-nitoquinoxaline-2,3-dione (CNQX) or
51
20 µM bicucullin, respectively, and analyzed by MiniAnalysis software (Synaptosoft,
Decatur, GA). The statistic analyses were performed using Kolmogorov-Smirnov 2
sample test.
52
CHPTER THREE
RESULTS Part I: Analyses of the Functions of the Palmitoyl Transferases GODZ and SERZ-β
in Knock-out Mice
3.1.1. Introduction and aim of study
GODZ and SERZ-β are close homologs and the only members of the DHHC family of
PATs that can palmitoylate the γ2 subunit of GABAARs and NL2 in HEK293T cells. To
analyze GODZ and SERZ-β in vivo, corresponding KO alleles as well as conditional
pseudo WT/floxed alleles were generated and their phenotypes characterized in mice and
cultured neurons.
3.1.2. Generation of fGODZ and fSERZ-β mutant and global KO mice
3.1.2.1. Design and generation of targeting vector
In order to allow analyses of loss of function alleles of GODZ and SERZ-β even if the
corresponding homozygous mutations are lethal, the Cre-loxP system was used to
generate conditional KO mice for GODZ and SERZ-β. Both proteins contain an Asp-His-
His-Cys cysteine-rich domain (DHHC-CRD) that has been implicated in PAT function
(Lobo et al., 2002) (Roth et al., 2002) and is encoded by exon 3 in both cases. Therefore,
to ensure complete ablation of gene function, the targeting vectors were constructed in
such a way that exons 3 and 4 of each gene were flanked by direct loxP repeats. The
downstream loxP site was introduced in the form of a PGK-neorselection cassette that
was flanked (floxed) by loxP sites (Fig. 3.1). This scheme should allow sequential
excision of the PGK-neor gene and of exons 3/4 of GODZ/SERZ-β by Cre-mediated
recombination of different pairs of loxP sites in the modified gene loci.
53
Figure 3.1. Targeting strategy for GODZ and SERZ-β a, b, Representation of the targeted genomic regions, targeting vectors, mutated alleles resulting from homologous recombination, floxed alleles before and after Cre mediated deletion of the PGK-neor cassettes, and the mutated alleles for GODZ (a) and SERZ-β (b). Rectangles represent exons with the DHHC domain-containing exons 3 of each gene shown in white and the PGK-neor gene cassettes in gray. The three triangles in each targeting vector represent the loxP sites. The locations of the probes used for genomic Southern analyses and primers used for RT-PCR are indicated by white bars and arrowheads, respectively. In case of both GODZ and SERZ-β, exon 3 was targeted for deletion.
54
3.1.2.2. Screening of positive clones
ES cells [BK4, male, a subclone from E14TG2a derived from 129/OlaHsd mice, (Ito et
al., 1995, Simpson et al., 1997)] were transfected with the linearized targeting vector for
either GODZ or SERZ-β. The genomic DNA was isolated from 270 neomycin resistant
embryonic stem cell clones for GODZ and 384 clones for SERZ-β to screen for the
subset that had undergone proper homologous recombination by Southern blot. Using
hybridization probes that annealed to the genomic DNA upstream of the 5’ end of the
linearlized targeting vectors, six (GODZ) and one (SERZ-β) promising ES clones were
identified, respectively (Fig. 3.2). Homologous recombination of each targeting construct
with its respective genomic target was confirmed by Southern blot using 3’ probes that
mapped to regions downstream of the region present in the targeting construct. The
genomic regions of each gene containing the three loxP sites and exons 3-4 of four
GODZ and one SERZ-β targeted ES cell clone were amplified by PCR and the sequences
confirmed. All five clones were used for blastocyst injection by the Penn State
Transgenic Animal Facility. High-grade chimeric mice (21-41% of offsprings) were
obtained for all five clones (Fig. 3.3). Between 50-83% of chimeric mice were males,
consistent with being derived mainly from targeted ES cells.
3.1.2.3. Establishing the fGODZ, fSERZ-β GODZ-/-, SERZ-β-/- and DKO mouse
lines
Promoter sequences contained within selective marker gene cassettes can interfere with
gene expression of the targeted loci (Pham et al., 1996, Uusi-Oukari et al., 2000). To
generate a functionally normal pseudo WT locus it is therefore essential to first eliminate
the PGK-neor cassette from the targeted allele. Towards this end, the high-grade male
chimeric mice were mated with female EIIa-Cre mice carrying the cre transgene under
the control of the adenovirus EIIa promoter, which drives expression of Cre recombinase
selectively during undifferentiated stages of oogenesis and in preimplantation embryos
(Dooley et al., 1989, Lakso et al., 1996). Southern blot analyses of offspring (F1) from
these matings carrying both the targeted gene loci (fGODZ-neo, fGODZ and GODZ KO
or fSERZ-β-neo, fSERZ-β and SERZ-β KO in Fig. 3.1) and the EIIa Cre transgene
55
Figure 3.2. Screening of G418-resistant ES cells a, b, Representative Southern blot screening of ES clones targeted for homologous recombination at the GODZ (a) and SERZ-β (b) loci. Genomic DNA was purified from G-418 resistant ES clones, digested with Bam HI (GODZ) or Kpn I (SERZ-β) and processed for Southern analyses using 5’ flanking probes. The positive clones showed additional restriction fragments of 3.8 kb (GODZ, clones No. 173 and 178 in this figure) and 1.5 kb (SERZ-β, clone No. 48 in this figure).
56
Figure 3.3. Chimeric mice. Four chimeric mice (agouti) acquired from GODZ embryonic stem cell clone No. 1 (original clone No. 155) and two control littermates (black, 9 days old).
57
revealed variable degrees of mosaicism for the different recombined GODZ and SERZ-β
alleles (Fig. 3.4a, Table 3.1, 3.2, also see Fig. 3.1 for nomenclature of alleles). To
segregate the different alleles and rid them of the EIIa Cre-transgene, these F1 mice were
crossed with WT 129 SvJ mice. The floxed alleles in F2 mice derived from female F1
mosaic mice were completely excised, in case of both GODZ and SERZ-β (Table 3.3). In
contrast, F2 mice that were derived from F1 males contained floxed pseudo WT loci that
had the PGK-neor cassette deleted but retained exons 3 and 4. The fGODZ/+ and fSERZ-
β/+ mice were then used to establish homozygous floxed GODZ and SERZ-β mice,
respectively. Analogously, GODZ+/- and SERZ-β+/- mice, which had exons 3 and 4 and
the PGK-neor cassette of the recombined allele deleted, were used to generate global
homozygous KO mice (GODZ-/- and SERZ-β-/-). Furthermore, double KO (DKO) mice
of GODZ and SERZ-β were obtained by interbreeding of these two lines.
Genomic locus floxed-neo floxed KO PGK-neor
A √ B √ √ √ C √ √ √ √D √ √ E √ √ √
Table 3.1: Loci contained in each mosaicism
Genomic
locus GODZ Cln. 1 GODZ Cln. 2 GODZ Cln. 3 GODZ Cln. 4 SERZ-β
A 13 4 3 7 5 B 1 1 0 0 2
C 3 0 2 1 0 D 7 3 6 4 16 E 1 0 1 0 9
WT 24 24 22 8 21 Total 49 32 34 20 53
Table 3.2: Number of offspring of Chimera x EIIaCre and GODZ/SERZ-β alleles present `
58
GODZ SERZ-β
Male x Female fGODZ GODZ KO GODZ +/+
fSERZ-β SERZ-β ΚΟ
SERZ-β +/+
129 SvJ x Mosaic D 0 42 50 0 8 9 Mosaic D x 129 SvJ 6 15 31 4 4 14
Table 3.3: Genotype of offspring of Mosaic D x 129 SvJ
3.1.2.4. Confirmation of deletion of exons 3/4 of GODZ and SERZ-β by Southern
hybridization and RT-PCR
The genotype of GODZ-/- and SERZ-β-/- mice was first confirmed by PCR using primer
pairs GZ1-GZ4 and GZ3-GZ4 for GODZ and SZ5-4 and SZ1-6 for SERZ-β. For further
confirmation, genomic Southern hybridization was performed using two different probes
for each GODZ and SERZ-β (Fig. 3.4a). For GODZ, 5’ probe was used to detect Bam HI
fragments with 4.7 kb and 3.8 kb in size. The WT GODZ allele showed one 4.7 kb Bam
HI fragment, whereas fGODZ-neo, fGODZ and GODZ KO allele showed an additional
3.8 kb Bam HI fragment. With probe GODZ-2, a 4.3 kb Nco I fragment was detected in
WT and fGODZ allele, whereas additional 3.8 and 2.3 kb Nco I fragments were detected
in fGODZ-neo and GODZ KO allele containing mice, respectively (Fig. 3.4a). For
SERZ-β, the 5’ probe detected one 8.7 kb Kpn I fragment for the WT allele and 1.5 and
8.7 kb Kpn I fragments for the fSERZ-β-neo, fSERZ-β and SERZ-β ΚΟ alleles.
Although the 1.5 kb Kpn I fragment was detected in fSERZ-β-neo, fSERZ-β and SERZ-
β ΚΟ mice, the 8.7 kb fragment was not recognizable in the case of WT mice due to
incomplete DNA digestion (Fig. 3.4a). With the 3’ hybridization probe, WT and fSERZ-
β alleles showed a 7.2 kb Bam HI fragment, whereas fSERZ-β-neo and SERZ-β KO
alleles showed additional 8.8 kb and 13.3 kb fragments, respectively. These results
indicated that all modified alleles for GODZ and SERZ-β were successfully generated in
mice, as intended. RT-PCR was employed to confirm deletion of exons 3/4 in GODZ and
SERZ-β transcripts (Fig. 3.4b, also see Fig. 3.1 for primer location). No PCR products
were detected from KO mice with the primer combination of GP1-GP2 or SP1-SP2
59
Figure 3.4. Detection and verification of GODZ and SERZ-β deletion mutations a, Southern blot analyses of genomic DNA from different GODZ and SERZ-β genotypes. Genomic DNA was purified from each animal, digested with Bam HI or Nco I to probe with 5’ probe (left upper) and probe GODZ 2 (left lower), respectively, for GODZ and with Kpn I or Bam HI to probe 5’ probe (right upper) and 3’ probe (right lower), respectively, for SERZ-β.
60
Bands representing each allele are indicated on the left. b, RT-PCR analyses of transcripts in WT, heterozygous and homozygous KO mice for GODZ (left) and SERZ-β (right). Primers used for RT-PCR are indicated in each panel. The location and 5’-3;’ direction of primers is indicated with arrowheads in figure 1. Bands representing WT and deleted alleles are indicated on the right. c, Immunofluorescent analyses of cultured cortical neurons of WT and homozygous KO mice. Cortical neurons prepared from WT, GODZ and SERZ-β KO embryos were double-immunostained for gephyrin (red, c1’, c2’), GODZ (green, c1, c2) or SERZ-β (green, c3, c4) at DIV 18. GODZ immunoreactivity observed in WT cortical neurons (c1) was absent in GODZ-/- neurons (c2). Similarly, SERZ-β immunoreactivity observed in WT cortical neurons (c3) was absent in SERZ-β -/- neurons (c4).
61
(G/SP1 and G/SP2 anneals to a region in exon 1 and 4, respectively), as expected if exon
4 is deleted. In contrast, the existence of transcripts containing exon 1 was confirmed by
PCR using G/SP1 with GP3 and SP3 primers that anneal to exon 6 of GODZ and SERZ-
β transcripts, respectively. These PCR reactions revealed truncated products
corresponding in size to mutated transcripts in which exons 3 and 4 were deleted as
expected. These results from Southern hybridization and RT-PCR unmistakably confirm
that exons 3 and 4 are absent from GODZ and SERZ-β KO mice.
3.1.2.5. Confirmation of loss of GODZ and SERZ-β protein in KO neurons
Deletion of exons 3 and 4 in each GODZ and SERZ-β allele leads to a frameshift in the
translational reading frame, thereby generating an abnormal and truncated protein. Since
the epitopes for GODZ and SERZ-β antibodies are located at the C-termini (exon 6), the
corresponding mutant proteins are not detectable. Consistent with this,
immunofluorescent staining for GODZ/SERZ-β was completely absent in the respective
mutant neuron cultures, save for weak unspecific staining (Fig. 3.4c).
3.1.3. Normal clustering of pre- and postsynaptic markers of GABAergic synapses
in GODZ KO and GODZ/SERZ-β KO cultures
Cultured cortical neurons transfected either GODZ-specific shRNA or dominant negative
GODZ showed deficits in postsynaptic clustering of GABAARs and reduced GABAergic
innervations (Fang et al., 2006). Based on these results, it was expected that GODZ KO
cortical neurons would show similar phenotypes. To assess the effects of deletion of
GODZ on the formation of inhibitory synapses, cultured cortical neurons prepared from
GODZ KO and WT mice were double stained for the presynaptic GABAergic marker
VIATT and postsynaptic GABAergic marker gephyrin (Fig. 3.5). Surprisingly, GODZ
KO neurons showed normal GABAergic innervations with unaltered punctate
immunoreactivity for gephyrin and VIATT compared to WT (105 ± 2.75%; n = 29-32 for
VIATT, 102.5 ± 3.6%; n = 29-32 for gephyrin) (Fig. 3.5b, c). The colocalization of
punctate gephyrin and VIATT staining was also unaffected (Fig. 3.5d, e). Furthermore,
deletion of GODZ did not interfere with the postsynaptic accumulation of γ2 subunit-
62
Figure 3.5. Normal clustering of pre- and postsynaptic markers of GABAergic synapses in GODZ KO cortical neurons WT and GODZ KO cortical neurons were double-immunostained for the GABAergic presynaptic marker VIATT (green) and postsynaptic gephyrin (red). a, GODZ KO cortical neurons (bottom) showed extensive immunoreactivity for VIATT (left) and gephyrin (middle) similar to WT (upper panels). Merged images are shown (right panels) to illustrate colocalization of pre- and postsynaptic markers. b, c, The average number of immunoreactive puncta for VIATT and gephyrin per 40 µm dendritic segment were determined. Quantitative analyses indicate unaltered numbers of immunoreactive puncta for VIATT (b), and gephyrin (c), respectively. d, e, The fraction of VIATT and gephyrin puncta that colocalized with gephyrin (d) and VIATT (e), respectively, was unchanged in GODZ KO compared to control WT cortical neurons. Data represents means ± S.E.
63
containing GABAARs as indicated by unaltered punctate γ2 subunit immunoreactivity in
GODZ KO compared to WT cortical cultures (Fig. 3.6a, c, d, number of puncta /40 µm
dendrite: γ2, 95 ± 6.1% of WT; n = 8-12; GAD, 98.8 ± 8.3% of WT; n = 9-12, p > 0.05).
Punctate γ2 subunit immunoreactivity remained perfectly colocalized with punctate GAD
immunoreactivity (Fig. 3.6e). The puncta size of γ2 and GAD was unchanged in GODZ
KO neurons compared to WT (Fig. 3.6g, h).
Suggestive of at least partial functional redundancy of GODZ and SERZ-β, DKO,
but not KO mice for either of GODZ or SERZ-β alone, showed postnatal lethality (see
below). We therefore predicted that DKO cultures would be more likely to exhibit
deficits in GABAergic synapses. However, similar to GODZ KO cultures DKO cortical
neurons showed normal punctate staining for the γ2 subunit and GAD (97.4 ± 3.2%; n =
30-46 for γ2 subunit, 96.2 ± 3.7%; n = 29-46 for GAD) (Fig. 3.6b, i, j). The puncta size of
γ2 (83.2 ± 3.2%, p < 0.01) and GAD (89.2 ± 3.1%, p < 0.05) is significantly reduced in
DKO neurons compared to WT (Fig. 3.6m, n).
3.1.4. Deficits in GABAAR postsynaptic clustering of GODZ KO and DKO cortical
neurons co-cultured with WT neurons
At first sight the normal clustering of pre- and postsynaptic markers of GABAergic
synapses in GODZ KO and DKO cultured neurons appeared to be in conflict with the
GABAergic deficits described in GODZ shRNA or GODZC157S transfected neurons (Fang
et al., 2006). However these conditions entailed analysis of transfected postsynaptic
GODZ deficient neurons that were surrounded by large untransfected and thus
functionally normal neurons. We wondered whether GABAergic deficits seen in
transfected neurons surrounded by normal untransfected neurons reflected an inability of
GODZ deficient neurons to compete with normal neurons for normal innervation. To
examine this possibility, GODZ KO neurons (10%) were co-cultured with GFP-
transgenic WT neurons (90%). The GFP signal was utilized to distinguish GFP-
transgenic WT from GODZ KO neurons. GFP-WT and GODZ KO pure culture were
used as controls. In sharp contrast to the pure GODZ KO cultures, GODZ KO neurons in
the co-culture showed drastically reduced numbers of immunoreactive puncta for the γ2
64
subunit (56.6 ± 9.6% of WT, n = 10-12 cells; p < 0.001), along with a corresponding
deficit in the punctate immunoreactivity for GAD (Fig. 3.6a-c, -40.8 ± 8.9%; n = 10-12;
p < 0.001). Similarly, a drastic reduction in immunoreactive puncta for γ2 to 49.7 ± 3.9%
of WT and GAD to 55.3 ± 4.0% of WT (n = 29-59; p < 0.001, for both markers) was
observed in co-cultures of DKO and WT neurons (Fig. 3.6f-h). These data indicate that
the GODZ KO and DKO cultures replicate the GABAergic deficit described for shRNA
and GODZ dominant negative construct transfected cultures described earlier (Fang et al.,
2006). The puncta size of γ2 and GAD was unchanged in GODZ KO neurons co-cultured
with WT neurons (Fig. 3.6g, h). In addition, the size of γ2 puncta was unchanged (Fig.
3.6m), whereas the size of GAD puncta was significantly reduced in DKO neurons co-
cultured with WT (87.1 ± 4.0%, p < 0.05) (Fig. 3.6n).
3.1.5. γ2 subunit containing GABAARs are required for efficient GABAergic innervation
It has been pointed that possible presynaptic deficits for GABAergic innervations are
mediated by the postsynaptic deficits in γ2 subunit-containing GABAARs in the GODZ
shRNA transfected neurons (Fang et al., 2006). Similar phenomena have been reported
by shRNA-mediated knock down of the γ2 subunit or γ2 subunit interacting trafficking
(Li et al., 2005, Yuan et al., 2008). By contrast GODZ KO and DKO neurons (analyzed
as pure rather than mixed cultures) showed normal GABAergic innervation as illustrated
by the punctuate staining for GAD-positive axon growing along dendrites,
indistinguishable from WT cultures (Fig. 3.6a1’-a1’’’, a2’-a2’’’, b1’-b1’’’, b2’-b2’’’). In
contrast, GODZ KO and DKO neurons co-cultured with an excess of WT neurons were
largely deprived of GABAergic innervation, with GAD positive axons of neighboring
WT neurons either passing by or growing across rather than along dendrites of GODZ
KO/DKO neurons (Fig. 3.6a3’-a3’’’, b3’-b3’’’). I hypothesized that under these
conditions γ2 subunit-deficient neurons were competing with WT neurons for
GABAergic innervation and that GABAergic axons that had a choice for some reason
would preferentially innervate neurons with normal levels of γ2 subunit containing
65
66
Figure 3.6. Deficits in GABAAR postsynaptic clustering of GODZ KO and DKO cortical neurons co-cultured with WT neurons GODZ KO (a, c-h) and DKO (b, i-n) cortical neurons were cultured on their own as a pure culture or co-cultured with GFP-WT neurons (10% KO/90% WT). The green fluorescence of GFP-positive WT neurons was used to distinguish them from KO neurons. The neurons were double-immunostained for the γ2 subunit of GABAARs (red, a1’, a2’, a3’, b1’, b2’, b3’) and GAD (blue, a1’’, a2’’, a3’’, b1’’, b2’’, b3’’). Merged images are shown (a1’’’, a2’’’, a3’’’, b1’’’, b2’’’, b3’’’) and boxed dendritic segments are enlarged in separate panels on the right. Arrow heads show that presynaptic WT neuron grow over the KO neurons (a, b). In pure GODZ KO
67
(a2’, a2’’) and DKO (b2’, b2’’) cultures, the numbers of puncta for γ2 and GAD were indistinguishable from corresponding values of WT neurons grown in a parallel (a1’, a1’’, c, d, b1’, b1’’, i, j). In contrast, GODZ KO (a3’, a3’’) and DKO (b3’, b3’’) neurons co-cultured with an excess of WT neurons showed significantly reduced numbers of puncta for both γ2 (GODZ KO; 56.6 ± 9.6%; n = 10-12; p < 0.001, DKO; 49.7 ± 3.9%; n = 30-59; p < 0.001) and GAD (GODZ KO; 59.2 ± 8.9%; n = 10-12; p < 0.001, DKO; 55.3 ± 4.0%; n = 29-59, p < 0.001) compared to corresponding values of WT neurons grown in pure culture (a1’, a1’’, c, d, b1’, b1’’, i, j). The fraction of γ2 puncta that colocalized with punctate GAD immunoreactivity was unchanged in pure cultures of GODZ KO (e) and DKO (k). Moreover, γ2 puncta colocalized with GAD in DKO neurons co-cultured with WT was reduced (82.6 ± 3.9%; n = 23-59, p < 0.001) (k). The fraction of GAD puncta that colocalized with γ2 was unchanged in GODZ KO and DKO compared to control WT cortical neurons, independent of whether the mutant neurons were analyzed in pure cultures or co-cultured with an excess of WT neurons (f, l). The size of puncta for γ2 (100.4 ± 5.1% in pure culture, 98.3 ± 5.4% in co-culture, p > 0.05 in both case) and GAD (89.4 ± 4.6% in pure culture, 105.2 ± 6.3% in co-culture, p > 0.05 in both case) was unchanged in GODZ KO neurons in pure culture or co-cultured with WT neurons (g, h). In contrast, DKO neurons in pure culture showed significantly reduced puncta size of γ2 (83.2 ± 3.2%, p < 0.01) and GAD (89.2 ± 3.1% of WT, p < 0.05) (m, n). The size of γ2 puncta was unchanged (95.1 ± 4.8% of WT, p > 0.05) (m), whereas the size of GAD puncta was significantly reduced in DKO neurons co-cultured with WT (87.1 ± 4.0% of WT, p < 0.05) (n).
68
GABAARs. To test this directly, I co-cultured GFP-transgenic γ2 subunit heterozygous
neurons (GFP-γ2+/-) with an excess of WT neurons and analyzed their GABAergic
innervation. As predicted, dendrites of GFP-tagged γ2+/- neurons showed significant
reductions in GABAergic innervation compared to WT neurons (density of GAD
immunoreactive puncta on dendrites of γ2+/- neurons, 75.7 ± 6.5% of WT, n = 10-12 p <
0.001), along with deficits in punctate immunoreactivity for postsynaptic GABAARs
(density of γ2 subunit immunoreactive puncta of γ2+/- neurons: 57.5 ± 5.2 % of WT, n =
9-12 p < 0.001) (Fig. 3.7). Virtually identical results were obtained when untagged γ2+/-
neurons were analyzed in cocultures containing an excess of GFP-tagged WT neurons
(not shown). The puncta size of γ2 subunit was unchanged (Fig. 3.7f), whereas the size of
GAD puncta was significantly increased in γ2+/- neurons co-cultured with WT (130.5 ±
9.3% of WT, p < 0.001) (Fig. 3.7g). Thus, γ2 subunit-containing GABAARs contribute to
normal GABAergic innervation, a property that is best evident under conditions where
GABAergic axons are forced to choose among target neurons that differ in the surface
expression of γ2 subunit-containing GABAARs.
3.1.6. Surface expression of γ2 subunit and NL2 in DKO
In addition to γ2 subunit of GABAARs, NL2 was also appeared as a selective substrate of
GODZ and SERZ-β in HEK293T cell PAT assay. To assess whether GODZ and SERZ-β
are required for normal surface expression of γ2 subunit of GABAARs and NL2, surface
biotinylation assay was performed in cultured DKO cortical neurons (Fig. 3.8). The γ2
subunit surface expression level in DKO neurons was unaltered compared to WT neurons
(109.5 ± 11.7% of WT, n = 5 for each genotype, p > 0.05) (Fig. 3.8a, b). Similarly, the
NL2 surface expression level was unaffected in DKO neurons (94.1 ± 11.6%, n = 4 for
each genotype, p > 0.05) (Fig. 3.8a, c). As a reference, GluR2/3 subunit of AMPARs was
also examined. The surface expression level of GluR2/3 was also unchanged in DKO
neurons compared to WT neurons (normalized by GluR2/3 total amount; 141.8 ± 17.5%,
n = 6 for each genotype, p > 0.05, normalized by β-tubulin total amount; 118.1 ± 9.0%, n
= 4 for each genotype, p > 0.05) (Fig. 3.8a, d).
69
Figure 3.7. Subtle deficits in γ2 subunit-containing GABAARs of γ2+/- neurons result in deficits in GABAergic innervation. a, Cortical neurons (DIV21) cultured from a WT embryo (top) and GFP-transgenic cortical neuron derived from a γ2+/- embryo cocultured with an 9:1 excess of WT neurons (bottom) were double labeled for the γ2 subunit (a1’, a2’) and GAD (a1’’, a2’’). Merged images are shown (a1’’’, a2’’’) and boxed dendritic segments are enlarged in separate panels on the right. Arrow heads show that axons of presynaptic WT neuron grow over the KO neurons (a). Mutant neurons (γ2+/-) were identified by the transgene encoded GFP fluorescence. γ2+/- neurons co-cultured with an excess of WT neurons showed significantly reduced numbers of puncta for both γ2 (54.4 ± 2.7 % of WT, n = 13-14 p < 0.001) and GAD (56.2 ± 4.8% of WT, n = 13-14 p < 0.001) compared to corresponding values of WT neurons grown in pure culture (a-c). γ2 puncta colocalized with GAD in γ2+/- neurons co-cultured with WT was reduced (87.7 ± 3.62% of WT; n = 13-14, p < 0.01) (d). In addition, GAD puncta colocalized with γ2 was reduced in γ2+/- neurons co-cultured with WT (91.4 ± 2.8% of WT; n = 13-14, p < 0.05) (e). The puncta size of γ2 subunit was unchanged (101.2 ± 7.3% of WT, p > 0.05) (f), whereas the size of GAD puncta was significantly increased in γ2+/- neurons co-cultured with WT (130.5 ± 9.3% of WT, p < 0.001) (g).
70
Figure 3.8. Analyses of surface expression of γ2 subunit of GABAARs and NL2 by surface biotinylation of cultured cortical neurons. Cortical neurons (DIV14-20) were surface biotinylated and extracts purified over protein A. a, Western blot analyses of the γ2 subunit of GABAARs, NL2, AMPAR GluR2/3 subunits and β-tubulin in aliquots of extracts from surface biotinylated neurons before (total) and after purification (surface) over protein A. b, c. Values of surface biotinylated γ2 subunit and NL2 protein fractions quantitated by western blots were normalized to tubulin (b) or NL2 (c) in aliquots of total extracts. The expression of the γ2 subunit in DKO neurons (b) was unchanged compared to WT neurons (109.5 ± 11.7% of WT, n = 5 for each genotype, p > 0.05). Similarly, surface levels of NL2 in DKO neurons (c) was unchanged compared to WT (94.1± 11.6%, n = 4 for each genotype, p >0.05). d, The surface level of GluR2/3 subunits normalized to total GluR2/3 in the same extracts. Surface GluR2/3 levels in DKO cortical neurons were unaltered compared to WT (118.1 ± 9.0% of WT, n = 4/genotype, p > 0.05). However, the total amount of GluR2/3 was reduced in DKO neurons compared to WT (72.8 ± 7.7% of WT, n = 4, p < 0.05).
71
Note that the total amount of GluR2/3 was downregulated in DKO neurons (72.8 ± 7.7%,
n = 4 for each genotype, p < 0.05), suggesting a compensatory mechanism at genomic
expression level.
3.1.7. Functional deficits of DKO neurons in inhibitory and excitatory
neurotransmission
To determine the effect of KO of GODZ and SERZ-β on the function of γ2 subunit
containing GABAARs and inhibitory synapses, mIPSC and GABA-induced whole cell
currents were analyzed in cultured cortical neurons prepared from DKO and control WT
mice (collaboration with Xia Wu and Dr. Gong Chen). In agreement with results from
immunofluorescent staining, the mIPSC and GABA-induced whole cell currents were
unaffected in DKO neurons compared to WT neurons (data not shown). To address
whether loss of GODZ and/or SERZ-β affect synaptic function of DKO neurons grown in
competition with WT neurons, DKO neurons were again analyzed in mixed cultures, this
time containing 30% DKO and 70% WT (GFP-transgenic) neurons. Whereas the GABA-
induced whole cell currents were unchanged (84.7 ± 14.8% of WT, n = 12/genotype, p >
0.05) (Fig. 3.9a), the frequency and amplitude of mIPSCs were significantly reduced in
DKO neurons compared to WT in the same culture (Median Inter-Event Interval; DKO,
1297.5 ms, WT, 1262.4 ms; n = 12/genotype; p < 0.05, Median for amplitude; WT 24.4
pA, DKO 21.4 pA, p < 0.0001) (Fig. 3.9c, d).
In addition to the γ2 subunit and NL2, potential substrates that are palmitoylated
by GODZ and SERZ-β in vitro include PSD-95, a scaffold protein at excitatory
postsynaptic sites (Fukata et al., 2004), and AMPAR GluR1-4 subunits (Hayashi et al.,
2005). Therefore, to assess whether GODZ and SERZ-β contribute to glutamatergic
synaptic function, we recorded mEPSCs and glutamate-induced whole cell current from
DKO neurons. Consistent with unaltered immunostaining for the γ2 subunit and GAD in
these cultures (Fig. 3.6f), neither mEPSCs nor glutamate-induced whole cell current were
changed in cultured DKO pure neurons (data not shown). Glutamate-evoked whole cell
currents in DKO neurons in co- culture were also not changed (91.1± 20.6%, n = 12 for
each genotype, p > 0.05) (Fig. 3.9b). In contrast, the frequency of mEPSCs was
72
Figure 3.9. Functional deficits of DKO neurons in inhibitory and excitatory neurotransmission. GABA- and glutamate-induced whole cell currents as well as GABAergic and glutamatergic miniature postsynaptic currents (mPSCs) were recorded from DKO cortical neurons (30%) co-cultured with an excess of GFP transgenic WT neurons (70%). a, b, GABA- (a) and glutamate-(b) induced whole cell currents of DKO neurons co-cultured with WT neurons (IGABA 3741 ± 651 pA, IGlu 931 ± 210 pA, n = 12) were indistinguishable from WT control neurons in the same culture (IGABA 4418 ± 770 pA, IGlu 1022 ± 234 pA, n = 12, p > 0.05). c-f, Cumulative probability for the frequencies and amplitudes of mIPSCs (c, d) and mEPSCs (e, f) are shown. The frequency (c) and amplitude (d) of mIPSCs were significantly reduced in DKO neurons (mean values: 0.15
73
± 0.08 Hz, 19.9 ± 1.9 pA, n = 12) compared to WT controls (0.24 ± 0.09 Hz, 22.5 ± 2.2 pA, n = 12, p < 0.05 for frequency, p < 0.001 for amplitude). In addition, the frequency of mEPSCs (e) was also reduced in DKO neurons (0.65 ± 0.18 Hz, n = 12) compared to WT controls (1.3 ± 0.51 Hz, n = 12, p < 0.001), whereas the amplitude was unaltered (DKO, 18.3 ± 2.4 pA, WT, 17.7 ± 2.1 pA, p > 0.05) (f).
74
significantly reduced in DKO neurons in co-culture (n = 12/genotype; Median for Inter-
Event Interval; WT, 280.1 ms; DKO, 509.0 ms, p < 0.001) (Fig. 3.9e). These results
suggest that GODZ and SERZ-β are essential for normal GABAergic inhibitory and
glutamatergic excitatory synaptic neurotransmission. Deficits in glutamatergic
transmission might reflect secondary deficits due to reduced GABAergic transmission,
which were not evident upon more acute knock down of GODZ/SERZ-β by shRNA or
dominant negative GODZ (Fang et al., 2006). Alternatively, they might represent
pleiotropic effects of GODZ/SERZ-β mediated by a multitude of other palmitoylated
GODZ/SERZ-β targets that directly or indirectly affect neural signaling.
3.1.8. Overt phenotype of GODZ-/-, SERZ-β-/- and DKO mice: Body weight,
lethality, fertility and posture
GODZ-/- and SERZ-β-/- mice are vital and fertile. Male, but not female GODZ KO mice
show a significant reduction in body weight compared to WT (male; 89.2 ± 2.5% of WT
at 1 month of age; n = 14-18; p < 0.001, 90.4 ± 1.7% of WT at 2 months of age; p <
0.01) (Fig. 3.10). GODZ/SERZ-β DKO mice exhibit a partially penetrant early postnatal
lethal phenotype and most pups died between postnatal day 1 and 2. Of a total of 439
progeny of which in theory 33.3% should have been homozygous mutants for both genes,
13% of the males and 21% of the female of the expected DKO mice survived for more
than one year. However, the DKO mice that survived to one year showed drastically
reduced body and brain weights compared to GODZ+/-, SERZ-β+/- or GODZ+/+, SERZ-
β-/- littermate controls (total weight 53.6 ± 1.7%; brain 74.7± 6.3%; n=3 for each
group) (Fig. 3.10). RNA tissue blot using 32P-labeled cDNA probes derived from 3’
untranslated regions indicated similar tissue specificity of GODZ and SERZ-β, with
GODZ mRNA more prominently expressed in brain, while SERZ-β mRNA more
abundant in heart and testes (Chaudhary and Skinner, 2002, Uemura et al., 2002). In
agreement with high level expression and an important function of GODZ and SERZ-β in
heart, DKO also showed decreased heart weight (44.4 ± 5.7% of WT; n = 3) (Fig. 3.10c).
75
Figure 3.10. Weight loss observed in GODZ KO and DKO mice a, GODZ male, but female, showed a significant reduction in body weight compared to WT (n = 14-18; 92.5 ± 2.5%; p < 0.001 at 1 month old, 90.4 ± 1.7%; p < 0.01 at 2 months old). b, DKO mice (female, one year old) showed significantly reduced body weight compared to its littermates (GODZ+/-, SERZ+/- or GODZ+/+, SERZ-/-) (53.6 ± 1.7%; p < 0.001; n = 3). c, The wet weight of brain and heart in DKO mice (female, one year old) was also significantly reduced (brain,74.7 ± 6.3%; p < 0.05, heart, 44.4 ± 5.7%; p < 0.01, n = 3). Error bars represent SE.
76
However, although DKO mice showed mostly an early postnatal lethal phenotype, the
male and female DKO mice who survived to adulthood were fertile.
Most but not all GODZ-/- mice show overt jumping behavior upon cage opening.
By contrast SERZ-β KO mice lacked an overt behavioral phenotype. This is in agreement
with the previous notion that GODZ rather than SERZ-β is likely to be the principal PAT
involved in trafficking of GABAARs (Fang et al., 2006). To explore the effects of KO of
GODZ on behavior, several behavioral paradigms were used and analyzed. GODZ KO
mice also show increased hind limb clenching when suspended by their tail, suggesting
increased muscle tension at any time examined from 1 to 10 months old (Fig. 3.11, Table
3.4). Of 28 WT mice examined, 2 (7.1%) showed spontaneous hindleg clenching upon
suspension by their tail. By comparison, 14 out of 36 GODZ KO mice (38.9%) showed
this phenotype. SERZ-β KO mice were indistinguishable from WT as only 3.1% showed
this phenotype. These spontaneous jumping in response to handling and excessive
hindleg clenching are reminiscent of hyperekplexia-like phenotypes (see discussion).
Genotype Sex Clenched Ambiguous Normal % with
unambiguous hindleg clenching
WT Male 1 1 11 7.1
38.9
3.1
Female 1 1 13 GODZ KO Male 8 3 9
Female 6 5 5 SERZ-β KO Male 0 1 17
Female 1 2 11
Table 3.4: Clenching phenotype of GODZ and SERZ-β KO mice
77
Figure 3.11. GODZ KO mice show an increased muscle tension GODZ KO mice clenched their hind limbs on suspension by their tail, indicating increased muscle tension. In WT mice, 2 out of 28 examined mice showed hind leg clenching. In comparison, 14 of a total of 36 GODZ KO mice showed this phenotype. (see also Table 3.4)
78
3.1.9. Behavioral characterization of GODZ KO mice
Although it has been pointed in our laboratory that male’s fighting stress associated with
establishing of a social hierarchy in each cage could effect on the outcomes of a
behavioral experiment, both males and females were used in the all of the behavioral
experiments, unless otherwise mentioned, to examine the overall phenotype of GODZ
KO mice
3.1.9.1. GODZ KO mice show hyperlocomotion in the Open Field test
In a forced Open Field paradigm, 21 different behavioral parameters were recorded.
Among these, GODZ KO mice showed statistically significant differences in several
parameters, with males showing consistently greater differences compared to WT than
females. This is in agreement with the notion that GODZ KO males, but not females,
showed a reduction in body weight, and with the greater penetrance of the lethal
phenotype observed in DKO males than females. Parameters in which both male and
female GODZ KO mice were significantly different from WT included the number of
horizontal movements in the second and third 5 min intervals of the 15 min Open Field
test (male GODZ KO compared to WT: 108.2 ± 3.2%; n = 14-18; p < 0.05 in the second
5 min interval, 119.2 ± 5.0%; p < 0.01 in the third 5 min, 109.8 ± 2.6%; p < 0.01 in the
total 15 min interval; females: 109.1 ± 2.1%; n = 9-16, p < 0.05 in the second 5 min),
109.4 ± 2.2%; 112.4 ± 1.8%; p < 0.01 in the first 10 min and total 15 min, respectively).
Increased locomotion of GODZ KO mice was also reflected in an increased center
distance traveled (male GODZ KO compared to WT: 121.8 ± 9.4%; p < 0.05 in the first
5 min, 224.1 ± 32.4%; p < 0.001 in the third 5min, 138.8 ± 12.8%; p < 0.01 in total 15
min: females: 138.8 ± 14.7%; 160.5 ± 25.0%; 140.4 ± 15.2%; p < 0.05 in the first and
second 5 min and the first 10 min, respectively) (Fig. 3.12). These results indicate that
GODZ KO mice are hyperlocomotive. This hyperlocomotion behavior is also reminiscent
of hyperekplexia-like phenotypes. On the other hand, GODZ KO mice showed normal
movement time, rest time, vertical activity and margin distance (data not shown).
79
Figure 3.12. GODZ KO mice show hyperlocomotion in the Open Field test GODZ KO mice (8 weeks old) were tested on an Open Field test. Out of 21 different parameters analyzed GODZ mice show significant differences compared to WT mice in the following parameters. Only the data from female mice are shown. a-d, The number of horizontal movement (n = 9 -16; 109.1 ± 2.1%; p < 0.05 in the second 5 min (a), 109.4 ± 2.2%; 112.4 ± 1.8%; p < 0.01 in the first 10 min and total 15 min (b), respectively, and the center distance (138.8 ± 14.7%; 160.5 ± 25.0%; 140.4 ± 15.2%; p < 0.05 in the first and second 5 min (c) and the first 10 min (d), respectively) shown here are the parameters in which both male and female of GODZ KO mice showed significant increase compared to WT mice. Error bars represent SE.
80
3.1.9.2. GODZ KO mice show normal level of anxiety
In a novel environment, mice show an innate fear of open areas. On first exposure to a
novel environment, they initially remain in the periphery close to the walls, a behavior
referred to as thigmotaxis. On the other hand, mice are also motivated to explore for food
and mating pairs. The resulting conflict between avoidance and approach of novel
environment is commonly used for assessing innate fear and anxiety under laboratory
conditions (Crawley, 1985). The open Field test described above indicated reduced
thigmotaxis of GODZ KO mice, a behavior that in some cases results from reduced
anxiety. To more directly address anxiety-like behavior, GODZ KO mice were analyzed
in the Elevated Plus Maze paradigm (Lister, 1987). However, the time spent in the open
arms (male; n = 12-18; 86.0 ± 19.3%: female; n = 8-16; 162.7 ± 31.5%, p > 0.05) and
the number of open arm (male; 131.3 ± 26.3%: female; 127.5 ± 17.7%, p > 0.05) or
closed arm entries (male; 118.3 ± 17.4%: female; 90.3 ± 12.5%, p > 0.05) were
unchanged in GODZ KO compared to WT mice (Fig. 3.13a-c). These results indicate
that reduced thigmotaxis in the Open Field test is due to increased locomotion entirely
and not due to altered anxiety-related behavior. Consistent with unaltered anxiety-related
behavior GODZ KO mice were also indistinguishable from WT in the Free Choice
Exploration paradigm, a second test used to assess anxiety-like behavior (Fig. 3.13d-h, n
= 9-16, novel units visited; 104.7 ± 23.1%, p > 0.05, familiar units visited; 117.9 ±
9.0%, p > 0.05, number of retractions; 110.0 ± 43.2%, p > 0.05, time spent in
novel units; 92.6 ± 19.3%, p > 0.05, time spent in familiar units; 104.9 ± 12.8%, p
> 0.05).
3.1.9.3. GODZ KO mice show enhanced Prepulse Inhibition
Prepulse Inhibition paradigm (PPI) has been utilized to assess sensorimotor gating, which
measures neural transmission of sensory information to motor systems (reviewed in
(Braff and Geyer, 1990, Braff et al., 2001)). In this paradigm, a startle response is elicited
by acoustic, electrical, tactile, or visual stimuli. The magnitude of the startle response is
reduced when the pulse stimulus is preceded by a weak pulse by 30 to 500 msec, a time
course that is too short to evoke conscious voluntary behavioral inhibition. This
81
Figure 3.13. GODZ KO mice show normal levels of anxiety GODZ KO mice were tested on the Elevated Plus Maze (EPM) (a-c, 12 weeks old) and Free Choice paradigm (d-h, 11 weeks old). Male and female GODZ KO mice showed normal levels of anxiety-related behavior in the EPM as indicated by the unaltered time spent on the open-arms (a), or in the unchanged number of open- (b) and closed-arm visits (c), compared to WT. In agreement, GODZ KO mice showed unaltered numbers of novel unit visit (d), familiar unit visit (e), retractions (f), and time spent in novel (g) and familiar units (h) in the Free choice paradigm, compared to WT. Error bars represent SE.
82
inhibition (“gating”) is termed prepulse inhibition (PPI). Deficits in PPI are a hallmark of
schizophrenia, bipolar disorder, as well as major depression and they serve as an
endophenotype of animal models of schizophrenia that is relatively easily tractable also
in rodents. Given that mutations in the GODZ-related PAT DHHC8 are associated with a
familial form of schizophrenia I subjected GODZ KO and littermate controls to
measurements of PPI. In this study, an acoustic stimulus consisting of a 120 dB startle
stimulus was randomly paired with a prepulse of 4, 8, or 12 dB above background level
(65 dB). Only females were tested. GODZ KO mice showed unaltered startle responses
to the 120 dB stimulus alone (n = 8-16, 62.2 ± 26.0%, p > 0.05), indicating normal
hearing and motor reflexes. In contrast, they showed hypersensitivity to a prepulse of 8
dB above background in the second block of the testing paradigm, resulting in significant
increased prepulse-mediated inhibition of the startle response (186.4 ± 21.3%, p < 0.05)
(Fig. 3.14). Similar increment of PPI was also observed in the assessment using
responces to the startle stimulus preceded by a prepulse of 4, 8 and 12 dB above
background noise through second and third block. This phenotype is the opposite of that
expected of an animal model of schizophrenia.
3.1.9.4. Other behavioral phenotypes
In order to test for possible changes in nociceptive sensitivity, GODZ KO mice were
subjected to a hot plate test. After they were placed on the hot plate, GODZ KO female,
but not male, mice showed a small but significant increase in the time to first hindpaw-
licking (Fig. 3.15), (female 127.6 ±7.5% of WT; n = 9-16; p < 0.05). Thus, GODZ
KO females suffer from subtle but significant deficits in reception or procession of a
nociceptive stimulus.
3.1.10. Upregulation of ZDHHC15 mRNA in brains of GODZ KO and DKO mice
As mentioned earlier, a fraction of DKO mice escape perinatal death and survive to
adulthood, suggesting that in DKO mice some of the other DHHC proteins might
compensate for the lack GODZ and SERRZ-β function. Using the γ2 subunit as a
substrate in the HEK293T cell PAT assays, DHHC15 showed a trace of palmitoylation
83
Figure 3.14. GODZ KO show enhanced Prepulse Inhibition GODZ KO mice (22 weeks of age, female) were compared to age and sex matched WT controls with respect to Pre-pulse inhibition (PPI) of the acoustic startle reflex. a, GODZ KO mice showed increased PPI compared to WT mice in the second block of the testing paradigm when the startle stimulus was preceded by a prepulse of 8 dB above background noise (186.4 ± 21.3%; n = 8-16; p < 0.05 ). Similar but insignificant tendencies were observed with a prepulse of 4 or 12 dB above background. b, Similar increment of PPI was also observed in the assessment using responces to the startle stimulus preceded by a prepulse of 4, 8 and 12 dB above background noise through second and third block. c, There was no significant change in the baseline startle response of GODZ KO compared to WT mice (n = 8-16, 137.8 ± 26.0%, p > 0.05). d-e, GODZ KO showed unaltered habituation over the testing paradigm (d) and T-max (e) compared to WT mice. f, GODZ KO mice used in PPI showed normal weight compared to WT. Data represents means ± S.E.
84
Figure 3.15. GODZ KO mice exhibit nociceptive sensory motor deficits GODZ KO mice (10 weeks of age) were analyzed for deficits in nociceptive sensory motor deficits using the hot plate test in 30 s testing interval. a, Upon placing on the hot plate they showed unaltered numbers of hind paw licking compared to age matched WT controls (the licking number was recorded in total testing interval) (GODZ KO males, n =11-18,128.2 ± 17.7% of WT controls; GODZ KO females, n = 9-16, 94.3 ± 15.8% of WT controls, p > 0.05 in each case). b, Female, but not male GODZ KO mice, showed a small but significant increase in the latency to start licking of hind their paws compared to controls (GODZ KO males, n = 11-18, 97.4 ± 6.6% of WT controls; GODZ females, n = 9-16, 127.6 ± 7.5% compared to controls, p < 0.05). Data represents means ± S.E.
85
activity albeit much weaker than GODZ and SERZ-β (Fang et al., 2006). To address
whether gene expression of ZDHHC15 and other GODZ-related transcripts was
upregulated in GODZ KO and DKO mice, I quantitated levels of ZDHHC 15, 21, and 25
transcripts in total RNA from brain. Preliminary analyses revealed that ZDHHC15 and
ZDHHC21 were expressed abundantly in brain, whereas ZDHHC 25 was not detectable
in any of the tissues analyzed (brain, heart, kidney, liver, lung, muscle and spleen) except
testis. Quantitative-RTPCR revealed that ZDHHC15 gene was significantly upregulated
in brain (cortex) of GODZ KO and DKO mice compared to WT (n = 4 for each genotype;
GODZKO; 129.3 ± 10.9% of WT, DKO; 207.1 ± 40.1% of WT, p < 0.05) (Fig.
3.16a, b). In contrast, no significance change was detectable for ZDHHC21 (n =
4/genotype; GODZ KO; 94.9 ± 11.5% of WT, DKO; 129.5 ± 19.3% of WT, p >
0.05) (Fig. 3.16c).
86
Figure 3.16. mRNA of ZDHHC15 is upregulated in brain of GODZ KO and DKO mice. Messenger RNA (mRNA) of ZDHHC15 and 21 of GODZ KO and DKO mice was quantified in brain using RT-PCR. House keeper gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for normalization. An average of triplicate PCR reactions was normalized by the average of the value of GAPDH that was amplified using the corresponding each mRNA (a) and total of four experiments using the same mRNA is shown (b, c) . The expression of ZDHHC 15 (a, b), but ZDHHC21 (a, c), was significantly increased in brain of GODZ KO and DKO compared to WT (GODZ KO; 129.3 ± 10.9% of WT, DKO; 207.1 ± 40.1% of WT, n = 4; p < 0.05).
87
Part II: GABAA receptor-dependent presentation of NL2 at inhibitory synapses
3.2.1. Introduction and aim of study
Previous analyses of neurons cultured from γ2 subunit KO mice revealed that loss of
postsynaptic GABAARs and gephyrin was paralleled by a drastic loss of punctate staining
for postsynaptic NL2. There are at least three possible mechanisms that could account for
this loss of postsynaptic NL2; 1) NL2 is normally trafficked through the secretory
pathway to the plasma membrane but fails to translocate to postsynaptic sites for some
other reasons, 2) NL2 is unable to move through the secretory pathway and thus trapped
intracellularly, perhaps in a complex with γ2 subunit deficient GABAARs, or 3) NL2 is
degraded before or after it reaches the plasma membrane. To address these possibilities, I
quantitated the expression of NL2 and GABAAR α1 and β2/3 subunits in γ2 KO cultures
and measured their expression at the cell surface.
3.2.2. The γ2 subunit of GABAARs is required for normal GABAAR and NL2
surface expression
To address whether γ2 subunit containing GABAARs are required for NL2 surface
expression, I employed surface biotinylation of cultured cortical neurons from γ2 subunit
KO mice. The total amount of NL2 in extracts of γ2 subunit KO cultures was
indistinguishable from controls (γ2 KO, 107.1 ± 38.8% of WT controls, n = 3/genotype,
p > 0.05) (Fig. 3.17a, b). In contrast, the surface expression level of NL2 was
significantly reduced in γ2 subunit KO neurons, independent of whether values
normalized to total NL2 (surface NL2, 48.7 ± 10.6% of WT) or β-tubulin (surface NL2,
57.2 ± 9.4%, n = 3, p < 0.01 for both comparisons) (Fig. 3.17a, b). In addition to NL2,
the surface expression levels of α1 and β2/3 subunits of GABAARs were also
significantly reduced in γ2 subunit KO mice (surface α1 subunit; 36.0 ± 4.8%; n =
5/genotype; p < 0.001, surface β2/3 subunit; 60.6 ± 12.0%; n = 4 for each genotype; p <
0.01), whereas the total amount of β2/3 subunit was unchanged (143.3 ± 24.4%; n =4 for
each genotype; p >0.05) (Fig. 3.17d, e). These results suggest that the γ2 subunit is
88
Figure 3.17. Reduced surface expression of NL2, α1 and β2/3 subunits of GABAARs in γ2 subunit KO neurons. a, b, The NL2 surface expression level was significantly reduced in γ2 subunit KO neurons independent of whether values were normalized to total NL2 (48.7 ± 10.6%) or β-tubulin (57.2 ± 9.4%, n = 3, p <0.01 for both comparisons), whereas the total expression normalized to β-tubulin was unchanged compared to WT (107.1 ± 38.8%, n = 3, p > 0.05). a, c, Neither the surface expression nor the total amount of GluR2/3 were significantly changed (total, 75.2 ± 13.6% of WT; surface, 112.4 ± 1.5% of WT, n = 4, p > 0.05 for both comparisons). d, e, The surface expression level of α1 and β2/3 subunits of GABAARs were also significantly reduced (α1, 36.0 ± 4.8% of WT, n = 5, p < 0.001; β2/3, 60.6 ± 12.0% of WT; n = 4; p < 0.01).
89
necessary for normal trafficking of NL2 and postsynaptic GABAARs to the cell surface
and that both proteins are trapped intracellularly, perhaps as part of a single complex. In
contrast, both the total amount and surface expression levels of GluR2/3 were unaltered
in γ2 subunit KO cultures (total amount; 75.2± 13.6%, n = 4 for each genotype, p > 0.05,
surface expression; 112.4 ± 1.5%, n = 4 for each genotype, p > 0.05) (Fig. 3.17a, c). This
is in marked contrast to GODZ/SERZ-β DKO neurons where the total expression of
AMPAR GluR2/3 was significantly reduced. This suggests that GODZ/SERZ-β
dependent expression of AMPARs is not simply compensatory in response to deficits in
GABAergic transmission.
90
CHAPTER FOUR
DISCUSSION
4.1. Overview of findings
Work presented here was designed to elucidate the function of GODZ and SERZ-β in
GABAAR clustering and GABAergic inhibitory synapse formation in vivo. GODZ KO
and DKO mice showed a significant reduction in body weight. In addition DKO mice
showed a partially penetrant early postnatal lethal phenotype. GODZ KO mice were
viable but showed hyperekplexia-like hyperlocomotion and enhanced prepulse inhibition,
indicative of altered sensorimotor gating. SERZ-β KO mice lacked an overt behavioral
phenotype. At the cellular level, GODZ KO and DKO mice showed deficits in
postsynaptic accumulation of GABAARs in cultured cortical neurons. However, this
phenotype was only seen in mixed cultures containing mutant and an excess of WT
neurons. The immunofluorescence phenotype of DKO neurons was confirmed
functionally by a reduction in the frequency and amplitude of mIPSCs. Furthermore, the
frequency of mEPSCs was also reduced. Loss of GODZ/SERZ-β in brain of mutant mice
was paralleled by a significant upregulation of transcripts for the closely related PAT
DHHC15, which may partly compensate for loss of GODZ and/or SERZ-β.
In a related project analyzing the functional relationship of GABAAR and NL2
with respect to their targeting to postsynaptic specialization, I found that the γ2 subunit is
required for normal expression of other GABAAR subunits and NL2 at the cell surface.
Thus, loss of postsynaptic GABAARs and NL2 in GODZ mutant neurons may in part
reflect their interdependence in intracellular trafficking, in addition to independent
deficits due to their reduced palmitoylation.
91
4.2. GODZ and SERZ-β are required for γ2 subunit postsynaptic clustering, but
dispensable for normal surface expression
In addition to the reduction of postsynaptic clustering of the γ2 subunit in GODZ
KO/DKO neurons co-cultured with WT neurons, the amplitude and frequency of mIPSCs
recorded from DKO neurons in co-culture were significantly reduced compared with WT
neurons. These results together indicate that GODZ KO and DKO neurons contain the
reduced number of GABAergic synapses and that the GABAergic synapses contain less
number of GABAARs compared to WT. In contrast, the GABA-evoked whole cell
current was unchanged in DKO neurons co-cultured with WT neurons. Consistent with
this result, γ2 subunit surface expression was unaltered in DKO compared to WT neurons.
These results suggest that GODZ/SERZ-β-mediated palmitoylation is dispensable for the
surface expression, but necessary for the translocation to the postsynaptic sites or the
stability at postsynaptic sites of γ2 subunit containing GABAARs (see below for the
possible mechanisms in discussion 8 and 9). This phenomenon is consistent with the fact
that no effects were observed in the postsynaptic clustering of γ2 subunit containing
GABAARs in pure culture of GODZKO and DKO cortical neurons. If the surface
expression of γ2 subunit containing GABAARs were reduced, the number of
immunoreactive puncta of the γ2 subunit could also be reduced even in pure KO cultures
because of the reduced availability of GABAARs on cell surface.
One of the reasons that the GODZ KO- or DKO-mediated deficits were only
observed in the competitive environment by mixing with excess WT neurons might be
because the velocity of GABAARs lateral movement is reduced in KOs. GABAARs
localize at extrasynaptic and synaptic sites and they exchange the location by lateral
diffusion (Thomas et al., 2005). Loss of palmitoylation may cause GABAARs to move
slower in plasma membrane; thereby it takes relatively longer time to establish the pre-
and postsynaptic connections than that between WT-WT connections. Alternatively, the
GABAARs at the site are not stable in KOs during and/or after establishing the pre- and
postsynaptic connections, resulting in the failing of the synapse formations. In both case,
competitive environment could greatly lead a disadvantage to KO neurons to establish
effective synapse formations as presynaptic WT neurons may preferentially innervate on
92
WT neurons that have more abundant and stable GABAARs available at synaptic sites. It
would be very interesting to assess whether the lateral movement of GABAARs at plasma
membrane is changed in GODZ/SERZ-β KOs using Florescence Recovery After
Photobleaching (FRAP). FRAP has been used to analyze dimensional lateral diffusion of
a fluorescently probed proteins in the cell membrane (reviewed in (Chen et al., 2006,
Henis et al., 2006)). To analyze the lateral movement of γ2 subunit containing GABAARs,
GODZ KO or DKO cortical cultures neurons will be transfected with pHGFP-γ2 subunit
and subjected to FRAP analyses. Due to the pH sensitivity of pHGFP tags, it allows to
analyze the lateral movement of tagged proteins specifically on the cell surface
(Miesenbock et al., 1998, Ashby et al., 2004). By focusing the bleaching at synaptic
terminal, it would provide a role of GODZ/SERZ-β-mediated palmitoylation on the
lateral movement of GABAARs required for synapse formation and maintenance.
4.3. DKO neuron exhibit deficit not only in the inhibitory but also the excitatory
neurotransmissions
In addition to the reduction in the amplitude and frequency of mIPSCs, the frequency of
mEPSCs was also reduced in DKO neurons in the co-culture. The miniature frequency is
usually representative for the presynaptical neurotransmitter release probability; however,
given the situation of mixed culture with 70% WT and 30% DKO, the presynaptic
neurons are most likely WT. Therefore, focusing on the postsynaptical deficit, there are
two possible mechanisms to explain this phenomenon: 1) a deficit in palmitoylation of
proteins in excytatorty synapse; 2) scaling/homeostatic effects by excitatory synapse. The
possibility of the first mechanism is based on the fact that GODZ/SERZ-β also
palmitoylate proteins in excitatory synapse such as PSD-95 (Fukata et al., 2004) and
AMPARs (Hayashi et al., 2005). The loss or reduced palmitoylation level of those
proteins might lead to the reduced postsynaptic clustering of AMPARs and other proteins
at excitatory synapse and the reduced mEPSC frequency. However, this mechanism does
not explain why the same phenomenon was not observed in neurons trasnfected with
GODZC157S, dominant negative form of GODZ, or with shRNA (Fang et al., 2006). The
93
inhibitory neurotransmission in those transfected neurons were significantly reduced, but
the excitatory neurotransmission unaffected (Fang et al., 2006).
The second mechanism, on the other hand, explains well the difference between
these two experiments. It has been known that balancing between excitatory and
inhibitory neurotransmission is critical for normal brain functions (reviewed in (Levinson
and El-Husseini, 2005a, Levinson and El-Husseini, 2005b, Akerman and Cline, 2007)).
For example, functional synapses are evenly distributed across dendritic surfaces and the
ratio of excitatory/inhibitory synapses on dendrite is highly correlated. (Liu, 2004).
Furthermore, studies in cultured neurons showed that the balance of excitatory and
inhibitory synaptic inputs can be homeostatically regulated in an activity-dependent
manner (Kilman et al., 2002, Liu, 2004). A prolonged response of GABAARs (Liu, 2004)
or a prolonged activity deprivation (O'Brien et al., 1998, Kilman et al., 2002) results in an
enhancement of excitatory synaptic strength and a reduction of inhibitory synaptic
strength. In contrast, a prolonged deprivation of inhibitory inputs leads an opposite effect
(Lissin et al., 1998, Liu, 2004). In current study of DKO neurons co-cultured with GFP-
WT, the number of GABAergic synapses as well as the GABAergic inhibitory
neurotransmission was significantly reduced. It can be expected that the excitatory
synapse formation is also reduced to compensate the reduced inhibition over the time of
neuronal maturation. In contrast to DKO neurons where no GODZ or SERZ-β is
available to begin with, the GABAergic synapses are acutely reduced in the neurons
transfected with GODZC157S or shRNA at DIV18 and analyzed two days later. It is likely
that the two days of GODZ/SERZ-β knockdown in matured WT neurons is enough for
the reduction in the number of GABAergic synapses, but not for the compensational
reduction in excitatory neurotransmission followed by the GABAersic deficits. To
differentiate the two mechanisms, it would be interesting to analyze whether the
excitatory neurotransmission is affected in the neurons trascfected with GODZC157S or
shRNA at earlier age and analyze after a longer time of period.
94
4.4. GODZ might be the major PAT for the γ2 subunit of GABAARs
Potsynaptic accumulation of the γ2 subunit in GODZ KO neurons co-cultured with WT
neurons was greatly reduced (Fig. 3.6). Based on the fact that DKO, but not GODZ KO,
mice showed severe lethal phenotype, DKO neurons were expected to show more severe
reduction in the postsynaptic clustering of γ2 containing GABAARs even in pure culture.
However, the phenotype of DKO neurons was indistinguishable from that of GODZ KO
neurons (Fig. 3.6f, g, h). Similar observations were made with previous experiments that
used GODZ shRNA and dominant negative GODZ (GODZC157S) to knockdown GODZ
and SERZ-β (Fang et al., 2006). Whereas GODZ shRNA is specific for GODZ,
GODZC157S can multimerize with both GODZ and SERZ-β and thus is likely to inhibit
the function of both GODZ and SERZ-β. Nevertheless, the effects of both treatments on
the structure and function of GABAergic synapses were similar (Fang et al., 2006). These
results suggest two possibilities. First, the compensatory mechanisms, such as an up-
regulating of DHHC15 expression, contribute to dampening the effects of the loss of
GODZ and SERZ-β. Second, GODZ is the major palmitoyl transferase of γ2 subunit of
GABAAR and SERZ-β might have a residual activity for γ2 subunit palmitoylation as
observed in vitro palmitoylation assay in HEK293T cells (Fang et al., 2006) and function
as a supplemental enzyme. To differentiate two possibilities, the same
immunofluorescent analyses but using SERZ-β KO neurons in co-culture or DKO
neurons transfected with DHHC15-specific shRNA is required. Alternatively, triple KO
mice of GODZ, SERZ-β and DHHC15 could be generated for the same analyses.
4.5. Compensation of loss of GODZ/SERZ-β function by other DHHC family
proteins
Whereas GODZ and SERZ-β KO mice are viable, DKO mice show perinatal lethality,
with only a fraction of mice surviving to adulthood. The causes of the lethal phenotype
could be, at least in part, the deficits in GABAergic synapses as observed in the reduction
of postsynaptic clustering of GABAARs and functional deficit in GABAergic and
glutamatergic neurotransmission. Although GODZ and SERZ-β are the only DHHC
proteins that palmitoylate the γ2 subunit of GABAARs and NL2 in vitro, the loss of
95
function of GODZ and SERZ-β in neurons may be compensated by other DHHCs.
DHHC15, one of the DHHC family proteins, is a good candidate as it has a trace activity
to palmitoylate γ2 subunit of GABAARs upon cotransfection into HEK293T cells.
Preliminary data from quantitative RT-PCR using mRNA purified from brain showed
that the expression of ZDHHC15 was increased in GODZ KO and DKO mice compared
with WT (Fig. 3.16a, b). In contrast, the expression of ZDHHC21 encoding another close
paralog of GODZ was unaltered in GODZ KO and DKO mice (Fig. 3.16a, c). It would be
very interesting to analyze whether the GABAergic deficit in the shRNA- or dominant
negative GODZ-transfected neurons can be rescued by cotransfection of ZDHHC15 gene.
Because GODZ and SERZ-β also palmitoylate other proteins such as PSD-95
(Fukata et al., 2004), AMPA receptors (AMPARs) (Hayashi et al., 2005), SNAP-25
(Fukata et al., 2004) and cysteine-string protein (Greaves et al., 2008), other possible
causes for KO mice phenotype should not be excluded. It could be because of the reduced
palmitoylation of PSD-95 and/or AMPARs, resulting in the deficit in glutamatergic
synapse, or the presynaptic deficit caused by the reduced palmitoylation level of SNAP-
25 or GAP43. However, it is unlikely as they are likely to be also palmitoylated by other
DHHC proteins. The loss of the palmitoylation of unidentified substrates by GODZ and
SERZ-β might also contribute to the phenotype. Given the fact that many proteins
implicated in neural development and synaptic functions are palmitoylated, it is very
important to be aware of the other possible substrate to assess the experimental results.
4.6. Behavioral phenotypes of GODZ KO mice
4.6.1. GODZ KO mice demonstrate reverse phenotype with NL2 transgenic mice
GODZ KO mice showed normal levels of anxiety-related behavior in the elevated plus
maze or the free choice paradigm. However, they showed reduced thigomotaxis as
indicated by the increased center distance traveled in the Open Field test compared to WT.
This phenotype is the inverse of that observed in NL2 transgenic mice. Moderately
elevated levels of NL2 in these mice resulted in an increase in the number of GABAergic
inhibitory synapses and an increase in the ratio of inhibitory/excitatory synapses
96
associated with marked elevated levels of anxiety-like behavior such as thigomotaxis
(Hines et al., 2008). The increased center distance traveled by GODZ KO mice is inverse
to the thigomotaxis behavior of NL2 overexpressing mice and suggestive of a deficit in
GABAergic inhibitory synapses in GODZ KO mice in vivo.
4.6.2. Hyperekplexia-like phenotype in GODZ KO ~ Possible link between
GODZ/SERZ-β with glycine receptors
Excessive hindleg clenching, spontaneous jumping in response to handling, and
hyperlocomotion in the Open Field test of GODZ KO mice are reminiscent of a
hyperekplexia-like phenotype seen in mice with mutations in glycine receptors or glycine
transporters (Becker et al., 2002). Hyperekplexia (aka startle disease) is a neurological
disorder, 80% of which is caused by diverse mutations in GLRA1, the gene encoding
glycine receptor subunit GlyR α1 (reviewed in (Zhou et al., 2002)). Three hyperekplexia-
like mouse models have been identified with different mutations in the GLRA1 gene
(spasmoid, caused by a missense mutation near N-terminus of the glycine receptor α1
subunit; oscillator, a microdeletion causing a frameshift and loss of M3-M4 loop and M4
domain of the same subunit) or GLRB gene (spastic; an insertion of a 7.1 kb
retrotransposon within intron 6 of GLRB) (Ryan et al., 1994, Kling et al., 1997)
(Kingsmore et al., 1994). The spasmoid mutation is thought to affect the interaction of
GlyRα1 with other components of the receptor complex. Spasmoid and spastic, as well as
GlyT2 KO mice display a hind leg clasping phenotype similar to GODZ KO mice.
Other mutations that cause hyperekplexia in human have been also found in the
genes encoding the glycine receptor β subunit (Rees et al., 2002), the presynaptic
sodium- and chloride-dependent glycine transporter 2 (GlyT2) (Gomeza et al., 2003,
Eulenburg et al., 2006, Rees et al., 2006), the inhibitory scaffold protein gephyrin (Reiss
et al., 2001, Rees et al., 2003) and the GDP-GTP exchange factor and gephyrin
interacting protein collybistin (Harvey et al., 2004). Gephyrin and collybistin are found at
both glycinergic and GABAergic synapses (Pfeiffer et al., 1982, Essrich et al., 1998, Kins
et al., 2000, Harvey et al., 2004). Collybistin has been proposed to play a role in actin
dynamics by binding to the Rho-type GTPase Cdc42 (Kins et al., 2000) and in trafficking
97
of proteins, including gephyrin and GABAARs to the subsynaptic actin cytoskeleton of
inhibitory synapses (reviewed in (Kneussel and Betz, 2000, Harvey et al., 2004, Luscher
and Keller, 2004)). The collybistin G55A mutation interferes with binding to gephyrin
and, upon overexpression in cultured neurons, has dominant negative effects on
postsynaptic clustering of GABAARs. It was suggested that this mutation is responsible
for the hyperekplexia phenotype by causing mislocalization of GABAARs and GlyRs in
brain stem and spinal cord (Harvey et al., 2004).
The hyperekplexia-like phenotype observed in GODZ KO mice might be due to
deficits in GABAergic inhibitory synapses in the brain stem and or spinal cord. However,
based on the similar functions of GABAergic and glycinergic inhibitory synapses and the
fact that mutations in collybistin and gephyrin implicated in the function of both types of
synapses can cause hyperekplexia, it can be hypothesized three possible mechanisms by
which GODZ (and possibly SERZ-β) deficits might lead to hyperekplexia. First,
preliminary analyses of glycinergic synapses in the retina indicate that NL2 is present not
only at GABAergic but also at glycinergic synapses (Dr. Rachel Wong, Washington U. St
Louis, personal communication). Thus, reduced palmitoylation of NL2 in GODZ KO and
DKO mice could contribute to deficits at both GABA and glycinergic synapses. Second,
GlyRs are perhaps palmitoylated by GODZ/SERZ-β and deficient in their palmitoylation
in KO mice lead a deficit in postsynaptic clustering of GlyRs and neurotransmission at
glycinergic synapse. There are Cys residues in the cytoplasmic loop between TM3 and 4
of α1 and β subunit of GlyRs. However, there is no apparent homology in this region
between these proteins. At last, given the fact that γ2 subunit of GABAARs and gephyrin
is interdependently required for their postsynaptic clustering (Essrich et al., 1998), GODZ
KO-mediated deficit of GABAAR postsynaptic accumulation might interfere with
gephyrin trafficking and its interacting protein collybistin indirectly. Their
mislocalization might lead to the deficit in GlyR trafficking, resulting in developing
hyperexplexia.
98
4.7. Technical limitation in the palmitoylation assay in neurons
To directly show whether palmitoylation levels of γ2 subunit of GABAARs and NL2 are
affected in GODZ KO, SERZ-β KO and DKO mice, an extensive trials of palmitoylation
assay using cultured cortical neurons was performed. Metabolic labeling with [3H]-
palmitate has been used to detect γ2 palmitoylation in cultured neurons (Keller et al.,
2004, Rathenberg et al., 2004). In this current experiment, γ2 subunit or NL2 were
needed to be immunoprecipitated to analyze the palmitoylation level specific for γ2
subunit and NL2. Due to the limited sensitivity of [3H] fluorography, it is critical to
immunoprecipitate a majority of target proteins. This is especially important for the
experiments where the palmitoylation levels of the target proteins are quantitatively
analyzed. For this reason, anti-γ2 antibody was also used in addition to anti-α1 antibody
which was used initially according to previous experiment (Keller et al., 2004). However,
it was unsuccessful to find the optimal protocol for γ2 immunoprecipitation using the
anti-γ2 subunit antibody. In addition, the mobility of γ2 subunit (~43 kDa) is similar to
that of IgG (50 kDa), giving rise to such a high background that masked the specific
signals for γ2 subunit. For NL2, an extensive trial of immunoprecipitation using anti-NL2
antibody (gift from N. Brose) led me to find the optimal condition for NL2
immunoprecipitation in cold experiment where all precipitated proteins were loaded on
one lane of SDS-PAGE. However, for the palmitoylation assay, the precipitated proteins
were divided to two portions; 75% is for [3H] fluorography and 25% is for Western blot
to verify the identity of immunoprecipitated [3H]-labeled proteins by probing with the
same anti-NL2 antibody. The change of the protein amount on one lane had great effects
on the NL2 signal. The 25% of immunoprecipitated protein showed no NL2 signals (100
kDa), which was not only because of insufficient amount of NL2 but also a smear
background around 80-90 kDa. This background was from IgG, probably a dimer of short
and long IgG molecule (~75 kDa). One reason of the unresolved dimer form of IgG in
SDS-PAGE was the low concentration of DTT used in loading buffer. In ordinal loading
buffer for SDS-PAGE contains 100-150 mM DTT, whereas 5 mM DTT was used in this
study as the thioester bond is known to be sensitive to reducing agents (Bizzozero, 1995).
99
However, the reason of the correlation of disappearance of NL2 signal with the IgG
background is not clarified.
It is always hampered to have a limitation in the availability of good quality of
antibody sufficient enough for immunoprecipitation. Now transgenic mice expressing
His-FLAG-YFP (HFY)-tagged γ2 and NL2 are available in our lab. The HFY-tag should
be able to facilitate affinity purification of γ2 subunit and NL2. Using this mice line to
cross with GODZ/SERZ-β KOs and acquire GODZKO or DKO with HFY-γ2 or HFY-
NL2, it is hoped that the palmitoylation level in KOs can be analyzed quantitatively
without further technical complications. Alternatively, it might be possible to generate
fluorescent reporters that respond upon palmitoylation by GODZ and SERZ-β. A GFP-
variant containing palmitoylation site from GAP43 has been invented to visualize the
localization of this palmitoylated GFP-variant into caveolae (Zacharias et al., 2002). It is
conceivable to generate a GFP-variant that contains the palmitoylation site from γ2
subunit of GABAARs or NL2 to verify their palmitoylation level in GODZ KO and DKO
neurons. Furthermore, the palmitoylation activity in GODZ KO and DKO can also be
assessed by immunefluorescence analyses of GFP-GAD65. GAD65 has been shown as a
substrate of GODZ in our lab (Fang et al, unpublished). Recent study showed that WT
GFP-GAD65 predominantly localized at Golgi-apparatus and presynaptic clusters in
neurons, whereas palmitoylation deficit mutant GFP-65(C30, 45A) localized at ER in
addition to Golgi (Kanaani et al., 2008). These data suggest that palmitoylation shifts
GAD65 from ER to Golgi and thereby to a post-Golgi vesicular pathway (Kanaani et al.,
2008). By analyzing the localization of transfected WT GFP-GAD65 or the
palmitoylation deficit mutant GFP-65(C30, 45A) in GODZ KO or DKO neurons, it
would be possible to assess whether palmitoylation activity is affected in those KOs. It is
expected that WT GFP-GAD65 would localize in ER and Golgi in KO neurons, whereas
the localization of GFP-65(C30, 45A) in ER and Golgi would not be changed in KO
compared to WT neurons.
100
4.8. Palmitoylation and lipid raft
Lipid raft is cholesterol and sphingolipid-rich microdomains in the membrane (Brown
and London, 1997, Brown, 1998). It has been thought to function as platforms where
proteins with affinity for rafts, including signaling proteins, interact with other proteins
efficiently (reviewed in (Simons and Toomre, 2000)). Lipid rafts are also involved in
trafficking/sorting of membrane proteins and secretory/endocytosis pathways (Brown and
London, 1998, Ikonen, 2001). Many neurological events such as synapse formation and
remodeling, synaptic transmission and neuronal polarity establishment are closely related
to the membrane dynamics. In cultured hippocampal neurons, lipid rafts exist abundantly
in dendrites (Hering et al., 2003). In addition, many neuronal proteins such as SNAP 25,
synaptotagmins, synaptophysin, AMPARs, NMDARs, and PSD-95 have been identified
as lipid raft-associated proteins in rat brain (Suzuki et al., 2001, Wong and Schlichter,
2004, Besshoh et al., 2005, Gil et al., 2006). Furthermore, depletion
cholesterol/sphingolipid leads to instability of AMPARs and loss of excitatory/inhibitory
synapses and dendritic spines, suggesting the importance of lipid rafts for normal
synaptic density and morphology.
GABAARs also associate with lipid rafts (Dalskov et al., 2005, Li et al., 2007). It
has been reported that there are two fraction of Triton X-100 insoluble GABAARs: 1)
high-density pool containing α, β, and γ2 subunits as well as gephyrin, 2) low-density
pool containing α and β associating with lipid rafts some of which contain δ subunit, but
not γ2 subunits or gephyrin (Li et al., 2007). After cholesterol depletion, the first pool
remains to be Triton X-100 insoluble, whereas the second pool becomes soluble in Triton
X-100. In addition, the lipid raft containing pool diffusely distributed on membrane
surface. Together, the authers suggested that lipid raft containing pool is mainly
extrasynaptic and the first pool is the postsynaptic complex of GABAergic synapses (Li
et al., 2007). Interestingly, the pool of NR1, NR2A and NR2B is reduced in lipid rafts
whereas increased in post-synaptic densities (PSDs) after ischemia, suggesting the close
interaction between lipid rafts and PSDs (Besshoh et al., 2005).
Many proteins have been reported to require palmitoylation to localize at lipid
rafts (for instance, (Wong and Schlichter, 2004, Salaun et al., 2005, Chakrabandhu et al.,
101
2007, Nini et al., 2007, Renner et al., 2007)). Some proteins such as CaMKIIalpha-
interacting protein BAALC1-6-8 and Herpes membrane-associated tegument protein
UL11 are need to be dual-acylated by N-terminal myristoylation and palmitoylation to
associate with lipid rafts (Wang et al., 2005, Koshizuka et al., 2007). It is also reported
that palmitoylation are necessary for two close homolog protein SNAP23 and SNAP25 to
associate with lipid raft. An additional cysteine residue in SNAP-23 increases affinity for
lipid rafts by three times compared with SNAP-25, suggesting the degree of
palmitoylation modify protein localization in lipid rafts (Salaun et al., 2005). On the other
hand, palmitoylation does not contribute or even inhibit some proteins to be translocated
to lipid rafts (Abrami et al., 2006, Pang et al., 2007). It is likely that the palmitoylation in
different protein has a distinct role in the association with lipid rafts and it could be
modified by other factors such as other acylation, protein-protein interactions, or other
protein trafficking mechanisms.
The effects of palmitoylation on the localization of γ2 subunit containing
GABAARs and NL2 to lipid rafts have not known yet. As described above, however, γ2
subunit containing GABAARs locate at PSD, not extrasynaptic lipid rafts (Li et al., 2007).
In the current study, the surface expression of γ2 subunit and NL2 were unchanged in
DKO cortical neurons, whereas the postsynaptic clustering of γ2 subunit containing
GABAARs were significantly reduced in DKO and GODZ KO neurons. These results
suggest that GODZ/SERZ-β-mediated palmitoylation of γ2 subunit and/or NL2 is
necessary for GABAARs to translocate to or stabilize at inhibitory postsynaptic sites.
From these points of views, one might hypothesize that non-palmitoylated GABAARs are
first targeted to extrasynaptic site via lipid raft association and transferred to postsynaptic
site by lateral movement that requires the GODZ/SERZ-β-mediated palmitoylation. In
other words, GODZ/SERZ-β-mediated palmitoylation might inhibit γ2 subunit containing
GABAARs and/or NL2 from the association with lipid rafts; thereby ensures to locate
them at postsynaptic sites. Alternatively, palmitoylation of GABAARs increase the
association with lipid rafts which contain ubiquitination machinery to be degraded (see
also discussion 9). It would be interesting to analyze whether γ2 subunit distribution is
changed in the three pools; high- and low- density pool of Triton X-100 insoluble
102
fractions and soluble fractions in GODZ and DKO neurons. The loss of palmitoylation by
GODZ/SERZ-β might lead more association of γ2 subunit containing GABAARs and/or
NL2 to lipid rafts.
4.9. Palmitoylation and ubiquitination
Ubiquitination of proteins by attachment of single- or polymeric-76 amino acids ubiquitin
has been known as a mechanism to regulate the protein in many way including
proteolysis through proteasomal pathway, protein localization and activity (Hochstrasser,
1996, Schnell and Hicke, 2003). Ubiquitination also regulates the synaptic strength by
changing the number of neurotransmitter receptors on the cell surface through the
endocytosis and contributes to neural activity-dependent accumulation of these receptors
(Colledge et al., 2003, Bingol and Schuman, 2004). Interestingly, this process is governed
by the cell activity (Ehlers, 2000, Lin et al., 2000).
Rapidly emerging evidences demonstrate that palmitoylation and ubiquitinaition
function in an opposite way. In yeast, for instance, Swf1 is a DHHC-CRD protein that
palmitoylates SNARE Tlg1 protein. In swf1∆, unpalmitoylated Tlg1 is recognized by
ubiquitin ligase Tul1 and degraded, suggesting a function of palmitoylation to protect the
protein from degradation (Valdez-Taubas and Pelham, 2005). Palmitoylation also plays
roles in the structural conformational change in TM domains to ensure the ER exit of the
SLP6 protein, a downstream protein of Wnt signaling (Abrami et al., 2008). The
palmitoylation-deficient SLP6 with two cysteine mutation at palmitoylation site undergo
monoubiquitination and is trapped in ER (Abrami et al., 2008). Furthermore, although the
palmitoylation has been thought to facilitate protein localization in a lipid rafts, the
palmitolation of anthrax toxin receptor TEM8 surprisingly prevents its association with
lipid raft where E3 ubiquitin ligase localizes and ubiquitination of the receptor occurs
(Abrami et al., 2006). Thus, palmitoylation counteracts the ubiquitination functions as it
might reduce the accessibility of the lysines in proximity of the palmitoylation site by E3
ubiquitin kinase, or keep the protein away from the membrane domain such as lipid rafts
containing the E3 ligase.
103
GABAAR β and γ2 subunits are known to be ubiquitinated at Lys residues in their
cytoplasmic loop. Similar to the other neurotransmissiton receptors, their ubiquitinaition
provides a mechanism to alter the efficacy of synaptic inhibition by removing GABAARs
in an activity-dependent manner (Saliba et al., 2007). In addition, GABAARs bind
directly to the ubiquitin-like protein Plic-1, a regulator of ubiquitin-dependent
proteasomal degradation. Plic-1 increases the stability of β3 subunit of GABAARs in ER
along with an increase in the abundance of poly-ubiquitinated receptor subunits (Saliba et
al., 2008). It also increases the rate of the insertion of GABAARs into membranes,
resulting in the increase of cell surface expression level of GABAARs (Bedford et al.,
2001, Saliba et al., 2008).
By analogy, GODZ/SERZ-β-mediated palmitoylation may counteract
ubiquitination effects on GABAARs. In preliminary screening using Split-Ubiquitin yeast
two hybrid system (Dualsystems Biotech, Switzerland) in our lab, Plic-1 was identified as
an interacting protein of GODZ (Shi et al, unpublished results). Using the Matchmaker
yeast two hybrid system (Clontech), TRIM32 is also identified as GODZ interacting
protein (Fang et al, unpublished results). TRIM32 is a member of a family of tripartite
motif (TRIM) genes containing a conserved RING finger domain and thus represent
putative ubiquitin E3 ligase (Reymond et al., 2001, Frosk et al., 2002, Kudryashova et al.,
2005). GODZ/SERZ-β-mediated palmitoylation might affect on the endocytosed
GABAARs from postsynaptic site through counteracting ubiquitin/UPS systems. It would
be very interesting to assess whether the ubiquitination level of GABAAR β and γ2
subunits is changed in GODZ KO/DKO neurons or in HEK293T cells transfected with
α2β3γ2 either alone or with GODZ in case a compensatory mechanism masks the effects
in KO neurons. To confirm that the effects of GODZ on ubiquitination is γ2 subunit
palmitoylation-dependent, the same experiment in HEK293T cells can also be carried out
using palmitoylation deficient mutant of γ2 or GODZ. In addition, the endocytosis rate of
these proteins also needs to be elucidated in GODZ KO/DKO neurons. The results of
these experiments will reveal a mechanism in which counteraction of palmitoylation and
ubiquitination affects on the synaptic strength of GABAergic inhibitory synapses.
104
4.10. Possible mechanism of cooperative function of NL2 and γ2 to induce inhibitory
synapse
In GABAARs γ2 KO neurons, the punctuate immunoreactivity of NL2 was dramatically
reduced. In this current study, the surface expression level of NL2, α1 and β2/3 subunit
of GABAARs were also significantly reduced, whereas the total amount of NL2 and β2/3
subunits in γ2 KO neurons unchanged compared with WT neurons. These results suggest
that γ2 subunit is required for NL2 to translocate at cell surface, as well as to postsynaptic
sites. In addition, analyses in our lab using HEK293T cells transfected with epitope-
tagged recombinant GABAAR subunits and NL2 revealed that NL2 coaggregates with the
β3 subunit only, α2β3γ2, α2β3 or α1β2 subunit combinations in the plasma membrane.
However, there was no significant coaggregation between γ2 subunit and NL2. These
results suggest that NL2 interacts with GABAARs in the plasma membrane in a γ2
subunit-independent manner. Furthermore, the expression of α2β3 and NL2 are sufficient
for the formation of functional GABAergic synapses in HEK293Tcells co-cultured with
neurons (Dong et al., 2007). Based on the role of NL2 in GABAergic synapse formation
and maturations (Scheiffele et al., 2000, Fu et al., 2003, Graf et al., 2004, Levinson et al.,
2005) together with those results, it can be hypothesized that indirect interaction between
NL2 and γ2 subunit through α or β subunits is required for the surface expression and
possibly postsynaptic clustering of the GABAARs-NL2 complex. This GABAAR-
dependent presentation of NL2 at the cell surface might promote interaction of the
postsynaptic membrane with GABAergic presynaptic nerve terminals, thereby ensuring
proper apposition of matching presynaptic site with postsynaptic GABAARs.
Recent studies by Chih et al. and Chubykin et al. nicely support this idea.
Although the overexpression of NL2 induced the formation of functional GABAergic
synapses (Chih et al., 2005, Chubykin et al., 2007), analyses of NL2 knock-out mice
showed that NL2 was dispensable for synaptic formation, but necessary for synaptic
maturation and functions in vivo (Varoqueaux et al., 2006). In addition, the NL2-induced
selective synaptogenesis requires synaptic activity (Chubykin et al., 2007). Thus, a model
proposed by this group divides phenomenon of synaptogenesis in two major processes.
First, transient synapses are constantly formed by other adhesion molecules such as
105
cadherins. These “immature synapses” are functional, but are stabilized and specified
further by the second process in activity-dependent manner (Chubykin et al., 2007). In
the second process, GABAAR coupled with NL2 might be the crucial player to define the
synapse specificities by receiving the synaptic neurotransmission from presynaptic
terminal.
Interestingly, knockdown of GODZ by shRNA in cortical neurons also results in
the significant reduction in the number of NL2 immunoreactive puncta. This
phenomenon could be through the deficits in postsynaptic clustering of γ2 subunit
containing GABAARs, which leads to the reduction in postsynaptic NL2 that is
interacting with GABAARs. Alternatively, the palmitoylation of NL2 per se is crucial for
postsynaptic clustering of the complex of GABAARs and NL2. It would be interesting to
analyze the immunoreactive puncta of γ2 subunit and NL2 in γ2 KO neurons transfected
with mutant γ2 subunit in which the palmitoylation site by GODZ and SERZ-β are
mutated, and the same scheme of experiment in NL2 KO neurons transfected with the
palmitoylation deficient NL2. This result would reveal whether the palmitoylation of
either γ2 subunit or NL2, or both, is necessary for GABAergic synapse formation.
4.11. Outlook
The present study has helped to strengthen our understanding of the role of
palmitoylation in the mechanism that regulates GABAergic neurotransmission, especially
in vivo. Localization and trafficking of GABAARs is known to be governed by complex
but sophisticated processes containing phosphorylation and ubiquitinaiton of GABAARs
and interaction with diverse regulatory proteins (Luscher and Keller, 2004, Jacob et al.,
2008). In current study, palmitoylation was shown to affect greatly on the postsynaptic
accumulation of GABAARs and thereby on the GABAergic neurotransmission, but not on
the surface expression level of GABAARs. Furthermore, deficit in GODZ/SERZ-β-
mediated palmitoylation caused to behavioral abnormalities. Thus, GODZ/SERZ-β-
mediated palmitoylation has a great impact on the normal brain functions. However,
further studies are needed to fully understand the role of GODZ/SERZ-β-mediated
palmitoylation in brain.
106
First, exploration of the other substrates of GODZ/SERZ-β is a must. Based on a
fact that a variety of proteins are palmitoylated in brain, GODZ and SERZ-β might
palmitoylate several proteins. It would help to assess the behavioral phenotype of these
KO mice and further understanding of the role of GODZ/SERZ-β-mediated
palmitoylation in brain function. Using yeast two-hybrid screening with GODZ as a bait,
several GODZ-interacting proteins have been identified in our lab (unpublished).
However, none of the proteins that have been reported as GODZ substrates were isolated
by this system except γ2 subunit of GABAARs. It could be due to the labile nature of
palmitoylation or the technical limitation as it restricts the localization of the bait protein.
It would be needed to use different portion of GODZ as bait and different Y2H systems.
Alternatively, it might be overcome by other methods such as co-immunoprecipitation of
crosslinked (cleavable) protein followed by a separation on SDS-PAGE and Mass-
spectrometry of each band.
Second, the relationship between GODZ/SERZ-β-mediated palmitoylation and
ubiquitination is also need to be clarified. Current study demonstrated that GODZ/SERZ-
β-mediated palmitoylation was dispensable for the surface expression of γ2 subunit of
GABAARs and NL2, whereas required for the postsynaptic clustering of γ2 subunit. As
discussed in detail above, palmitoylation might inhibit ubiquitination thereby protect γ2
subunit from protein degradation. Thus, it is necessary to assess the ubiquitination level
of γ2 subunit in GODZ KO or DKO neurons.
Third, the effects of GODZ/SERZ-β-mediated palmitoylation on the presynaptic
site are also needed to be examined. In this study, it was shown that the presynaptic
innervations were impaired by the GODZ deficit in postsynaptic site. Most recent study
reported that the level of presynaptic proteins such as vesicular GABA transporter
(VGAT) and vesicular glutamate transporter (VGluT) were increased in the transgenic
mice that express NL2 slightly higher than WT level (Hines et al., 2008). In the current
study, the total amount of NL2 was unchanged in DKO neurons. However, preliminary
study in our lab showed that postsynaptic clustering of NL2 in GODZ deficient neurons
by shRNA transfection was significantly reduced. Similar phenotype is expected to be
observed in GODZ KO or DKO neurons. This deficit may indirectly affect on
107
presynaptic elements by probably reducing the expression level of the presynaptic
proteins as expected opposition effect of the NL2 transgenic mice. The protein level of
VGAT and VGluT are needed to be quantified using brain extract from GODZ KO and
DKO mice.
Most interestingly, revealing of the mechanism in which γ2 subunit is required for
the NL2 trafficking seems to be critical to understand GABAergic synapse formation.
Current study showed that NL2 was trapped intracellularly in γ2 KO neurons along with
α and β subunits that coaggregated with NL2 in overexpressing heterologous cells. It is
inconsistent with the previous observation that coexpression of α and β subunit in
heterologous cells are sufficient to assemble GABA-gated ion channels on the cell
surface, whereas γ2 subunit is required for postsynaptic clustering of GABAARs (Whiting,
1999). It could be due to the overexpression system and/or basic difference in the
molecular machinery between neuron and the heterologous cells. These data led a
hypothesis that GABAARs and NL2, that is selective cell adhesive molecule required for
the maturation of inhibitory synapse, might be presented interdependently on the cell
surface to facilitate proper apposition of pre- and postsynaptic terminal. This hypothesis,
though, is based on the direct or indirect interaction between α and/or β subunit with
NL2. It is necessary to find out whether they interact each other or through other protein.
Furthermore, from the study done in DKO neurons, GODZ-mediated palmitoylation
might not play a role in the NL2-GABAAR interdependency for surface expression.
However, it could facilitate synapse maturation or stability as observed the presynaptic
innervation was declined by the postsynaptic deficient of GODZ.
At last, a new insight of the GODZ function is emerging. Upon overexpression in
Xenopus oocyte, GODZ mediated Ca2+ transport (G. A. Quamme, personal
communication). In addition, this transport is modulated by palmitoylation as 2-
bromopalmitate, general inhibitor of palmitoylation, inhibits Ca2+ transport by about 50%.
Our preliminary data also showed that Ca2+ influx was dramatically reduced in DKO
neurons compared to WT neurons. HIP14, another DHHC protein, has been also shown
as a “chanzyme’ that transport Mg2+ in palmitoylation dependent-manner (Goytain et al.,
2008). GODZ mainly exists in Golgi-apparatus with less so at underneath the plasma
108
membrane. It would be very interesting to address where GODZ mediate Ca2+ transport,
and furthermore, whether this Ca2+ transport is required for the regulation of trafficking
of GABAARs and NL2.
This study took an advantage of the co-culture technique to set up a competitive
environment for KO neurons. This technique provided a detail molecular phenotype of
KO neurons that was not detectable in pure culture. Similarly other studies often lack KO
phenotype in pure culture. For example, knock-down of NLs by RNAi led to a reduction
in synaptic density (Chih et al., 2005), whereas no deficit was observed in the
electrophysiological features and morphological characterizations in neurons prepared
from triple KO mice of NL1, 2 and 3 (Varoqueaux et al., 2006). This could be the
compensational effects by other systems as detailed in discussion in this study, or lack of
competition that exist in RNAi experiment but in pure KO neurons. Thus, it might be
hasty to draw a conclusion based on the results acquired from studies performed in pure
culture. This co-culture technique would be very useful to address the role of the protein
of interest in KO mice in future.
109
BIBLIOGRAPHY
Abrami, L., Kunz, B., Iacovache, I. and van der Goot, F. G., 2008. Palmitoylation and ubiquitination regulate exit of the Wnt signaling protein LRP6 from the endoplasmic reticulum. Proc Natl Acad Sci U S A. 105, 5384‐5389.
Abrami, L., Leppla, S. H. and van der Goot, F. G., 2006. Receptor palmitoylation and ubiquitination regulate anthrax toxin endocytosis. J Cell Biol. 172, 309‐320.
Abu, J., Batuwangala, M. and Symonds, P., 2008. Expression of RAR beta2 gene by real‐time RT‐PCR: Differential expression in normal subjects compared to cervical cancer patients normalised against GAPDH as a housekeeping gene. Eur J Obstet Gynecol Reprod Biol.
Akerman, C. J. and Cline, H. T., 2007. Refining the roles of GABAergic signaling during neural circuit formation. Trends Neurosci. 30, 382‐389.
Alldred, M. J., Mulder‐Rosi, J., Lingenfelter, S. E., Chen, G. and Luscher, B., 2005. Distinct gamma2 subunit domains mediate clustering and synaptic function of postsynaptic GABAA receptors and gephyrin. J Neurosci. 25, 594‐603.
Argos, P., Landy, A., Abremski, K., Egan, J. B., Haggard‐Ljungquist, E., Hoess, R. H., Kahn, M. L., Kalionis, B., Narayana, S. V., Pierson, L. S., 3rd and et al., 1986. The integrase family of site‐specific recombinases: regional similarities and global diversity. Embo J. 5, 433‐440.
Ashby, M. C., Ibaraki, K. and Henley, J. M., 2004. It's green outside: tracking cell surface proteins with pH‐sensitive GFP. Trends Neurosci. 27, 257‐261.
Badner, J. A. and Gershon, E. S., 2002. Meta‐analysis of whole‐genome linkage scans of bipolar disorder and schizophrenia. Mol Psychiatry. 7, 405‐411.
Banker, G. a. G., K (Ed.), 1998. Culturing nerve cells, 2nd Edition. MITT, Cambridge. Barnard, E. A., Skolnick, P., Olsen, R. W., Mohler, H., Sieghart, W., Biggio, G., Braestrup, C.,
Bateson, A. N. and Langer, S. Z., 1998. International Union of Pharmacology. XV. Subtypes of gamma‐aminobutyric acidA receptors: classification on the basis of subunit structure and receptor function. Pharmacol Rev. 50, 291‐313.
Bartels, D. J., Mitchell, D. A., Dong, X. and Deschenes, R. J., 1999. Erf2, a novel gene product that affects the localization and palmitoylation of Ras2 in Saccharomyces cerevisiae. Mol Cell Biol. 19, 6775‐6787.
Baulac, S., Huberfeld, G., Gourfinkel‐An, I., Mitropoulou, G., Beranger, A., Prud'homme, J. F., Baulac, M., Brice, A., Bruzzone, R. and LeGuern, E., 2001. First genetic evidence of GABA(A) receptor dysfunction in epilepsy: a mutation in the gamma2‐subunit gene. Nat Genet. 28, 46‐48.
Becker, L., von Wegerer, J., Schenkel, J., Zeilhofer, H. U., Swandulla, D. and Weiher, H., 2002. Disease‐specific human glycine receptor alpha1 subunit causes hyperekplexia phenotype and impaired glycine‐ and GABA(A)‐receptor transmission in transgenic mice. J Neurosci. 22, 2505‐2512.
Bedford, F. K., Kittler, J. T., Muller, E., Thomas, P., Uren, J. M., Merlo, D., Wisden, W., Triller, A., Smart, T. G. and Moss, S. J., 2001. GABA(A) receptor cell surface number and subunit stability are regulated by the ubiquitin‐like protein Plic‐1. Nat Neurosci. 4, 908‐916.
Benowitz, L. I. and Routtenberg, A., 1997. GAP‐43: an intrinsic determinant of neuronal development and plasticity. Trends Neurosci. 20, 84‐91.
110
Benson, D. L., Colman, D. R. and Huntley, G. W., 2001. Molecules, maps and synapse specificity. Nat Rev Neurosci. 2, 899‐909.
Besshoh, S., Bawa, D., Teves, L., Wallace, M. C. and Gurd, J. W., 2005. Increased phosphorylation and redistribution of NMDA receptors between synaptic lipid rafts and post‐synaptic densities following transient global ischemia in the rat brain. J Neurochem. 93, 186‐194.
Bingol, B. and Schuman, E. M., 2004. A proteasome‐sensitive connection between PSD‐95 and GluR1 endocytosis. Neuropharmacology. 47, 755‐763.
Bizzozero, O. A., 1995. Chemical analysis of acylation sites and species. Methods Enzymol. 250, 361‐379.
Bogdanov, Y., Michels, G., Armstrong‐Gold, C., Haydon, P. G., Lindstrom, J., Pangalos, M. and Moss, S. J., 2006. Synaptic GABA(A) receptors are directly recruited from their extrasynaptic counterparts. Embo J. 25, 4381‐4389.
Braff, D. L. and Geyer, M. A., 1990. Sensorimotor gating and schizophrenia. Human and animal model studies. Arch Gen Psychiatry. 47, 181‐188.
Braff, D. L., Geyer, M. A. and Swerdlow, N. R., 2001. Human studies of prepulse inhibition of startle: normal subjects, patient groups, and pharmacological studies. Psychopharmacology (Berl). 156, 234‐258.
Brickley, S. G., Cull‐Candy, S. G. and Farrant, M., 1999. Single‐channel properties of synaptic and extrasynaptic GABAA receptors suggest differential targeting of receptor subtypes. J Neurosci. 19, 2960‐2973.
Brose, N., 1999. Synaptic cell adhesion proteins and synaptogenesis in the mammalian central nervous system. Naturwissenschaften. 86, 516‐524.
Brown, D. A. and London, E., 1997. Structure of detergent‐resistant membrane domains: does phase separation occur in biological membranes? Biochem Biophys Res Commun. 240, 1‐7.
Brown, D. A. and London, E., 1998. Functions of lipid rafts in biological membranes. Annu Rev Cell Dev Biol. 14, 111‐136.
Brown, R. E., 1998. Sphingolipid organization in biomembranes: what physical studies of model membranes reveal. J Cell Sci. 111 ( Pt 1), 1‐9.
Brunig, I., Scotti, E., Sidler, C. and Fritschy, J. M., 2002. Intact sorting, targeting, and clustering of gamma‐aminobutyric acid A receptor subtypes in hippocampal neurons in vitro. J Comp Neurol. 443, 43‐55.
Calero, G., Gupta, P., Nonato, M. C., Tandel, S., Biehl, E. R., Hofmann, S. L. and Clardy, J., 2003. The crystal structure of palmitoyl protein thioesterase‐2 (PPT2) reveals the basis for divergent substrate specificities of the two lysosomal thioesterases, PPT1 and PPT2. J Biol Chem. 278, 37957‐37964.
Chakrabandhu, K., Herincs, Z., Huault, S., Dost, B., Peng, L., Conchonaud, F., Marguet, D., He, H. T. and Hueber, A. O., 2007. Palmitoylation is required for efficient Fas cell death signaling. Embo J. 26, 209‐220.
Chakravarti, A., 2002. A compelling genetic hypothesis for a complex disease: PRODH2/DGCR6 variation leads to schizophrenia susceptibility. Proc Natl Acad Sci U S A. 99, 4755‐4756.
Chamoun, Z., Mann, R. K., Nellen, D., von Kessler, D. P., Bellotto, M., Beachy, P. A. and Basler, K., 2001. Skinny hedgehog, an acyltransferase required for palmitoylation and activity of the hedgehog signal. Science. 293, 2080‐2084.
Charych, E. I., Yu, W., Miralles, C. P., Serwanski, D. R., Li, X., Rubio, M. and De Blas, A. L., 2004. The brefeldin A‐inhibited GDP/GTP exchange factor 2, a protein involved in vesicular
111
trafficking, interacts with the beta subunits of the GABA‐A receptors. J Neurochem. [Epub ahead of print].
Chaudhary, J. and Skinner, M. K., 2002. Identification of a novel gene product, Sertoli cell gene with a zinc finger domain, that is important for FSH activation of testicular Sertoli cells. Endocrinology. 143, 426‐435.
Chen, L., Chetkovich, D. M., Petralia, R. S., Sweeney, N. T., Kawasaki, Y., Wenthold, R. J., Bredt, D. S. and Nicoll, R. A., 2000. Stargazin regulates synaptic targeting of AMPA receptors by two distinct mechanisms. Nature. 408, 936‐943.
Chen, Y., Lagerholm, B. C., Yang, B. and Jacobson, K., 2006. Methods to measure the lateral diffusion of membrane lipids and proteins. Methods. 39, 147‐153.
Chen, Z. W., Chang, C. S., Leil, T. A., Olcese, R. and Olsen, R. W., 2005. GABA(A) receptor‐associated protein regulates GABAA receptor cell‐surface number in Xenopus laevis oocytes. Mol Pharmacol. 68, 152‐159.
Chen, Z. W. and Olsen, R. W., 2007. GABAA receptor associated proteins: a key factor regulating GABAA receptor function. J Neurochem. 100, 279‐294.
Chih, B., Engelman, H. and Scheiffele, P., 2005. Control of excitatory and inhibitory synapse formation by neuroligins. Science. 307, 1324‐1328.
Chih, B., Gollan, L. and Scheiffele, P., 2006. Alternative splicing controls selective trans‐synaptic interactions of the neuroligin‐neurexin complex. Neuron. 51, 171‐178.
Cho, K. O., Hunt, C. A. and Kennedy, M. B., 1992. The rat brain postsynaptic density fraction contains a homolog of the Drosophila discs‐large tumor suppressor protein. Neuron. 9, 929‐942.
Christopherson, K. S., Sweeney, N. T., Craven, S. E., Kang, R., El‐Husseini Ael, D. and Bredt, D. S., 2003. Lipid‐ and protein‐mediated multimerization of PSD‐95: implications for receptor clustering and assembly of synaptic protein networks. J Cell Sci. 116, 3213‐3219.
Chubykin, A. A., Atasoy, D., Etherton, M. R., Brose, N., Kavalali, E. T., Gibson, J. R. and Sudhof, T. C., 2007. Activity‐dependent validation of excitatory versus inhibitory synapses by neuroligin‐1 versus neuroligin‐2. Neuron. 54, 919‐931.
Colledge, M., Snyder, E. M., Crozier, R. A., Soderling, J. A., Jin, Y., Langeberg, L. K., Lu, H., Bear, M. F. and Scott, J. D., 2003. Ubiquitination regulates PSD‐95 degradation and AMPA receptor surface expression. Neuron. 40, 595‐607.
Craven, S. E., El‐Husseini, A. E. and Bredt, D. S., 1999. Synaptic targeting of the postsynaptic density protein PSD‐95 mediated by lipid and protein motifs. Neuron. 22, 497‐509.
Crawley, J. N., 1985. Exploratory behavior models of anxiety in mice. Neurosci Biobehav Rev. 9, 37‐44.
Crestani, F., Lorez, M., Baer, K., Essrich, C., Benke, D., Laurent, J. P., Belzung, C., Fritschy, J. M., Luscher, B. and Mohler, H., 1999. Decreased GABAA‐receptor clustering results in enhanced anxiety and a bias for threat cues. Nat Neurosci. 2, 833‐839.
Dalskov, S. M., Immerdal, L., Niels‐Christiansen, L. L., Hansen, G. H., Schousboe, A. and Danielsen, E. M., 2005. Lipid raft localization of GABA A receptor and Na+, K+‐ATPase in discrete microdomain clusters in rat cerebellar granule cells. Neurochem Int. 46, 489‐499.
de Planque, M. R. and Killian, J. A., 2003. Protein‐lipid interactions studied with designed transmembrane peptides: role of hydrophobic matching and interfacial anchoring. Mol Membr Biol. 20, 271‐284.
Dean, C., Scholl, F. G., Choih, J., DeMaria, S., Berger, J., Isacoff, E. and Scheiffele, P., 2003. Neurexin mediates the assembly of presynaptic terminals. Nat Neurosci. 6, 708‐716.
112
Dong, N., Qi, J. and Chen, G., 2007. Molecular reconstitution of functional GABAergic synapses with expression of neuroligin‐2 and GABAA receptors. Mol Cell Neurosci. 35, 14‐23.
Dooley, T. P., Miranda, M., Jones, N. C. and DePamphilis, M. L., 1989. Transactivation of the adenovirus EIIa promoter in the absence of adenovirus E1A protein is restricted to mouse oocytes and preimplantation embryos. Development. 107, 945‐956.
Dumoulin, A., Rostaing, P., Bedet, C., Levi, S., Isambert, M. F., Henry, J. P., Triller, A. and Gasnier, B., 1999. Presence of the vesicular inhibitory amino acid transporter in GABAergic and glycinergic synaptic terminal boutons. J Cell Sci. 112 ( Pt 6), 811‐823.
Duncan, J. A. and Gilman, A. G., 1998. A cytoplasmic acyl‐protein thioesterase that removes palmitate from G protein alpha subunits and p21(RAS). J Biol Chem. 273, 15830‐15837.
Ehlers, M. D., 2000. Reinsertion or degradation of AMPA receptors determined by activity‐dependent endocytic sorting. Neuron. 28, 511‐525.
El‐Husseini, A. E., Schnell, E., Chetkovich, D. M., Nicoll, R. A. and Bredt, D. S., 2000. PSD‐95 involvement in maturation of excitatory synapses. Science. 290, 1364‐1368.
el‐Husseini Ael, D. and Bredt, D. S., 2002. Protein palmitoylation: a regulator of neuronal development and function. Nat Rev Neurosci. 3, 791‐802.
El‐Husseini Ael, D., Schnell, E., Dakoji, S., Sweeney, N., Zhou, Q., Prange, O., Gauthier‐Campbell, C., Aguilera‐Moreno, A., Nicoll, R. A. and Bredt, D. S., 2002. Synaptic strength regulated by palmitate cycling on PSD‐95. Cell. 108, 849‐863.
Essrich, C., Lorez, M., Benson, J. A., Fritschy, J. M. and Luscher, B., 1998. Postsynaptic clustering of major GABAA receptor subtypes requires the gamma 2 subunit and gephyrin. Nat Neurosci. 1, 563‐571.
Eulenburg, V., Becker, K., Gomeza, J., Schmitt, B., Becker, C. M. and Betz, H., 2006. Mutations within the human GLYT2 (SLC6A5) gene associated with hyperekplexia. Biochem Biophys Res Commun. 348, 400‐405.
Fang, C., Deng, L., Keller, C. A., Fukata, M., Fukata, Y., Chen, G. and Luscher, B., 2006. GODZ‐mediated palmitoylation of GABA(A) receptors is required for normal assembly and function of GABAergic inhibitory synapses. J Neurosci. 26, 12758‐12768.
Fernandez‐Hernando, C., Fukata, M., Bernatchez, P. N., Fukata, Y., Lin, M. I., Bredt, D. S. and Sessa, W. C., 2006. Identification of Golgi‐localized acyl transferases that palmitoylate and regulate endothelial nitric oxide synthase. J Cell Biol. 174, 369‐377.
Fritschy, J.‐M. and Mohler, H., 1995. GABAA receptor heterogeneity in the adult rat brain: differential regional and cellular distribution of seven major subunits. J Comp Neurol. 359, 154‐194.
Fritschy, J.‐M., Paysan, J., Enna, A. and Mohler, H., 1994. Switch in the expression of rat GABAA‐receptor subtypes during postnatal development: an immunohstochemical study. J Neurosci. 14, 5302‐5324.
Fritschy, J. M., Johnson, D. K., Mohler, H. and Rudolph, U., 1998. Independent assembly and subcellular targeting of GABAA‐receptor subtypes demonstrated in mouse hippocampal and olfactory neurons in vivo. Neurosci Lett. 249, 99‐102.
Frosk, P., Weiler, T., Nylen, E., Sudha, T., Greenberg, C. R., Morgan, K., Fujiwara, T. M. and Wrogemann, K., 2002. Limb‐girdle muscular dystrophy type 2H associated with mutation in TRIM32, a putative E3‐ubiquitin‐ligase gene. Am J Hum Genet. 70, 663‐672.
Fu, Z., Washbourne, P., Ortinski, P. and Vicini, S., 2003. Functional excitatory synapses in HEK293 cells expressing neuroligin and glutamate receptors. J Neurophysiol. 90, 3950‐3957.
Fukata, M., Fukata, Y., Adesnik, H., Nicoll, R. A. and Bredt, D. S., 2004. Identification of PSD‐95 palmitoylating enzymes. Neuron. 44, 987‐996.
113
Gauthier‐Campbell, C., Bredt, D. S., Murphy, T. H. and El‐Husseini Ael, D., 2004. Regulation of dendritic branching and filopodia formation in hippocampal neurons by specific acylated protein motifs. Mol Biol Cell. 15, 2205‐2217.
Gil, C., Cubi, R., Blasi, J. and Aguilera, J., 2006. Synaptic proteins associate with a sub‐set of lipid rafts when isolated from nerve endings at physiological temperature. Biochem Biophys Res Commun. 348, 1334‐1342.
Glykys, J., Peng, Z., Chandra, D., Homanics, G. E., Houser, C. R. and Mody, I., 2007. A new naturally occurring GABA(A) receptor subunit partnership with high sensitivity to ethanol. Nat Neurosci. 10, 40‐48.
Gomeza, J., Ohno, K., Hulsmann, S., Armsen, W., Eulenburg, V., Richter, D. W., Laube, B. and Betz, H., 2003. Deletion of the mouse glycine transporter 2 results in a hyperekplexia phenotype and postnatal lethality. Neuron. 40, 797‐806.
Goytain, A., Hines, R. M. and Quamme, G. A., 2008. Huntingtin‐interacting proteins, HIP14 and HIP14L, mediate dual functions:Palmitoyl acytransferase and Mg2+ transport. J Biol Chem.
Graf, E. R., Kang, Y., Hauner, A. M. and Craig, A. M., 2006. Structure function and splice site analysis of the synaptogenic activity of the neurexin‐1 beta LNS domain. J Neurosci. 26, 4256‐4265.
Graf, E. R., Zhang, X., Jin, S. X., Linhoff, M. W. and Craig, A. M., 2004. Neurexins induce differentiation of GABA and glutamate postsynaptic specializations via neuroligins. Cell. 119, 1013‐1026.
Greaves, J. and Chamberlain, L. H., 2007. Palmitoylation‐dependent protein sorting. J Cell Biol. 176, 249‐254.
Greaves, J., Salaun, C., Fukata, Y., Fukata, M. and Chamberlain, L. H., 2008. Palmitoylation and membrane interactions of the neuroprotective chaperone cysteine‐string protein. J Biol Chem.
Gu, H., Marth, J. D., Orban, P. C., Mossmann, H. and Rajewsky, K., 1994. Deletion of a DNA polymerase beta gene segment in T cells using cell type‐specific gene targeting. Science. 265, 103‐106.
Gupta, P., Soyombo, A. A., Shelton, J. M., Wilkofsky, I. G., Wisniewski, K. E., Richardson, J. A. and Hofmann, S. L., 2003. Disruption of PPT2 in mice causes an unusual lysosomal storage disorder with neurovisceral features. Proc Natl Acad Sci U S A. 100, 12325‐12330.
Haltia, M., Rapola, J. and Santavuori, P., 1973. Infantile type of so‐called neuronal ceroid‐lipofuscinosis. Histological and electron microscopic studies. Acta Neuropathol. 26, 157‐170.
Harvey, K., Duguid, I. C., Alldred, M. J., Beatty, S. E., Ward, H., Keep, N. H., Lingenfelter, S. E., Pearce, B. R., Lundgren, J., Owen, M. J., Smart, T. G., Luscher, B., Rees, M. I. and Harvey, R. J., 2004. The GDP‐GTP exchange factor collybistin: an essential determinant of neuronal gephyrin clustering. J Neurosci. 24, 5816‐5826.
Hayashi, S. and McMahon, A. P., 2002. Efficient recombination in diverse tissues by a tamoxifen‐inducible form of Cre: a tool for temporally regulated gene activation/inactivation in the mouse. Dev Biol. 244, 305‐318.
Hayashi, T., Rumbaugh, G. and Huganir, R. L., 2005. Differential regulation of AMPA receptor subunit trafficking by palmitoylation of two distinct sites. Neuron. 47, 709‐723.
Henis, Y. I., Rotblat, B. and Kloog, Y., 2006. FRAP beam‐size analysis to measure palmitoylation‐dependent membrane association dynamics and microdomain partitioning of Ras proteins. Methods. 40, 183‐190.
114
Herd, M. B., Haythornthwaite, A. R., Rosahl, T. W., Wafford, K. A., Homanics, G. E., Lambert, J. J. and Belelli, D., 2008. The expression of GABAA beta subunit isoforms in synaptic and extrasynaptic receptor populations of mouse dentate gyrus granule cells. J Physiol. 586, 989‐1004.
Hering, H., Lin, C. C. and Sheng, M., 2003. Lipid rafts in the maintenance of synapses, dendritic spines, and surface AMPA receptor stability. J Neurosci. 23, 3262‐3271.
Hines, R. M., Wu, L., Hines, D. J., Steenland, H., Mansour, S., Dahlhaus, R., Singaraja, R. R., Cao, X., Sammler, E., Hormuzdi, S. G., Zhuo, M. and El‐Husseini, A., 2008. Synaptic imbalance, stereotypies, and impaired social interactions in mice with altered neuroligin 2 expression. J Neurosci. 28, 6055‐6067.
Hochstrasser, M., 1996. Ubiquitin‐dependent protein degradation. Annu Rev Genet. 30, 405‐439. Hofmann, K., 2000. A superfamily of membrane‐bound O‐acyltransferases with implications for
wnt signaling. Trends Biochem Sci. 25, 111‐112. Huang, K. and El‐Husseini, A., 2005. Modulation of neuronal protein trafficking and function by
palmitoylation. Curr Opin Neurobiol. 15, 527‐535. Ichtchenko, K., Nguyen, T. and Sudhof, T. C., 1996. Structures, alternative splicing, and neurexin
binding of multiple neuroligins. J Biol Chem. 271, 2676‐2682. Ikonen, E., 2001. Roles of lipid rafts in membrane transport. Curr Opin Cell Biol. 13, 470‐477. Ito, M., Oliverio, M. I., Mannon, P. J., Best, C. F., Maeda, N., Smithies, O. and Coffman, T. M.,
1995. Regulation of blood pressure by the type 1A angiotensin II receptor gene. Proc Natl Acad Sci U S A. 92, 3521‐3525.
Iwasato, T., Datwani, A., Wolf, A. M., Nishiyama, H., Taguchi, Y., Tonegawa, S., Knopfel, T., Erzurumlu, R. S. and Itohara, S., 2000. Cortex‐restricted disruption of NMDAR1 impairs neuronal patterns in the barrel cortex. Nature. 406, 726‐731.
Jacob, T. C., Bogdanov, Y. D., Magnus, C., Saliba, R. S., Kittler, J. T., Haydon, P. G. and Moss, S. J., 2005. Gephyrin regulates the cell surface dynamics of synaptic GABAA receptors. J Neurosci. 25, 10469‐10478.
Jacob, T. C., Moss, S. J. and Jurd, R., 2008. GABA(A) receptor trafficking and its role in the dynamic modulation of neuronal inhibition. Nat Rev Neurosci. 9, 331‐343.
Jin, M., Yuan, Q., Li, S. and Travis, G. H., 2007. Role of LRAT on the retinoid isomerase activity and membrane association of Rpe65. J Biol Chem. 282, 20915‐20924.
Kanaani, J., Diacovo, M. J., El‐Husseini Ael, D., Bredt, D. S. and Baekkeskov, S., 2004. Palmitoylation controls trafficking of GAD65 from Golgi membranes to axon‐specific endosomes and a Rab5a‐dependent pathway to presynaptic clusters. J Cell Sci. 117, 2001‐2013.
Kanaani, J., el‐Husseini Ael, D., Aguilera‐Moreno, A., Diacovo, J. M., Bredt, D. S. and Baekkeskov, S., 2002. A combination of three distinct trafficking signals mediates axonal targeting and presynaptic clustering of GAD65. J Cell Biol. 158, 1229‐1238.
Kanaani, J., Patterson, G., Schaufele, F., Lippincott‐Schwartz, J. and Baekkeskov, S., 2008. A palmitoylation cycle dynamically regulates partitioning of the GABA‐synthesizing enzyme GAD65 between ER‐Golgi and post‐Golgi membranes. J Cell Sci. 121, 437‐449.
Kandasamy, S. K. and Larson, R. G., 2006. Molecular dynamics simulations of model trans‐membrane peptides in lipid bilayers: a systematic investigation of hydrophobic mismatch. Biophys J. 90, 2326‐2343.
Kanematsu, T., Jang, I. S., Yamaguchi, T., Nagahama, H., Yoshimura, K., Hidaka, K., Matsuda, M., Takeuchi, H., Misumi, Y., Nakayama, K., Yamamoto, T., Akaike, N. and Hirata, M., 2002.
115
Role of the PLC‐related, catalytically inactive protein p130 in GABAA receptor function. Embo J. 21, 1004‐1011.
Kang, R., Swayze, R., Lise, M. F., Gerrow, K., Mullard, A., Honer, W. G. and El‐Husseini, A., 2004. Presynaptic trafficking of synaptotagmin I is regulated by protein palmitoylation. J Biol Chem. 279, 50524‐50536.
Kang, Y., Zhang, X., Dobie, F., Wu, H. and Craig, A. M., 2008. Induction of GABAergic postsynaptic differentiation by alpha‐neurexins. J Biol Chem. 283, 2323‐2334.
Keller, C. A., Yuan, X., Panzanelli, P., Martin, M. L., Alldred, M., Sassoe‐Pognetto, M. and Luscher, B., 2004. The gamma2 subunit of GABA(A) receptors is a substrate for palmitoylation by GODZ. J Neurosci. 24, 5881‐5891.
Kilman, V., van Rossum, M. C. and Turrigiano, G. G., 2002. Activity deprivation reduces miniature IPSC amplitude by decreasing the number of postsynaptic GABA(A) receptors clustered at neocortical synapses. J Neurosci. 22, 1328‐1337.
Kingsmore, S. F., Giros, B., Suh, D., Bieniarz, M., Caron, M. G. and Seldin, M. F., 1994. Glycine receptor beta‐subunit gene mutation in spastic mouse associated with LINE‐1 element insertion. Nat Genet. 7, 136‐141.
Kins, S., Heinrich Betz, H. and Kirsch, J., 2000. Collybistin, a newly identified brain‐specific GEF, induces submembrane clustering of gephyrin. Nature Neuroscience. 3, 22‐29.
Kittler, J. T., Chen, G., Honing, S., Bogdanov, Y., McAinsh, K., Arancibia‐Carcamo, I. L., Jovanovic, J. N., Pangalos, M. N., Haucke, V., Yan, Z. and Moss, S. J., 2005. Phospho‐dependent binding of the clathrin AP2 adaptor complex to GABA(A) receptors regulates the efficacy of inhibitory synaptic transmission. Proc Natl Acad Sci U S A. 102, 14871‐14876.
Kittler, J. T., Chen, G., Kukhtina, V., Vahedi‐Faridi, A., Gu, Z., Tretter, V., Smith, K. R., McAinsh, K., Arancibia‐Carcamo, I. L., Saenger, W., Haucke, V., Yan, Z. and Moss, S. J., 2008. Regulation of synaptic inhibition by phospho‐dependent binding of the AP2 complex to a YECL motif in the GABAA receptor gamma2 subunit. Proc Natl Acad Sci U S A. 105, 3616‐3621.
Kittler, J. T., Delmas, P., Jovanovic, J. N., Brown, D. A., Smart, T. G. and Moss, S. J., 2000. Constitutive endocytosis of GABAA receptors by an association with the adaptin AP2 complex modulates inhibitory synaptic currents in hippocampal neurons. J Neurosci. 20, 7972‐7977.
Kittler, J. T., McAinsh, K. and Moss, S. J., 2002. Mechanisms of GABAA receptor assembly and trafficking: implications for the modulation of inhibitory neurotransmission. Mol Neurobiol. 26, 251‐268.
Kittler, J. T., Rostaing, P., Schiavo, G., Fritschy, J. M., Olsen, R., Triller, A. and Moss, S. J., 2001. The subcellular distribution of GABARAP and its ability to interact with NSF suggest a role for this protein in the intracellular transport of GABAA receptors. Mol Cell Neurosci. 18, 13‐25.
Kittler, J. T., Thomas, P., Tretter, V., Bogdanov, Y. D., Haucke, V., Smart, T. G. and Moss, S. J., 2004. Huntingtin‐associated protein 1 regulates inhibitory synaptic transmission by modulating gamma‐aminobutyric acid type A receptor membrane trafficking. Proc Natl Acad Sci U S A. 101, 12736‐12741.
Kling, C., Koch, M., Saul, B. and Becker, C. M., 1997. The frameshift mutation oscillator (Glra1(spd‐ot)) produces a complete loss of glycine receptor alpha1‐polypeptide in mouse central nervous system. Neuroscience. 78, 411‐417.
Knabl, J., Witschi, R., Hosl, K., Reinold, H., Zeilhofer, U. B., Ahmadi, S., Brockhaus, J., Sergejeva, M., Hess, A., Brune, K., Fritschy, J. M., Rudolph, U., Mohler, H. and Zeilhofer, H. U., 2008.
116
Reversal of pathological pain through specific spinal GABAA receptor subtypes. Nature. 451, 330‐334.
Kneussel, M. and Betz, H., 2000. Clustering of inhibitory neurotransmitter receptors at developing postsynaptic sites: the membrane activation model. Trends Neurosci. 23, 429‐435.
Kneussel, M., Brandstätter, J. H., Gasnier, B., Feng, G., Sanes, J. R. and Betz, H., 2001. Gephyrin‐independent clustering of postsynaptic GABAA receptor subtypes. Mol Cell Neurosci. 17, 973‐982.
Kneussel, M., Brandstätter, J. H., Laube, B., Stahl, S., Müller, U. and Betz, H., 1999. Loss of postsynaptic GABAA receptor clustering in gephyrin‐deficient mice. J Neurosci. 19, 9289‐9297.
Kornau, H. C., Seeburg, P. H. and Kennedy, M. B., 1997. Interaction of ion channels and receptors with PDZ domain proteins. Curr Opin Neurobiol. 7, 368‐373.
Korpi, E. R., Grunder, G. and Luddens, H., 2002. Drug interactions at GABA(A) receptors. Prog Neurobiol. 67, 113‐159.
Koshizuka, T., Kawaguchi, Y., Nozawa, N., Mori, I. and Nishiyama, Y., 2007. Herpes simplex virus protein UL11 but not UL51 is associated with lipid rafts. Virus Genes. 35, 571‐575.
Kudryashova, E., Kudryashov, D., Kramerova, I. and Spencer, M. J., 2005. Trim32 is a ubiquitin ligase mutated in limb girdle muscular dystrophy type 2H that binds to skeletal muscle myosin and ubiquitinates actin. J Mol Biol. 354, 413‐424.
Lakso, M., Pichel, J. G., Gorman, J. R., Sauer, B., Okamoto, Y., Lee, E., Alt, F. W. and Westphal, H., 1996. Efficient in vivo manipulation of mouse genomic sequences at the zygote stage. Proc Natl Acad Sci U S A. 93, 5860‐5865.
Lakso, M., Sauer, B., Mosinger, B., Jr., Lee, E. J., Manning, R. W., Yu, S. H., Mulder, K. L. and Westphal, H., 1992. Targeted oncogene activation by site‐specific recombination in transgenic mice. Proc Natl Acad Sci U S A. 89, 6232‐6236.
Laurie, D. J., Seeburg, P. H. and Wisden, W., 1992. The distribution of 13 GABAA receptor subunit mRNAs in the rat brain. II. Olfactory bulb and cerebellum. J Neurosci. 12, 1063‐1076.
Lee, J. D. and Treisman, J. E., 2001. Sightless has homology to transmembrane acyltransferases and is required to generate active Hedgehog protein. Curr Biol. 11, 1147‐1152.
Leil, T. A., Chen, Z. W., Chang, C. S. and Olsen, R. W., 2004. GABA(A) receptor‐associated protein traffics GABA(A) receptors to the plasma membrane in neurons. J Neurosci. 24, 11429‐11438.
Levi, S., Logan, S. M., Tovar, K. R. and Craig, A. M., 2004. Gephyrin is critical for glycine receptor clustering but not for the formation of functional GABAergic synapses in hippocampal neurons. J Neurosci. 24, 207‐217.
Levinson, J. N., Chery, N., Huang, K., Wong, T. P., Gerrow, K., Kang, R., Prange, O., Wang, Y. T. and El‐Husseini, A., 2005. Neuroligins mediate excitatory and inhibitory synapse formation: involvement of PSD‐95 and neurexin‐1beta in neuroligin‐induced synaptic specificity. J Biol Chem. 280, 17312‐17319.
Levinson, J. N. and El‐Husseini, A., 2005a. Building excitatory and inhibitory synapses: balancing neuroligin partnerships. Neuron. 48, 171‐174.
Levinson, J. N. and El‐Husseini, A., 2005b. New players tip the scales in the balance between excitatory and inhibitory synapses. Mol Pain. 1, 12.
Lewis, D. A., Hashimoto, T. and Volk, D. W., 2005. Cortical inhibitory neurons and schizophrenia. Nat Rev Neurosci. 6, 312‐324.
117
Li, R. W., Yu, W., Christie, S., Miralles, C. P., Bai, J., Loturco, J. J. and De Blas, A. L., 2005. Disruption of postsynaptic GABA receptor clusters leads to decreased GABAergic innervation of pyramidal neurons. J Neurochem. 95, 756‐770.
Li, X., Serwanski, D. R., Miralles, C. P., Bahr, B. A. and De Blas, A. L., 2007. Two pools of Triton X‐100‐insoluble GABA(A) receptors are present in the brain, one associated to lipid rafts and another one to the post‐synaptic GABAergic complex. J Neurochem. 102, 1329‐1345.
Lin, J. W., Ju, W., Foster, K., Lee, S. H., Ahmadian, G., Wyszynski, M., Wang, Y. T. and Sheng, M., 2000. Distinct molecular mechanisms and divergent endocytotic pathways of AMPA receptor internalization. Nat Neurosci. 3, 1282‐1290.
Linder, M. E. and Deschenes, R. J., 2003. New insights into the mechanisms of protein palmitoylation. Biochemistry. 42, 4311‐4320.
Lissin, D. V., Gomperts, S. N., Carroll, R. C., Christine, C. W., Kalman, D., Kitamura, M., Hardy, S., Nicoll, R. A., Malenka, R. C. and von Zastrow, M., 1998. Activity differentially regulates the surface expression of synaptic AMPA and NMDA glutamate receptors. Proc Natl Acad Sci U S A. 95, 7097‐7102.
Lister, R. G., 1987. The use of a plus‐maze to measure anxiety in the mouse. Psychopharmacology (Berl). 92, 180‐185.
Liu, G., 2004. Local structural balance and functional interaction of excitatory and inhibitory synapses in hippocampal dendrites. Nat Neurosci. 7, 373‐379.
Liu, H., Abecasis, G. R., Heath, S. C., Knowles, A., Demars, S., Chen, Y. J., Roos, J. L., Rapoport, J. L., Gogos, J. A. and Karayiorgou, M., 2002. Genetic variation in the 22q11 locus and susceptibility to schizophrenia. Proc Natl Acad Sci U S A. 99, 16859‐16864.
Liu, Y. L., Fann, C. S., Liu, C. M., Chang, C. C., Yang, W. C., Wu, J. Y., Hung, S. I., Chan, H. Y., Chen, J. J., Hsieh, M. H., Hwang, T. J., Faraone, S. V., Tsuang, M. T., Chen, W. J. and Hwu, H. G., 2007. HTF9C gene of 22q11.21 region associates with schizophrenia having deficit‐sustained attention. Psychiatr Genet. 17, 333‐338.
Lobo, S., Greentree, W. K., Linder, M. E. and Deschenes, R. J., 2002. Identification of a Ras palmitoyltransferase in Saccharomyces cerevisiae. J Biol Chem. 277, 41268‐41273.
Luscher, B. and Keller, C. A., 2004. Regulation of GABAA receptor trafficking, channel activity, and functional plasticity of inhibitory synapses. Pharmacol Ther. 102, 195‐221.
Lydiard, R. B., 2003. The role of GABA in anxiety disorders. J Clin Psychiatry. 64 Suppl 3, 21‐27. Magee, A. I. and Courtneidge, S. A., 1985. Two classes of fatty acid acylated proteins exist in
eukaryotic cells. Embo J. 4, 1137‐1144. Malizia, A. L., 2002. Receptor binding and drug modulation in anxiety. Eur
Neuropsychopharmacol. 12, 567‐574. Mansouri, M. R., Marklund, L., Gustavsson, P., Davey, E., Carlsson, B., Larsson, C., White, I.,
Gustavson, K. H. and Dahl, N., 2005. Loss of ZDHHC15 expression in a woman with a balanced translocation t(X;15)(q13.3;cen) and severe mental retardation. Eur J Hum Genet. 13, 970‐977.
Marsden, K. C., Beattie, J. B., Friedenthal, J. and Carroll, R. C., 2007. NMDA receptor activation potentiates inhibitory transmission through GABA receptor‐associated protein‐dependent exocytosis of GABA(A) receptors. J Neurosci. 27, 14326‐14337.
Micchelli, C. A., The, I., Selva, E., Mogila, V. and Perrimon, N., 2002. Rasp, a putative transmembrane acyltransferase, is required for Hedgehog signaling. Development. 129, 843‐851.
Miesenbock, G., De Angelis, D. A. and Rothman, J. E., 1998. Visualizing secretion and synaptic transmission with pH‐sensitive green fluorescent proteins. Nature. 394, 192‐195.
118
Missler, M., 2003. Synaptic cell adhesion goes functional. Trends Neurosci. 26, 176‐178. Missler, M. and Sudhof, T. C., 1998. Neurexins: three genes and 1001 products. Trends Genet.
14, 20‐26. Mitchell, D. A., Vasudevan, A., Linder, M. E. and Deschenes, R. J., 2006. Protein palmitoylation by
a family of DHHC protein S‐acyltransferases. J Lipid Res. 47, 1118‐1127. Mizokami, A., Kanematsu, T., Ishibashi, H., Yamaguchi, T., Tanida, I., Takenaka, K., Nakayama, K.
I., Fukami, K., Takenawa, T., Kominami, E., Moss, S. J., Yamamoto, T., Nabekura, J. and Hirata, M., 2007. Phospholipase C‐related inactive protein is involved in trafficking of gamma2 subunit‐containing GABA(A) receptors to the cell surface. J Neurosci. 27, 1692‐1701.
Mor, A. and Philips, M. R., 2006. Compartmentalized Ras/MAPK signaling. Annu Rev Immunol. 24, 771‐800.
Mukai, J., Liu, H., Burt, R. A., Swor, D. E., Lai, W. S., Karayiorgou, M. and Gogos, J. A., 2004. Evidence that the gene encoding ZDHHC8 contributes to the risk of schizophrenia. Nat Genet. 36, 725‐731.
Murphy, K. C., Jones, L. A. and Owen, M. J., 1999. High rates of schizophrenia in adults with velo‐cardio‐facial syndrome. Arch Gen Psychiatry. 56, 940‐945.
Nadolski, M. J. and Linder, M. E., 2007. Protein lipidation. Febs J. 274, 5202‐5210. Nam, C. I. and Chen, L., 2005. Postsynaptic assembly induced by neurexin‐neuroligin interaction
and neurotransmitter. Proc Natl Acad Sci U S A. 102, 6137‐6142. Nezil, F. A. and Bloom, M., 1992. Combined influence of cholesterol and synthetic amphiphillic
peptides upon bilayer thickness in model membranes. Biophys J. 61, 1176‐1183. Nini, L., Waheed, A. A., Panicker, L. M., Czapiga, M., Zhang, J. H. and Simonds, W. F., 2007. R7‐
binding protein targets the G protein beta 5/R7‐regulator of G protein signaling complex to lipid rafts in neuronal cells and brain. BMC Biochem. 8, 18.
Nusser, Z., Sieghart, W., Benke, D., Fritschy, J.‐M. and Somogyi, P., 1996. Differential synaptic localization of two major γ‐aminobutyric acid type A receptor α subunits on hippocampal pyramidal cells. PNAS. 93, 11939‐11944.
Nymann‐Andersen, J., Wang, H., Chen, L., Kittler, J. T., Moss, S. J. and Olsen, R. W., 2002. Subunit specificity and interaction domain between GABA(A) receptor‐associated protein (GABARAP) and GABA(A) receptors. J Neurochem. 80, 815‐823.
O'Brien, R. J., Kamboj, S., Ehlers, M. D., Rosen, K. R., Fischbach, G. D. and Huganir, R. L., 1998. Activity‐dependent modulation of synaptic AMPA receptor accumulation. Neuron. 21, 1067‐1078.
Orban, P. C., Chui, D. and Marth, J. D., 1992. Tissue‐ and site‐specific DNA recombination in transgenic mice. Proc Natl Acad Sci U S A. 89, 6861‐6865.
Otani, K., Ujike, H., Tanaka, Y., Morita, Y., Kishimoto, M., Morio, A., Uchida, N., Nomura, A. and Kuroda, S., 2005. The ZDHHC8 gene did not associate with bipolar disorder or schizophrenia. Neurosci Lett. 390, 166‐170.
Pang, D. J., Hayday, A. C. and Bijlmakers, M. J., 2007. CD8 Raft localization is induced by its assembly into CD8alpha beta heterodimers, Not CD8alpha alpha homodimers. J Biol Chem. 282, 13884‐13894.
Patterson, S. I. and Skene, J. H., 1999. A shift in protein S‐palmitoylation, with persistence of growth‐associated substrates, marks a critical period for synaptic plasticity in developing brain. J Neurobiol. 39, 423‐437.
Paysan, J., Bolz, J., H., M. and Fritschy, J. M., 1994. GABAA receptor α1 subunit: an ealy marker for area specifrication in developing rat cerebral cortex. J Comp Neurol. 350, 133‐149.
119
Petryshen, T. L., Middleton, F. A., Tahl, A. R., Rockwell, G. N., Purcell, S., Aldinger, K. A., Kirby, A., Morley, C. P., McGann, L., Gentile, K. L., Waggoner, S. G., Medeiros, H. M., Carvalho, C., Macedo, A., Albus, M., Maier, W., Trixler, M., Eichhammer, P., Schwab, S. G., Wildenauer, D. B., Azevedo, M. H., Pato, M. T., Pato, C. N., Daly, M. J. and Sklar, P., 2005. Genetic investigation of chromosome 5q GABAA receptor subunit genes in schizophrenia. Mol Psychiatry. 10, 1074‐1088, 1057.
Pfeiffer, F., Graham, D. and Betz, H., 1982. Purification by affinity chromatography of the glycine receptor of rat spinal cord. J Biol Chem. 257, 9389‐9393.
Pham, C. T., MacIvor, D. M., Hug, B. A., Heusel, J. W. and Ley, T. J., 1996. Long‐range disruption of gene expression by a selectable marker cassette. Proc Natl Acad Sci U S A. 93, 13090‐13095.
Philibert, R. A., Winfield, S. L., Sandhu, H. K., Martin, B. M. and Ginns, E. I., 2000. The structure and expression of the human neuroligin‐3 gene. Gene. 246, 303‐310.
Prange, O., Wong, T. P., Gerrow, K., Wang, Y. T. and El‐Husseini, A., 2004. A balance between excitatory and inhibitory synapses is controlled by PSD‐95 and neuroligin. Proc Natl Acad Sci U S A. 101, 13915‐13920.
Rathenberg, J., Kittler, J. T. and Moss, S. J., 2004. Palmitoylation regulates the clustering and cell surface stability of GABAA receptors. Mol Cell Neurosci. 26, 251‐257.
Rees, M. I., Harvey, K., Pearce, B. R., Chung, S. K., Duguid, I. C., Thomas, P., Beatty, S., Graham, G. E., Armstrong, L., Shiang, R., Abbott, K. J., Zuberi, S. M., Stephenson, J. B., Owen, M. J., Tijssen, M. A., van den Maagdenberg, A. M., Smart, T. G., Supplisson, S. and Harvey, R. J., 2006. Mutations in the gene encoding GlyT2 (SLC6A5) define a presynaptic component of human startle disease. Nat Genet. 38, 801‐806.
Rees, M. I., Harvey, K., Ward, H., White, J. H., Evans, L., Duguid, I. C., Hsu, C. C., Coleman, S. L., Miller, J., Baer, K., Waldvogel, H. J., Gibbon, F., Smart, T. G., Owen, M. J., Harvey, R. J. and Snell, R. G., 2003. Isoform heterogeneity of the human gephyrin gene (GPHN), binding domains to the glycine receptor, and mutation analysis in hyperekplexia. J Biol Chem. 278, 24688‐24696.
Rees, M. I., Lewis, T. M., Kwok, J. B., Mortier, G. R., Govaert, P., Snell, R. G., Schofield, P. R. and Owen, M. J., 2002. Hyperekplexia associated with compound heterozygote mutations in the beta‐subunit of the human inhibitory glycine receptor (GLRB). Hum Mol Genet. 11, 853‐860.
Reiss, J., Gross‐Hardt, S., Christensen, E., Schmidt, P., Mendel, R. R. and Schwarz, G., 2001. A mutation in the gene for the neurotransmitter receptor‐clustering protein gephyrin causes a novel form of molybdenum cofactor deficiency. Am J Hum Genet. 68, 208‐213.
Renner, U., Glebov, K., Lang, T., Papusheva, E., Balakrishnan, S., Keller, B., Richter, D. W., Jahn, R. and Ponimaskin, E., 2007. Localization of the mouse 5‐hydroxytryptamine(1A) receptor in lipid microdomains depends on its palmitoylation and is involved in receptor‐mediated signaling. Mol Pharmacol. 72, 502‐513.
Resh, M. D., 1999. Fatty acylation of proteins: new insights into membrane targeting of myristoylated and palmitoylated proteins. Biochim Biophys Acta. 1451, 1‐16.
Resh, M. D., 2006. Palmitoylation of ligands, receptors, and intracellular signaling molecules. Sci STKE. 2006, re14.
Reymond, A., Meroni, G., Fantozzi, A., Merla, G., Cairo, S., Luzi, L., Riganelli, D., Zanaria, E., Messali, S., Cainarca, S., Guffanti, A., Minucci, S., Pelicci, P. G. and Ballabio, A., 2001. The tripartite motif family identifies cell compartments. Embo J. 20, 2140‐2151.
120
Roth, A. F., Feng, Y., Chen, L. and Davis, N. G., 2002. The yeast DHHC cysteine‐rich domain protein Akr1p is a palmitoyl transferase. J Cell Biol. 159, 23‐28.
Roth, A. F., Wan, J., Bailey, A. O., Sun, B., Kuchar, J. A., Green, W. N., Phinney, B. S., Yates, J. R., 3rd and Davis, N. G., 2006. Global analysis of protein palmitoylation in yeast. Cell. 125, 1003‐1013.
Rudolph, U. and Antkowiak, B., 2004. Molecular and neuronal substrates for general anaesthetics. Nat Rev Neurosci. 5, 709‐720.
Ryan, S. G., Buckwalter, M. S., Lynch, J. W., Handford, C. A., Segura, L., Shiang, R., Wasmuth, J. J., Camper, S. A., Schofield, P. and O'Connell, P., 1994. A missense mutation in the gene encoding the alpha 1 subunit of the inhibitory glycine receptor in the spasmodic mouse. Nat Genet. 7, 131‐135.
Salaun, C., Gould, G. W. and Chamberlain, L. H., 2005. The SNARE proteins SNAP‐25 and SNAP‐23 display different affinities for lipid rafts in PC12 cells. Regulation by distinct cysteine‐rich domains. J Biol Chem. 280, 1236‐1240.
Saliba, R. S., Michels, G., Jacob, T. C., Pangalos, M. N. and Moss, S. J., 2007. Activity‐dependent ubiquitination of GABA(A) receptors regulates their accumulation at synaptic sites. J Neurosci. 27, 13341‐13351.
Saliba, R. S., Pangalos, M. and Moss, S. J., 2008. The ubiquitin‐like protein Plic‐1 enhances the membrane insertion of GABAA receptors by increasing their stability within the endoplasmic reticulum. J Biol Chem.
Sara, Y., Biederer, T., Atasoy, D., Chubykin, A., Mozhayeva, M. G., Sudhof, T. C. and Kavalali, E. T., 2005. Selective capability of SynCAM and neuroligin for functional synapse assembly. J Neurosci. 25, 260‐270.
Scheffer, I. E. and Berkovic, S. F., 2003. The genetics of human epilepsy. Trends Pharmacol Sci. 24, 428‐433.
Scheiffele, P., Fan, J., Choih, J., Fetter, R. and Serafini, T., 2000. Neuroligin expressed in nonneuronal cells triggers presynaptic development in contacting axons. Cell. 101, 657‐669.
Schmidt, M. F., Bracha, M. and Schlesinger, M. J., 1979. Evidence for covalent attachment of fatty acids to Sindbis virus glycoproteins. Proc Natl Acad Sci U S A. 76, 1687‐1691.
Schmitz, C., van Kooten, I. A., Hof, P. R., van Engeland, H., Patterson, P. H. and Steinbusch, H. W., 2005. Autism: neuropathology, alterations of the GABAergic system, and animal models. Int Rev Neurobiol. 71, 1‐26.
Schmucker, D., Clemens, J. C., Shu, H., Worby, C. A., Xiao, J., Muda, M., Dixon, J. E. and Zipursky, S. L., 2000. Drosophila Dscam is an axon guidance receptor exhibiting extraordinary molecular diversity. Cell. 101, 671‐684.
Schnell, J. D. and Hicke, L., 2003. Non‐traditional functions of ubiquitin and ubiquitin‐binding proteins. J Biol Chem. 278, 35857‐35860.
Schweizer, C., Balsiger, S., Bluethmann, H., Mansuy, M., Fritschy, J. M., Mohler, H. and Luscher, B., 2003. The γ2 subunit of GABAA receptors is required for maintenance of receptors at mature synapses. Mol Cell Neurosci. 24, 442‐450.
Shapiro, L., Love, J. and Colman, D. R., 2007. Adhesion molecules in the nervous system: structural insights into function and diversity. Annu Rev Neurosci. 30, 451‐474.
Sieghart, W., Fuchs, K., Tretter, V., Ebert, V., Jechlinger, M., Hoger, H. and Adamiker, D., 1999. Structure and subunit composition of GABA(A) receptors. Neurochem Int. 34, 379‐385.
Sieghart, W. and Sperk, G., 2002. Subunit composition, distribution and function of GABA(A) receptor subtypes. Curr Top Med Chem. 2, 795‐816.
121
Simons, K. and Toomre, D., 2000. Lipid rafts and signal transduction. Nat Rev Mol Cell Biol. 1, 31‐39.
Simpson, E. M., Linder, C. C., Sargent, E. E., Davisson, M. T., Mobraaten, L. E. and Sharp, J. J., 1997. Genetic variation among 129 substrains and its importance for targeted mutagenesis in mice. Nat Genet. 16, 19‐27.
Smotrys, J. E. and Linder, M. E., 2004. Palmitoylation of intracellular signaling proteins: regulation and function. Annu Rev Biochem. 73, 559‐587.
Song, J. Y., Ichtchenko, K., Sudhof, T. C. and Brose, N., 1999. Neuroligin 1 is a postsynaptic cell‐adhesion molecule of excitatory synapses. Proc Natl Acad Sci U S A. 96, 1100‐1105.
Soriano, P., Montgomery, C., Geske, R. and Bradley, A., 1991. Targeted disruption of the c‐src proto‐oncogene leads to osteopetrosis in mice. Cell. 64, 693‐702.
Strittmatter, S. M., Valenzuela, D., Kennedy, T. E., Neer, E. J. and Fishman, M. C., 1990. G0 is a major growth cone protein subject to regulation by GAP‐43. Nature. 344, 836‐841.
Sudo, Y., Valenzuela, D., Beck‐Sickinger, A. G., Fishman, M. C. and Strittmatter, S. M., 1992. Palmitoylation alters protein activity: blockade of G(o) stimulation by GAP‐43. Embo J. 11, 2095‐2102.
Sundstrom‐Poromaa, I., Smith, D. H., Gong, Q. H., Sabado, T. N., Li, X., Light, A., Wiedmann, M., Williams, K. and Smith, S. S., 2002. Hormonally regulated alpha(4)beta(2)delta GABA(A) receptors are a target for alcohol. Nat Neurosci. 5, 721‐722.
Suzuki, T., Ito, J., Takagi, H., Saitoh, F., Nawa, H. and Shimizu, H., 2001. Biochemical evidence for localization of AMPA‐type glutamate receptor subunits in the dendritic raft. Brain Res Mol Brain Res. 89, 20‐28.
Tabuchi, K. and Sudhof, T. C., 2002. Structure and evolution of neurexin genes: insight into the mechanism of alternative splicing. Genomics. 79, 849‐859.
Tanaka, K., Kitagawa, Y. and Kadowaki, T., 2002. Drosophila segment polarity gene product porcupine stimulates the posttranslational N‐glycosylation of wingless in the endoplasmic reticulum. J Biol Chem. 277, 12816‐12823.
Taniguchi, H., Gollan, L., Scholl, F. G., Mahadomrongkul, V., Dobler, E., Limthong, N., Peck, M., Aoki, C. and Scheiffele, P., 2007. Silencing of neuroligin function by postsynaptic neurexins. J Neurosci. 27, 2815‐2824.
Thomas, P., Mortensen, M., Hosie, A. M. and Smart, T. G., 2005. Dynamic mobility of functional GABA(A) receptors at inhibitory synapses. Nat Neurosci. 8, 889‐897.
Tretter, V., Jacob, T. C., Mukherjee, J., Fritschy, J. M., Pangalos, M. N. and Moss, S. J., 2008. The clustering of GABA(A) receptor subtypes at inhibitory synapses is facilitated via the direct binding of receptor alpha 2 subunits to gephyrin. J Neurosci. 28, 1356‐1365.
Tsutsumi, R., Fukata, Y. and Fukata, M., 2008. Discovery of protein‐palmitoylating enzymes. Pflugers Arch.
Uemura, T., Mori, H. and Mishina, M., 2002. Isolation and characterization of Golgi apparatus‐specific GODZ with the DHHC zinc finger domain. Biochem Biophys Res Commun. 296, 492‐496.
Uji, A., Matsuda, M., Kukita, T., Maeda, K., Kanematsu, T. and Hirata, M., 2002. Molecules interacting with PRIP‐2, a novel Ins(1,4,5)P3 binding protein type 2: Comparison with PRIP‐1. Life Sci. 72, 443‐453.
Ullrich, B., Ushkaryov, Y. A. and Sudhof, T. C., 1995. Cartography of neurexins: more than 1000 isoforms generated by alternative splicing and expressed in distinct subsets of neurons. Neuron. 14, 497‐507.
122
Ushkaryov, Y. A., Petrenko, A. G., Geppert, M. and Sudhof, T. C., 1992. Neurexins: synaptic cell surface proteins related to the alpha‐latrotoxin receptor and laminin. Science. 257, 50‐56.
Uusi‐Oukari, M., Heikkila, J., Sinkkonen, S. T., Makela, R., Hauer, B., Homanics, G. E., Sieghart, W., Wisden, W. and Korpi, E. R., 2000. Long‐range interactions in neuronal gene expression: evidence from gene targeting in the GABA(A) receptor beta2‐alpha6‐alpha1‐gamma2 subunit gene cluster. Mol Cell Neurosci. 16, 34‐41.
Valdez‐Taubas, J. and Pelham, H., 2005. Swf1‐dependent palmitoylation of the SNARE Tlg1 prevents its ubiquitination and degradation. Embo J. 24, 2524‐2532.
Varoqueaux, F., Aramuni, G., Rawson, R. L., Mohrmann, R., Missler, M., Gottmann, K., Zhang, W., Sudhof, T. C. and Brose, N., 2006. Neuroligins determine synapse maturation and function. Neuron. 51, 741‐754.
Varoqueaux, F., Jamain, S. and Brose, N., 2004. Neuroligin 2 is exclusively localized to inhibitory synapses. Eur J Cell Biol. 83, 449‐456.
Vesa, J., Hellsten, E., Verkruyse, L. A., Camp, L. A., Rapola, J., Santavuori, P., Hofmann, S. L. and Peltonen, L., 1995. Mutations in the palmitoyl protein thioesterase gene causing infantile neuronal ceroid lipofuscinosis. Nature. 376, 584‐587.
Wallace, R. H., Marini, C., Petrou, S., Harkin, L. A., Bowser, D. N., Panchal, R. G., Williams, D. A., Sutherland, G. R., Mulley, J. C., Scheffer, I. E. and Berkovic, S. F., 2001. Mutant GABA(A) receptor gamma2‐subunit in childhood absence epilepsy and febrile seizures. Nat Genet. 28, 49‐52.
Wallner, M., Hanchar, H. J. and Olsen, R. W., 2003. Ethanol enhances alpha 4 beta 3 delta and alpha 6 beta 3 delta gamma‐aminobutyric acid type A receptors at low concentrations known to affect humans. Proc Natl Acad Sci U S A. 100, 15218‐15223.
Wang, H., Bedford, F. K., Brandon, N. J., Moss, S. J. and Olsen, R. W., 1999. GABAA‐receptor‐associated protein links GABAA receptors and the cytoskeleton. Nature. 397, 69‐72.
Wang, H. and Olsen, R. W., 2000. Binding of the GABA(A) receptor‐associated protein (GABARAP) to microtubules and microfilaments suggests involvement of the cytoskeleton in GABARAP‐GABA(A) receptor interaction. J Neurochem. 75, 644‐655.
Wang, Q., Liu, L., Pei, L., Ju, W., Ahmadian, G., Lu, J., Wang, Y., Liu, F. and Wang, Y. T., 2003. Control of synaptic strength, a novel function of Akt. Neuron. 38, 915‐928.
Wang, X., Tian, Q. B., Okano, A., Sakagami, H., Moon, I. S., Kondo, H., Endo, S. and Suzuki, T., 2005. BAALC 1‐6‐8 protein is targeted to postsynaptic lipid rafts by its N‐terminal myristoylation and palmitoylation, and interacts with alpha, but not beta, subunit of Ca/calmodulin‐dependent protein kinase II. J Neurochem. 92, 647‐659.
Washbourne, P., Cansino, V., Mathews, J. R., Graham, M., Burgoyne, R. D. and Wilson, M. C., 2001. Cysteine residues of SNAP‐25 are required for SNARE disassembly and exocytosis, but not for membrane targeting. Biochem J. 357, 625‐634.
Whiting, P. J., 1999. The GABA‐A receptor gene family: new targets for therapeutic intervention. Neurochem Int. 34, 387‐390.
Wisden, W., Laurie, D. J., Monyer, H. and Seeburg, P. H., 1992. The distribution of 13 GABAA receptor subunit mRNAs in the rat brain. I. Telencephalon, diencephalon, mesencephalon. J Neurosci. 12, 1040‐1062.
Wong, W. and Schlichter, L. C., 2004. Differential recruitment of Kv1.4 and Kv4.2 to lipid rafts by PSD‐95. J Biol Chem. 279, 444‐452.
123
Xu, W., Schluter, O. M., Steiner, P., Czervionke, B. L., Sabatini, B. and Malenka, R. C., 2008. Molecular dissociation of the role of PSD‐95 in regulating synaptic strength and LTD. Neuron. 57, 248‐262.
Xue, L., Gollapalli, D. R., Maiti, P., Jahng, W. J. and Rando, R. R., 2004. A palmitoylation switch mechanism in the regulation of the visual cycle. Cell. 117, 761‐771.
Yamagata, M., Sanes, J. R. and Weiner, J. A., 2003. Synaptic adhesion molecules. Curr Opin Cell Biol. 15, 621‐632.
Yeh, D. C., Duncan, J. A., Yamashita, S. and Michel, T., 1999. Depalmitoylation of endothelial nitric‐oxide synthase by acyl‐protein thioesterase 1 is potentiated by Ca(2+)‐calmodulin. J Biol Chem. 274, 33148‐33154.
Yuan, X., Yao, J., Norris, D., Tran, D. D., Bram, R. J., Chen, G. and Luscher, B., 2008. Calcium‐modulating cyclophilin ligand regulates membrane trafficking of postsynaptic GABA(A) receptors. Mol Cell Neurosci. 38, 277‐289.
Zacharias, D. A., Violin, J. D., Newton, A. C. and Tsien, R. Y., 2002. Partitioning of lipid‐modified monomeric GFPs into membrane microdomains of live cells. Science. 296, 913‐916.
Zhang, F. L. and Casey, P. J., 1996. Protein prenylation: molecular mechanisms and functional consequences. Annu Rev Biochem. 65, 241‐269.
Zhou, L., Chillag, K. L. and Nigro, M. A., 2002. Hyperekplexia: a treatable neurogenetic disease. Brain Dev. 24, 669‐674.
Vita Shoko Murakami
Education 2003 – 2008 The Pennsylvania State University, University Park, PA, USA Ph.D in Neuroscience candidate, Thesis advisor: Bernhard Lüscher
1995 – 1997 Tokyo University of Agriculture and Technology, Tokyo, Japan MS in Agricultural Science
1991 – 1995 Tokyo University of Agriculture and Technology, Tokyo, Japan BS in Agricultural Science Professional Experience 2003-2008 Graduate Assistant, The Pennsylvania State University 2004-2005 Teaching Assitant, The Pennsylvania State University 2002-2003 Research Assistant, Fernando Nottebohm Lab, The Rockefeller University 1999-2002 Research Assistant , Seth Darst Lab, The Rockefeller University Publications Li, X., Wang, X. J., Tannenhauser, J., Podell, S., Mukherjee, P., Hertel, M., Biane, J., Masuda, S., Nottebohm, F., Gaasterland, T. (2007). Genomic resources for songbird research and their use in characterizing gene expression during brain development. Proc Natl Acad Sci U S A. 104, 6834-9 Chlenov, M., Masuda, S., Murakami, K. S., Nikiforov, V., Darst, S. A., Mustaev, A. (2005). Structure and function of lineage-specific sequence insertions in the bacterial RNA polymerase beta' subunit. J Mol Biol. 353, 138-54 Masuda S, Murakami KS, Wang S, Anders Olson C, Donigian J, Lenon F, Darst SA, Campbell EA. (2004). Crystal Structures of the ADP and ATP Bound Forms of the Bacillus Anti-sigma Factor SpoIIAB in Complex with the Anti-anti-sigma SpoIIAA. J Mol Biol. 340(5):941-56. Murakami, K. S, Masuda. S & Darst, S. A. (2004). Crystallographic analysis of Thermus aquaticus RNA polymerase holoenzyme and a holoenzyme/promoter DNA complex. Methods Enzymol. 370, 42-53. Murakami, K. S., Masuda, S., Darst, S. A. (2002) Structural Basis of Transcription Initiation: T. aquaticus RNA Polymerase Holoenzyme at 4 Å Resolution. Science. 296, 1280-1284. Murakami, K. S., Masuda, S., Campbell, E. A., Muzzin, O., Darst, S. A. (2002) Structural Basis of Transcription Initiation: An RNA Polymerase Holoenzyme/DNA Complex at 6.5 Å Resolution. Science. 296, 1285-1290. Campbell, E. A., Masuda, S., Sun, J. L., Muzzin, O., Olson, C., A., Wang, S., and Darst, S. A. (2002). Crystal structure of the Bacillus stearothermophilus anti-σ factor SpoIIAB with the sporulation σ factor σF. Cell. 108, 795-807. Kageyama, Y., Masuda, S., Hirose, S. and Ueda, H. (1997) Temporal regulation of the mid-prepupal gene FTZ-F1: DHR3 early late gene product is one of the plural positive regulators. Genes to Cells 2, 559-569. Kunst, F. et al. (1997) The complete genome sequence of the gram-positive bacterium Bacillus subtilis. Nature 390, 249-256. Homuth, G., Masuda, S., Mogk, A., Kobayashi, Y. and Schumann, W. (1997) The dnaK operon of Bacillus subtilis is heptacistronic. J. Bacteriol. 179, 1153-1164. Mizuno, M., Masuda, S., Takemaru, K., Hosono, S., Sato, T., Takeuchi, M. and Kobayashi, Y. (1996). Systematic sequencing of the 283 kb 210 degrees-232 degrees region of the Bacillus subtilis genome containing the skin element and many sporulation genes. Microbiology. 142, 3103-3111.