Loyola University ChicagoLoyola eCommons
Dissertations Theses and Dissertations
2009
Regulation of Transglutaminase by 5-HT2AReceptor Signaling and CalmodulinYing DaiLoyola University Chicago
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Recommended CitationDai, Ying, "Regulation of Transglutaminase by 5-HT2A Receptor Signaling and Calmodulin" (2009). Dissertations. Paper 86.http://ecommons.luc.edu/luc_diss/86
LOYOLA UNIVERSITY CHICAGO
REGULATION OF TRANSGLUTAMINASE BY
5-HT2A RECEPTOR SIGNALING AND CALMODULIN
A DISSERTATION SUBMITTED TO
THE FACULTY OF THE GRADUATE SCHOOL
IN CANDIDACY FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
PROGRAM IN NEUROSCIENCE
BY
YING DAI
CHICAGO, IL
DECEMBER 2009
Copyright by Ying Dai, 2009 All rights reserved.
iii
ACKNOWLEDGEMENTS
Over five years of study at two great Universities make it impossible for me to
extend my gratitude to all of the people who have assisted me throughout my doctoral
education. First of all, I wish to express my gratitude to all the members of my
dissertation committee: Dr. George Battaglia, Dr. Thackery Gray, Dr. Nancy Muma,
Dr. Edward J. Neafsey and Dr. Karie Scrogin. Their kind encouragement, perceptive
criticism, and willing assistance made this dissertation possible. I sincerely thank my
advisor, Dr. Nancy Muma, who has supervised my doctoral research, overseen the
planning of the project and given my work minute consideration for the last five years.
Without her mentoring and prodding, I would never have made it this far.
I appreciate the help of many KU faculties who have supported me during my
dissertation studies. I would especially like to extend my gratitude to Dr. Qian Li and
Dr. Stephen Fowler, for their invaluable guidance and friendship during this process.
I would also like to thank the faculty and staff of Neuroscience Program and my
laboratory colleagues who have provided me with tremendous support in my graduate
studies at Loyola University Chicago. In particular, I would like to thank Dr. Lydia
DonCarlos, Peggy Richied, Nichole Dudek, Ju Shi, Cuihong Jia, Bozena Zemaitaitis
and Fran Garcia.
Lastly, I want to take this opportunity to thank my loving parents, Yiquan Dai and
iv
Zhiyang Song, who always support me and believe in me. Even though they are
thousands of miles away, their constant love has given me much needed
encouragement and inspiration throughout all of my work in graduate school.
I am grateful to the financial support from High-Q/CHDI Foundation and NIH
(Grant# MH068612) that made the research in this dissertation possible.
v
Dedicated to my parents—
Your love and support have made me the person I am today.
vi
TABLE OF CONTENTS
ACKNOWLEDGEMENTS iii LIST OF TABLES viii LIST OF FIGURES ix LIST OF ABBREVIATIONS xi ABSTRACT xiv CHAPTER ONE: BACKGROUND 1
INTRODUCTION 1
REVIEW OF RELEVANT LITERATURE 7 Transglutaminases (TGases), a family of enzymes 7 Physiological functions of TGases 8 Regulatory mechanisms and substrates of TGases 9 5-HT2A receptor signaling and small G protein transamidation 11 Small G proteins 18 Calmodulin (CaM) 23 Pathological role of TGases in neurodegenerative diseases 25 Alzheimer’s disease (AD) and TGase 25 Parkinson’s disease (PD) and TGase 26 Huntington's disease (HD) and TGase 27
CHAPTER TWO: TRANSGLUTAMINASE-CATALYZED TRANSAMIDATION: A NOVEL MECHANISM FOR RAC1 ACTIVATION BY 5-HT2A RECEPTOR STIMULATION 34
ABSTRACT 34
INTRODUCTION 35
MATERIALS AND METHODS 37
RESULTS 44
DISCUSSION 66 CHAPTER THREE: PHOSPHOLIPASE C, CALCIUM AND CALMODULIN SIGNALING ARE REQUIRED FOR 5-HT2A RECEPTOR-MEDIATED TRANSAMIDATION OF RAC1 BY TRANSGLUTAMINASE 73
vii
ABSTRACT 73
INTRODUCTION 74
MATERIALS AND METHODS 76
RESULTS 80
DISCUSSION 97
CHAPTER FOUR: STRIATAL EXPRESSION OF A CALMODULIN FRAGMENT IMPROVED MOTOR FUNCTION, WEIGHT LOSS AND NEUROPATHOLOGY IN THE R6/2 MOUSE MODEL OF HUNTINGTON’S DISEASE 104
ABSTRACT 104
INTRODUCTION 105
MATERIALS AND METHODS 107
RESULTS 116
DISCUSSION 134 CHAPTER FIVE: GENERAL CONCLUSION 141
REVIEW OF RESULTS AND SIGNIFICANCE 141
LIMITATIONS OF THE PRESENT STUDIES 152
FUTURE STUDIES: DISRUPTING CAM-HTT INTERACTION AS A
POTENTIAL THERAPEUTIC STRATEGY IN HUMANS 149
CONCLUSIONS 151 REFERENCES LIST 155 VITA 179
viii
LIST OF TABLES
Table Page
1. Quantitative analysis of neuropathology 133
ix
LIST OF FIGURES Figure Page
1. Molecular switch of the small G-proteins 19
2. Proposed involvement of TGase and CaM in the formation of htt- containing aggregates 33
3. Serotonin-induced increase of Rac1 transamidation in A1A1v cells 52
4. Serotonin receptor specificity on induction of Rac1 transamidation in A1A1v cells 53
5. The effects of cell differentiation on Rac1 transamidation 55
6. Rac1 activity was transiently increased after serotonin treatment 57
7. Dose-dependent effects of cystamine on Rac1 transamidation 58
8. Rac1 activity is inhibited by cystamine 59
9. Knockdown of TGase2 by siRNAs prevents Rac1 transamidation 61
10. Transamidation of serotonin to Rac1 is mediated by TGase 63
11. DOI stimulates transamidation of Rac1 to serotonin to a similar extent as those treated with serotonin 65
12. Effect of PLC inhibition on DOI-induced Ca2+ signals and TGase- modified Rac1 in A1A1v cells 86
13. Pre-incubation with a Ca2+ chelator attenuated the DOI-mediated elevation of cytosolic Ca2+ and TGase-modified Rac1 88
14. The Ca2+ ionophore mimiced the DOI-induced increase in cytosolic Ca2+ and TGase-catalyzed transamidation of Rac1 90
15. The CaM inhibitor W-7 caused a dose-dependent reduction in DOI- stimulated TGase-modified Rac1 92
x
16. Rac1 activity-related domains and potential TGase-targeted amino acid residues 94
17. Schematic representation showing 5-HT2A receptor signaling-mediated transamidation of Rac1 by TGase 96
18. Effect of CaM-fragment on body weight and survival 124
19. Video-based gait analysis on the treadmill 125
20. Expression of CaM-fragment enhanced the locomotor activity in R6/2 mice 126
21. CaM-fragment expression delayed the onset of the rotarod defects in R6/2 mice 127
22. TGase-modified htt in R6/2 mice striatum was reduced by CaM- fragment expression 128
23. Histological evaluation of neuropathology 129
24. Expression of CaM-fragment in mouse striatum did not significantly affect the activity CaM kinase II or TGase 131
25. Regulation of TGase by 5-HT2A receptor signaling and CaM 153
xi
LIST OF ABBREVIATIONS 5-HT serotonin
5-HT2A R serotonin 2A receptor
AA arachidonic acid
AAV adeno-associated virus
ANOVA analysis of variance
BDNF brain-derived neurotrophic factor
BSA bovine serum albumin
Ca2+ calcium
CaM Calmodulin
CaMKII Ca2+-calmodulin dependent kinase type II
DAG diacylglycerol
DAPI 4',6-diamidino-2-phenylindole
DOI (-)-1-(2,5-dimethoxy-4-iodophenyl)-2-amino-propane HC1
ECL enhanced chemiluminescence
EDTA ethylenediaminetetraacetic acid
EGTA ethylene glycol tetraacetic acid
ELISA enzyme-linked immunosorbent assay
ER endoplasmic reticulum
xii
FBS fetal bovine serum
G protein guanine nucleotide-binding protein
GDP guanosine diphosphate
GFP green fluorescent protein
GPCR G protein-coupled receptor
GTP guanosine triphosphate
GTPyS Guanosine-5'-O-(thiotriphosphate)
HD Huntington's disease
HEK 293 human embryonic kidney cells
HRP horseradish peroxidase
htt huntingtin protein
IOD integrated optical density
IP3 inositol 1,4,5-triphosphate
PBS phosphate-buffered saline
PIP2 phophatidyl inositol-4,5-bisphosphate
PKA cAMP-dependent protein kinase
PKC protein kinase C
PLC phospholipase C
PMSF phenylmethylsulphonyl fluoride
SEM standard error of the mean
SSRI selective serotonin reuptake inhibitor
TBS Tris-buffered saline
xiii
TGase transglutaminase
WT wild type
xiv
ABSTRACT
Transglutaminase (TGase), nature’s biological glue, catalyzes the
post-translational modification of proteins by formation of intra- and intermolecular
protein cross-links or by primary amine incorporation. TGase has various
physiological functions, such as skin-barrier formation and blood clot stabilization,
whereas increasing evidence indicates they may also involved in neurodegenerative
diseases including Huntington’s disease (HD), Alzheimer disease’s, and progressive
supranuclear palsy. Mutant huntingtin (htt) and small G proteins (e.g. Rac 1) are
potential substrates of TGases. The purpose of this dissertation was to characterize the
mechanisms by which 5-HT2A receptor signaling and calmodulin (CaM) regulate
TGase-catalyzed transamidation of Rac1 and htt in cultured neuronal cells and HD
transgenic animals, respectively.
5-HT2A receptors are G-protein coupled receptors which are widely expressed in
the brain, peripheral vasculature, platelets and skeletal muscle. They are involved in
diverse physiological functions, from platelet aggregation to neuroendocrine release.
In A1A1v cells, a rat cortical cell line, stimulation of 5-HT2A receptor induces small G
protein Rac1 transamidation and activation. An inhibitory agent or knockdown of
TGase by siRNA revealed that TGase is responsible for transamidation and activation
of Rac1. Moreover, serotonin was identified as an amine that becomes transamidated
xv
to Rac1 by TGase. The classical signal transduction pathway of 5-HT2A receptors is
Gq/11-coupled activation of phospholipase C (PLC), which hydrolyzes
phosphatidylinositol 4,5-bisphosphate (PIP2) to inositol 1,4,5-trisphosphate (IP3) and
diacyglycerol. IP3 mobilizes Ca2+ from the endoplasmic reticulum and thereby
increases intracellular Ca2+ and consequently, it may enhance TGases enzymatic
activity. Inhibition of PLC or manipulation of intracellular Ca2+ by a chelating agent
suppressed 5-HT2A receptor-mediated Rac1 transamidation, whereas an elevation in
intracellular Ca2+ via an ionophore can mimic 5-HT2A receptor-induced cytosolic Ca2+
increases and is sufficient to induce TGase-catalyzed Rac1 transamidation. Moreover,
a CaM inhibitor decreased 5-HT2A receptor-stimulated Rac1 modification by TGase in
a dose-dependent manner. These results suggest that PLC, Ca2+ and CaM signaling
are required for 5-HT2A receptor-mediated transamidation of Rac1 by TGase.
In the next study, we propose that interrupting CaM interactions with htt is
therapeutically beneficial in HD. This hypothesis was based on the observations that
CaM regulates TGase-modification of mutant htt in cells and co-localizes with TGase
and htt in intranuclear inclusions in HD cortex. Furthermore the association of CaM
with mutant htt was demonstrated by affinity purification and coimmunoprecipitation
approaches. Our previous studies demonstrated that in HEK293 and SH-SY5Y cells,
expression of a CaM-fragment, consisting of amino acids 76-121 of CaM, decreased
binding of CaM to mutant htt, decreased TGase-modified htt, decreased cytotoxicity
associated with mutant htt and normalized intracellular calcium release. In this study,
xvi
an adeno-associated virus (AAV) that expresses the CaM-fragment was injected into
the striatum of HD transgenic R6/2 mice and littermate control mice. The HD mice
with CaM-fragment expression had significantly reduced body weight loss and
improved motor function compare to HD control mice. Without affecting the activity
of CaM-dependent enzymes such as CaM-dependent kinase II, CaM-fragment
specifically reduced TGase-modified htt, the percentage of htt-positive nuclei and the
size of intranuclear htt aggregates in HD mouse striatum. Thus, disrupting CaM-htt
interaction with CaM-fragment may provide a new therapeutic strategy for HD
patients.
The data presented here support our hypothesis that stimulation of 5-HT2A
receptors induces TGase-catalyzed Rac1 transamidation and activation by PLC and
Ca2+/CaM signaling, whereas disruption of CaM-htt interaction inhibits
TGase-catalyzed modification of htt and provides neuronal protection in HD. These
results further suggest that CaM regulates the TGase transamidation reaction and
TGase modification of htt is involved in the formation and stabilization of
htt-aggregates and may play a role in the pathogenesis of HD.
1
CHAPTER ONE
BACKGROUND
INTRODUCTION
Transglutaminases (TGases, EC 2.3.2.13) are a family of calcium
(Ca2+)-dependent enzymes that catalyze the formation of a covalent bond between
peptide-bound glutamine residues and either peptide-bound lysine residues or mono-
or polyamines (Folk and Finlayson, 1977, Folk et al., 1980, Walther et al., 2003).
TGases are widely distributed in the human body with various physiological functions,
such as extracellular matrix organization, skin-barrier formation and blood
coagulation (Griffin et al., 2002;Lorand and Graham, 2003). TGases are subject to
transcriptional regulation by retinoic acid and steroid hormones (Fujimoto et al.,
1996;Ou et al., 2000), and require the binding of Ca2+ for their activity (Burgoyne and
Weiss, 2001).
Serotonin signaling may also regulate TGases activity possibly by increasing
cytosolic Ca2+ (Walther et al., 2003, Guilluy et al., 2007). Stimulation of 5-HT2A
receptors activate phospholipase C (PLC) through Gq/11, leading to an accumulation
of inositol 1,4,5-trisphosphate (IP3) and consequent release of Ca2+ from the
endoplasmic reticulum. In platelets, activation of 5-HT2A receptors stimulate
TGase-catalyzed transamidation of serotonin to small G proteins, RhoA and Rab4,
2
making them constitutively active (Walther et al., 2003). TGase-dependent RhoA
transamidation and activation by serotonin stimulation was also observed in vascular
smooth muscle cells (Guilluy et al., 2007). Neuronal differentiation of SH-SY5Y cells
induced by retinoic acid increases the expression and activation of TGases, resulting
in transamination of putrescine to RhoA and activation of RhoA (Singh et al., 2003).
Small G proteins are a family of monomeric 20~30 kDa GTP-binding proteins,
which are homologous to the alpha subunit of heterotrimeric G-proteins. Small G
proteins regulate a wide variety of processes in the cell, including growth, cellular
differentiation, cell movement and lipid vesicle transport (Matozaki et al., 2000).
Rac1 belongs to the Rho family of small G proteins, a subgroup of the Ras
superfamily. Members of the Rho family are involved primarily in the regulation of
cytoskeletal organization (Etienne-Manneville and Hall, 2002). Bearing 5 glutamine
residues (Gln 2,61,74,141,162) and 17 lysine residues in the amino acid sequence
(Matos et al., 2000), Rac1 might serve as a suitable substrate of TGases.
TGase-catalyzed transamination of small G proteins may result in constitutive
activation of the proteins (Walther et al., 2003). Investigating the regulatory
mechanisms of TGase-modification of small G proteins and the functional
consequences in a neuronal cell line may further explore the role of TGase in neuronal
differentiation and neurite outgrowth.
Calmodulin (CaM), another Ca2+-binding protein, has also been shown to play
a role in regulating TGase. CaM is a 17 kDa protein that activates a host of enzymes
3
during Ca2+ binding (Cheung, 1982). CaM coimmunoprecipitated with TGase in
transfected cells (Zainelli et al., 2004) and increased TGase activity in human
erythrocyte (Billett and Puszkin, 1991), platelets and chicken gizzard (Plank et al.,
1983). Recently, a series of studies showed that the activity of TGase is critical for the
pathology of a number of neurodegenerative diseases, such as Huntington's disease
(HD)(Karpuj and Steinman, 2004;Violante et al., 2001). CaM may also be involved in
HD based on its interaction with both TGase and huntingtin (htt) (Bao et al., 1996,
Zainelli et al., 2004).
HD is an autosomal dominant neurodegenerative disorder with progressive
neurological symptoms and mental decline such as chorea, rigidity, gait disturbance,
abnormal posturing, seizure, depression and dementia (Harper, 1991). HD occurs in
individuals whose HTT gene has more than 36 CAG repeats, resulting in a mutant
protein with abnormal expansions of the polyglutamine domain in the N-terminus.
The pathological hallmark of HD is intranuclear inclusions and cytoplasmic
aggregates composed of mutant htt protein (DiFiglia et al., 1997;Ross et al., 1998). In
vitro and in cell culture, mutant htt is an excellent substrate for TGases (Gentile et al.,
1998;Kahlem et al., 1998;Karpuj et al., 1999). The mRNA, protein, and enzymatic
activity of TGases are elevated in HD brain (Karpuj et al., 1999;Lesort et al.,
1999;Zainelli et al., 2003). Increasing evidence indicates that TGases may contribute
to the formation of mutant htt aggregates in HD (Cooper et al., 1999). Htt protein
colocalizes with TGase2 and its product, -( -glutamyl) lysine covalent bond in
4
intranuclear inclusions in the frontal cortex of HD (Zainelli et al., 2004). On the other
hand, our lab and others reported CaM associates with mutant htt as demonstrated by
coimmunoprecipitation (Zainelli et al., 2004) and affinity purification (Bao et al.,
1996). Furthermore, CaM colocalizes with TGase2 and with htt in HD intranuclear
inclusions (Zainelli et al., 2004). Taken together, these studies suggest that mutant htt
may bind to CaM resulting in increases TGase-modification to htt. This modification
may stabilize htt monomers or polymers and contribute to htt-containing aggregate
formation and cytotoxicity.
Both TGases and CaM have been explored as therapeutic targets for HD.
Administration of the TGase inhibitor, cystamine, extended survival, improved motor
performance and increased neuroprotective gene transcription in HD transgenic mice
(Dedeoglu et al., 2002, Karpuj et al., 2002a). TGases2 ablation in HD transgenic mice
results in a drastic reduction in -( -glutamyl) lysine bond levels and neuronal death
in the cortex and striatum (Mastroberardino et al., 2002). CaM inhibitor, W5, can
decrease TGases-catalyzed cross-linking of htt in cells transfected with TGase2 and
mutant htt (Zainelli et al., 2004). However, direct inhibition of CaM or TGase may not
be the best therapeutic approach, since both enzymes have a lot of other biological
functions and long-term inhibition might cause untoward effects. A new strategy is to
identify the htt-binding domain in CaM, and express this CaM fragment in HD
models. It might disrupt CaM-htt interaction by competing with endogenous CaM,
and thereby decrease TGase-modified htt, htt-aggregates and cell death. This
5
therapeutic strategy has been tested in cell models of HD and will next be tested in an
HD transgenic mouse model.
We hypothesize that stimulation of 5-HT2A receptors induces
TGase-catalyzed Rac1 transamidation and activation by increasing intracellular
Ca2+, whereas disruption of CaM-htt interaction inhibits TGase-catalyzed
modification of htt and provides neuronal protection in HD. Three specific aims
are proposed to test this hypothesis.
Specific Aim 1: To determine if the activity and transamidation of Rac1 by
TGase are increased by 5-HT2A receptor signaling.
The first specific aim is to determine if the activity and transamidation of Rac1
are increased by 5-HT2A receptor signaling in a rat cortical cell line, A1A1v cells. We
will use either a TGase inhibitor or siRNA targeting TGase to determine if Rac1
transamidation and activation are mediated TGase. In addition, we will determine if
serotonin is incorporated into Rac1 by TGase following 5-HT2A receptor stimulation.
Lastly, we will test if DOI, a 5-HT2A/2c receptor agonist, selectively increases
TGase-catalyzed transamination of Rac1 in vivo.
Specific Aim 2: To determine if 5-HT2A receptor-regulated transamination of
Rac1 by TGase is dependent on an increase of intracellular Ca2+ mediated by PLC
signaling, and if calmodulin regulate TGase-modification of Rac1.
The second specific aim designed is to explore the downstream mechanistic
pathways in 5-HT2A receptor-mediated Rac1 transamidation. We will determine if
6
inhibition of PLC prevents the increase of intracellular Ca2+ and transamination of
Rac1 in response to activation of 5-HT2A receptors. A Ca2+ chelator or Ca2+ ionophore
will be used to manipulate intracellular Ca2+ and determine if the increase in
intracellular Ca2+ is necessary and sufficient to induce Rac1 transamination. We will
also determine if inhibiting calmodulin prevents 5-HT2A receptor-stimulated
transamidation of Rac1.
Specific Aim 3: To determine if disrupting the interaction of calmodulin and
huntingtin decreases TGase-modified huntingtin and huntingtin-aggregates, improves
motor function and increases survival of HD transgenic mice.
We will use a viral vector to deliver a calmodulin-fragment, which can
interrupt calmodulin and mutant huntingtin binding, to the striatum of R6/2 HD
transgenic mice, then monitor survival and body weight, evaluate motor performance
by behavioral tests such as DigiGait, Actometer and Rotarod. We will also measure
neuropathological changes such as intranuclear htt-aggregates and striatal atrophy by
immunofluorescence and Nissl staining. The specificity of calmodulin-fragment will
be tested by measuring calmodulin-dependent protein kinase II (CaMKII) and TGase
activity.
7
REVIEW OF RELEVANT LITERATURE
In the literature review, the family of TGases will be introduced first,
followed by their physiological functions. The next section will focus on regulatory
mechanisms and substrates of TGases. The regulation of TGases-mediated
transamidation of small G protein by 5-HT2A receptor signaling will be highlighted.
Rac1, as a potential substrate of TGases, will be presented. Regulation of TGases by
calmodulin will also be discussed in this section. Lastly, the pathological role of
TGases in neurodegenerative diseases including Alzheimer disease, Parkinson disease
and especially Huntington’s disease will be reviewed.
Transglutaminases (TGases), a family of enzymes
TGases (EC 2.3.2.13) are a family of Ca2+-dependent enzymes. Once activated,
TGases catalyze the cross-linking of proteins via the γ-carboxamide group of
peptide-bound glutamine and the є-amino group of peptide-bound lysine, forming a
inter- or intramolecular isodipeptide bond (Griffin et al., 2002). The enzyme can also
covalently link biogenic amines and polyamines, such as spermine or serotonin
(5-Hydroxytryptamine; 5-HT), to a peptide-bound glutamine residue (Dale et al.,
2002;Folk et al., 1980).
To date, nine isoforms of transglutaminase have been identified, each encoded
by different but structurally and functionally related related gene. Family members
include keratinocyte transglutaminase (TGase1), tissue transglutaminase (TGase2),
epidermal transglutaminase (TGase3), prostate transglutaminase (TGase4),
8
transglutaminase X (TGase5), transglutaminase Y (TGase6), transglutaminase Z
(TGase7), factor XIII-A subunit (fibrin-stabilizing factor) and ATP-binding
erythrocyte membrane protein band 4.2 (B4.2) (Lorand and Graham, 2003).
At least 4 isoforms, TGase1, 2, 3 and TGase7, are expressed in normal human
brain tissue (Kim et al., 1999, Grenard et al., 2001) and mutant huntingtin protein is
identified as a substrate of the first three TGase isoforms (Zainelli et al., 2005).
However, of the nine known human isozymes, tissue TGase (TGase2) is ubiquitously
expressed and most well characterized.
Physiological functions of TGases
TGases are widely distributed in various tissues and are involved in various
physiological functions such as blood clot formation, wound healing, skinbarrier
formation, extracellular matrix assembly, cell death, and cell differentiation (Griffin et
al., 2002, Lorand and Graham, 2003).
For example, following exposure to thrombin and Ca2+, fibrin stabilizing
factor (factor XIII) is activated and converted to factor XIII-A containing only the A
subunit that, in turn, catalyzes the formation of Nε(γ-glutamyl)lysine
protein-to-protein side chain bridges within the clot network. Introduction of these
covalent crosslinks greatly stabilize the fibrin and augments its resistance to
fibrinolytic enzymes (Lorand, 2001).
In addition to cytoplasmic and nuclear localization, a significant part of the
TGase2 protein pool is present on the cell surface. Cell surface TGase2 acts as a
9
coreceptor of integrins, coordinating binding of extracellular matrix (ECM) proteins
(Zemskov et al., 2006). It also crosslinks the fibronectin (as well as other ECM
proteins) to improve cell adhesion (Aeschlimann and Thomazy, 2000). TGase2
functions to stabilize the ECM by forming large polymeric structures that are resistant
to proteolytic/chemical degradation and mechanical stresses (Griffin et al., 2002).
The homozygous null TGase2 mice are viable and have normal size and
weight with no severe phenotype (De Laurenzi and Melino, 2001, Nanda et al., 2001),
suggesting that other TGase isoforms may compensate for the lack of TGase2 to some
extent. However, TGase2-/- mice do have altered fibroblast function, defective wound
healing (Nanda et al., 2001), decreased phagocytic clearance of apoptotic cells
(Szondy et al., 2003, Rose et al., 2006), mild glucose intolerance and hyperglycemia
likely due to reduced insulin secretion (Bernassola et al., 2002). Therefore, it is
plausible that under disease or stress, TGase2 play an essential role in tissure repair or
healing and that further testing of these mice will reveal how TGase2 is critical in
other physiologic states.
Regulatory mechanisms and substrates of TGases
TGases are Ca2+-dependent enzymes containing a cysteine in their active site
that is unmasked only in the presence of Ca2+ (Hand et al., 1993) and Ca2+ activation
of TGase is further regulated by other signal modulators such as GTP (Monsonego et
al., 1998), phospholipids (Griffin et al., 2002, Beninati and Piacentini, 2004), tumor
necrosis factor alpha (Chen et al., 2000), nitric oxide (Bernassola et al., 1999) and
10
CaM (Puszkin and Raghuraman, 1985, Zainelli et al., 2004).
An elevation in intracellular Ca2+ is the required element for activation of
TGases. Elevations in intracellular Ca2+ needed to activate TGases may arise via
extracellular influx, release from intracellular stores, or decreased levels of other Ca2+
binding proteins (Lorand and Conrad, 1984). The binding of Ca2+ to TGases induces
conformational changes and exposes the active site to substrate proteins (Hand et al.,
1993, Casadio et al., 1999).
TGase2 has been reported to be a GTP-binding protein with GTPase activity
(Achyuthan and Greenberg, 1987). GTP binding to the enzyme can inhibit its
transamidation activity at physiological Ca2+ levels (Bergamini et al., 1987). Binding
of GTP to TGase keeps the enzyme in a conformation such that the C-terminus blocks
the enzyme's catalytic core (Monsonego et al., 1998, Casadio et al., 1999).
Interestingly, an elevation in cytosolic Ca2+ will overcome GTP inhibition and activate
TGase (Bergamini, 1988).
Other regulatory effects may also occur through the interaction of TGase with
phospholipids, nitric oxide and CaM. When phospholipid vesicles are in the
crystalline-liquid transition state, they can interact with and inhibit TGase activity.
The inhibition may be due to the hydrophobic environment provided by the
phospholipid hydrocarbon chain when the enzyme is inserted into the bilayer (Fesus
et al., 1983). Exposure to tumor necrosis factor alpha causes fibronectin
multimerization in lung endothelial matrix by increasing TGase activity (Chen et al.,
11
2000). Nitric oxide has an inhibitory effect on both TGase2 and coagulation factor
XIII, probably via S-nitrosylation of their crucial thiol groups (Bernassola et al.,
1999). CaM increased TGase activity in human erythrocyte (Billett and Puszkin,
1991), platelets and chicken gizzard (Plank et al., 1983). A CaM inhibitor can
decrease TGases-catalyzed cross-linking of htt in cells transfected with TGase2 and
mutant htt (Zainelli et al., 2004).
Small G proteins have emerged as the new substrates of TGase. It has
been reported that neuronal differentiation of SH-SY5Y cells induced by retinoic
acid increases the expression and activation of TGases, resulting in transamidation of
putrescine to RhoA and activation of RhoA (Singh et al., 2003). In platelets and aortic
smooth muscle cells, TGases can catalyze the incorporation of serotonin into
small GTPases ("transamidation"), such as RhoA and Rab4. This process of
transamidation renders these small GTPases constitutively active (Walther et al.,
2003, Guilluy et al., 2007). Activation of 5-HT2A receptors, which increases Ca2+
availability, induces serotonin transamidation to small GTPases (Walther et al.,
2003). Therefore, the next section will introduce the 5-HT2A receptor, including
its structure, signal transduction, distribution and pathophysiological roles.
5-HT2A receptor signaling and small G protein transamidation
Overview and classification of 5-HT receptors
As one of the most complex families of neurotransmitter receptors, 5-HT
receptors have at least 16 distinct members cloned, identified and classified into 7
12
different subfamilies according to their ligand recognition profiles, signal transduction
mechanisms, and structural characteristics (Humphrey et al., 1993, Hoyer et al., 1994,
Hoyer et al., 2002). Except for the 5-HT3 receptor, which is a ligand-gated ion
channel, the rest of 5-HT receptors belong to the G protein-coupled receptor (GPCR)
superfamily.
The 5-HT1 receptor class includes 5 members, 5-HT1A, 5-HT1B, 5-HT1D,
5-HT1F and 5-HT1F, which are classically coupled to the Gi/o proteins that
negatively regulate adenylyl cyclase and induces the opening of K+ channels. They
are expressed both pre and post-synaptically and involved in the regulation of
serotonin release (Hoyer et al., 2002).
The 5-HT2 family consists of 5-HT2A, 5-HT2B and 5-HT2C receptors that
primarily activate phospholipase C (PLC) via coupling with Gq/11. Activation of
5-HT2A receptors also mediates neuronal depolarization, a result of the closing of K+
channels (Aghajanian and Marek, 1999, Lambe and Aghajanian, 2001).
The 5-HT3 receptor is unique among the currently known subtypes not only
because it is one of the first 5-HT receptors identified, corresponding to the M
receptor (Gaddum and Picarelli, 1957), but also because it belongs to the ligand-gated
channel superfamily instead of coupling to G proteins or second messenger systems.
The 5-HT3 receptor mediates the rapid excitatory electrophysiological response due
to a transient inward current, subsequent to the opening of nonselective cation
channels (Na+, Ca2+ influx, K+ efflux) (Peters et al., 1992, Hoyer et al., 2002).
13
The 5-HT4, 5-HT6 and 5-HT7 receptors are single members in different families
that mainly stimulate adenylyl cyclase through the activation of Gαs proteins
(Raymond et al., 2001).
The 5-HT5 family (5-HT5A and 5-HT5B receptors) is neither coupled to adenylyl
cyclase nor PI-PLC. The effector systems of this family remain unclear, but some
evidence indicates 5-HT5A receptor is implicated in the action of LSD (Grailhe et al.,
1999, Nelson, 2004).
5-HT2A receptor distribution and pathophysiological roles
5-HT2A receptors are widely distributed in central and peripheral tissues. In the
central nervous system (CNS), these receptors have been found mainly in the cortical
areas (neocortex, entorhinal and pyriform cortex, claustrum), caudate nucleus, nucleus
accumbens, olfactory tubercle and hippocampus (Hoyer et al., 1986, Pazos et al.,
1987). In the cortex the great majority of labeled cells were pyramidal neurons
(Wright et al., 1995, Willins et al., 1997). In the periphery, 5-HT2A receptors are
located in platelets (de Chaffoy de Courcelles et al., 1987), vascular smooth muscle
(Cohen et al., 1981), and uterine smooth muscle (Wilcox et al., 1992), inducing
vasoconstriction and platelet aggregation. Studies that have investigated the
subcellular location of the 5-HT2A receptor indicate that the majority of 5-HT2A
receptors in the human cerebellum and rat CNS are in cytosol, rather than the cellular
membrane (Cornea-Hebert et al., 1999, Eastwood et al., 2001).
The 5HT2A receptor has been shown to be involved in a number of different
14
processes including endocrine modulation (Van de Kar et al., 2001), pain modulation
(Sommer, 2004), thermoregulation (Ootsuka and Blessing, 2006), cognitive function
and memory (Meneses, 2002, Williams et al., 2002). In addition, 5-HT2A receptor
signaling is implicated in a number of disorders including hypertension,
atherosclerosis, and different psychiatric diseases such as anxiety, stress, suicide
behavior and schizophrenia (Roth, 1994, Weisstaub et al., 2006).
Serotonin is one of many neurotransmitters that influence the activation of
hypothalamic-pituitary-adrenal (HPA) axis and stimulate secretion of oxytocin,
adrenocorticotrophic hormone (ACTH), corticosterone (Van de Kar et al., 2001,
Hemrick-Luecke and Evans, 2002, Zhang et al., 2002). Serotonergic terminals from
the medial raphe nucleus make synaptic interaction with the corticotrophin releasing
factor (CRF) synthesizing neurons in the paraventricular nucleus of rat hypothalamus
(Liposits et al., 1987). Moreover, 5-HT2A receptors are found in hypothalamic
paraventricular nucleus and in both CRF synthesizing neurons and
oxytocin-containing neurons (Van de Kar et al., 2001, Zhang et al., 2002). When CRF
is released, it induces ACTH secretion from the anterior pituitary gland, which in turn
stimulates the release of corticosterone from the adrenal cortex. Administration of
DOI, a 5-HT2A/2C receptor agonist increases plasma levels of oxytocin, ACTH,
corticosterone in rats. The 5-HT2A receptor selective antagonist, MDL 100,907, but
not the 5-HT2C receptor antagonist SB242084 can dose-dependently inhibit the
neuroendocrine effects of DOI (Van de Kar et al., 2001, Zhang et al., 2002).
15
5-HT2A receptors are the target of some therapeutic agents for a variety of
psychiatric disorders. Atypical antipsychotic drugs such as clozapine display potent
5-HT2A receptors antagonism with relatively weak blockade at D2 dopamine (DA)
receptors, various effects at D1 receptors (Meltzer et al., 1989, Arora and Meltzer,
1994). Moreover, the clinical response to the selective serotonin reuptake inhibitors
(SSRI) in treatment-resistant patients is augmented by atypical antipsychotic drugs
and some antidepressants (Ostroff and Nelson, 1999, Carpenter et al., 2002,
Marangell et al., 2002). The common feature of these agents is that they are able to
occupy 5-HT2 receptors and to block the responses mediated by those receptors, in
particular by 5-HT2A receptors (Marek et al., 2003). These reports support a role for
5-HT2A receptors in antipsychotic drug action.
5-HT2A receptors not only mediated the effects of some antipsychotic drugs, but
also are involved in pathophysiology of psychiatric disorders. Abnormalities in
5-HT2A receptors have been proposed to be implicated in the pathology of
schizophrenia (Woolley and Shaw, 1954, Dean, 2003). Direct evidence supporting this
hypothesis came from radioligand binding studies in post-mortem schizophrenia
patients, which suggest there is a decrease in the density of cortical 5-HT2A receptors
in those patients (Arora and Meltzer, 1991, Dean and Hayes, 1996). The levels of
mRNA encoding the 5-HT2A receptors were also reduced in the superior temporal
gyrus of schizophrenic subjects (Hernandez and Sokolov, 2000). In depressed patients
and suicide victims, the postmortem studies indicated an elevated level of 5-HT2A
16
receptor in the cortex, amygdala and hippocampus (Arango et al., 1990, Hrdina et al.,
1993, Pandey et al., 2002). However, positron emission tomography (PET) and
radioligand binding studies detected a significant decrease in 5-HT2A receptor density
in hippocampus of both young and old depressed patients (Mintun et al., 2004,
Sheline et al., 2004). Furthermore, 5-HT2A receptors are also involved in modulating
anxiety-related behaviors in humans and rodents. For example, global disruption of
5-HT2A receptor signaling in htr2a–/– mice reduces the inhibition in conflict anxiety
paradigms, whereas selective restoration of 5-HT2A receptor signaling to the cortex
normalized conflict anxiety behaviors (Weisstaub et al., 2006).
5-HT2A receptor structure and signal transduction
The serotonin 5-HT2A receptor gene is located on human chromosome 13q14-q21
and comprises 471 amino acid in rats, mice, and humans (Hoyer et al., 2002). It
contains three exons, separated by two introns, with the coding region of the gene
spanning 1.4 kb (Sanders-Bush et al., 2003).
The 5-HT2A receptor is coupled via the Gq/11 proteins to the phospholipase С
(PLC) signaling cascade. PLC hydrolyzes phosphatidyl-inositol 4,5-bisphosphate
(PIP2) to diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). IP3 mobilizes
Ca2+ from the endoplasmic reticulum and thereby leads to an increase of cytosolic
Ca2+ concentration (Roth et al., 1998).
In addition to the major pathways mentioned above, extensive evidence suggests
that 5-HT2A receptors couple to other effector pathways. 5-HT2A receptor stimulation
17
also induces the activation of phospholipase A2 and the release of the second
messenger arachidonic acid, which is involving Gβγ-mediated ERK1/2 activation and
Gα12/13-coupled, Rho-mediated p38 activation (Felder et al., 1990, Kurrasch-Orbaugh
et al., 2003). Through interaction with ADP-ribosylation factor 1 (ARF1) via its
C-terminal domain, 5-HT2A receptor is able to signal through the phospholipase D
(PLD) pathway (Mitchell et al., 1998, Robertson et al., 2003).
5-HT2A receptor is known to trigger MAPK activation via PKC/Raf-1 pathway
(Hershenson et al., 1995, Watts, 1996). The JAK/STAT signaling pathway is activated
by a number of G protein coupled receptors including 5-HT2A receptors
(Guillet-Deniau et al., 1997, Singh et al., 2009).
Depending on the cell types, 5-HT2A receptor is able to increase or diminish
cyclic adenosine monophosphate (cAMP) accumulation. It increases cAMP in a cell
line derived from embryonic rat cortex (A1A1 cells) by protein kinase C-dependent
and calcium/calmodulin-dependent mechanisms (Berg et al., 1994a) and in FRTL-5
thyroid cells through a pertussis toxin-sensitive mechanism (Tamir et al., 1992).
However, in rat renal mesangial cells, 5-HT2A receptor activation can inhibit adenylyl
cyclase activity and forskolin-stimulated cAMP accumulation (Garnovskaya et al.,
1995).
Because transamidation of small G proteins (e.g. RhoA and Rab4) mediated by
5-HT2A receptor signaling leads to platelet α-granule release and aggregation (Walther
et al., 2003), and TGase-dependent Rho A transamidation also inhibits contraction of
18
vascular smooth muscle cells, a brief introduction of small G proteins especially Rac1
whose transamidation is extensively studied in this dissertation will be presented in
the next section.
Small G proteins
Small G-proteins, short for small guanine nucleotide-binding proteins, are
monomeric G proteins with molecular masses of 20–30 kDa. They are homologous to
the alpha subunit of heterotrimeric G-proteins, but they can function on their own by
cycling between an inactive GDP-bound state and an active GTP-bound state. The
GTP/GDP cycling is controlled by many regulator molecules such as
GTPase-activating proteins (GAPs), guanine nucleotide exchange factors (GEFs) and
GDP dissociation inhibitors (GDIs) (Hakoshima et al., 2003). GTP hydrolysis is
accelerated by GAPs, resulting in inactivation of the cognate G-protein.
GAP-dependent signaling is antagonized by GEF, which activate small G proteins by
promoting the nucleotide exchange of GTP for GDP. Furthermore, small G proteins
are regulated by GDIs, which maintain small G proteins in their inactive GDP-bound
form, in addition to preventing their association to the plasma membrane (Figure 1).
19
Figure 1. Molecular switch of the small G-proteins. The activation of small G protein
is mediated by three types of regulatory proteins: GDI (guanine nucleotide
dissociation inhibitor), GAP (GTPase activating protein), and GEF (guanine
nucleotide exchange factors).
20
Biological functions of Rac1
Rac1, short for Ras-related C3 botulinum toxin substrate 1, belongs to the Rho
family of small G proteins, a subgroup of the Ras superfamily. Binding to a variety of
downstream target proteins (effectors), members of the Rho family (e.g. RhoA, Rac1,
Cdc42) are associated with a wide array of cellular processes such as cytoskeletal
organization, vesicular transport, cell cycle progression, cell adhesion and migration,
neuronal differentiation and a variety of enzymatic activities (Etienne-Manneville and
Hall, 2002, Burridge and Wennerberg, 2004).
Rac1 is best known as a regulator for the assembly of the actin cytoskeleton,
thereby playing a role in neurite outgrowth and neuronal differentiation. Expression of
a constitutively active mutant of Rac1 in neuroblastoma cells leads to the formation of
neurites (Leeuwen et al., 1997), and production of filopodia and lamellipodia in the
developing growth cone (Kozma et al., 1997). In contrast, over-expression of
dominant negative Rac1 in SH-SY5Y cells blocks retinoic acid (RA)-induced neurite
outgrowth and expression of neuronal markers, suggesting that activation of Rac1
regulates neuronal differentiation (Pan et al., 2005). Rac1 play an essential role in
fibroblasts cell growth in response to mitogenic stimulation since microinjection of
Rac1 stimulated cell cycle progression through G1 and subsequent DNA synthesis.
This effect can be blocked by microinjection of dominant negative forms of Rac1
(Olson et al., 1995). In growth factor-stimulated fibroblasts and neuronal cells, Rac1
was transiently activated in a broad area of the plasma membrane, followed by a
21
localized activation at nascent lamellipodia (Kurokawa et al., 2005). Consistent with
this finding, Rac1 is required to stimulate the formation of lamellipodia and
membrane ruffles.
Rac1 is also involved in multiple cell death pathways. Depending on the
interaction with other signaling molecules and on the type of cells, it becomes
pro-apoptotic (Embade et al., 2000, Aznar and Lacal, 2001, Harrington et al., 2002) or
anti-apoptotic (Nishida et al., 1999, Deshpande et al., 2000, Murga et al., 2002). For
example, neurotrophin receptor p75 activates Rac1, which in turn activates c-Jun
N-terminal kinase (JNK) and causes apoptosis in neuronal cells (Harrington et al.,
2002). On the other hand, Rac1 protects endothelial cells from tumor necrosis
factor-alpha-induced apoptosis (Deshpande et al., 2000) and promotes COS7 cell
survival by the activation of phosphatidylinositol 3-kinase (PI3K) and Akt (Murga et
al., 2002).
Structure of Rac1
The Rho family are monomeric globular proteins consisting of less than 300
amino acids (Wennerberg and Der, 2004). Like all members of the Rho family, Rac1
functions as a conformational switch by cycling active GTP and inactive GDP-bound
forms. Rho GTPases share a common GTPase domain, which carries out the basic
function of nucleotide binding and hydrolysis. The GTPase domain consists of a
six-stranded β-sheet surrounded by α-helices (Vetter and Wittinghofer, 2001). Most
typical Rho proteins consist only of the GTPase domain and short N- and C-terminal
22
extensions. C-terminal post-translational modifications such as isoprenoid addition
facilitate specific subcellular location and association with specific membranes, which
is crucial for their function (Wennerberg and Der, 2004). The differences between the
GDP- and GTP- bound structural forms of RhoA are confined primarily to two
segments, referred to as switch I and switch II, and this feature is probably shared by
all Rho GTPases (Ihara et al., 1998, Vetter and Wittinghofer, 2001).
Bearing five glutamine residues in the amino acid sequence (Matos et al., 2000),
Rac1 might serve as a suitable substrate of TGases. The transamidation of primary
amines to RhoA and Rab4 by TGases renders these small G proteins constitutively
active (Walther et al., 2003, Guilluy et al., 2007). Similarly, post-translational
modifications, such as transamidation and phosphorylation of Rac1 may affect its
ability to interact with regulatory proteins (e.g. GAPs, GEFs and GDIs) or to convert
GTP back to GDP, resulting in increased activation of Rac1 and stimulation of its
signaling cascade in the cells.
The Conserved Domain Database (CDD) (http://www.ncbi.nlm.nih.gov/
entrez/query.fcgi? db=cdd) provides an interactive tool to identify conserved domains
present in protein sequences. The residues, which regulate Rac1 activity after
post-translational modifications, are most likely to be located at GTP/Mg2+ binding
sites, and GAPs, GEFs and GDIs interaction sites in the Rac1 sequence. Two
glutamine residues (Q61, Q74) and three lysine residues (K5, K16, and K116),
potential tagets of TGase-catalyzed modification, are identified within these
23
activity-related domains using CDD search of the Rac1 sequence (PSSM-Id: 57957).
In line with this finding, it has been reported that site-specific deamidation of a Gln
residue in the Rho family of G proteins (Q61 in Rac and Cdc42, Q63 in RhoA) by
CNF-1 inhibits both intrinsic and GAPs-stimulated GTP hydrolysis activity, resulting
in constitutive activation of these proteins (Flatau et al., 1997, Schmidt et al., 1997).
CNF-1 has also been shown to possess in vitro TGase activity. In the presence of
primary amines, RhoA is transamidated in vitro at Q63 by CNF-1 and at positions 52,
63 and 136 by guinea pig liver TGase (Schmidt et al., 1998). Similarly, in addition to
Q61 which is critical in regulating Rac1 activity, the other four glutamine residues in
Rac1 may also be modified by TGase.
Calmodulin (CaM)
The other regulator molecule of TGase that will be focused on this dissertation is
CaM. It is a 17 kDa, dumbbell shaped, Ca2+ binding protein that is ubiquitously
expressed in cells (Babu et al., 1985). There are two EF hand motifs at each end of its
dumbbell-like structure, separated by a helical linker region. The C-terminal EF hand
motifs have three to five fold higher affinity for Ca2+ than those in the N-terminal
(Chin and Means, 2000). Upon Ca2+ binding to the EF hand motifs, both N-terminal
and C-terminal domains adopt an open confirmation, exposing hydrophobic surfaces,
which then interact with a variety of proteins triggering events such as the release of
autoinhibitory domains (for CaM-dependent kinases and calcineurin), active site
remodeling (anthrax adenylyl cyclase), and CaM-induced dimerization of membrane
24
proteins (Bachs et al., 1994, Hoeflich and Ikura, 2002). However, CaM can interact
with target proteins in both Ca2+ dependent as well as independent manners. In the
absence of Ca2+, the N-terminal domain adopts a closed confirmation whereas the
C-terminal domain is in a semi-open state with partial exposure of a hydrophobic
patch. Thus the C-terminal domain of CaM could interact with target proteins even in
the absence of Ca2+ (Swindells and Ikura, 1996).
The temporal and spatial associations between changes in Ca2+ and the function of
CaM have been identified in numerous studies. Changes in intracellular Ca2+
concentration induce dynamic compartmentalization and mobilization of CaM
including translocation from the cytosol to the nucleus (Luby-Phelps et al., 1995,
Deisseroth et al., 1998, Craske et al., 1999). CaM activation is coupled to elevation in
intracellular Ca2+ and also correlates with the spatial pattern of increased Ca2+ (Hahn et
al., 1992).
CaM not only acts as a ubiquitous transducer of intracellular Ca2+ signals but also
modulates intracellular Ca2+ concentrations. CaM can act as intracellular Ca2+ buffer,
regulate the plasma membrane Ca2+ ATPase, ryanodine receptors, IP3 receptors and
cyclic nucleotide gated Ca2+ channels (Baimbridge et al., 1992, Liu et al., 1994, Patel et
al., 1997, Balshaw et al., 2001).
Various downstream substrates of CaM have been studied extensively and the
studies presented here focus on delineating the role of Ca2+/CaM in regulation of
TGase-catalyzed transamidation activity. CaM increased TGase activity in human
25
erythrocyte (Billett and Puszkin, 1991), platelets and chicken gizzard (Plank et al.,
1983). In the presence of CaM, TGases are activated at lower Ca2+ concentrations than
the concentration that is normally needed for activation (Billett and Puszkin, 1991). In
cells transfected with TGase2 and mutant htt, CaM co-immunoprecipitates with both
TGase2 and mutant htt. A CaM inhibitor can decrease TGases-catalyzed cross-linking
of htt (Zainelli et al., 2004). Furthermore, fluorescence confocal microscopy
demonstrated that CaM colocalizes with TGase2 and with htt in HD intranuclear
inclusions (Zainelli et al., 2004), suggesting that CaM may play a pivotal role in HD.
Pathological role of TGases in neurodegenerative diseases
Protein aggregation and inclusion bodies are the common features of several
neurodegenerative diseases including Alzheimer’s disease, Parkinson’s disease, and
Huntington’s disease. Selkoe and colleagues were the first to propose the role for
TGases in neurodegeneration. They demonstrated that TGases could catalyze the
formation of insoluble polymers composed of human neurofilament proteins by
forming covalently crosslink (Selkoe et al., 1982). Increasing evidence suggests
TGases may be involved in protein aggregation in those neurodegenerative diseases
(Ross and Poirier, 2004, Muma, 2007).
Alzheimer’s disease (AD) and TGase
Neurofibrillary tangles (NFTs), deposits of tau filaments in the neuronal cell
body, are one of the most important pathologic characteristics of AD. Numerous
groups have shown that tau is cross-linked by TGases in vitro, forming insoluble
26
filamentous polymers (Dudek and Johnson, 1993, Appelt and Balin, 1997, Murthy et
al., 1998, Norlund et al., 1999). In post-mortem studies, it has been reported that
TGase2 colocalizes with paired helical filaments (PHFs), which are composed of tau,
in AD brain (Appelt et al., 1996). Our laboratory and others further demonstrated that
PHF-tau from AD brain contains TGase-catalyzed gamma-glutamyl-lysine
isodipeptide bonds (Norlund et al., 1999), and there is extensive co-localization of
TGase-catalyzed isodipeptide bonds and PHF-tau (Citron et al., 2002, Singer et al.,
2002), providing circumstantial evidence for the involvement of TGases in AD
pathology. Moreover, PHF-tau co-localizes with TGase-catalyzed cross-links in brain
regions devoid of NFT in stage II AD, which would be expected to contain them in
stage III, suggesting TGase as a contributing factor in NFT formation induces tau
cross-links in an early stage of AD (Singer et al., 2002).
Furthermore, TGase protein expression and cross-linking activity are
increased in AD brain and cerebrospinal fluid (CSF) as compared to controls (Kim et
al., 1999, Bonelli et al., 2002). Interestingly, TGase activity are only increased in the
brain regions with abundant neurofibrillary pathology, but not in the cerebellum, a
region spared of NFT pathology (Johnson et al., 1997).
Parkinson's disease (PD) and TGases
Multiple lines of evidence suggest that TGase-catalyzed bonds are associated
with Lewy bodies, the major inclusion body in PD. In vitro and in cell culture, TGase
is also capable of crosslinking a-synuclein, a major component of Lewy bodies in PD,
27
to form intramolecular cross-links and high molecular weight polymers (Jensen et al.,
1995, Junn et al., 2003). Moreover, Glu79 and Lys80 in a-synuclein have been
identified as the TGase-reactive residues and Lys80 residue is located in a conserved
sequence in the synuclein gene family (Jensen et al., 1995). Immunohistochemistry
studies demonstrate that TGase-catalyzed cross-links are elevated in dopaminergic
neurons in PD substantia nigra, and co-localize with α-synuclein in Lewy bodies
(Junn et al., 2003, Andringa et al., 2004). On Western blots, the TGase-catalyzed
cross-link bond comigrates with α-synuclein from PD substantia nigra (Citron et al.,
2002, Andringa et al., 2004).
Huntington's disease (HD) and TGase
HD is an autosomal dominant neurodegenerative disorder characterized by
cognitive and behavioral disturbance, involuntary movements (chorea), striatal and
cortical neurodegeneration and neuronal inclusions (Gusella and MacDonald, 2000).
HD is caused by expansion of CAG repeats in exon1 of HTT gene, which is translated
into an expanded polyglutamine (polyQ) stretch at the N-terminus of the huntingtin
protein (htt) (Huntington’s Disease Collaborative Research Group, 1993).
Consequently, N-terminal proteolytic fragments of mutant htt form intranuclear and
cytoplasmic aggregates in neurons, the prominent neuropathologic hallmarks of HD.
This CAG “triplet” is normally repeated about 20 times, but an increase in the number
of repeats to 40 or more results in the expression of the disease (Duyao et al., 1993,
Huntington’s Disease Collaborative Research Group, 1993). Additionally, there is an
28
inverse correlation between age of onset of HD symptoms and the number of CAG
repeats (Duyao et al., 1993).
Several lines of evidence suggest that TGase may be involved in the
pathophysiology of HD. It was reported firstly by Cariello and colleagues (Cariello et
al., 1996) that TGase activity is above maximum control levels in some HD patients
and is correlated with the CAG repeat length. In vitro and in cell culture, htt is an
excellent substrate for TGase (Gentile et al., 1998, Kahlem et al., 1998, Karpuj et al.,
1999). The rate of the cross-linking reaction increases over an order of magnitude
with repeat lengths of over 40 glutamines. Additionally, TGase-catalyzed
cross-linking of mutant htt resulted in the formation of htt polymers (Kahlem et al.,
1998). TGase mRNA, protein levels, and activity are selectively elevated in HD
affected brain regions (Karpuj et al., 1999, Lesort et al., 1999, Lesort et al., 2002,
Zainelli et al., 2003).
TGase has become a therapeutic target for HD in both preclinical experiments
and clinical trials. Knocking-out TGase2 in an HD transgenic mouse model, R6/1
mice, results in improved motor performance and increases survival (Mastroberardino
et al., 2002). R6/2 mice treated with cystamine, a TGase inhibitor, showed extended
survival, improved body weight and motor performance and delayed
neuropathological sequela (Dedeoglu et al., 2002, Karpuj et al., 2002a). In the other
mouse model of HD, the YAC 128 mouse, treatment with cystamine ameliorates
striatal volume loss and decreases striatal neuronal atrophy (Van Raamsdonk et al.,
29
2005). Cysteamine, the dimer of cystamine, is an orphan drug approved for the
treatment of nephropathic cystinosis in humans. In 2008, researchers at Raptor
Pharmaceuticals Corp began with Phase II clinical trials of cysteamine for HD.
Distribution and functions htt protein
As a 350 kDa protein composed of 3,144 amino acids, htt is expressed
ubiquitously throughout the body. The highest concentrations are found in the brain
and testes, with moderate amounts in the liver, heart, and lungs (Walker, 2007). In the
brain, htt has the highest expression in the neocortex, cerebellar cortex, striatum, and
hippocampus (Fusco et al., 1999). Although htt is found primarily in the cytoplasm,
smaller amounts have also be detected in other cellular compartments, including the
nucleus (Hoogeveen et al., 1993, Kegel et al., 2002)
Several studies have revealed the role of wildtype htt in development. Complete
knockout of the mouse Htt gene is embryonic lethal between embryonic days 7.5 and
8.5 (Duyao et al., 1995, Nasir et al., 1995, Zeitlin et al., 1995). It was later found
that htt contributes to the formation of the nervous system. Mice carrying reduced
levels of wild-type htt display profound malformations of the cortex and striatum
(White et al., 1997). Several lines of evidence indicate that wild-type htt facilitates
neuronal survival as well. For example, overexpression of wildtype htt exhibits
neuroprotection against ischemic injury, excitotoxicity, and caspase activation
(Rigamonti et al., 2000, Zhang et al., 2003, Leavitt et al., 2006), where as disruption
of the mouse homologue of HTT gene results in a progressive degenerative neuronal
30
phenotype and sterility (O'Kusky et al., 1999, Dragatsis et al., 2000). Moreover, htt
may also participate in the regulation of apoptosis, control of BDNF production,
vesicular and mitochondrial transport, neuronal gene transcription, and synaptic
transmission (Cattaneo et al., 2005).
Similar to wildtype htt, mutant htt with expanded polyglutamine stretch is found
in the cytoplasm as well as the nucleus. In HD, cleavage of the full-length mutant htt
is critical for the formation of htt-containing aggregates which are a prominent
pathological feature in neurons in the cortex and striatum (DiFiglia et al., 1997, Sapp
et al., 1997). Htt has been defined as a caspase substrate with numerous sites for
specific caspase activity, such as amino acids 552 (caspase 2), amino acids 513, 552,
530, and 589 (caspase 3), and amino acid 586 (caspase 6) (Wellington et al., 1998,
Wellington et al., 2000). In HD and in transgenic mouse models, the activity of
caspases 3 and 9 is increased (Wellington et al., 2000, Zeron et al., 2004) and
inhibition of caspase 3, 6 or 9 activity decreases toxicity in models of HD (Wellington
et al., 2000, Tang et al., 2005).
While mutant htt is primarly cytosolic, the cleavage products of mutant htt,
N-terminal fragment with the polyglutamine stretch, can translocate into the nucleus
and mediate toxic functions of htt in HD (Hodgson et al., 1999, Graham et al., 2006).
Mutant htt can induce neurodegeneration by an apoptotic mechanism in a cell model
(Saudou et al., 1998). Proteasomal activity is decreased and the proteasome is unable
to process the polyglutamine repeat in htt-transfected cells and HD knock-in mouse
31
brains (Zhou et al., 2003, Venkatraman et al., 2004), suggesting that mutant htt may
disturb normal proteasome function. Also, mutant htt appears to interfere with other
cellular mechanisms such as gene transcription. It has been reported that mutant htt
causes decreased transcription of the brain-derived neurotrophic factor (BDNF) gene
(Zuccato et al., 2001), which is is necessary for survival of striatal neurons. cAMP
response element (CRE)-mediated transcription, which promotes the expression of
several pro-survival genes, are also impaired by mutant htt (Steffan et al., 2000,
Wyttenbach et al., 2001).
Although some evidence suggests that htt-associated toxicity is linked to soluble
htt and its protein–protein interactions (Petersen et al., 1999), it may also be correlated
with the formation of toxic aggregate species, which are found in the neuronal
intranuclear inclusions (NIIs) and cytoplasmic aggregates in the cortex and striatum
of HD brains (Davies et al., 1997, DiFiglia et al., 1997, Sapp et al., 1997).
Interestingly, htt-containing aggregates are predominately nuclear in juvenile HD
cases, while they are mainly localized to the neural processes in adult-onset cases
(DiFiglia et al., 1997).
CaM-regulated TGase-modification of htt
We previously reported that CaM associates with htt and TGase2 as
demonstrated by coimmunoprecipitation in transfeced cells and fluorescence
colocalization in intranuclear inclusions in HD brains (Zainelli et al., 2004). CaM has
been shown to increase TGase activity in different cells and tissues (Puszkin and
32
Raghuraman, 1985, Billett and Puszkin, 1991). Inhibition of CaM results in decreased
TGase-catalyzed modifications of htt in cells transfected with mutant htt and TGase2.
Using affinity purification, mutant htt with an expanded glutamine repeat binds to
CaM with a higher affinity than wild-type htt, suggesting in vivo interaction between
CaM and htt as well (Bao et al., 1996). These data, along with the observation that
TGase mRNA, protein, and activity are elevated in HD (Karpuj et al., 1999;Lesort et
al., 1999;Zainelli et al., 2003), and TGase2 colocalizes with both htt protein and
TGase-catalyzed cross-links in HD intranuclear inclusions (Zainelli et al., 2003),
implicate CaM and TGase in htt-aggregate formation in HD (Figure 2).
Hypothetically, interaction of mutant htt with both CaM and TGase results in an
increase in TGase-modified htt and consequent htt stabilization and aggregate
formation.
33
Figure 2. Proposed involvement of TGase and CaM in the formation of htt-containing
aggregates. When there are more than 35 glutamines (Gln) repeat in mutant htt,
protein conformation is changed. Some proteases such as caspase 3 and calpain can
cleave mutant htt and generate a toxic N-terminal htt fragment, which can be
transported to nucleus and neurites. We propose that N-terminal htt associated with
CaM at a specific binding domain, and htt were modified by TGase. This modification
may stabilize htt monomers or polymers and contribute to cytotoxicity and formation
of htt-containing aggregates.
34
CHAPTER TWO
TRANSGLUTAMINASE-CATALYZED TRANSAMIDATION: A NOVEL MECHANISM FOR RAC1 ACTIVATION BY 5-HT2A RECEPTOR
STIMULATION
(Published in J Pharmacol Exp Ther. 2008; 326(1):153-62)
ABSTRACT
Transglutaminase (TGase)-induced activation of small G proteins via 5-HT2A
receptor signaling leads to platelet aggregation (Walther et al., 2003). We hypothesize
that stimulation of 5-HT2A receptors in neurons activates TGase, resulting in
transamidation of serotonin to a small G protein, Rac1, thereby constitutively
activating Rac1. Using immunoprecipitation and immunoblotting, we show that in a
rat cortical cell line, A1A1v cells, serotonin increases TGase-catalyzed transamidation
of Rac1. This transamidation occurs in both undifferentiated and differentiated cells.
Treatment with a 5-HT2A/2C receptor agonist, 2,5-dimethoxy-4-iodoamphetamine
(DOI), but not the 5-HT1A receptor agonist, 5-hydroxy-2-dipropylamino tetralin
(DPAT), increases transamidation of Rac1 by TGase. In A1A1v cells, 5-HT2A
receptors mediate the transamidation reaction since expression of 5-HT2C receptors
was not detectable and the selective 5-HT2A receptor antagonist blocked
transamidation. Time course studies demonstrate that transamidation of Rac1 is
significantly elevated after 5 and 15 minutes of serotonin treatment, but returns to
35
control levels after 30 minutes. The activity of Rac1 is also transiently increased
following serotonin stimulation. Inhibition of TGase by cystamine or siRNA reduces
TGase-modification of Rac1 and cystamine also prevents Rac1 activation. Serotonin
itself is bound to Rac1 by TGase following 5-HT2A receptor stimulation as
demonstrated by co-immunoprecipitation experiments and a dose-dependent decrease
of serotonin-associated Rac1 by cystamine. These data support the hypothesis that
Rac1 activity is transiently increased due to TGase-catalyzed transamidation of
serotonin to Rac1 via stimulation of 5-HT2A receptors. Activation of Rac1 via TGase
is a novel effector and second messenger of the 5-HT2A receptor signaling cascade in
neurons.
INTRODUCTION
5-HT2A receptors are G-protein coupled receptors which are widely expressed
in the brain, peripheral vasculature, platelets and skeletal muscle. 5-HT2A receptors
are involved in diverse physiological functions, from platelet aggregation to
neuroendocrine release (Van de Kar et al., 2001, Walther et al., 2003).
Pathophysiologically, 5-HT2A receptor signaling is implicated in a number of
disorders including hypertension, atherosclerosis, anxiety and depression (Roth, 1994,
Weisstaub et al., 2006). The classical signal transduction pathway of 5-HT2A receptors
is Gq/11-coupled activation of phospholipase C (PLC) (Sanders-Bush et al., 2003),
which hydrolyzes phosphatidylinositol 4,5-bisphosphate to inositol
36
1,4,5-trisphosphate (IP3) and diacyglycerol. IP3 mobilizes calcium from the
endoplasmic reticulum and thereby increases intracellular Ca2+ (Julius, 1991).
TGases (EC 2.3.2.13) are a family of Ca2+-dependent enzymes. Once activated,
TGases can catalyze the cross-linking of proteins via the γ-carboxamide group of
peptide-bound glutamine and the є-amino group of peptide-bound lysine, forming an
inter- or intramolecular isodipeptide bond (Griffin et al., 2002). TGases can also
covalently link biogenic amines and polyamines, such as serotonin or spermine, to a
peptide-bound glutamine residue in a transamidation reaction (Folk et al., 1980, Dale
et al., 2002). In platelets, activation of 5-HT2A receptors stimulates TGase-catalyzed
transamidation of serotonin to small G proteins, such as RhoA and Rab4, rendering
them constitutively active (Walther et al., 2003). Furthermore, neuronal differentiation
of SH-SY5Y cells induced by retinoic acid increases the expression/activation of
TGases, resulting in transamidation of putrescine to RhoA and activation of RhoA
(Singh et al., 2003).
Rac1 belongs to the Rho family of small G proteins, a subgroup of the Ras
superfamily. Members of the Rho family (e.g. RhoA, Rac1, Cdc42) are associated
with a wide array of cellular processes such as cytoskeletal organization, vesicular
transport, cell cycle progression, cell adhesion and migration, neuronal differentiation
and a variety of enzymatic activities (Etienne-Manneville and Hall, 2002, Burridge
and Wennerberg, 2004). Like other small G proteins, Rac1 functions as a molecular
switch which cycles between two conformational states: a GTP-bound, active state
37
and a GDP bound, inactive state. The GTP/GDP cycling is controlled by many
regulator molecules such as GTPase-activating proteins (GAPs), guanine nucleotide
exchange factors (GEFs) and GDP dissociation inhibitors (GDIs) (Hakoshima et al.,
2003). Post-translation modification of Rac1 in the regulator-interaction sites or in the
GTP-hydrolyzing domain may have an enormous effect on its activation cycle.
Bearing five glutamine residues in the amino acid sequence (Matos et al., 2000), Rac1
might serve as a suitable substrate of TGases resulting in the transamidation of
primary amines to Rac1 and rendering this small G protein constitutively active.
A1A1v cells derived from embryonic rat cortex (Scalzitti et al., 1998) provide
an excellent experimental model to study 5-HT2A receptor signaling. In the present
study, we use A1A1v cells to test whether 5-HT2A receptor stimulation increases
TGase-catalyzed transamidation and activation of Rac1 since they endogenously
express 5-HT2A/1A receptors, Gαq/11, PLC β, TGase 2, the serotonin transporter, and
small G proteins including Rac1, RhoA and Cdc42.
MATERIALS AND METHODS
Cell Culture
A1A1v cells, a rat cortical cell line, were grown on 100 mm2 plates coated with
poly-L-ornithine (Sigma, St Louis, MO) and maintained in 5% CO2 at 33°C, in
Dulbecco’s modified Eagle medium (DMEM) (Fisher Scientific, Pittsburgh, PA)
containing 10% fetal bovine serum (FBS) (Fisher Scientific, Pittsburgh, PA). Cells
38
were induced to differentiate by incubation at 37°C for 4 days. Before each
experiment, cells were maintained in DMEM with 10% charcoal-treated FBS for 48
hours. Charcoal adsorption removes most but not all serotonin in the medium. The
maximal final concentration of serotonin in the medium is approximately 3 nM
(Unsworth and Molinoff, 1992). Cells from passages 8-15 were used for all
experiments.
Drugs
The following drugs were used in this study: (-)-1-(2,5-dimethoxy-4-
lodophenyl)-2-aminopropane HCl (DOI) and serotonin (Sigma, St Louis, MO).
(R)-(+)-8-hydroxy-2-dipropylamino tetralin hydrobromide (DPAT) (Tocris, Ellisville,
MO). 2-aminoethyl disulfide dihydrochloride (cystamine) (MP biomedicals, Costa
Mesa, CA). α-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenyl)ethyl]-4-piperidine
methanol (MDL 100907) (a gift from Hoechst Marion Roussel Research Institute,
Cincinnati, OH). Serotonin was dissolved in 10 µM HCl and MDL 100907 was
dissolved in a minimal volume of Dimethyl sulfoxide (DMSO) then diluted with
saline. The remaining drugs were dissolved in purified water. All compounds were
further diluted (at least 1:100) in cell culture media before they were applied to the
cells and washed away prior to lysing the cells.
Immunoprecipitation of TGase-modified Protein
A1A1v cells were harvested and lysed using lysis buffer A (25 mM Tris-HCl, pH 7.5,
250 mM NaCl, 5 mM EDTA, 1% Triton X-100 and 1:1000 protease inhibitor cocktail
39
(Sigma, St Louis, MO) containing 104 µM AEBSF, 0.08 µM aprotinin, 2 µM
leupeptin, 4 µM bestatin, 1.5 µM pepstatin A and 1.4 µM E-64). Protein
concentration was determined using the BCA Protein Assay kit (Pierce, Rockford, IL).
Immunopurification of proteins containing TGase-catalyzed bonds was performed
using 81D4 mAb (mouse IgM) prebound to Sepharose beads (Covalab, Lyon, France)
using a protocol developed by Covalab and as described previously (Norlund et al.,
1999, Zainelli et al., 2005). The 81D4 antibody is well characterized and has been
previously shown to be specific for the N epsilon-(gamma-L-glutamy)-L-lysine
isopeptide and N epsilon-(gamma-L-glutamy)-L-lysine isopeptide cross-link
generated by TGase (Sarvari et al., 2002, Thomas et al., 2004). The 81D4 antibody
has been extensively used to demonstrate increases in TGase-catalyzed bonds (Citron
et al., 2002, Junn et al., 2003, Andringa et al., 2004). The use of the 81D4 antibody in
a competitive ELISA assay was shown to result in isodipeptide cross-link
measurements that correlate well to those measured by HPLC analysis but provide
more sensitivity than the HPLC approach (Sarvari et al., 2002). Transfection of cells
to over-express TGases, including TGase 1, 2 and 3, and a substrate protein such as
mutant huntingtin protein results in increased presence of the isodipeptide cross-link
in mutant huntingtin protein as demonstrated with immunoprecipitation with the 81D4
antibody (Zainelli et al., 2005).
Briefly, 20µl of sepharose-81D4 beads were washed three times in TBS/0.1%
Tween 20 with gentle shaking for 15 minutes, followed by adding 200µg cell lysate (1
40
μg/μl) to the washed beads and incubating for 2 hours at 37°C. After incubation, the
pellets were washed four times in TBS/0.1% Tween 20 for 15 minutes. Then 20µl of
loading buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 0.1% bromophenol blue, 10%
glycerol and 5% β-mercaptoethanol) was added to the washed pellets followed by 5
minutes incubation at 90°C. The samples were then centrifuged at 9,000 × g for 2
minutes and the supernatant was transferred and stored at -80°C until immunoblot
analysis.
Immunoprecipitation of Rac1
200 µg of protein from each sample was brought up to a total volume of 100 µl with
IP buffer (50 mM Tris-HCl, pH 7.4, 10 mM EGTA, 100 mM NaCl, 0.5% Triton-X
100 and 0.1% protease inhibitor cocktail), then the samples were precleared using
10µl of recombinant protein G (rProtein G) agarose (Invitrogen, Carlsbad, CA) for 1
hour. The samples were centrifuged at 9,000 × g for 10 minutes and the supernatant
was incubated for 1.5 hour at 4ºC with either 2 µg of Rac1 antibody (Upstate, Lake
Placid, NY) or 2 µg of normal mouse IgG (Santa Cruz, Santa Cruz, CA) as a control
for non-specific binding. The immuno-complexes were precipitated using 20 µl of
rProtein G agarose at 4ºC for 1 hour. The agarose-immuno complexes were washed
three times in IP buffer and centrifuged after each wash at 100 × g for 3 minutes. After
the last wash, bound proteins were eluted by adding 2× PAGE sample buffer and
heating for 5 minutes at 90ºC. The samples were centrifuged at 13,000 × g for 5
minutes and the supernatant was transferred and stored at -80°C until immunoblot
41
analysis.
Immunoblot
Immunoaffinity purified proteins and cell lysates were separated on 12%
SDS-polyacrylamide gels and then electrophoretically transferred to nitrocellulose
membranes. Membranes were then incubated in blocking buffer (5% non-fat dry milk,
0.1% Tween 20, 1× TBS) for 1 hour at room temperature. Membranes were incubated
overnight at 4°C with primary antibodies on a shaker. Primary antibodies (Upstate
Biotechnology, NY: anti-Rac1, mouse IgG, 1:700; anti-Na+/K+ ATPase, mouse IgG, 1:
10000; Abcam, Cambridge, MA: anti-serotonin, rabbit IgG, 1:1000; BD Pharmingen,
San Jose, CA: anti-5-HT2C receptor, mouse IgG, 1:300; Cell Signaling, Danvers, MA:
anti-phosphorylated ERK1/2 at residues Thr202/Tyr204, mouse IgG, 1:1,000;
anti-ERK1/2, rabbit IgG, 1:1,000; MP Biomedicals, Aurora, OH: anti-actin, mouse
IgG, 1:20,000; CovalAb, Lyon, France: 81D4, mouse IgM, 1: 500; NeoMarkers,
Fremont, CA: TG100, mouse IgG, 1:100) were diluted in antibody buffer (1% non-fat
dry milk, 0.1% Tween 20, 1× TBS). The next day, membranes were washed with
TBS/0.1% Tween 20 and then incubated with goat-anti-mouse or goat-anti-rabbit
secondary antibody conjugated to HRP (Jackson ImmunoResearch, West Grove, PA)
diluted in antibody buffer. Membranes were washed and signal was detected using
enhanced chemiluminescence (ECL) western blotting detection reagents (Amersham
Biosciences, Piscataway, NJ). Using Scion Image for Windows (Scion, Frederick,
MD), immunoblots were quantified by calculating the integrated optical density (IOD)
42
of each protein band on the film.
Expression and Purification of Glutathione S-Transferase (GST)-PAK1
The pGEX-2T plasmid was used to express Rac1 interactive domain of human PAK1
(residues 51–135) fused to GST. The BL21 (DE3) E. coli (Invitrogen life technologies,
Carlsbad, CA) were transformed with plasmid expressing GST-PAK1 and grown
overnight at 37°C on LB-ampicillin plates. Then a single colony was used to inoculate
25ml LB-ampicillin (100 µg/ml) which was shaken overnight at 37°C. 10 ml of the
overnight culture was used to inoculate 500ml LB-ampicillin which was grown for 3
h at 37°C while shaking. After induction with 0.4 mM isopropyl
β-D-1-thiogalactopyranoside (IPTG) for 2 h at 30-32ºC, bacteria were lysed in lysis
buffer B (50 mM Hepes, pH 7.6, 1% Triton X-100, 100 mM NaCl, 5 mM MgCl2, 10%
glycerol, 1 mM PMSF and 0.1% protease inhibitor cocktail). The lysates were
sonicated and centrifuged for 15 minutes at 9,000 × g at 4ºC. Then the supernatant
was added to glutathione-Sepharose 4B (Amersham Biosciences, Piscataway, NJ) and
the mixture was incubated at 4ºC for 2 hours. The beads were then centrifuged at 4ºC
at 2,000 × g and were washed four times with lysis buffer B. The beads were
resuspended in lysis buffer B and were stored at -80ºC for at most two weeks.
Rac1 Activity Assay
A1A1v cells were treated for 5 or 15 minutes with 14 µM serotonin or vehicle (10 µM
HCl). The cells were harvested in lysis buffer C (50 mM Tris–HCl, pH 7.4, 100 mM
NaCl, 10 mM MgCl2, 1% Triton X-100, 0.1% SDS, 1 mM PMSF and 0.1% protease
43
inhibitor cocktail). Cell lysates were centrifuged at 14,500 × g for 20 minutes at 4°C
and 200 µl of the supernatant was incubated with 20 µl of GST-PAK1-Sepharose
beads for 40 minutes at 4°C under constant agitation. The beads were washed three
times with lysis buffer C, and 2× Laemmli sample buffer was added to the washed
beads followed by 5 minutes incubation at 90°C. The samples were then centrifuged
at 9,000 × g for 2 minutes and equivalent amounts of proteins in the supernatants
were loaded on 12% SDS-PAGE as well as 20 µg cell lysates (in order to detect total
Rac1 protein levels), followed by immunoblot analysis as described above.
Small Interfering RNA
To induce TGase2 gene silencing, two siRNA duplexes to target the coding sequence
of rat TGase2 mRNA were designed and synthesized by QIAGEN (Germantown,
MD). The target sequence is 5’-AAGAGCGAGATGATCTGGAAT-3’ for siRNA1
and is 5’- AGAGCCAACCACCTGAACAAA-3’ for siRNA2. The sense strand of
each siRNA was labeled at 3' end with Alexa Fluor 488 to monitor transfection
efficiency. At 30~50% confluence, A1A1v cells were transfected with siRNA at a
final concentration of 90 nM using Lipofectamine™ 2000 (Qiagen, Germantown, MD)
according to the manufacturer’s instructions. 24 hours after transfection, siRNA-lipid
complexes were removed by changing medium. Transfection efficiency was assessed
based on the percentage of fluorescent cells observed under Nikon Eclipse TE 2000U
microscopy. 48 hours after transfection, cells were stimulated with DOI or vehicle for
5 minutes, and then cell lysates were collected to measure TGase2 expression and
44
TGase-modified Rac1 by immunoblot and immunoprecipitation. Cells incubated with
Lipofectamine™ 2000 alone were used as the nontransfected control.
Statistical Analyses
All data are presented as group mean ± the standard error of the mean (SEM) and
analyzed by one-way or two-way ANOVA. Post hoc tests were conducted using
Newman-Keuls Multiple Comparison Test. SYSTAT 11 (Systat Software, Inc., San
Jose, CA) and GraphPad Prism 5.0 (GraphPad Software, Inc., San Diego, CA) were
used for all statistical analyses. A probability level of p<0.05 was considered to be
statistically significant for all statistical tests.
RESULTS
Serotonin treatment induces Rac1 transamidation in A1A1v cells
In order to determine whether TGase-catalyzed transamidation of Rac1 is increased
after serotonin treatment, we treated A1A1v cells with 14 µM serotonin for 5, 15 or
30 minutes. The transamidation of Rac1 is significantly elevated after 5 or 15 minutes
of serotonin treatment by approximately 2~2.5 fold as compared to vehicle
(HCl)-treated cells (Fig. 3). However, there is no significant difference in the amount
of TGase-modified Rac1 in cells treated with serotonin for 30 minutes as compared to
vehicle-treated cells (Fig. 3A, B).
5-HT2A receptor stimulation increases the transamidation of Rac1
In order to explore the serotonin receptor specificity for induction of TGase-catalyzed
45
transamidation, A1A1v cells were stimulated with 14 µM serotonin, 3 µM DOI
(5-HT2A/2C receptor agonist), 10 nM DPAT (5-HT1A receptor agonist) or 10 µM HCl
(vehicle) for 15 minutes. Treatment with serotonin or DOI significantly (p<0.05)
increases the amount of TGase-modified Rac1 2-fold as compared to vehicle-treated
cells, whereas treatment with DPAT has no effect on the amount of TGase-modified
Rac1 compared to vehicle-treated cells (Fig. 4A, B). Expression of 5-HT2A receptors
in A1A1v cells has been previously confirmed using anti-sense oligodeoxynucleotide
strategies and radioligand binding assays (Scalzitti et al., 1998). Here, 5-HT2C
receptor expression in undifferentiated and differentiated A1A1v cells was examined
by immunoblot analysis. Choroid plexus tissue from the fourth ventricle of a rat was
used as a positive control and HEK 293 cell lysates were used as a negative control.
We found that 5-HT2C receptors are expressed in rat choroid plexus, but 5-HT2C
receptor expression was not detected in either HEK293 cells or undifferentiated or
differentiated A1A1v cells (Fig. 4C). To exclude the possibility that the lack of
involvement of the 5-HT1A receptors in Rac1 transamidation is not due to insufficient
concentration of DPAT, we treated cells with 14 µM serotonin and increasing
concentrations of DPAT (1 nM, 10 nM, and 100 nM) for 5 minutes, and then detected
ERK phosphorylation by immunoblot. Treatment with 14 µM serotonin and 10 nM or
100 nM DPAT significantly increased phosphorylation of ERK (Fig. 4D, E),
indicating 10 nM DPAT is enough to activate 5-HT1A receptors in A1A1v cells. To
further confirm that the effect of DOI on Rac1 transamidation in A1A1v cells is due
46
to stimulation of the 5-HT2A receptors, cells were pretreated with 100 nM MDL
100907 (a selective 5-HT2A receptor antagonist) for 15 minutes, and then stimulated
with DOI for 5 minutes. We found the pretreatment of MDL 100907 significantly
reduced DOI-induced Rac1 transamidation without itself having any effect on Rac1
transamidation (Fig. 4F).
DOI increases Rac1 transamidation in both undifferentiated and differentiated
A1A1v cells
After differentiation of A1A1v cells, the cytoskeleton undergoes a dramatic
reorganization and the cells acquire a neuronal-like cell shape with long processes
similar to axons and dendrites compared to undifferentiated cells (Fig. 5A). Rho
family GTPases are critical regulators of the actin cytoskeleton organization
(Etienne-Manneville and Hall, 2002, Burridge and Wennerberg, 2004). This prompted
us to explore the effects of cell differentiation on Rac1 transamidation stimulated by
5-HT2A receptor activation. Stimulation of 5-HT2A receptors with DOI increased
TGase-catalyzed transamidation of Rac1 in both undifferentiated and differentiated
cells (Fig. 5B). Treatment with DOI in A1A1v cells significantly increased the amount
of TGase-modified Rac1 by approximately 2~2.5 fold as compared to vehicle-treated
cells (p<0.01). In vehicle-treated groups, the amount of TGase-modified Rac1 in
differentiated cells was similar to the amount in undifferentiated cells, indicating that
cell differentiation has no effect on basal level of Rac1 transamidation. Although the
amount of TGase-modified Rac1 in DOI-treated differentiated cells was increased
47
compared to DOI-treated undifferentiated cells, this increase was not significant,
suggesting that cell differentiation also does not have a significant effect on
DOI-induced transamidation of Rac1 (Fig. 5C).
The activity of Rac1 is transiently increased following serotonin stimulation
Racl is GDP-bound in the inactive state and GTP-bound when activated. Activated
Rac1 binds to its downstream effectors such as PAK1, therefore a GST-PAK1 fusion
protein purified from E. coli was used to determine if serotonin stimulation increases
the activated from of Rac1. A1A1v cells were treated with serotonin for 5 or 15
minutes, the cells were harvested and cell lysates were incubated with GST-PAK1
prebound to glutathione-Sepharose beads. Then equivalent amounts of the purified
proteins were resolved by SDS-PAGE. Immunoblot analysis was performed using an
antibody against Rac1. As shown in Fig. 6, the activity of Rac1 is increased by 80
percent after 5 minutes of serotonin treatment, but returns back to baseline after 15
minutes in the presence of serotonin, indicating that Rac1 becomes transiently
activated after serotonin treatment in A1A1v cells. In addition, there is no significant
change in the total amount of Rac1 in the cell lysate after serotonin treatment (Fig.
6A), suggesting that the activity increase is not due to synthesis of new Rac1.
TGase inhibition reduces Rac1 transamidation and activity
The activity of Rac1 is under the direct control of a large set of regulatory proteins. To
exam whether the serotonin-stimulated increase of Rac1 activity is due to
TGase-catalyzed transamidation, A1A1v cells were treated with increasing
48
concentrations (0 µM, 100 µM, 500 µM and 1000 µM) of the TGase inhibitor,
cystamine, for 1 h, followed by treatment with 14 µM serotonin for 5 minutes.
Precipitation of TGase-modified proteins and the activated Rac1 were performed as in
previous experiments. Then immunopurified proteins and activated Rac1 were
examined on immunoblots using anti-Rac1 and 81D4 antibodies. We found that
pretreatment with cystamine significantly decreases Rac1 transamidation (Fig. 7) and
activity (Fig. 8) in a dose-dependent manner. Treatment with 100 µM, 500 µM or
1000 µM of cystamine decreases the amount of TGase-modified Rac1 by
approximately 50%, 70% or 80%, respectively, as compared to untreated cells (Fig.
7A, C). A similar dose-dependent decrease in TGase-modified Rac1 was also
observed when cells were treated with serotonin for 15 minutes following cystamine
inhibition (data not shown). Treatment with 500 µM or 1000 µM of cystamine
decreases the amount of activated Rac1 by approximately 20% or 40%, respectively,
as compared to untreated cells (Fig. 8A, C). These results indicate that
TGase-catalyzed transamidation of Rac1 contributes to the increase in Rac1 activity
upon serotonin stimulation. The ubiquitously expressed Na+/K+ ATPase is a
well-established plasma membrane marker and its function underlies essentially all of
mammalian cell physiology (Kaplan, 2002). In order to verify that the dose-dependent
decrease of Rac1 transamidation and activation was not due to cystamine-induced
total Rac1 reduction or cellular toxicity, cell lysates from the same experiment were
examined on western blots with antibodies for Rac1 or Na+/K+ ATPase. There is no
49
significant difference in total Rac1 or Na+/K+ ATPase levels between
cystamine-treated and untreated cells, indicating that the concentrations of cystamine
were not toxic to the cells (Fig. 7B, 8B).
Next, a second and more specific approach was used to determine the
significance of TGase in Rac1 transamidation; we designed two siRNA duplexes to
inhibit endogenous TGase expression. Several isoenzymes of TGase are found in the
brain, of which TGase2 is the most abundant, therefore siRNAs were developed to
silence rat TGase2 gene. At 24 hours post-transfection, transfection efficiency of both
siRNA reached about 90%. 48 hours after transfection, siRNA1 and siRNA2
transfection of A1A1v cells resulted in 95% and 65% down-regulation of TGase2
protein expression, respectively (data not shown). Reprobing of membrane with
anti-Rac1 antibody revealed that neither siRNA changed Rac1 protein level,
indicating no off-target effect on Rac1 (Fig. 9A). DOI induced TGase-modification of
Rac1 was significantly reduced by knocking down TGase2 expression with siRNA1
or siRNA2. As compared to DOI-stimulated nontransfected cells, siRNA1 and
siRNA2 transfection caused 70% and 30% decreases in TGase-modified Rac1
responding to DOI, respectively (Fig. 9B, C). These results confirm that 5-HT2A
receptor-mediated Rac1 transamidation is dependent on TGase2 expression in A1A1v
cells.
TGase-mediated transamidation of serotonin to Rac1
TGase-modified Rac1 did not show a significant upward shift on immunoblots
50
compared to native Rac1 in cell lysates (data not shown), indicating that the molecular
weight of Rac1 does not significantly increase after modification by TGase. Therefore,
a small amine such as serotonin is most likely incorporated into Rac1 upon
stimulation of 5-HT2A receptors. To test this hypothesis, we stimulated A1A1v cells
with serotonin or vehicle for 5 minutes then cells were harvested and cell lysates were
used to immunoprecipitate Rac1 with an anti-Rac1 antibody, or immunoprecipitate
TGase-modified proteins with the 81D4 antibody. The immunopurified proteins were
examined on immunoblots using antibodies directed against serotonin.
Immunoprecipitation of Rac1 and probing for serotonin reveals an association
between Rac1 and serotonin in A1A1v cells (Fig. 10A). However we were unable to
detect serotonin in TGase-modified proteins (Fig. 10A). This may be due to a
compromise of the serotonin antibody epitope during the immunoprecipitation of
TGase-modified proteins, since this procedure uses higher temperatures and longer
incubation times compared to the Rac1 immunoprecipitation procedure. Therefore in
order to confirm that serotonin is associated with Rac1 by a TGase-catalyzed covalent
bond, we treated cells with increasing concentrations (0 μM, 100 μM, 500 μM, 1000
μM) of cystamine for 1 hour followed by stimulating the cells with serotonin for 5
minutes. Then we immunoprecipitated Rac1 and detected associated serotonin by
immunoblot, as described above. We found that treatment with cystamine decreases
the serotonin-associated Rac1 in a dose-dependent manner (Fig. 10B, D), thus
supporting the hypothesis that serotonin is incorporated into Rac1 by a
51
TGase-catalyzed covalent bond. To compare the effects of different agonists on the
incorporated serotonin, cells were exposed to either serotonin or DOI for 5 min. Then
Rac1 was immunoprecipitated and the incorporated serotonin was detected by
immunoblot. There was no significant difference in serotonin incorporation between
serotonin and DOI-treated cells. Pretreatment with cystamine also inhibited
DOI-induced serotonin incorporation (Fig. 11).
52
Fig.3 Serotonin-induced increase of Rac1 transamidation in A1A1v cells. (A) After 5,
15 or 30 minutes of 14 µM serotonin treatment, TGase-modified proteins were
immunoprecipitated with 81D4 antibody coupled to sepharose. TGase-modified Rac1
was then detected on immunoblots with anti-Rac1 antibody. (B) Quantitation of
immunoblots for 3 separate experiments. Data shown are the mean IOD ± SEM and
normalized to vehicle-treated control levels. One-way ANOVA (F(3,8)=7.31, p<0.05)
indicates a significant difference among groups. Newman-Keuls Multiple Comparison
Test indicates p<0.05* as compared to vehicle treatment.
53
Fig. 4 Serotonin receptor specificity on induction of Rac1 transamidation in A1A1v
cells. (A) Cells were stimulated with 14 µM serotonin, 3 µM DOI (5-HT2A/2C receptor
agonist), 10 nM DPAT (5-HT1A receptor agonist) or vehicle for 15 minutes.
TGase-modified Rac1 was detected by immunoprecipitation and immunoblot analysis.
(B) Quantitation of immunoblots for 3 separate experiments. Data shown are the
mean IOD ± SEM and normalized to vehicle-treated control levels. One-way ANOVA
(F(3,8)=10.05, p<0.01) indicates a significant difference among various treatments.
Newman-Keuls Multiple Comparison Test indicates p<0.05* as compared to vehicle
treatment. (C) Immunoblot analysis of 5-HT2C receptor expression in undifferentiated
(UD) and differentiated (DI) A1A1v cells. Rat choroid plexus was used as a positive
54
control and HEK 293 cell was used as a negative control. (D) A1A1v cells were
treated with vehicle, 14 µM serotonin or DPAT (1 nM to 100 nM) for 5 minutes. Cell
lysates were then resolved by SDS-PAGE, followed by immunoblotting with an
anti-phosphorylated ERK antibody. Equal loading was verified by reprobing the same
membrane with antibodies to total ERK and actin. (E) Quantitation of immunoblots
for 3 separate experiments. Data shown are the mean IOD ± SEM and normalized to
serotonin-treated control levels. Each phospho-ERK value was normalized using the
corresponding total ERK value to control for the possibility of differential loading.
One-way ANOVA (F(4,10)=18.12, p<0.001) indicates a significant difference among
various treatments. Newman-Keuls Multiple Comparison Test *indicates p<0.05 as
compared to vehicle treatment and #indicates p<0.05 as compared to serotonin
treatment. (F) Upper, cells were stimulated with vehicle, 14 µM serotonin, 3 µM DOI.
Some cells were pretreated with 100 nM MDL 100907 prior to treatment with DOI or
treated with MDL100907 alone. TGase-modified Rac1 was detected by
immunoprecipitation and immunoblot analysis. Lower, quantitation of immunoblots
for 3 separate experiments. Data shown are the mean IOD ± SEM and normalized to
vehicle-treated control levels. One-way ANOVA (F(4,10)=38.96, p<0.001) indicates a
significant difference among various treatments. Newman-Keuls Multiple
Comparison Test indicates p<0.05* as compared to vehicle treatment and p<0.05# as
compared to DOI treatment.
55
Fig. 5 The effects of cell differentiation on Rac1 transamidation. (A) A1A1v cells
were incubated at 37°C for 4 days to induce differentiation. Compared to
undifferentiated cells (left), differentiated cells (right) develop a neuron-like cell
shape with long neurites. (B) Cells were treated with 3 µM DOI for 15 minutes,
followed by immunoprecipitation and immunoblot analysis. Stimulation of 5-HT2A
receptors by DOI increased the TGase-catalyzed transamidation of Rac1 in both
undifferentiated and differentiated cells. (C) Quantitation of immunoblots for 3
separate experiments. Data shown are the mean IOD ± SEM and normalized to
vehicle-treated control levels in undifferentiated cells. Two-way ANOVA indicates a
56
significant main effect of DOI treatment (F(1,8)=49.48, p<0.01). However, there was
no significant main effect of cell differentiation on TGase-modified Rac1 (F(1,8)=4.07,
p=0.08). The interaction between DOI treatment and cell differentiation was also not
significant for TGase-modified Rac1 (F(1,8)=0.34, p=0.58). Newman-Keuls
Multiple Comparison Test indicates p<0.01* as compared to vehicle treatment in
undifferentiated cells, p<0.01# as compared to vehicle treatment in differentiated cells.
Scale bar, 105 µm.
57
Fig. 6 Rac1 activity was transiently increased after serotonin treatment. (A) Top ,
activated Rac1 was purified using GST-PAK1 coupled to glutathione-Sepharose beads
and detected on immunoblot with an antibody against Rac1. Middle, the total amount
of Rac1 in the cell lysates used for pull-down of activated Rac1 was monitored by
immunoblot. Bottom, Ponceau staining of GST-PAK1 documents equal amounts of
protein loading in each lane. (B) Quantitation of immunoblot for 3 separate
experiments. Data shown are the mean IOD ± SEM and normalized to vehicle-treated
control levels. The two-way ANOVA reveals a significant (F(1,8)=8.64, p<0.05) main
effect for serotonin treatment, and a significant (F(1,8)=6.32, p<0.05) interaction
between serotonin treatment and time course. Newman-Keuls Multiple Comparison
Test indicates p<0.05* as compared to 5 minutes of serotonin treatment.
58
Fig.7 Dose-dependent effects of cystamine on Rac1 transamidation. (A)
Immunoprecipitation and immunoblot analyses reveal that cystamine causes a
dose-dependent (100 to 1000 µM) inhibition of the transamidation of Rac1 as
indicated by less intense bands. (B) Cells lysates from the same experiment were
examined on immunoblots with antibodies for Rac1 and Na+/K+ ATPase, which
indicate that cystamine has no effect of on total Rac1 and cell viability, respectively.
(C) Quantitation of effects of cystamine on Rac1 transamidation in 3 separate
experiments. Data shown are the mean IOD ± SEM and normalized to untreated
control levels. One-way ANOVA (F(3,8)=19.41, p<0.001) indicates a significant effect
of cystamine on Rac1 transamidation. Newman-Keuls Multiple Comparison Test
indicates p<0.05* as compared to untreated cells.
59
Fig.8 Rac1 activity is inhibited by cystamine. (A) Top , pull-down and immunoblot
analyses reveal that cystamine causes a dose-dependent (100 to 1000 µM) inhibition
of the activation of Rac1. Middle, the same membrane was reprobed with antibody
81D4, directed against TGase-catalyzed covalent bonds. Bottom, Ponceau staining of
GST-PAK1 (~35 kDa) documents equal amounts of protein loading in each lane. (B)
Cell lysates from the same experiment were examined on immunoblots with an
antibody for Na+/K+ ATPase. (C) Quantitation of the effects of cystamine on Rac1
activation in 3 separate experiments. Data shown are the mean IOD ± SEM and
normalized to untreated control levels. One-way ANOVA (F(3,8)=20.94, p<0.001)
60
indicates a significant effect of cystamine on Rac1 activity. Newman-Keuls Multiple
Comparison Test indicates p<0.05* as compared to untreated cells, p<0.001** as
compared to untreated cells.
61
Fig.9 Knockdown of TGase2 by siRNAs prevents Rac1 transamidation. (A) Cells
were incubated with 90nM of TGase2-specific siRNAs for 24 hours, and then treated
with 3 µM of DOI or vehicle for 5 minutes at 48 hours post-transfection. Immunoblot
analyses confirmed that transfection of siRNA1 or siRNA2 significantly inhibited
TGase2 protein expression compared to nontransfected control (NT). The membranes
were stripped and reprobed with anti-Rac1 and anti-actin antibodies. (B)
TGase-modified Rac1 was detected by immunoprecipitation and immunoblot analysis.
DOI induced TGase-modification of Rac1 were significantly decreased in
siRNAs-transfected cells compared to NT cells. (C) Quantitation of effects of siRNAs
62
on Rac1 transamidation in 3 separate experiments. Data shown are the mean IOD ±
SEM and normalized to DOI-stimulated nontransfected cells. Two-way ANOVA
indicates a significant main effect of transfection (F(2,12)=24.68, p<0.0001), a
significant main effect of DOI treatment (F(1,12)=63.23, p<0.0001) and a significant
interaction between transfection and DOI treatment (F(2,12)=9.42, p<0.01) on
TGase-modified Rac1. Newman-Keuls Multiple Comparison Test indicates p<0.01*
or p<0.001** as compared to vehicle treatment in nontransfected cells, p<0.01# or
p<0.001## as compared to DOI treatment in nontransfected cells.
63
Fig.10 Transamidation of serotonin to Rac1 is mediated by TGase. (A)
Immunoprecipitation of Rac1, but not TGase-modified proteins, reveals an
association between Rac1 and serotonin in A1A1v cells upon 5 minutes of 14 µM
serotonin stimulation. (B) Immunoprecipitation and immunoblot analysis reveal that
cystamine causes a dose-dependent (100 to 1000 µM) reduction of the
serotonin-associated Rac1. Normal mouse IgG is used as a negative control (NC) for
immunoprecipitation. (C) Cell lysates from the same experiment were examined on
immunoblots with an antibody for Na+/K+ ATPase, which indicates cystamine has no
effect of on cell viability. (D) Quantitation of the effects of cystamine on
64
serotonin-induced transamidation of Rac1. Data shown are the mean IOD ± SEM and
normalized to untreated control levels. One-way ANOVA (F(3,8) = 7.15, p<0.05)
indicate a significant difference in serotonin-associated Rac1 among cells treated with
different concentration of cystamine. Newman-Keuls Multiple Comparison Test
indicates p<0.05* as compared to untreated cells.
65
Fig.11 DOI stimulates transamidation of Rac1 to serotonin to a similar extent as those
treated with serotonin. (A) Cells were stimulated with vehicle, 14 µM 5-HT or 3 µM
DOI for 5 minutes. Some cells were pretreated with 500 µM cystamine (Cys) for 1
hour prior to stimulation with DOI. Transamidation of serotonin to Rac1 was detected
by immunoprecipitation and immunoblot analysis. (B) Quantitation of immunoblot
for 3 separate experiements. Data shown are the mean IOD ± SEM and normalized to
serotonin-treated levels. One-way ANOVA (F(3,8)=19.80, p<0.001) indicates a
significant difference in Rac1-incorporated serotonin among groups. Newman-Keuls
Multiple Comparison Test * indicates p<0.05 as compared to vehicle-treated cells and
# indicates p<0.05 as compared to serotonin-treated cells.
66
DISCUSSION
In platelets, 5-HT2A receptor activation causes TGase-catalyzed
transamidation of RhoA and Rab4, leading to activation of these proteins and platelet
aggregation (Walther et al., 2003). In aplysia ganglia, serotonin-treatment induces the
activation of Cdc42 and its downstream effector PAK to regulate the actin
cytoskeleton (Udo et al., 2005). In the present study, we extend these findings to a rat
cortical cell model and find that 5-HT2A receptor stimulation increased
TGase-catalyzed transamidation of Rac1 and Rac1 activity, both of which can be
suppressed by TGase inhibition. In elucidating the mechanism further, we identify
serotonin as an amine that becomes transamidated to Rac1 by TGase. Activation of
Rac1 via TGase is a novel effector and second messenger of the 5-HT2A receptor
signaling pathway in neurons.
To exam the serotonin receptor specificity on induction of Rac1
transamidation, we treated cells with different serotonin receptor agonists and found
that the 5-HT2A/2C receptor agonist DOI, but not the 5-HT1A receptor agonist DPAT
induces a significant increase in TGase-modified Rac1 with a similar magnitude to
that observed in serotonin-treated cells. Since there is no 5-HT2C receptor expression
in A1A1v cells and MDL 100907 reversed the effect of DOI on Rac1 transamidation
(Fig. 4), serotonin or DOI-induced Rac1 transamidation is selectively mediated by
activation of 5-HT2A receptors in A1A1v cells.
Previous studies demonstrated that TGase-mediated transamidation of the Rho
67
family of small G proteins blocked the GTP-hydrolyzing activity of these proteins,
rendering these small GTPases constitutively active for their respective signaling
pathways (Masuda et al., 2000, Walther et al., 2003). Based on these results, we
hypothesized that 5-HT2A receptor-stimulated transamidation of Rac1 by TGase will
result in prolonged activation of Rac1. Unexpectedly, we found a transient increase in
both Rac1 activity and TGase-catalyzed transamidation which returned to baseline
levels following continuous exposure to serotonin (Figs. 3 and 6). The transient
response may due to selective degradation of transamidated Rac1. Mammalian cells
contain multiple proteolytic systems for degradation of various classes of intracellular
proteins. Most abnormal proteins (mutant or misfolded) are degraded in the
ubiquitin–proteasome pathway. Rac1 is normally a stable protein, but activation by
cytotoxic necrotizing factor-1 (CNF-1) in rat bladder carcinoma cells (804G) and
human umbilical vein endothelial cells resulted in increased sensitization to
ubiquitination and proteasomal degradation (Doye et al., 2002, Munro et al., 2004).
Further, dominant-positive forms of Rac1 appeared more susceptible to
ubiquitin-mediated proteasomal degradation compared to both dominant-negative and
wild-type Rac1(Doye et al., 2002). Activated and transamidated Rac1 may be targeted
for proteasomal degradation, resulting in transient activation of Rac1. However, there
is no significant change in total Rac1 protein 15 min after serotonin stimulation (Fig.
4A), suggesting that activated or transamidated Rac1 may only account for small
fraction of total Rac1.
68
In this study, we found differences in the time course for Rac1 transamidation
and for its increased activity. After serotonin stimulation for 15 minutes, Rac1 activity
is reduced to baseline (Fig. 6) while TGase-catalyzed transamidation of Rac1 still
remains at a relatively high level (Fig. 3). These results suggest that transamidation
may not only occur at the GTPase activity-related residues, but also at residues which
do not have an effect on Rac1 activity. Essentially, transamidation of Rac1 at some
sites may induce activation of the small G proteins, while transamidation at other sites
may not affect the GTPase activity of Rac1. If activated Rac1 is more sensitive to
proteasomal degradation, the activated Rac1 may be degraded earlier than
non-activated Rac1 that is transamidated at sites not involved in activation.
Cystamine has been shown to decrease TGase activity and TGase-catalyzed
є-(γ-glutamyl) lysine bonds in cultured cells and animal models of neurodegenerative
diseases (Karpuj et al., 2002a, Ientile et al., 2003, Zainelli et al., 2005). As a primary
amine, cystamine is a potential substrate for TGase, and acts as a competitive
inhibitor for TGase by blocking access to the active site of the enzyme for the
glutamine residues in proteins which would otherwise participate in forming
є-(γ-glutamyl) lysine bonds (Lorand et al., 1979). In our study, pretreatment with
cystamine reduced both serotonin-stimulated Rac1 transamidation (Fig. 7A, C) and
activation (Fig. 8A, C) in a dose-dependent manner. Those observations were further
supported by the use of TGase2-specific siRNAs, which significantly suppressed DOI
induced TGase-modification of Rac1 (Fig. 9B, C). These results suggest that
69
cystamine prevented Rac1 transamidation through TGase inhibition, and that
transamidation of Rac1 by TGase may contribute to the increase in Rac1 activity.
Post-translational modifications, such as transamidation and phosphorylation
of Rac1 may affect its ability to interact with regulatory proteins (e.g. GAPs, GEFs
and GDIs) or to convert GTP back to GDP, resulting in increased activation of Rac1
and stimulation of its signaling cascade in the cells. The Conserved Domain Database
(CDD) (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=cdd) provides an
interactive tool to identify conserved domains present in protein sequences. We
searched the CDD for GTP/Mg2+ binding sites, and GAPs, GEFs and GDIs interaction
sites in the Rac1 sequence (PSSM-Id: 57957) because they will be most likely to
modify Rac1 activity after TGase-catalyzed transamidation at these sites. We
identified two glutamine residues (Gln61, Gln74) located within these domains. It has
been reported that site-specific deamidation of a Gln residue in the Rho family G
proteins (Gln61 in Rac and Cdc42, Gln63 in RhoA) by CNF-1 inhibits both intrinsic
and GAPs-stimulated GTP hydrolysis activity, resulting in constitutive activation of
these proteins (Flatau et al., 1997, Schmidt et al., 1997). CNF-1 has also been shown
to possess in vitro TGase activity. In the presence of primary amines, RhoA is
transamidated in vitro at Gln63 by CNF-1 and at positions 52, 63 and 136 by guinea
pig liver TGase (Schmidt et al., 1998). Similarly, in addition to Gln61 which is critical
in regulating Rac1 activity, the other four glutamine residues in Rac1 may also be
modified by TGase. It is not surprising, therefore, that the same dosage of cystamine
70
prevents Rac1 transamidation and its activation to different extents (Fig. 7C, 8C).
Walther et al. found that in platelets TGase covalently cross-links serotonin to
RhoA or Rab4 at a position in the phosphate-binding site that is conserved in the
sequences of all Ras-related small GTPases (Walther et al., 2003). It has also been
shown that polyamines such as putrescine, spermidine and spermine could be
incorporated into Rac1 by bacterial TGase in vitro (Masuda et al., 2000). In our study,
co-immunoprecipitation assays demonstrated that serotonin is associated with Rac1 in
A1A1v cells after serotonin stimulation (Fig. 10A). To explore the association
mechanism further, we inhibited TGase by cystamine and found a reduction of
serotonin-associated Rac1 with increasing concentrations of cystamine (Fig. 10B, D),
indicating that serotonin is incorporated into Rac1 by a TGase-catalyzed bond. Since
both serotonin and DOI stimulation can lead to serotonin incorporation to similar
levels (Fig. 11), the serotonin bound to 5-HT2A receptors is not likely the source of
Rac1-incorporated serotonin; the serotonin bound to Rac1 may originate from
endogenously synthesized serotonin or serotonin transported into the cell from serum
in the cell culture media.
The first signal transduction mechanism identified for the 5-HT2A receptor was
Gq/11 mediated activation of PLC, leading to increased accumulation of IP3 and an
increase in intracellular Ca2+ (Boess and Martin, 1994). In addition to activation of
PLC, extensive evidence suggests that 5-HT2A receptors couple to other effector
pathways, such as phospholipase A2/arachidonic acid cascade (Berg et al., 1994b),
71
Mitogen-Activated Protein Kinase signaling (Watts, 1998) and phospholipase
D/protein kinase C pathway (Mitchell et al., 1998). On the other hand, TGases are
subjected to transcriptional regulation by retinoic acid and steroid hormones
(Fujimoto et al., 1996, Ou et al., 2000), and require the binding of Ca2+ for their
activity (Burgoyne and Weiss, 2001). Evidence indicates that TGase activity can be
significantly enhanced in response to increased intracellular Ca2+ through IP3
generation (Zhang et al., 1998). In aplysia, serotonin mediated activation of Cdc42
was dependent on PLC and phosphatidylinositol 3-kinase (PI3K) (Udo et al., 2005).
In RA-induced neuronal differentiation of SH-SY5Y cells, TGase-catalyzed
transamidation is required for activation of RhoA (Singh et al., 2003), whereas
activation of Rac1 is mediated by PI3K in a transamidation-independent manner (Pan
et al., 2005). Further studies are needed to elucidate the underlying molecular
mechanisms by which the 5-HT2A receptor signaling regulates TGase-catalyzed
activation of Rac1.
The present study provides the first evidence in neurons that stimulation of
5-HT2A receptors induces an increase in TGase-catalyzed transamidation of serotonin
into Rac1 and constitutive activation of Rac1. Further studies using alternative
approaches to measure Rac1 transamidation, such as mass spectroscopy, are needed to
confirm this hypothesis. Rac1 activation via 5-HT2A receptor stimulation may have an
important functional impact given the plethora of pathways that utilize this small G
protein. For example, Rac1 is best known as a regulator for the assembly of the actin
72
cytoskeleton, thereby playing a role in neurite outgrowth and neuronal differentiation.
We demonstrate that stimulation of 5-HT2A receptor can increase the transamidation
of Rac1 in both undifferentiated and differentiated A1A1v cells, suggesting that
transamidation and constitutive activation of this signal transducer are not altered by
neuronal differentiation of cells. However, the effect of 5-HT2A receptor-mediated
activation of Rac1 on cytoskeleton organization, cell cycle progression,
transcriptional activation or other crucial cellular functions in neurons has yet to be
explored.
34
CHAPTER THREE:
PHOSPHOLIPASE C, CALCIUM AND CALMODULIN SIGNALING ARE
REQUIRED FOR 5-HT2A RECEPTOR-MEDIATED TRANSAMIDATION OF
RAC1 BY TRANSGLUTAMINASE
ABSTRACT
Increasing evidence indicates that small G proteins undergo transglutaminase
(TGase)-catalyzed transamindation and activation in neurons, platelets, and smooth
muscle cells. We have previously reported that 5-HT2A receptor stimulation increased
TGase-catalyzed transamidation and activation of Rac1 small G protein in A1A1v
cells, a rat embryonic cortical cell line. In this study, we explore the signaling
pathways involved in 5-HT2A receptor-mediated Rac1 transamidation. In cells
pretreated with phospholipase C (PLC) inhibitor U73122, the 5-HT2A receptor
specific agonist, DOI-stimulated increase in intracellular Ca2+ concentration and
TGase-modified Rac1 were significantly attenuated as compared to those pretreated
with U73343, an inactive analog. Addition of the membrane-permeant Ca2+ chelator,
BAPTA-AM strongly reduced TGase-catalyzed Rac1 transamindation upon DOI
stimulation. Conversely, when cells were treated with Ca2+ ionophore, ionomycin at a
concentration that produced an elevation of cytosolic Ca2+ to a level comparable to
cells treated with DOI, ionomycin mediated an increase in TGase-modified Rac1
73
74
without 5-HT2A receptor activation. Moreover, calmodulin (CaM) inhibitor W-7,
significantly decreased Rac1 transamindation in a dose-dependent manner in
DOI-treated cells. To identify potential TGase-targeting residues that could render
Rac1 constitutively active upon transamidation, a search in NCBI Conserved Domain
Database was conducted, which revealed that two glutamine residues (Q61, Q74) and
three lysine residues (K5, K16, and K116) are located within activity-related domains.
Taken together, these results indicated that 5-HT2A receptor-coupled PLC activation
and subsequent Ca2+/CaM signaling are necessary in TGase-catalyzed Rac1
transamidation, and an increase in intracellular Ca2+ is sufficient to induce Rac1
transamination in A1A1 cells.
INTRODUCTION
Rac1 (Ras-related C3 botulinum toxin substrate 1), one of the most extensively
characterized members in the Rho family of small G proteins, was initially discovered
as a substrate for ADP-ribosylation induced by the C3 component of botulinum toxin
(Didsbury et al., 1989). Subsequently, Rac1 and its downstream effectors were
identified as key signaling molecules in various cell functions, such as cytoskeleton
reorganization, cell transformation, axonal guidance, and cell migration
(Etienne-Manneville and Hall, 2002, Bosco et al., 2009).
Like all members of the Rho superfamily, Rac1 functions as a molecular switch,
cycling between an inactive GDP-bound state and an active GTP-bound state. Rho
75
small G proteins can be activated by different signal transduction pathways initiated
by extracellular factors such as platelet-derived growth factor, insulin or epidermal
growth factor (Bishop and Hall, 2000). Serotonin has also been shown to induce
activation via transglutaminase (TGase)-catalyzed transamidation of small G proteins
in neurons, platelets, and aortic smooth muscle cells (Walther et al., 2003, Guilluy et
al., 2007, Dai et al., 2008). TGases catalyze the transamidation of protein-bound
glutamines and primary amines such as serotonin in a Ca2+-dependent manner
(Ahvazi et al., 2002). We previously reported that the effect of serotonin on Rac1
transamidation in A1A1v cells, a rat cortical cell line, is due to stimulation of the
5-HT2A receptors (Dai et al., 2008). However, the underlying molecular mechanisms
by which the 5-HT2A receptor signaling regulates TGase-catalyzed transamidation and
activation of Rac1 are still unclear.
5-HT2A receptor activation can increase IP3 via Gq/11-mediated activation of
phospholipase C (PLC) (Conn and Sanders-Bush, 1984, de Chaffoy de Courcelles et
al., 1985). PLC plays an important role in intracellular signal transduction by
hydrolyzing phosphatidylinositol 4,5-bisphosphate (PIP2), a membrane phospholipid,
and thereby generating two second messengers: inositol trisphosphate (IP3) which
diffuses through the cytosol and releases Ca2+ from intracellular endoplasmic
reticulum stores and diacylglycerol (DAG) (Boess and Martin, 1994). It has been
reported that TGase activity can be significantly enhanced in response to increased
intracellular Ca2+ through inositol 1,4,5-trisphosphate (IP3) generation (Zhang et al.,
76
1998). Moreover, serotonin-mediated activation of small G protein cdc42 was
dependent on PLC signaling pathways (Udo et al., 2005).
Calmodulin (CaM) has been shown to increase TGase enzymatic activity. In the
presence of CaM, a membrane-associated erythrocyte TGase is activated at
physiological and lower than physiological Ca2+ concentrations (Billett and Puszkin,
1991). Moreover, in the presence of Ca2+, CaM enhanced TGase activity 3-fold in
human platelets and the chicken gizzard (Puszkin and Raghuraman, 1985). A CaM
inhibitor prevented TGase-catalyzed cross-linking of huntingtin in cells co-transfected
with mutant huntingtin and TGase (Zainelli et al., 2004). Taken together, we
hypothesize that TGase-catalyzed Rac1 modification upon 5-HT2A receptor
stimulation is dependent on PLC-mediated increases in intracellular Ca2+ and is
regulated by CaM. To test this hypothesis, we examined the effects of PLC inhibition,
CaM inhibition, and manipulation of intracellular Ca2+ by means of a Ca2+ ionophore
or a chelating agent on TGase-modified Rac1 in response to 5-HT2A receptor
activation in A1A1v cells.
MATERIALS AND METHODS
Cell Culture
A1A1v cells, a rat cortical cell line, were grown on 100 mm2 plates coated with
poly-L-ornithine (Sigma, St Louis, MO) and maintained in 5% CO2 at 33°C, in
Dulbecco’s modified Eagle medium (DMEM) (Fisher Scientific, Pittsburgh, PA)
77
containing 10% fetal bovine serum (FBS) (Fisher Scientific, Pittsburgh, PA). Before
each experiment, cells were maintained in DMEM with 10% charcoal-treated FBS for
48 hours. Charcoal adsorption removes most but not all serotonin in the medium. The
maximal final concentration of serotonin in the medium is approximately 3 nM
(Unsworth and Molinoff, 1992). Cells from passages 8-15 were used for all
experiments.
Drugs
The following drugs were used in this study: (-)-1-(2,5-dimethoxy-4-lodophenyl)-
2-aminopropane HCl (DOI) (Sigma, St Louis, MO), U73122 (Tocris Bioscience
Ellisville, MO), U73343 (Sigma, St Louis, MO), BAPTA AM (Invitrogen, Carlsbad,
CA), ionomycin (Invitrogen, Carlsbad, CA), and W-7 hydrochloride (Tocris
Bioscience Ellisville, MO).
Measurement of intracellular Ca2+ concentration
Cells were grown to 90% confluence in black-sided 96-well plates (Fisher Scientific,
Pittsburgh, PA). Cells were washed twice with modified Kreb's medium (135 mM
NaCl, 5.9 mM KCl, 1.5 mM CaCl2, 1.2 mM MgCl2, 11.5 mM D-glucose, 11.6 mM
Hepes, pH 7.3) and then incubated in the same medium with 5 µM Fura-2 AM
(Molecular Probes, Carlsbad, CA), 0.1% bovine serum albumin, and 0.02% Pluronic
F127 detergent (Poenie et al., 1986) for 60 min at 33°C incubator in the dark. After
loading, the cells were washed twice and incubated in the dark in modified Kreb's
78
medium or pretreated with drugs for 30 min. Following 1 min of equilibration, the
cells were stimulated with a single injection of 3 µM DOI, and the response was
recorded until a plateau was reached (about 4 min). Fura-2 fluorescence (at 510 nm)
was measured with a BioTek fluorescence plate reader. After background fluorescence
was subtracted, the ratio of fluorescence at 340 nm excitation to that at 380 nm
excitation was calculated and used as an index of the intracellular Ca2+ concentration
(the 340/380 nm fluorescence ratio is positively correlated with the absolute values of
intracellular Ca2+ concentration).
Immunoprecipitation of TGase-modified Protein
A1A1v cells were harvested and lysed using lysis buffer A (25 mM Tris-HCl, pH 7.5,
250 mM NaCl, 5 mM EDTA, 1% Triton X-100 and 1:1000 protease inhibitor cocktail
(Sigma, St Louis, MO) containing 104 µM AEBSF, 0.08 µM aprotinin, 2 µM
leupeptin, 4 µM bestatin, 1.5 µM pepstatin A and 1.4 µM E-64). Protein
concentration was determined using the BCA Protein Assay kit (Pierce, Rockford, IL).
Immunopurification of proteins containing TGase-catalyzed bonds was performed
using 81D4 mAb (mouse IgM) prebound to Sepharose beads (Covalab, Lyon, France)
using a protocol developed by Covalab and as described previously (Norlund et al.,
1999, Zainelli et al., 2005). The 81D4 antibody has been extensively used to
demonstrate increases in TGase-catalyzed bonds (Citron et al., 2002, Junn et al., 2003,
Andringa et al., 2004). The use of the 81D4 antibody in a competitive ELISA assay
was shown to result in isodipeptide cross-link measurements that correlate well with
79
those measured by HPLC analysis but provide more sensitivity than the HPLC
approach (Sarvari et al., 2002). Transfection of cells to over-express TGases,
including TGase 1, 2 and 3, and a substrate protein such as mutant huntingtin protein
results in increased presence of the isodipeptide cross-link in mutant huntingtin
protein as demonstrated with immunoprecipitation with the 81D4 antibody (Zainelli et
al., 2005).
Briefly, 20µl of sepharose-81D4 beads were washed three times in TBS/0.1%
Tween 20 with gentle shaking for 15 minutes, followed by adding 200µg cell lysate (1
μg/μl) to the washed beads and incubating for 2 hours at 37°C. After incubation, the
pellets were washed four times in TBS/0.1% Tween 20 for 15 minutes. Then 20µl of
loading buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 0.1% bromophenol blue, 10%
glycerol and 5% β-mercaptoethanol) was added to the washed pellets followed by 5
minutes incubation at 90°C. The samples were then centrifuged at 9,000 × g for 2
minutes and the supernatant was transferred and stored at -80°C until immunoblot
analysis.
Immunoblot
Immunoaffinity purified proteins and cell lysates were separated on 12%
SDS-polyacrylamide gels and then electrophoretically transferred to nitrocellulose
membranes. Membranes were then incubated in blocking buffer (5% non-fat dry milk,
0.1% Tween 20, 1× TBS) for 1 hour at room temperature. Membranes were incubated
overnight at 4°C with primary antibodies on a shaker. Primary antibodies (Upstate
80
Biotechnology, NY: anti-Rac1, mouse IgG, 1:700; anti-Na+/K+ ATPase, mouse IgG, 1:
10000) were diluted in antibody buffer (1% non-fat dry milk, 0.1% Tween 20, 1×
TBS). The next day, membranes were washed with TBS/0.1% Tween 20 and then
incubated with goat-anti-mouse or goat-anti-rabbit secondary antibody conjugated to
HRP (Jackson ImmunoResearch, West Grove, PA) diluted in antibody buffer.
Membranes were washed and signal was detected using enhanced chemiluminescence
(ECL) western blotting detection reagents (Amersham Biosciences, Piscataway, NJ).
Using Scion Image for Windows (Scion, Frederick, MD), immunoblots were
quantified by calculating the integrated optical density (IOD) of each protein band on
the film.
Statistical Analyses
Data are presented as mean ± the standard error of the mean (SEM) and analyzed by
one- or two-way ANOVA. Post hoc tests were conducted using Bonferroni's multiple
comparison tests. GraphPad Prism 5.0 (GraphPad Software Inc., San Diego, CA) was
used for all statistical analyses. A probability level of p < 0.05 was considered to be
statistically significant for all statistical tests.
RESULTS
PLC inhibitor decreases the response of intracellular Ca2+ and TGase-modified
Rac1 to 5-HT2A receptor activation
We previously reported that 5-HT2A receptor stimulation induced
81
TGase-catalyzed Rac1 transamidation and activation in A1A1v cells (Dai et al., 2008).
To test the dependence of the intracellular Ca2+ response and Rac1 transamidation on
PLC activation, cells were pretreated with 5 µM of the PLC inhibitor, U73122, or an
inactive analog, U73343, for 30 min prior to the stimulation of 5-HT2A receptors by 3
µM DOI. U73122 inhibits both PLC-mediated hydrolysis of PIP2 to IP3 and the
coupling of G protein-PLC activation (Bleasdale et al., 1990). Changes in intracellular
Ca2+ were monitored by the 340/380 nm fluorescence ratio of the Fura-2 loaded cells.
DOI-stimulation induced a robust increase (by at least 200%) in the intracellular Ca2+
concentration within cells pretreated with U73343, where as PLC inhibition by
U73122 reduced the intracellular Ca2+ response to DOI (Fig. 12A). TGase-modified
Rac1 was evaluated by immunoprecipitation using the 81D4 antibody directed against
the TGase-catalyzed covalent bond and immunoblots in parallel experiments. There
were significant increases in TGase-modified Rac1 within DOI-stimulated cells, but
pretreatment with U73122 significantly attenuated DOI-induced Rac1 modification by
TGase (Fig. 12B, C). These results suggest a specific role of PLC upstream of Rac1
transamidation in A1A1v cells. Two-way ANOVA indicates a significant main effect
of DOI [F(1,8) =108.3; p<0.001] and U73122 [F(1,8) =29.99; p<0.001] on
TGase-modified Rac1. There was also a significant interaction between DOI and
U73122 [F(1,8) =17.40; p<0.01]. The ubiquitously expressed Na+/K+ ATPase is a well
established plasma membrane marker, and its function underlies essentially all of
mammalian cell physiology (Kaplan, 2002). To verify that the PLC inhibitor-mediated
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the decrease in Rac1-transamidation by TGase was not due to U73122-induced
cellular toxicity, cell lysates from the same experiment were examined on immunoblot
with antibodies for Na+/K+ ATPase. There are no significant differences in Na+/K+
ATPase levels among cells with different treatments (Fig. 12B). Similar
measurements were used in all of the following experiments.
Ca2+ chelator inhibits DOI-stimulated modification of Rac1 by TGase
In order to examine whether TGase-modified Rac1 specifically depends on an
increase in intracellular Ca2+ upon 5-HT2A receptor activation, A1A1v cells were
pretreated with increasing concentrations of the membrane-permeant Ca2+ chelator,
BAPTA-AM (0, 5, 10, 20 µM) for 30 min, then the cells were challenged by DOI.
Dose-dependent reductions in the DOI-mediated Ca2+ response were observed by
measuring Fura-2 fluorescence (Fig. 13A). Since 20 µM BAPTA caused the most
significant inhibition in Ca2+ increase, it was used in the parallel experiments that
evaluated TGase-modified Rac1. DOI-stimulation in cells without
BAPTA-pretreatment significantly increases the amount of TGase-modified Rac1
about 2-fold compared with vehicle-stimulated cells, whereas pretreatment with
BAPTA has a notable inhibitory effect on DOI-induced Rac1 modification by TGase
(Fig. 13B, C), suggesting that an increase in intracellular Ca2+ is required for
DOI-mediated Rac1 transamidation. Two-way ANOVA indicates a significant main
effect of DOI [F(1,8) =37.09; p<0.001] and BAPTA [F(1,8) =9.85; p<0.05] on
TGase-modified Rac1. There was also a significant interaction between DOI and
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BAPTA [F(1,8) =9.50; p<0.05].
Ca2+ ionophore induces TGase-modified Rac1 without 5-HT2A receptor
activation
To determine if an increase in intracellular Ca2+ is sufficient to enhance Rac1
modification by TGase, a Ca2+ ionophore, ionomycin was used to produce an
elevation of cytosolic Ca2+ concentration without 5-HT2A receptor activation.
Ionomycin is an effective and specific Ca2+ carrier, so it has been used in studies of
Ca2+ flux across plasma and ER membranes. To determine the concentration of
ionomycin that raises the levels of cytosolic Ca2+ to levels comparable to 3µM DOI
(the concentration of DOI used to induce TGase-modified Rac1), serial dilutions of
ionomycin (from 1000 nM to 12.5 nM) were tested for their ability to induce
increases in intracellular Ca2+. As shown in Fig. 14A, 25 nM ionomycin was able to
produce a comparable increase in cytosolic Ca2+ in comparison to 3 µM DOI, so this
concentration was used in the parallel experiments to measure the effects on
TGase-modified Rac1. After cells were treated with either 3 µM DOI or 25 nM
ionomycin for 15 min, ionomycin mediated an increase in TGase-modified Rac1 to
levels similar to those induced by DOI. One-way ANOVA indicated there is no
significant difference in TGase-modified Rac1 between DOI- and ionomycin-treated
cells. Taken together, these data suggest an increase in intracellular Ca2+ via an
ionophore can mimic 5-HT2A receptor-induced cytosolic Ca2+ increases and is
sufficient to induce TGase-catalyzed Rac1 transamidation.
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TGase-modified Rac1 is reduced by CaM inhibition in a dose-depend manner
TGase is not only a Ca2+-dependent enzyme, but its activity is also positively
regulated by CaM. Moreover, Ca2+ signaling in cells is mainly mediated by CaM, a
potent Ca2+-binding protein. The CaM inhibitor, W-7, binds with high affinity to CaM
in the presence of Ca2+ and thereby inhibits the activation of CaM-dependent enzymes
(Asano and Hidaka, 1984). To test whether CaM modulates TGase-catalyzed
transamidation of Rac1, cells were pre-incubated for 1 h with 0, 0.5 or 5 µM W-7
hydrochloride, and then stimulated with 3 µM DOI or vehicle for 15 min. We found
that in DOI-stimulated cells, the levels of TGase-modified Rac1 were significantly
higher than in vehicle-stimulated cells, whereas pretreatment with W-7 decreased
DOI-stimulated Rac1 modification by TGase in a dose-dependent manner (Fig. 15).
Two-way ANOVA indicates a significant main effect of DOI [F(1,12) =144.6;
p<0.001] and W-7 [F(2,12) =8.152; p<0.01] on TGase-modified Rac1. There was also
a significant interaction between DOI and W-7 [F(2,12) =5.01; p<0.05].
Searching potential TGase-targeting residues at the activity-related domains in
Rac1
We have previously reported that TGase-catalyzed transamidation of Rac1 was
able to transiently increase Rac1 activity (Dai et al., 2008). Like other small G
proteins, Rac1 acts as a molecular switch and cycles between the GDP-bound inactive
and GTP-bound active forms. There are at least three types of regulators for small G
proteins: GTPase activating protein (GAP) which accelerates GTP hydrolysis,
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resulting in inactivation of small G proteins; guanine nucleotide exchange factors
(GEF) which stimulates conversion from the GDP-bound to the GTP-bound form; and
GDP dissociation inhibitor (GDI) which inhibits this reaction. Post-translational
modifications, such as TGase-catalyzed transamidation, of Rac1 at the conserved
domains that interact with these regulators may affect its ability to convert GTP to
GDP, resulting in increased activation. The Conserved Domain Database (available on
http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml) provides an interactive tool to
identify conserved domains present in protein sequences. We searched the Conserved
Domain Database for GTP/Mg2+ binding sites, and GAP, GEF, and GDI interaction
sites in the Rac1 sequence (accession numbers: NP_008839) since TGase-catalyzed
modifications at theses sites would more likely lead to functional changes that would
impact on Rac1 activity (Fig. 16A). TGase-catalyzed transamidation may not only
incorporate an amine into the glutamine residue of the acceptor protein, but also form
intra-molecular cross-links between glutamine and lysine residues within the protein
(Hand et al., 1993). Two glutamine residues (Q61, Q74) and three lysine residues (K5,
K16, and K116) are located at the activity-related domains and are highlighted in
Rac1 structure (Fig. 16B).
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Figure 12. Effect of PLC inhibition on DOI-induced Ca2+ signals and TGase-modified
Rac1 in A1A1v cells. (A) After pretreatment with 5 µM U73122 or U73343 (negative
control) for 30 min, and stimulation with either 3 µM DOI or vehicle (Ve), changes in
the fluorescence ratio (340/380 nm) were recorded from cells loaded with Fura-2. A
representative example from three independent experiments conducted in triplicate is
shown. Arrows indicate the time of drug application. (B) Upper,
immunoprecipitation (IP) and immunoblot (IB) analyses revealed that U73122
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pretreatment inhibited the increase in TGase-modified Rac1 upon DOI stimulation.
Lower, cells lysates from the same experiment were examined on IB with Na+/K+
ATPase antibody, which indicated that U73122 had no effect of on cell viability. (C)
Quantitation of effects of U73122 and DOI on TGase-modified Rac1 in three separate
experiments. Data shown were the mean IOD±S.E.M., and they were normalized to
U73343- and Ve- treated cells. Two-way ANOVA followed by Bonferroni test: * and
*** indicated p<0.05 and 0.001 as compared to U73343- and Ve- treated cells,
respectively; # indicated p<0.001 as compared to U73343- and DOI- treated cells.
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Figure 13. Pre-incubation with a Ca2+ chelator attenuated the DOI-mediated elevation
of cytosolic Ca2+ and TGase-modified Rac1. (A) Dynamic changes in fluorescence
ratio (340/380 nm) in Fura-2 loaded A1A1v cells. The Ca2+ response to exposure to 3
µM DOI was gradually reduced with the increasing concentrations of BAPTA. A
representative example of three independent experiments conducted in triplicate is
shown. (B) Upper, cells were pretreated with 20 µM BAPTA or the vehicle for
BAPTA (Ve1) for 30 mins, and then stimulated with 3 µM DOI or the vehicle for DOI
(Ve2) for 15 mins. TGase-modified Rac1 was detected by IP and IB analysis. BAPTA
inhibited the increase in TGase-modified Rac1in DOI- stimulated cells. Lower, cells
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lysates from the same experiment were examined on IB with Na+/K+ ATPase
antibody. There was no significant difference among various treatments. (C)
Quantitation of effects of BAPTA and DOI on TGase-modified Rac1 in three separate
experiments. Data shown were the mean IOD±S.E.M., and they were normalized to
Ve1- and Ve2- treated cells. Two-way ANOVA followed by Bonferroni test: *
indicated p<0.01 as compared to Ve1- and Ve2- treated cells; # and ## indicated
p<0.05 and p<0.01 as compared to Ve1- and DOI- treated cells, respectively.
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Figure 14. The Ca2+ ionophore mimiced the DOI-induced increase in cytosolic Ca2+
and TGase-catalyzed transamidation of Rac1. (A) Fura-2 loaded cells were stimulated
with either 3µM DOI or different concentrations of ionomycin. The changes in the
fluorescence ratio (340/380 nm) were recorded. A representative example from three
independent experiments conducted in triplicate is shown. (B) Upper, cells were
treated for 15 mins with 3µM DOI, 25 nM ionomycin or vehicles (Ve). Ionomycin
caused an increase in TGase-modified Rac1 comparble to that induced by DOI. Lower,
cell viability, as indicated by Na+/K+ ATPase levels was not significantly different
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among the various treatments. (C) Quantitation of effects of ionomycin and DOI on
TGase-modified Rac1 in three separate experiments. Data shown were the mean
IOD±S.E.M., and were normalized to Ve-1-treated cells. One-way ANOVA followed
by Bonferroni test: * indicates p<0.01 as compared to Ve-1-treated cells; # indicates
p<0.01 as compared DOI-treated cells.
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Figure 15. The CaM inhibitor W-7 caused a dose-dependent reduction in
DOI-stimulated TGase-modified Rac1. (A) Cells were pretreated with increasing
concentrations of W-7 for 1 h and then stimulated with either 3 µM DOI or vehicle
(Ve). TGase-modified Rac1 and Na+/K+ ATPase were measured by IP and/or IB. W-7
decreased the levels of TGase-modified Rac1 upon DOI stimulation, but did not
significantly affect Na+/K+ ATPase at the concentrations applied. (B) Quantitation of
effects of W-7 on TGase-modified Rac1 in three separate experiments. Data shown
were the mean IOD±S.E.M., and were normalized to Ve-stimulated cells without W-7
pretreatment. Two-way ANOVA followed by Bonferroni test: * and ***indicate
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p<0.05 and p<0.001 as compared to Ve-stimulated cells without W-7 pretreatment,
respectively; # and ## indicate p<0.01 and p<0.001 as compared to DOI-stimulated
cells without W-7 pretreatment, respectively.
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Figure 16. Rac1 activity-related domains and potential TGase-targeted amino acid
residues. (A) Amino acid sequence alignment of human Rac1. Conserved
activity-related sites were identified by searching NCBI Conserved Domain Database
(CDD). # indicates a GTP/Mg2+ binding site; * indicates GAP interaction sites; +
indicates GEF interaction sites; & indicated GDI interaction sites. Two glutamine
residues (Q61, Q74) and three lysine residues (K5, K16, and K116), highlighted in
gray, are directly localized in these potential activity-related domains. (B) Rac1
structure adapted from http://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?
uid=56843. The potential TGase-targets, glutamine and lysine residues at Rac1
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activity-related domains are highlighted in yellow.
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Figure 17. Schematic representation showing 5-HT2A receptor signaling-mediated
transamidation of Rac1 by TGase. DOI stimulates Gq/11 proteins-coupled 5-HT2A
receptors, leading to the activation of PLC, which in turn hydrolyses PIP2 into IP3. IP3
releases Ca2+ from ER and results in the activation of Ca2+-dependent proteins, such
as CaM and TGase. Positively regulated by both Ca2+ and CaM, TGase catalyzes
transamidation of Rac1 to bioamines, such as serotonin transported into the cytoplasm
by SERT. Abbreviations: 5-HT2AR, 5-HT2A receptor; CaM, calmodulin; ER,
endoplasmic reticulum; PLC, phospholipase C; PIP2, phophatidyl
inositol-1,4-bisphosphate; IP3, inositol-1,4,5-triphosphate; SERT, serotonin
transporter; TGase, transglutaminase; α, β, γ, G protein α, β, γ subunits
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DISCUSSION
Several lines of evidence indicate Gq/11-protein-coupled receptors, such as
bradykinin or endothelin-1 receptor, are also able to stimulate the activation of Rac1
(van Leeuwen et al., 1999, Clerk et al., 2001), but the mechanisms by which Rac1
becomes activated through these receptors remains poorly characterized. Nevertheless,
it is worth noting that the activation of Rac1 is highly regulated by physiological
stimuli, and aberrant activation occurs under many pathological conditions such as
Salmonella invasion, tumor cell migration, and retinal degeneration (Bourguignon et
al., 2000, Belmonte et al., 2006, Brown et al., 2007). Therefore, further studies that
delineate signaling pathways involved in Rac1 activation may uncover new potential
therapeutic targets for these diseases. We have previously demonstrated that the
5-HT2A receptor, another Gq/11-protein-coupled receptor, is responsible for
TGase-catalyzed transamidation and activation of Rac1 (Dai et al., 2008). In the
present study, we investigated the mechanisms involved in Rac1 transamidation in
response to 5-HT2A receptor stimulation. Our experimental results identified that
PLC-mediated increase in intracellular Ca2+ and subsequent CaM activation are
required for 5-HT2A receptor-mediated Rac1 transamidation. 5-HT2A receptor
stimulation activates PLC which catalyzes the hydrolysis of PIP2 into IP3. IP3
mobilizes Ca2+ from ER stores and thereby increases the intracellular Ca2+ and leads
to the activation of CaM. Our data suggest that both Ca2+ and CaM activate TGase,
which catalyzes transamidation of Rac1 to bioamines, such as serotonin (Fig. 17).
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Except for the 5-HT3 receptor, which is a ligand-gated ion channel, the other
5-HT receptors belong to the G protein-coupled receptor (GPCR) superfamily. G
proteins such as Gα12 and Gα13, have been shown to transduce signals from GPCR to
activate Rho small G proteins (Suzuki et al., 2003). One of the GEFs for Rho,
p115RhoGEF, has served as a direct link between Gα12/Gα13 and Rho (Hart et al.,
1998, Kozasa et al., 1998). However, the signaling pathways between 5-HT2A
receptors and Rac1 transamidation and activation are not well understood. 5-HT2A
receptor is primarily coupled to Gq/11 and activates various isoforms of PLC (Conn
and Sanders-Bush, 1984, de Chaffoy de Courcelles et al., 1985). It also induces the
activation of phospholipase A2 (PLA2) and the release of arachidonic acid (Felder et
al., 1990, Kurrasch-Orbaugh et al., 2003). Moveover, through interaction with
ADP-ribosylation factor 1 (ARF1) via its C-terminal domain, 5-HT2A receptor is able
to signal through the phospholipase D (PLD) pathway (Mitchell et al., 1998,
Robertson et al., 2003). Our results suggest that 5-HT2A receptor activation triggers
TGase-catalyzed Rac1 transamidation at least predominantly through a pathway
involving PLC (Fig.12).
Although it has been reported that PLC activates Rac in a manner dependent on
Iba1, a Ca2+-binding protein specifically expressed in macrophage/microglia
(Kanazawa et al., 2002), Rac1 transamidation and activation in A1A1v cells, a
neuronal cell line, could be mediated by other signaling pathways. Cleavage of PIP2
by PLC yields two important second messengers, IP3 and DAG, to initiate a variety of
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cellular functions. Since Ca2+ chelation reduces DOI-stimulated Rac1 transamidation
to basal levels (Fig.13), IP3-induced Ca2+ release from ER rather than DAG signaling
plays a pivotal role in TGase-catalyzed Rac1 transamidation. These results are in
agreement with an earlier study suggesting that in thrombin- and collagen-stimulated
human blood platelets, PLC activity and an increase in intracellular Ca2+ concentration
were required for Rac activation, whereas PKC inhibition had no effect (Soulet et al.,
2001). Increases in intracellular Ca2+ concentration upon 5-HT2A receptor stimulation
is achieved by releasing intracellular Ca2+ stores and/or by activating Ca2+ channels,
depending on the cell of interest. 5-HT2A receptor-induced contraction of rat aorta
may require the concerted action of voltage-gated Ca2+ channels and phosphoinositide
turnover (Nakaki et al., 1985, Roth et al., 1986, Watts, 1998). More interestingly,
stimulation of 5-HT2A receptors on astrocytes caused Ca2+ influx through voltage
independent Ca2+ channels which were dependent of the IP3 production and
subsequent Ca2+ release from internal stores (Eberle-Wang et al., 1994, Hagberg et al.,
1998). Therefore, both Ca2+ mobilization from internal stores and influx of
extracellular Ca2+ could contribute to the PLC/IP3-mediated intracellular Ca2+
increase.
CaM and TGase are Ca2+ dependent proteins. CaM activation is coupled to
intracellular Ca2+ elevation and also correlates with the spatial pattern of increased
Ca2+ (Hahn et al., 1992). Upon Ca2+ binding to the EF hand motifs of CaM, both N-
and C-terminal domains adopt an open confirmation, exposing hydrophobic surfaces,
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which then interact with a variety of target proteins including TGase (Puszkin and
Raghuraman, 1985, Zainelli et al., 2004). TGase contains a cysteine in their active site
that is unmasked only in the presence of Ca2+ (Hand et al., 1993) and Ca2+ activation
of TGase is further regulated by other signal modulators such as GTP (Monsonego et
al., 1998) and CaM (Puszkin and Raghuraman, 1985, Zainelli et al., 2004). In order
for TGase to become receptive to the glutamine substrate, Ca2+ first must bind to
TGase, inducing its conformational change which permits the glutamine substrate
binding to the catalytic center (Folk, 1983, Greenberg et al., 1991). A rise of Ca2+
concentration increases TGase activity in a dose dependent manner in vitro and in
cells (Siefring et al., 1978, Shin et al., 2004, Li et al., 2009). Consistent with these
results, the elevation of intracellular Ca2+ induced via a Ca2+ ionophore lead to
increased TGase-catalyzed Rac1 transamidation (Fig. 14).
In addition to Ca2+, CaM also interacts with TGase and regulates its
cross-linking activity. CaM has been shown to coimmunoprecipitate with TGase2 in
transfected cells (Zainelli et al., 2004). CaM increased TGase activity in human
erythrocyte (Billett and Puszkin, 1991), platelets, chicken gizzard (Puszkin and
Raghuraman, 1985) and rat liver (Hand et al., 1985). Interestingly, exogenous CaM
with a binding site on the α-subunits of the hexadecameric phosphorylase kinase
molecule (αβγδ)4, stimulates TGase-catalyzed incorporation of putrescine into the β-
and γ-subunits, but inhibits incorporation of amines into the α-subunit (Nadeau and
Carlson, 1994). Some of the CaM antagonists, such as dansylcadaverine, inhibit TGase
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(Lorand et al., 1979) and its protein cross-linking ability. We have previously reported
that a CaM antagonist or inhibitory CaM fragments, attenuated TGase-modified mutant
huntingtin in cell and animal models of Huntington's disease (Zainelli et al., 2004, Dai
et al., 2009, Dudek et al., 2009). CaM antagonists and Ca2+-chelators also have been
shown to depress the motile progression of sperm cells possibly mediated by
TGase-catalyzed cross-linking of the contractile proteins (Cariello and Nelson, 1985).
Our present results revealed that CaM inhibitor, W-7, reduced TGase-modified-Rac1 in
dose-dependent fashion (Fig. 15), indicating that TGase-catalyzed Rac1 transamidation,
at least in part, was positively regulated by CaM. Moreover, in the presence of 5-HT2A
receptor agonist, CaM has been shown to interact with 5-HT2A receptor at the sites
that are important for G protein coupling, suggesting a putative role for CaM in
regulating 5-HT2A receptor function (Turner and Raymond, 2005). Thus, one could
not exclude the possibility that the effect of CaM inhibition is mediated by alterations
in 5-HT2A receptor signaling.
The significance of TGase in Rac1 transamidation was confirmed by TGase
siRNA and inhibitor in our previous studies (Dai et al., 2008). In addition to small G
proteins such as Rac1 and RhoA (Walther et al., 2003, Dai et al., 2008) that could
undergo TGase-catalyzed transamidation to serotonin (serotonylation), more recently,
arterial delete cytoskeletal proteins including actin, myosin heavy chain and filamin A
have been identified as serotonylated proteins (Watts et al., 2009). However, serotonin
may not be the only amine that could be incorporated in these proteins. Neuronal
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differentiation of SH-SY5Y cells induced by retinoic acid increases the
expression/activation of TGases, resulting in transamidation of putrescine to RhoA and
activation of RhoA (Singh et al., 2003). Moreover, spermidine and putrescine are
suitable substrates for transglutamination of RhoA in vitro (Schmidt et al., 2001). In
addition to amine incorporation, another important TGase-catalyzed modification of
small G proteins is deamidation of glutamine residue. The site-specific deamidation of
Gln53 and Gln61 of RhoA and Ras, respectively, by the bacterial toxin TGases
inhibits the GTPase activity and renders small G proteins constitutively active (Lorand
and Graham, 2003).
In addition to TGase-catalyzed reactions, other Ca2+/CaM dependent pathways
may also contribute to Rac1 activation. For example, platelet-derived growth factor
(PDGF) stimulates phosphorylation of Tiam1, the Rac1-specific GEF, and this
phosphorylation was significantly reduced by Ca2+ chelation, inhibition of Ca2+/CaM
dependent kinase II (CaMKII), or disruption of PLC-γ expression in Swiss 3T3
fibroblasts (Fleming et al., 1998). Targeted CaMKII knockdown using siRNA resulted
in the inhibition of wound-induced Rac activation and vascular smooth muscle cell
migration (Mercure et al., 2008).
In conclusion, our study provides important new insights into the signaling
mechanisms underlying 5-HT2A receptor-induced Rac1 transamidation and activation
by TGase in A1A1v cells, and highlights the necessary role of PLC-mediated Ca2+
mobilization and CaM in TGase-catalyzed Rac1 transamidation. As an activating
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signal for both TGase and CaM, an elevation of intracellular Ca2+ is sufficient to
induce Rac1 transamidation. Accumulating evidence suggests that Rho family of
small G proteins is subject to TGase-mediated modification and activation. The
essential role of Rac1 in cytoskeletal organization is well characterized, while the
biological functions of Rac1 transamidation by TGase of in neurons have yet to be
explored.
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CHAPTER FOUR
STRIATAL EXPRESSION OF A CALMODULIN FRAGMENT IMPROVED MOTOR FUNCTION, WEIGHT LOSS AND NEUROPATHOLOGY IN THE R6/2
MOUSE MODEL OF HUNTINGTON’S DISEASE
(Published in J Neurosci. 2009; 29(37):11550-9)
ABSTRACT
Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder,
caused by a polyglutamine expansion in the huntingtin protein (htt). Increasing
evidence suggests that transglutaminase (TGase) plays a critical role in the
pathophysiology of HD possibly by stabilizing monomeric, polymeric and aggregated
htt. We previously reported that in HEK293 and SH-SY5Y cells expression of a
calmodulin (CaM)-fragment, consisting of amino acids 76-121 of CaM, decreased
binding of CaM to mutant htt, TGase-modified htt and cytotoxicity associated with
mutant htt and normalized intracellular calcium release. In this study, an
adeno-associated virus (AAV) that expresses the CaM-fragment was injected into the
striatum of HD transgenic R6/2 mice. The CaM-fragment significantly reduced body
weight loss and improved motor function as indicated by improved rotarod
performance, longer stride length, lower stride frequency, fewer low mobility bouts
and longer travel distance than HD controls. A small but insignificant increase in
survival was observed in R6/2 mice with CaM-fragment expression.
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105
Immunoprecipitation studies show that expression of the CaM-fragment reduced
TGase-modified htt in the striatum of R6/2 mice. The percentage of htt-positive nuclei
and the size of intranuclear htt aggregates were reduced by the CaM-fragment without
striatal volume changes. The effects of CaM-fragment appear to be selective, as
activity of another CaM-dependent enzyme, CaM-dependent kinase II, was not altered.
Moreover, inhibition of TGase-modified htt was substrate-specific since overall
TGase activity in the striatum was not altered by treatment with the CaM-fragment.
Taken together, these results suggest that disrupting CaM-htt interaction may provide
a new therapeutic strategy for HD.
INTRODUCTION
Patients with Huntington’s disease (HD) present symptoms such as chorea,
irregular gait, reduced motor coordination and weight loss (Harper, 1991). HD is a
progressive and fatal neurological disorder caused by expansion of CAG repeats in
HTT gene, conferring a toxic gain of function to the huntingtin (htt) protein
(Huntington’s Disease Collaborative Research Group, 1993). It is characterized
neuropathologically by intranuclear inclusions and cytoplasmic aggregates composed
of htt with an expanded polyglutamine domain (DiFiglia et al., 1997, Ross et al.,
1998). Currently, there are no effective treatments to prevent or slow the progression
of the disease.
Increasing evidence indicates that TGases may contribute to the
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pathophysiology of HD (Cooper et al., 1999, Karpuj et al., 2002b). TGases, a family
of Ca2+-dependent enzymes, catalyze a covalent bond between peptide-bound
glutamine residues and either lysine-bound peptide residues or mono- or polyamines
(Folk et al., 1980, Griffin et al., 2002). In cell culture, mutant htt is an excellent
substrate for TGases (Gentile et al., 1998, Kahlem et al., 1998, Zainelli et al., 2005).
The mRNA, protein and enzymatic activity of TGases are elevated in HD brain
(Karpuj et al., 1999, Lesort et al., 1999, Zainelli et al., 2003). TGase 2 ablation in HD
transgenic mice resulted in a drastic reduction in -(γ-glutamyl) lysine bond levels
and neuronal death in the cortex and striatum (Mastroberardino et al., 2002).
Administration of the TGase inhibitor, cystamine, extended survival and improved
motor performance in HD transgenic mice (Karpuj et al., 2002a).
Calmodulin (CaM) increased TGase activity in human erythrocyte (Billett and
Puszkin, 1991), platelets and chicken gizzard (Puszkin and Raghuraman, 1985). CaM
activates a host of enzymes upon Ca2+ binding (Cheung, 1982) and associates with
mutant htt as demonstrated by affinity purification (Bao et al., 1996) and
immunoprecipitation (Zainelli et al., 2004). Moreover, CaM colocalizes with htt and
TGase 2 in HD intranuclear inclusions (Zainelli et al., 2004). Since a CaM inhibitor
decreased TGase-catalyzed cross-linking of htt in cells (Zainelli et al., 2004), we
tested the ability of fragments of CaM to interrupt the interaction of CaM and mutant
htt and decrease the deleterious effects of TGase in HEK-293 cells (Dudek et al.,
2008). A CaM-fragment, containing amino acids 76-121, reduced binding of CaM to
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mutant htt in vitro and in HEK-293 cells. AAV-mediated expression of the
CaM-fragment significantly decreased TGase-modified htt, and mutant htt-associated
cytotoxicity in differentiated SH-SY5Y cells, a neuroblastoma cell line, which stably
express mutant htt. Importantly, CaM-fragment did not alter the total activity of
TGase or another CaM-dependent enzyme, CaM kinase II, suggesting that
CaM-fragment has specific effects on the CaM-htt interaction (Dudek et al., 2009). In
the present study, we assessed the therapeutic potential of CaM-fragment in the R6/2
mouse model of HD. R6/2 transgenic mice express exon 1 of the human HD gene
with an increased CAG repeat. Consequently, they develop a progressive neurological
phenotype and pathological changes that resemble many features of HD (Mangiarini
et al., 1996, Li et al., 2000). Survival, body weight, motor performance and
neuropathological features were monitored to determine whether AAV-mediated
expression of the CaM-fragment has beneficial effects on the course of HD in R6/2
mouse model.
MATERIALS AND METHODS
Animals. Male R6/2 transgenic mice (with 100-115 CAG repeats in the
transgene) and wild-type littermate mice were purchased from Jackson Laboratories
(Bar Harbor, ME) at 6 weeks of age. The mice had free access to food and water in an
environment controlled for temperature and humidity and a 12 h light/dark cycle. The
behavioral tests were conducted in the light part of the cycle. All procedures were
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conducted in accordance with the National Institutes of Health Guide for the Care and
Use of Laboratory Animals as approved by the University of Kansas Institutional
Animal Care and Use Committee.
Adenoassociated Virus (AAV) Injections. Recombinant AAV serotype 2 vectors
were generated as described in our previous studies (Dudek et al., 2009). Three
resultant vectors expressed each of the following: GFP and the CaM-fragment
(containing amino acids 76-121 of calmodulin), GFP and a scrambled peptide
(containing the same amino acids of the CaM-fragment but in a randomly scrambled
sequence), and GFP alone. The titers of final AAV products were ranged from 2~5 ×
1014 vector genomes per ml. Bilateral striatal injections of GFP-expressing AAV were
performed in 7-week-old mice (1 µl per each side with 200 nl/min infusion rate at
coordinates of anterior/posterior 0.5 mm, lateral/medial ±2 mm and dorsal/ventral -3
mm relative to bregma (Franklin et al., 1997)). Mice were randomly divided into five
groups (11~14 mice for each group) with a defined genotype and treatment: CaM-HD
(R6/2 mice injected with AAV expressing the CaM-fragment), Vec-HD (R6/2 mice
injected with empty vector AAV expressing GFP alone), Scr-HD (R6/2 mice injected
with AAV expressing a scrambled version of the CaM-fragment), CaM-WT
(wild-type mice injected with AAV expressing CaM-fragment) and Vec-WT
(wild-type mice injected with empty vector AAV).
Body weight and survival. Body weights were measured twice a week.
Survival was checked twice daily at 8 h intervals. Brains were removed after death
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and snap frozen in 2-methylbutane/dry ice bath and stored at -80°C.
Behavioral tests. Three behavioral tests were performed between 10 and 14
weeks of age at weekly intervals. Rotarod performance, gait dynamics and
locomotion were examined every Wednesday, Friday and Sunday, respectively.
Gait dynamics. The DigiGait imaging system (Mouse Specifics, Inc., Boston,
MA) was utilized for gait analyses as previously described (Hampton et al., 2004).
Briefly, mice were placed on a motorized transparent treadmill belt moving at a speed
of 14 cm/s. Digital images of paw placement were recorded at 150 Hz through a video
camera mounted below the animal. The proprietary software analyzed the resulting
digital images to generate a set of periodic waveforms that described the paw area and
movement of each limb relative to the treadmill belt through consecutive strides. Gait
data were pooled from all four paws and used to determine numerous gait dynamic
measures including stride length and frequency, variability of stance width and paw
area at peak stance.
Locomotion. To accurately measure locomotor behaviors including the total
distance traveled and the number of low mobility bouts (defined as remaining
continuously in a virtual circle of 15 mm radius for 10 sec), a force-plate actometer
(constructed in Dr. Fowler’s laboratory, University of Kansas) was used as described
previously (Fowler et al., 2001). Mice were placed in a 28 cm × 28 cm force-plate
actometer for a 30-min recording once a week. When the animal moves on the plate,
its movements are sensed by four supporting force transducers positioned at the
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corners of the plate. The signals are processed by specialized computer algorithms
written in house to yield measurements of travel distance and bouts of low mobility.
Rotarod performance. A rotarod apparatus (MED-Associates, St. Albans, VT)
was used to measure fore- and hindlimb motor coordination and balance. The mice
were given a training session at 9 weeks of age (four trials per day for 3 consecutive
days) to acclimate them to the rotarod apparatus. During the test period, each mouse
was placed on the rotarod with increasing speed, from 4 rpm to 40 rpm in 300 seconds.
The latency to fall off the rotarod within this time period was recorded. Mice were
tested on the rotorod once a week. Each mouse received two consecutive trials and the
mean latency to fall was used in the analysis.
Immunoprecipitation (IP). Mouse brains were bisected mid-saggitally. One
half was used in IP, CaM Kinase II Activity and TGase assays. The other half was
used for immunohistochemistry assays. To prepare striatal homogenates for IP, a half
brain was cut into 300-μm coronal sections with a cryostat (Leica, Nussloch,
Germany). Striatum was punched out from five consecutive sections and
homogenized in lysate buffer (10 mM Tris-HCl, pH 7.5, 0.14 M NaCl, 1 mM EDTA,
and 1:1000 protease inhibitor mixture) in a Tissue Tek homogenizer. Protein
concentration was determined using the BCA Protein Assay kit (Pierce Chemical,
Rockford, IL) and tissue homogenates containing 150 µg of protein were centrifuge at
12,000 × g for 5 min at 4°C to separate the insoluble fraction. After removal of the
supernatant, the insoluble fraction was resuspended in 60 µl of 95% formic acid and
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incubated in a shaking water bath at 37°C for 40 min. The formic acid was then
evaporated using a CentriVap Speed Vacuum (Labconco, Kansas City, MO) for 1.5 h
at 45°C. The pellets were resuspended in 150 µl of IP wash buffer (10 mM Tris-HCl,
pH 7.5, 0.14 M NaCl, and 0.1% Tween 20) and sonicated on ice at 10 x 5 sec pulse.
Immunopurification of proteins containing TGase-catalyzed bonds was performed
using 81D4 monoclonal antibody prebound to Sepharose beads (Covalab, Lyon,
France) using a protocol developed by Covalab and as described previously (Norlund
et al., 1999, Zainelli et al., 2005).
Immunoblot. Immunoaffinity-purified proteins were separated on 10%
SDS-polyacrylamide gels and then electrophoretically transferred to nitrocellulose
membranes. Membranes were then incubated in blocking buffer (5% nonfat dry milk,
0.1% Tween 20, and 1× TBS) for 1 h at room temperature. Membranes were
incubated overnight at 4°C with primary antibody on a shaker. Primary antibody
(Chemicon International, Temecula, CA: anti-Huntingtin amino acids 1-82, mouse
IgG, 1:500) was diluted in antibody buffer (1% nonfat dry milk, 0.1% Tween 20, and
1× TBS). The next day, membranes were washed with TBS/0.1% Tween 20, and then
they were incubated with goat anti-mouse secondary antibody conjugated to
horseradish peroxidase (Jackson ImmunoResearch Laboratories Inc., West Grove, PA)
diluted in antibody buffer. Membranes were washed, and signal was detected using
enhanced chemiluminescence Western blotting detection reagents (GE Healthcare,
Chalfont, St.Giles, UK). Using Scion Image for Windows (Scion Corporation,
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Frederick, MD), immunoblots were quantified by calculating the integrated optical
density (IOD) of each protein band on the film.
Evaluation of htt aggregates by immunofluorescence. Sections of frozen
mouse brain tissue (20 µm thick) were mounted on glass slides, and then fixed in 4%
paraformaldehyde. After fixation, the sections were washed in PBS (pH 7.4), and then
nonspecific binding was blocked with 5% normal goat serum (NGS). Sections were
incubated overnight with primary antibody: MAB5374 (mouse IgG, 1:100, directed
against human huntingtin amino acids 1~256) in 1% NGS + 1X PBS. Next, sections
were washed and incubated for 1h with secondary antibody: goat anti-mouse IgG, Fcγ
fragment specific, conjugated to DyLight 649 (1:400) (Jackson ImmunoResearch,
West Grove, PA). Next, coverslips were mounted on tissue sections with Prolong
Gold antifade reagent with DAPI (Invitrogen, Carlsbad, CA). As a control for
nonspecific labeling with secondary antibody, omission of the primary antibody was
also performed on a slide from each case. Htt-aggregate positive nuclei and size of
htt-aggregates were measured in each of ten 215 ×215 µm microscope fields by
Olympus IX-81 microscope (Olympus, Japan), in each of five rostrocaudally spaced
sections in the striatum of 5 mice from the three groups of R6/2 mice (wild-type
littermates did not show htt immunoreactivity). At least 450 nuclei per mouse were
obtained and the percentage of nuclei containing htt-aggregates and area of
htt-aggregates were calculated by CellProfiler software (Whitehead Institute,
Cambridge, MA) and a mean value was obtained for each group.
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Measurement of striatal volume by Nissl staining. Cryostat sections (20 µm
thick) were postfixed in 4% paraformaldehyde, then dehydrated in graded alcohols.
After delipidated in 1:1 alcohol/chloroform for 1.5 h, sections were rehydrated
through graded alcohols and stained with 0.2% aqueous solution of cresyl violet
(Sigma, St. Louis, MO) for 5 min, followed by a brief rinse with water and
dehydration in graded alcohols. Sections were cleared in xylene (two changes, 5
min each) and cover-slipped with Permount (Fisher Scientific, Fair Lawn, NJ). The
volume of the striatum was measured according to the principle of Cavalieri (Cyr et
al., 2005) (volume=s1d1+s2d2…sndn, where s=surface area and d=distance between
two sections). We considered 15 coronal levels of the striatal sections (from Bregma
1.7 mm, with an interval of 200 μm between the sections) for the volumetric
measurement study. Image capturing was performed by using an inverted microscope
(Olympus IX-81) coupled to a digital camera (Hamamatsu EMCCD, Japan). The
montage image of a coronal brain section was constructed by Slidebook (Intelligent
Imaging Innovations, Denver, CO). The surface area of striatum in each section was
measured using NIH Image J [http://rsbweb.nih.gov/ij/]. Data were expressed as the
average Cavalieri volume ±SEM (mm3) of 4~5 mice per group.
CaM Kinase II Activity Assay. CaM kinaseII enzyme activity was analyzed
using the SignaTECT Calcium/Calmodulin-Dependent Protein Kinase Assay System
(Promega, Madison, WI), according to the manufacturer's protocol. Mouse striatum
was homogenized in the extraction buffer (20mM Tris-HCl pH 8.0, 2mM EDTA,
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2mM EGTA, 2mM DTT, 1mM PMSF and 1:1000 Protease Inhibitor Cocktail (Sigma,
St Louis, MO)). The homogenate was centrifuged at 350 × g for 5 min at 4°C and the
supernatant was mixed with [γ-32P]ATP (at 3000Ci/mmol, 10mCi/ml), 50 µM
biotinylated CaM kinase II substrate, reaction buffer containing 50mM Tris-HCl (pH
7.5), 10mM MgCl2 and 0.5mM DTT, with or without control buffer containing 1mM
EGTA. After incubation at 30°C for 2 min, the reaction was terminated by adding 7.5
M guanidine hydrochloride and spotted to a streptavidin-impregnated membrane.
Membranes were washed and the retained radioactive substrate of CaM kinase II was
quantitated by liquid scintillation counting (Beckman, Fullerton, CA). Radioactive
counts were used as an index of endogenous CaM kinase II activity in the sample.
TGase II Activity Assay. TGase activity was measured by detecting the
incorporation of a biotinylated TGase amine substrate, 5-(biotinamido)pentylamine
(Pierce, Rockford, IL) into N,N’-dimethylcasein (Calbiochem, San Diego, CA) as
previously described (Dudek et al., 2009). 96-well Immulon 4 HBX plates (Dynatech,
Franklin, MA) were coated with 100 µl of N,N’-dimethylcasein (20 mg/ml) in 0.05M
sodium bicarbonate, and stored at 4°C overnight. After the unbound
N,N’-dimethylcasein was discarded, the plate was washed with PBS and blocked with
nonfat dry milk (2% milk in PBS) for 1 hour at 37°C, followed by 3 washes with PBS.
Striatum from the various mice were homogenized in 0.1 M Tris-HCl pH 8.3, 1 mM
EDTA, 1 mM PMSF, and protease inhibitor cocktail. Striatal homogenates (15 µg of
protein/sample) were added to the plate with 0.1 M Tris-HCl pH 8.5, 0.15 M NaCl, 5
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mM DTT, 0.5 mM biotin-labeled pentylamine, and 5 mM CaCl2. The mixture was
incubated for 1 hour at 37°C. The plate was washed with TBS and 0.001% Tween
followed by incubation with streptavidin conjugated to HRP (Jackson
ImmunoResearch, West Grove, PA) for 1 hour at room temperature. After five washes,
1 x TMB substrate solution (eBioscience, SanDiego, CA) was added and color was
allowed to develop for 3 min. Then the reaction was stopped with 3N sulfuric acid
and the absorbance was read at 450nm. For GTP-inhibited TGase enzymatic activity,
the assay was performed in the presence of 500 µM GTPγS (Sigma-Aldrich, St Louis,
MO). Negative controls were run in the absence of pentylamine or dimethylcasein.
Each assay also included a standard curve of varying amounts of guinea pig TGase
(Sigma, St. Louis, MO). Each sample was measured in triplicate. TGase activity was
converted to a percentage control based on the mean value for the Vec-WT mice.
Statistics. All data are presented as mean ± S.E.M. and analyzed by one- or
two-way ANOVA with repeated measures. Post hoc tests were conducted using
Bonferroni's multiple comparison tests. There were one or two mice dead after week
13 in each HD group, so the last observed value was used for the subsequent missing
values. Survival time was demonstrated by Kaplan-Meier survival curves and
analyzed by a log-rank test. GB-STAT 10.0 (Dynamic Microsystems, Inc., Silver
Spring, MD) and GraphPad Prism 5.0 (GraphPad Software Inc., San Diego, CA) were
used for all statistical analyses. A probability level of p < 0.05 was considered to be
statistically significant for all statistical tests.
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RESULTS
AAV-mediated delivery of CaM-fragment in R6/2 mice attenuated body weight
loss
Body weight was monitored from 7 weeks of age onwards. All groups of mice were of
similar initial body weight (22.9±0.3 g, n=10~14 mice in each group). Changes in
body weight were expressed as a percentage of body weight measured at 7 weeks of
age (Fig. 18a). Body weight in all groups slowly increased or remained unchanged
until week 10. Thereafter, mice in the two wild-type (WT) groups continued gaining
body weight. The Vec-HD (R6/2 mice injected with empty vector AAV expressing
GFP alone) and Scr-HD mice (R6/2 mice injected with AAV expressing a scrambled
version of the CaM-fragment) had profound weight loss over the duration of the
observation period, whereas CaM-HD mice (R6/2 mice injected with AAV expressing
the CaM-fragment) maintained their body weight, or slightly increased their body
weight (Fig. 18a). One-way ANOVA [F(4,50)=82.48; p<0.0001] followed by
Bonferroni's multiple comparison test indicated that mice in CaM-HD group had less
change in body weight than those in the Vec-HD group at 12-16 weeks of age (p <
0.05), and there was no significant difference in body weight change between Scr-HD
and Vec-HD at each time point. Similarly, body weight change in the CaM-WT (WT
mice injected with AAV expressing the CaM-fragment) was not significantly different
from the Vec-WT (WT mice injected with empty vector AAV expressing GFP alone).
CaM-fragment expression did not significantly increase R6/2 mice survival
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Although the first death took place on day 88, 78 and 67 and mean survival time was
112, 102.9, 99.7 days in CaM-HD, Vec-HD and Scr-HD mice, respectively,
suggesting a small (about 10%) increase in life span of CaM-HD mice, Kaplan-Meier
survival curves demonstrated no statistically significant effect of CaM-fragment
expression on the mortality in R6/2 mice (Fig. 18b). By log-rank comparison, the
three groups of HD mice did not significantly differ from each other (p > 0.05). None
of the mice in WT groups died during the observation period (from 6 to 27 weeks of
age).
CaM-fragment expression increases stride length and lowers stride frequency in
R6/2 mice
Gait dynamics were characterized using the DigiGait Imaging System. Generally,
there is a clear difference between WT mice and Vec-HD or Scr-HD mice starting
from week 11 in all the parameters measured, whereas the difference between WT
mice and CaM-HD mice is not significant in stride length and frequency. At a speed
of 14 cm/s, WT mice maintained a regular alternating gait at a consistent stride length
of 5.8±0.3 cm and stride frequency of 2.5±0.1 steps/sec, whereas Vec-HD and Scr-HD
mice walked at a gradually reduced stride length and higher stride frequency. For
stride length (Fig. 19a), the two-way ANOVA with repeated measures indicated a
significant main effect of group [F(4,126)= 35.77; p<0.0001] and week [F(3,126)= 12.12;
p<0.0001]. There was also a significant interaction between group and week [F(12,126)=
5.71; p<0.0001]. The post hoc Bonferroni test indicated that stride length of CaM-HD
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mice is indistinguishable from the two HD control groups at 10 weeks of age, but
CaM-HD mice displayed a longer stride length as compared to Vec-HD or Scr-HD
mice at week 11~13. Stride length of CaM-HD mice was not significantly shorter than
WT mice until at week 13. For stride frequency (Fig. 19b), two-way ANOVA
indicated significant differences among groups [F(4,126)=11.99; p<0.0001], but there is
no significant effect of week [F(3,126)= 0.202; p=0.895] or interaction between group
and week [F(12,126)= 1.368; P=0.190]. The Bonferroni test indicated that CaM-HD
mice had a significantly lower stride frequency than Vec-HD at week 11-13. Although
Vec-HD and Scr-HD were statistically indistinguishable at any time point, there was a
statistically significant difference between CaM-HD and Scr-HD at week 12. There
was no significant difference between CaM-HD and WT mice in stride frequency.
Analysis of some other gait dynamics revealed differences between genotypes. For
example, the measure of stance width variation between steps was greater (Fig. 19c)
and paw area at peak stance was smaller (Fig. 19d) in HD mice than in WT mice, but
the CaM-fragment had no beneficial effects on these gait indices. For stance width
variation, two-way ANOVA followed by Bonferroni test indicated significant
differences between HD and WT groups [F(4,126)=13.61; p<0.0001], but no significant
differences among HD groups or WT groups (P>0.05). There is no significant effect
of week [F(3,126)= 1.21; p=0.31] although interaction between group and week is
significant [F(12,126)= 1.97; P=0.03]. Similar results were obtained in the analysis of
paw area at peak stance [Group factor: F(4,126)=10.42; p<0.0001], whereas week effect
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is significant [F(3,126)= 13.68; p<0.0001] and interaction between group and week is
not [F(12,126)= 1.55; P=0.11].
CaM-HD mice had fewer low mobility bouts and longer travel distance than
Scr-HD or Vec-HD mice
Force-plate actometers were used to measure the locomotor activity of R6/2 and WT
mice. The number of low mobility bouts (LMB, defined as remaining continuously in
a virtual circle of 15 mm radius for 10 sec) is presented in Fig. 20a. Two-way ANOVA
with repeated measures indicated a significant effect of group [F(4,144) =27.75, p <
0.001] and a significant effect of week [F(3, 144) = 3.179, p < 0.05], but the interaction
between group and week was not significant. The number of LMB gradually
increased in HD mice without CaM-fragment expression, whereas CaM-HD mice,
similar to WT mice, maintained LMB at a relatively lower level. At week 12 and 13,
the Bonferroni post hoc test detected significantly fewer LMB in CaM-HD mice as
compared to either Scr-HD or Vec-HD mice. Total travel distance in a 30 min session
provided another index of locomotor activity of HD mice (Fig. 20b). Two-way
ANOVA with repeated measures indicated a significant effect of group [F(4,138) = 3.40,
p = 0.02], but the effect of week [F(3,138) = 0.17, p=0.92] and interaction between
group and week [F(12,138) =1.12, p=0.35] were not significant. Consistent with reduced
LMB in HD mice treated with CaM-fragment, CaM-fragment expression also
increased the locomotor activity in HD mice as indicated by a significantly greater
travel distance in CaM-HD mice than in either Scr-HD (at week 12,13) or Vec-HD
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mice (at week 12). There was no significant difference between CaM-HD and WT
mice in distance traveled in 30 min.
CaM-fragment improved performance on the rotarod in R6/2 mice
Rotarod was used to measure fore- and hindlimb motor coordination and balance
starting at 10 weeks of age (i.e., three weeks after the AAV injections). At ten weeks
of age, the two groups of WT mice were able to stay on the rod during the 300 sec
time period measured, whereas none of the three groups of HD mice could finish the
test without falling in 300 sec. Even at this early time point after injection of the AAV,
the mean latency to fall was significantly differed among the HD groups, 245.9±32.0,
150.2±29.5, 98.1±19.8 sec in CaM-HD, Vec-HD and Scr-HD mice, respectively. After
10 weeks of age, there was a progressive decline in performance of all HD mice, but
the CaM-HD mice still achieved better performance than the other two HD groups
(Fig 21). Using analysis using two-way ANOVA with repeated measurements, we
found a significant main effect of group [F(4,188) = 49.35, p < 0.0001], a significant
main effect of week [F(4,188) = 18.86, p < 0.0001] and a significant interaction between
group and week [F(16,188) = 4.714, p < 0.0001] on latency to fall off the rotarod.
Bonferroni post hoc test indicated that CaM-HD mice had longer latency to fall as
compared to Scr-HD mice at 10-14 weeks of age and Vec-HD mice at 10-11 weeks of
age (p < 0.05).
CaM-fragment expression reduced TGase-modified htt in R6/2 mice striatum
Cell culture studies indicated that AAV-mediated CaM-fragment expression can
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significantly inhibit the formation of TGase-modified htt (Dudek et al., 2009). Here
we used striatal homogenates from HD or WT mice to detect TGase-catalyzed
cross-linking of htt. Immunopurification of proteins containing є-(γ-glutamyl) lysine
bonds was performed using 81D4 mAb prebound to Sepharose beads.
TGase-modified htt was then detected on immunoblots as shown in the Fig. 22a.
TGase-modified htt was only detected in HD but not WT mice. One-way ANOVA
[F(2,15) =18.44, p<0.0001] followed by Bonferroni's comparison test found there was a
significant reduction (by approximate 50%) in TGase-modified htt in the striatum of
CaM-HD mice as compared to Scr-HD or Vec-HD mice (Fig. 22b).
CaM-fragment expression decreases the percentage of htt-positive nuclei and the
size of htt aggregates in the striatum of R6/2 mice
Striatal intranuclear and neuropil inclusions containing mutant htt are prominent
neuropathological hallmarks of HD and may play an important role in disease
progression. In R6/2 mice, intranuclear aggregates are much larger than neuropil
aggregates and amenable to quantification, so we examined the effect of
CaM-fragment on intranuclear htt aggregates in the striatum of R6/2 mice. In
CaM-HD mice, the percentage of htt-positive nuclei was lower and the size of
intranuclear aggregates appeared smaller than in the control virus-treated HD mice
(Fig. 23a). These observations were confirmed by quantification of nuclear aggregates
(Table 1). We found a significant reduction in the percentage of striatal nuclei
containing htt-aggregates (approximately 18%) and the nuclear aggregate size
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(approximately 35%) in CaM-HD mice as compared with either Vec- or Scr-HD mice.
GFP fluorescence was observed in coronal brain sections from AAV-injected mice,
indicating a widespread infection and expression of CaM-fragment in the striatum
(Fig. 23a). Both human HD and R6/2 mouse brains are characterized by atrophy of
the striatum. To exam whether the CaM-fragment delivery decreases gross striatal
atrophy, we used Nissl staining and the Cavalieri principle to estimate striatal volumes.
There was no statistically significant difference in mean striatal volumes between
CaM-HD and the two groups of control HD mice. However, all three groups of HD
mice had significantly smaller striatal volumes as compared with WT littermates (Fig.
23b, Table 1). As expected, striatal neuronal atrophy was observed in HD mice in
high-power images.
CaM Kinase II activity was not affected by CaM-fragment expression
To test if expression of the CaM-fragment would interfere with other CaM-dependent
enzymes, we measured the activity of calmodulin-dependent protein kinase II (CaM
kinase II) in mouse striatal homogenates. We found no significant differences in CaM
kinase II activity among the various HD or WT mouse groups in either the presence or
absence of EGTA, a calcium chelator (Fig. 24a). However, EGTA caused a general
decrease in CaM kinase II activity in all mouse groups. Two-way ANOVA indicated a
significant main effect of EGTA [F(1,50)=72.12; p<0.0001, but there was no significant
effect of mouse group [F(4,50)=0.543; p=0.705] or an interaction between EGTA and
group [F(4,50)=0.966; p=0.435].
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CaM-fragment did not change total TGase activity
In order to explore whether the reduced TGase-modified htt in CaM-HD mice is due
to a substrate-dependent inhibition or a general decrease in TGase enzymatic activity,
we compared total TGase activity in the striatum among the different groups of mice.
There is a significant increase in TGase activity in the three groups of HD mice as
compared to CaM-WT or Vec-WT mice (p<0.01). Expression of the CaM-fragment
did not have an effect on TGase activity in either HD or WT mice. In the presence of
GTPγS, which specifically inhibits TGase activity, TGase activity is dramatically
reduced (at least 60%) in all groups (Fig. 24b). Two-way ANOVA indicated a
significant main effect of GTPγS [F(1,50)=428.149; p<0.0001] and mouse group
[F(4,50)= 5.535; p<0.01], and there was a significant interaction between GTPγS and
mouse group [F(4,50)= 5.032; p<0.01]. The activity of guinea pig liver TGase was
measured in each assay in the range from 0.01 to 0.5 milliunits/ml and resulted in a
linear correlation with an R-square value from 0.95 to 0.99. The activity of TGase in
the striatal samples fell within this linear range for guinea pig liver TGase.
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Fig.18 Effect of CaM-fragment on body weight and survival. (a) Changes in body
weight were expressed as a percentage of body weight measured at 7 week of age.
One-way ANOVA and Bonferroni's test found that the change in body weight was
significantly smaller in CaM-HD than in Vec-HD (p< 0.05) starting from week 12,
and there is no significant difference between Scr-HD and Vec-HD. * indicates p<0.05
(CaM-HD vs. Vec-HD). (b) Kaplan-Meier survival curves showed the first death was
at day 88, 78 and 67 in CaM-HD, Vec-HD and Scr-HD mice, respectively. By
log-rank comparison, three groups of HD mice did not differ from each other (p>0.05).
(n=9~14 in each group)
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Fig.19 Video-based gait analysis on the treadmill. Mice were placed on a treadmill
belt moving at a speed of 14 cm/s. Gait data were pooled from all four paws.
CaM-HD exhibited a significantly greater stride length (a), and a lower stride
frequency (b). There is no significant difference between CaM-HD and Vec-HD or
Scr-HD in stance width variability (c) and paw area at peak stance (d). Two-way
ANOVA with repeated measures followed by Bonferroni test. * indicates p<0.05
(CaM-HD vs. Vec-HD), # indicates p<0.05 (CaM-HD vs. Scr-HD), n=6-14 in each
group
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Fig.20 Expression of CaM-fragment enhanced the locomotor activity in R6/2 mice.
Spontaneous locomotor activities of R6/2 and WT control mice were recorded in a
force-plate actometer apparatus for 30 min. (a) CaM-HD mice displayed a
significantly fewer number of low mobility bouts than the two HD control groups at
week 12-13. (b) Distance traveled by CaM-HD mice was significantly longer than
Scr-HD at week 12-13 and Vec-HD at week 12. Two-way ANOVA with repeated
measures followed by Bonferroni test. * indicates p<0.05 (CaM-HD vs. Vec-HD), #
indicates p<0.05 (CaM-HD vs. Scr-HD), n=9-14 in each group
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Fig.21 CaM-fragment expression delayed the onset of the rotarod defects in R6/2
mice. Mice were placed on a rotating rod with increasing speed, from 4 rpm to 40 rpm
in 300 seconds. The latency to fall off the rotarod within this time period was
recorded. Two-way ANOVA with repeated measures followed by Bonferroni test
found that CaM-HD mice had significantly longer latency to fall than Scr-HD starting
from week 10, and than Vec-HD at week 10-11. * indicates p<0.05 (CaM-HD vs.
Vec-HD), # indicates p<0.05 (CaM-HD vs. Scr-HD), n=9-14 in each group
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Fig.22 TGase-modified htt in R6/2 mice striatum was reduced by CaM-fragment
expression. (a) The insoluble fraction from mouse striatal homogenates was dissolved
with formic acid. Immunopurification (IP) of proteins containing є-(γ-glutamyl)
lysine bonds was performed using 81D4 mAb prebound to Sepharose beads.
Immunopurified proteins were then examine on immunoblots (IB) using an antibody
against htt. (b) Quantitation of immunoblots. Data shown are the mean IOD± S.E.M.,
and they are normalized to CaM-HD mice. * indicates p<0.05 as compared to
CaM-HD (n=5 in each group at 13-14 weeks of age).
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Fig. 23 Histological evaluation of neuropathology (a) Top and middle,
immunofluorescent labeling of striatum in R6/2 mice at 14 weeks of age.
Htt-aggregates were labeled with htt antibody MAB5374 (red), the nuclei were
labeled with DAPI (blue). The composite images show that the percentage of
htt-positive nuclei and the size of nuclear htt-aggregates were decreased in CaM-HD
as compared to the control-HD mice. Scale bar=30µm. Bottom, photomicrographs of
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GFP protein distribution in the brain of representative AAV-injected animals indicated
that the same vector-derived CaM-fragment was expressed in the striatum. Scale
bar=1 mm. (b) Top , montage images of Nissl-stained brain coronal sections from
CaM-HD, Vec-HD and CaM-WT mice at the level when the corpus callosum starts to
merge in the middle. Scale bar=1 mm. Bottom, high magnification of micrograph of
the dorsomedial aspect of the striatum from the sections above. There is marked
neuronal atrophy with small angulated neurons in Vec-HD mouse, with relative
preservation of neuronal size in CaM-HD mouse. Scale bar=50 µm.
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Fig. 24 Expression of CaM-fragment in mouse striatum did not significantly affect the
activity CaM kinase II or TGase. (a) Mouse striatal homogenates were used to
measure CaM kinase II activity. Two-way ANOVA indicates a significant main effect
of EGTA. However, there was no significant main effect of mouse group. The
interaction between EGTA and group was also not significant. Bonferroni posttest
indicates no significant differences in CaM kinase II activity among the various
groups in either the presence or absence of EGTA. (b) The presence of the
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CaM-fragment did not significantly change the levels of TGase activity in either HD
or WT mice. However, a significant elevation of TGase activity in three groups of HD
mice was observed as compared with WT mice. When 500 µM GTPγS was added,
there is a dramatic reduction in all mouse groups. Two-way ANOVA indicates a
significant main effect of GTPγS and mouse group, and there is a significant
interaction between GTPγS and group. Bonferroni posttest *indicates p<0.01 as
compared to Vec-WT; # indicates p<0.01 as compared to CaM-HD in the absence of
GTPγS. Each column represents the mean ± SEM. (n=5 in each group at 13-14 weeks
of age).
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134
DISCUSSION
Although there have been enormous strides in understanding the molecular
and mechanistic pathways that mediate the progression of HD, an effective treatment
to slow or prevent the disease-associated physical and mental decline has yet to be
discovered. A number of reports in cultured cells have shown that interfering peptides
can selectively inhibit pathological interactions between mutant htt and other proteins,
and therefore are a potential therapeutic strategy for HD (Nagai et al., 2000, Dudek et
al., 2008, 2009). However, only a few studies have been performed to verify the
efficacy of these peptides in vivo (Kazantsev et al., 2002, Tang et al., 2009). We
previously found that plasmid or AAV-mediated delivery of CaM-fragment
significantly attenuated TGase-modified htt, htt-associated cytotoxicity and
intracellular Ca2+ disturbances without interfering with the activity of other
CaM-dependent enzymes in HD cell models (Dudek et al., 2008, 2009). We now
report that expression of the CaM-fragment in the striatum ameliorated body weight
loss, improved motor performance of R6/2 mice, reduced TGase-modified htt and
decreased the number and size of nuclear htt-aggregates in the striatum.
Striatal delivery of CaM-fragment significantly delayed the onset of
movement abnormalities and improved motor function as measured by analysis of
gait, rotarod performance and locomotor behavior. It has been reported that gait
deficiencies in R6/2 mice became apparent from 8 weeks of age. As visualized in the
footprint analysis, R6/2 mice displayed a significantly shorter stride length, a
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staggering movement and a gait that lacked a uniform step pattern compared to WT
littermates (Carter et al., 1999). Striatal injection of AAV-CaM-fragment conferred
significant improvements in stride length and frequency measurements, and a mild,
insignificant increase in paw area at peak stance in R6/2 mice. Decreased motor
function on the rotarod is the most commonly reported measure among animal models
of HD (Schilling et al., 1999, von Horsten et al., 2003). Three weeks after
CaM-fragment expression, R6/2 mice started to show significantly improved rotarod
performance and this beneficial effect was maintained from 10 weeks of age onward.
Although motor abnormalities such as bradykinesia (abnormally slow movements)
and pronounced hypoactivity were frequently observed in HD animal models
(Mangiarini et al., 1996, Keene et al., 2002), there have been relatively few
quantitative studies on spontaneous locomotor activity of R6/2 mice. In the present
experiments using a force-plate actometer, progressive locomotor deterioration
(assessed by number of low mobility bouts and travel distance) was observed in R6/2
mice injected with control AAV, whereas delivery of AAV expressing CaM-fragment
to R6/2 mice striatum significantly improved their locomotor deficits.
In comparison to the TGase inhibitor cystamine, which prolonged the life span
of R6/2 mice by 12%~19% (Dedeoglu et al., 2002, Karpuj et al., 2002a), there was a
non-significant increase in the mean survival time by approximately 10% in the
CaM-HD mice. Differences in other mechanisms of action could possibly account for
the different outcomes. In addition to inhibiting TGase, cystamine increased
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transcription of neuroprotective genes, increased glutathione levels, and ameliorated
apoptosis (Dedeoglu et al., 2002, Karpuj et al., 2002a). In contrast, that
CaM-fragment decreased TGase-modified htt, normalized intracellular calcium
release, and reduced mutant htt-associated cytotoxicity but had no effect on TGase
activity (Dudek et al., 2008, 2009). Secondly, the different outcomes could be due to
the different brain regions treated. The CaM-fragment was expressed in the striatum
only while the cystamine-treatment was systemically delivered and could affect other
brain regions that display neuropathology in HD such as the frontal cortex. Rather
than unspecific inhibition of TGase by cystamine, disrupting the CaM-htt interaction,
as with the CaM-fragment, is a promising new approach for the treatment HD.
As a result of its expanded N-terminal polyglutamine region, mutant htt is
processed and deposited as a component of insoluble protein aggregates that persist in
neuronal nuclei, perikarya, and processes (DiFiglia et al., 1997). Although some
evidence suggests that htt-associated toxicity is linked to soluble htt and its
protein–protein interactions (Petersen et al., 1999), htt aggregates act as a phenotypic
readout that can reflect pathologically relevant processes such as htt cleavage,
misfolding, and sequestration (Chopra et al., 2007). A number of compounds which
are neuroprotective in R6/2 mice, such as the TGase inhibitor cystamine (Dedeoglu et
al., 2002), the antioxidant coenzyme Q10 (Ferrante et al., 2002) and the energy buffer
creatine (Ferrante et al., 2000), significantly suppress htt aggregates. Our present
results revealed that CaM-fragment significantly reduced TGase-modified htt in the
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insoluble fraction, the percentage of htt-positive nuclei and intranuclear htt-aggregate
size in the striatum, which may contribute to the benefits of CaM-fragment. These
findings support the hypothesis that CaM-regulated TGase modification of htt
contributes to the formation and stabilization of htt-aggregates and may play a role in
the pathogenesis of HD. However, it is difficult to speculate whether there would be
an associated increase in soluble or oligomeric htt in the R6/2 mouse striatum with
CaM-fragment expression. There is evidence that TGase modifications stabilize
proteins (Tucholski et al., 1999), so inhibiting TGase modifications to htt may
increase clearance of the htt protein.
R6/2 mice developed significant striatal atrophy as compared to WT
littermates, a pathological characteristic associated with the gradual
neurodegeneration occurring in the mice. Although there is no significant difference
in striatal volume between CaM-HD and control-HD mice by light microscopy, we
could not exclude the possibility that expression of CaM-fragment would reduce the
striatal atrophy if a more sensitive technology (MR scanning) was applied. Moreover,
if striatal injections of AAV were performed at an earlier age, there might be more
notable beneficial results including increases in survival and striatal volume.
Similar to increases in TGase protein level and enzymatic activity in human
HD brain (Karpuj et al., 1999, Lesort et al., 1999), our results show that TGase
activity is elevated in R6/2 mice compared with WT littermates. Interestingly, striatal
delivery of CaM-fragment did not change the overall activity of TGase in either R6/2
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mice or WT mice. These results are consistent with our previous studies in cell culture
(Dudek et al., 2009), suggesting that CaM-fragment may specifically affect the
CaM-mutant htt interaction and selectively alter TGase-catalyzed modifications of
mutant htt without interfering with the activity of TGase on other substrate proteins.
Furthermore, CaM-fragment expression did not affect CaM kinase II activity,
indicating that CaM-fragment had no effect on the activity of other CaM-dependent
enzymes. Most importantly, expression of CaM-fragment in WT mice had no effect
on any of the behavioral tests nor striatal histopathology, suggesting there is minimal,
if any, deleterious effect of long-term expression of CaM-fragment.
A prominent hypothesis on the pathogenesis of HD is that the aberrant
interactions of the extended polyglutamine repeats (polyQs) of mutant htt, either with
other proteins or with each other result in htt-aggregate formation (Wanker, 2000).
Glutamine repeats may function as polar zippers and expansion of polyQs may
increase the affinity of htt for its protein binding partners (Perutz et al., 1994). The
principal proteins that are known to interact with htt are: htt associated protein 1 (Li et
al., 1995), htt interacting protein (Kalchman et al., 1997), type 1 inositol
1,4,5-trisphosphate receptor (InsP3R1) (Tang et al., 2003) and calmodulin (Bao et al.,
1996, Zainelli et al., 2004). As revealed by our previous in vitro assay (Dudek et al.,
2009), the neuroprotective effects of CaM-fragment may depend on its ability to
competitively bind to mutant htt thereby disrupting the CaM-mutant htt interaction.
First, this disruption may result in the CaM and mutant htt no longer in close
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proximity to each other, thus the TGase activity in htt modification and aggregate
formation may be reduced. Second, the association between mutant htt and
CaM-fragment may prevent the further interaction of mutant htt with other proteins,
such as InsP3R1, an intracellular Ca2+ release channel. Since mutant htt selectively
associates with and activates InsP3R1(Tang et al., 2003), disrupting their interaction
should stabilize neuronal Ca2+ homeostasis. This hypothesis is supported by in vitro
experiments that indicated the CaM-fragment (Dudek et al., 2008) or
InsP3R-fragment (Tang et al., 2009) attenuates mutant htt-associated intracellular
Ca2+ disturbances.
Although it will be advantageous to confirm the results from the current study
using a different HD animal model, special care should be taken to choose a different
strain other than R6/2 mice. Instead of body weight loss, YAC128 mice have
significantly (27%) increased body weight at 12 months of age (Van Raamsdonk et al.,
2006, Van Raamsdonk et al., 2007), which in turn can cause bias in most behavioral
tests. Obvious or diffuse nuclear htt accumulation is not detectable in the striatum or
cortices of BAC HD mice even in 18-month-old mice (Gray et al., 2008).
Intergenerational instability and somatic instability of the HD CAG repeat have been
observed in HdhQ111 knock-in mice in all three background strains: C57BL/6, FVB/N
and 129Sv (Lloret et al., 2006). The phenotype of N171-82Q mice is more variable
than that of R6/2 mice, and therefore a much larger number of mice are necessary to
provide adequate power (Hersch and Ferrante, 2004).
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Many therapeutic targets for HD today are based on the pathogenesis
hypotheses, from mitochondrial dysfunction to transcriptional perturbation to TGase
hyperactivity (Beal and Ferrante, 2004). However, since clinical trials of
mechanism-based therapies for HD have been limited by insufficient statistical power
and insignificant improvement (Handley et al., 2006), current clinical treatments are
symptomatic. The major neurological symptoms associated with HD include
disordered voluntary movements: uncoordinated, arrhythmic, and slow fine motor
movements; rigidity; and gait disturbances (Harper, 1991). Our in vivo results
demonstrated that viral delivery of CaM-fragment to the striatum of R6/2 mice
significantly improved motor coordination and balance, enhance locomotor activity
and alleviated gait disturbances, suggesting the possibility of a better quality of life
for HD patients if this new therapeutic strategy is successfully translated to the
clinical trials.
The present studies provide, for the first time, in vivo evidence that
CaM-fragment has significant efficacy in improving the behavioral deficits and
neuropathological phenotype in the HD animal model. The positive effects of
CaM-fragment in R6/2 mice provide further evidence that CaM-regulated TGase
modification of htt may contribute to HD pathogenesis. More importantly, these
studies have identified a novel therapeutic strategy for treating HD patients.
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141
CHAPTER FIVE
GENERAL CONCLUSION
REVIEW OF RESULTS AND SIGNIFICANCE
In chapter two, we identified a novel effector mechanism attributed to 5-HT2A
receptor signaling. This chapter described the effect of 5-HT2A agonists (5-HT or DOI)
on transamidation and activation of Rac1 in a cell line derived from rat embryonic
cortex. The transamidation is mediated by TGase, as suggested by a TGase inhibitor,
cystamine or knockdown of TGase with siRNA. The transamidation of Rac1
contributed to its transient activation, since TGase inhibition significantly reduced
both transamidation and activation. Moreover, serotonin was identified as an amine
that becomes transamidated to Rac1 by TGase.
TGase-catalyzed transamidation of small G proteins not only occurred in
platelets, vascular smooth muscle cells and cells derived from cervical cancer (Singh
et al., 2001, Walther et al., 2003, Guilluy et al., 2007), but also occurred in neuronal
cell lines, such as SH-SY5Y neuroblastoma cell line (Singh et al., 2003) as well as
A1A1v cell line derived from rat embryonic cortex (Dai et al., 2008). One of the
consequences of small G protein transamidation was constitutive activation. Members
of the Rho family, particularly Rac1, are associated with a wide array of cellular
142
processes such as cytoskeletal organization, vesicular transport, cell cycle progression,
cell adhesion and migration (Etienne-Manneville and Hall, 2002, Burridge and
Wennerberg, 2004) and pathological conditions such as Salmonella invasion, tumor
cell migration, and retinal degeneration (Bourguignon et al., 2000, Belmonte et al.,
2006, Brown et al., 2007). The physiological functions of TGase-catalyzed
transamidation and activation of small G proteins in the nervous system remains
poorly understood. Recent studies have demonstrated that activation of TGase and
transamidation of RhoA to putrescine induced by retinoic acid in SH-SY5Y cells,
results in activation of ERK1/2, JNK1, and p38 MAP kinases, which may stimulate
transcription factors and induce gene expression for neuronal differentiation, as
indicated by neurite outgrowth and neuronal marker expression (Singh et al., 2003).
In addition to small G proteins, cytoskeletal proteins such as intermediate filaments in
neurons, have also been shown to undergo TGase-catalyzed transamidation in vitro
(Selkoe et al., 1982, Miller and Anderton, 1986) and in human cerebral cortex
(Norlund et al., 1999). We demonstrate that stimulation of 5-HT2A receptors can
increase the transamidation of Rac1 in both undifferentiated and differentiated A1A1v
cells, suggesting that transamidation and constitutive activation of this signal
transducer are not altered by neuronal differentiation of cells. The functional
consequences of transamidation of Rac1 by TGase especially in neurons needs to be
further explored.
The signaling pathways involved in Rac1 transamidation have been
143
investigated in chapter three. The different components in the potential signaling
pathways have been targeted by the antagonists and /or agonists. We found inhibition
PLC decreased intracellular Ca2+ concentration as well as TGase-modified Rac1 upon
5-HT2A receptor activation. The crucial role of Ca2+ signaling in Rac1 transamidation
was confirmed by manipulation of intracellular Ca2+ by means of a Ca2+ chelating
agent or an ionophore. When cells were pretreated with a Ca2+ chelator,
TGase-catalyzed Rac1 transamidation was significantly reduced. Conversely,
treatment with a Ca2+ ionophore was sufficient to induce an increase in Rac1
transamidation by TGase. Moreover, the transamidation reaction was also regulated
by CaM, a Ca2+ binding protein, as demonstrated by a CaM antagonist which
depresses the stimulatory effect of 5-HT2A receptor activation on Rac1 transamidation.
Searching the Conserved Domain Database revealed that two glutamine residues (Q61,
Q74) and three lysine residues (K5, K16, and K116) are located at the activity-related
domains. These results provided new insights into the signaling mechanisms
underlying 5-HT2A receptor-induced Rac1 transamidation and activation by TGase in
A1A1v cells. The present study indicated that 5-HT2A receptor-coupled PLC
activation and subsequent Ca2+/CaM signaling are necessary in TGase-catalyzed Rac1
transamidation, and identified potential TGase-targeting residues that could render
Rac1 constitutively active upon transamidation.
In the studies presented in chapter four, we shifted our focus from Rac1 to
mutant htt as another substrate of TGase-catalyzed transamidation reaction. Our
144
previous studies in the cell models of HD screened various CaM fragments based on
their ability to decrease TGase-modified htt and cytotoxicity, as well as normalize
intracellular Ca2+ release. In this study, an AAV that expresses a CaM fragment was
delivered into the striatum of HD transgenic R6/2 mice. Ameliorated body weight loss
and improved motor performance were observed in those mice as compared to control
virus-injected HD mice. CaM-fragment expression also specifically reduced
TGase-modified htt, the percentage of htt-positive nuclei and the size of intranuclear
htt aggregates in the striatum.
Although the life span of CaM-fragment treated HD mice was not
significantly prolonged (there was a non-significant increase in the mean survival
time by ~10%), our results demonstrated that CaM-fragment expression in the
striatum of HD mice significantly improved motor coordination and balance, enhance
locomotor activity and alleviated gait disturbances. The gene mutation that causes
HD was identified in 1993 (Huntington’s Disease Collaborative Research Group,
1993), but there is currently no effective therapy and the disease inevitably leads to
death within 10 to 15 years of symptom onset (Bates, 2003). Medications are
available to help manage the signs and symptoms associated with HD, but will not
stop the progressive physical and mental decline. AAV-mediated gene therapy for HD
by preventing mutant htt expression or interupting its interaction with other proteins
has emerged as an attractive new strategy. The AAV-mediated knock-down of htt via
bilateral injections into the striatum of HD transgenic mice resulted in a reduction in
145
neuronal intranuclear inclusions, and improvements on behavioral phenotypes and
histological deficits (Harper et al., 2005, Rodriguez-Lebron et al., 2005). Disruption
interaction mutant htt with either CaM (Dai et al., 2009) or type 1 IP3 receptor (Tang
et al., 2009) by AAV expressing competitive fragments alleviated motor deficits and
improved neuropathological abnormalities. Our results demonstrate the importance of
CaM-htt association for HD pathogenesis and suggested that CaM fragment is a
potential therapeutic agent for HD.
According to our in vivo studies, wild-type mice injected with the AAV
expressing CaM-fragment did not differ from those injected with AAV vector in terms
of body weight, any of the behavioral tests, striatal histopathology, nor CaM KII or
TGase enzyme activity up to 29 weeks old, suggesting there are minimal, if any,
deleterious effects of long term expression of CaM-fragment. The CaM-fragment
might not be able to disrupt the binding of CaM to other proteins to inhibit other
biologic functions. The CaM target database
(http://calcium.uhnres.utoronto.ca/ctdb/flash.htm) provides a program that searches
for possible CaM-binding motifs in a protein sequence (Yap et al., 2000). The
database currently includes the CaM-binding sites identified in over 300 proteins. No
putative binding sites were located in the sequence of CaM-fragment suggesting that
the interruption of binding of CaM to mutant huntingtin protein is selective.
LIMITATIONS OF THE PRESENT STUDIES
There are certain limitations that should be noted when interpreting the data of
146
present studies.
We did not have direct evidence to establish a causal relationship between
Rac1 transamidation and activation. Based on the facts that inhibition of TGase
caused decreases in both Rac1 transamidation and activation, and that two glutamine
residues (Gln61, Gln74) located in Rac1 activity-related domain are potential targets
of transglutaminase, we are only able to conclude that 5-HT2A receptor stimulation
increased TGase-catalyzed transamidation of Rac1, which may contribute to the
increase in Rac1 activity. Although retinoic acid promoted activation of RhoA by
TGase-catalyzed transamidation in neuroblastoma SH-SY5Y cells (Singh et al., 2003),
transamidation-independent activation of Rac1 was observed in SH-SY5Y cells
stimulated with retinoic acid (Pan et al., 2005). Further experiments could be carried
out to explore if other mechanisms are also involved in 5-HT2A receptor-mediated
Rac1 activation in A1A1v cells.
Although the TGase inhibitor cystamine decreased the serotonin-associated
Rac1 in co-immunoprecipitation experiments, we could not confirm a direct
association between Rac1 and serotonin via a TGase-catalyzed covalent bond.
Another common binding partner could link serotonin to Rac1. However, this
possibility is undermined by the fact that serotonin-associated Rac1 did not show a
significant upward shift on immunoblots compared with native Rac1 in cell lysates,
indicating that the molecular weight of serotonin-associated Rac1 does not
significantly increase. Therefore, the binding partner, if it exists, should also be a
147
small molecule likely an amine.
The attempt to replicate 5-HT2A receptor-mediated Rac1 transamidation in rat
cortex failed. In Sprague Dawley rats that were given subcutaneously injections of
DOI (2.5 mg/kg) and sacrificed 5 or 15 minutes later, there is no significant increase
in TGase-modified Rac1 in the frontal cortex compared to saline-treated rats. The
discrepancy between cell-based and in vivo studies could be attributed to the
following possibilities. The DOI dosage and route of administration in rats may not be
able to mimic the circumstances under which 5-HT2A receptor stimulation induces
Rac1 transamidation in A1A1v cells. Moreover, the time course of reactions for
DOI-mediated Rac1 transamidation may be different between cultured cells and
whole animals. Lastly, the cultured cells grow in an environment that lack
intercellular communication or neurotransmitter cross-talk, which differs from the
normal surrounding milieu presenting in the brain.
In our studies that evaluated protective effects of CaM-fragment in R6/2
mice, immunofluorescence staining demonstrated that the percentage of htt-positive
nuclei and the size of intranuclear htt aggregates in the striatum were reduced by
CaM-fragment expression. Although TGase-modified htt in the striatal homogenates
was decreased in CaM-expressing HD mice as compared to the other two groups of
control HD mice (as indicated in immunoprecipitation and immunoblotting
experiments), we were unable to confirm that the reduction of htt aggregates was due
to decreased TGase-modification of htt. Our original experimental design that aimed
148
to measure the colocalization of htt aggregates with TGase-modified covalent bonds
could help to test this hypothesis. However, the only commercially available antibody
directed against TGase-modified covalent bonds (81D4) is raised in mouse. High
levels of background staining were observed with the 81D4 antibody in R6/2 mouse
tissues, likely due to the binding of secondary anti-mouse antibody to endogenous
mouse tissue Igs and other components. This technical difficulty compromised our
ability to explore the role of TGase-modified covalent bonds in the formation of htt
aggregation in HD mice.
Our previous in vitro experiment indicated that CaM-fragment can directly
inhibit the binding of mutant htt and CaM (Dudek et al., 2009). In this in vivo study,
since the striatum of age matched HD mice were used in immunofluorescence
staining, Nissil staining, immunoprecipitation, TGase and CaM activity assay, we did
not have enough striatal homogenates to confirm the hypothesis that striatal
CaM-fragment expression disrupted mutant htt-CaM association in vivo. The other
experiment that could support the hypothesis that the CaM-fragment can directly
disrupt the interaction between mutant htt and CaM is co-immunoprecipitation to
demonstrate the binding of CaM-fragment to mutant htt. However, we have not been
able to find a specific antibody directed against this CaM-fragment. Moreover, due to
the small size of the CaM-fragment (consisting of 46 amino acids and weighing
5,158.6 daltons), detection of the proteins on immunoblots was difficult.
As for the timing of the delivery of AAV expressing CaM-fragment, our
149
original plan was to inject AAV to R6/2 mice at a much younger age, but the youngest
mice we could purchase from Jackson Laboratories were at 6 weeks of age due to the
genotyping process. Therefore, we chose the new R6/2 strain
(B6CBA-Tg(HDexon1)62Gpb/3J) which has fewer CAG repeats in exon 1 (100-115
CAG repeats) over the previous strain (B6CBA-Tg(HDexon1)62Gpb/ 1J) with
154-159 CAG repeats. The new strain exhibited a concomitant decrease in severity
and delayed onset in the expected neurological phenotype (Jackson Laboratories,
http://jaxmice.jax.org/strain/002810.html), which actually gave CaM fragment more
time to perform its function. However, if striatal injection of AAV was performed at a
younger age, we might have more beneficial results including significant increases in
survival and striatal volume.
FUTURE STUDIES: DISRUPTING CAM-HTT INTERACTION AS A
POTENTIAL THERAPEUTIC STRATEGY IN HUMANS
Currently there is no cure for HD. On August 15, 2008 the U.S. Food and
Drug Administration (FDA) approved the use of tetrabenazine to treat Huntington’s
chorea (the involuntary writhing movements), making it the first drug approved for
use in the United States to treat the disease. Tetrabenazine works mainly as a
Vesicular Monoamine Transporter (VMAT)-inhibitor (Zheng et al., 2006) and thereby
increases the early metabolic degradation of dopamine. Other agents targeting
dopamine system have also been used to ameliorate chorea and treat irritability,
hallucinations and delusions. Those agents include dopamine antagonists such as
150
haloperidol, fluphenazine and pimozide (Caine and Shoulson, 1983, Girotti et al.,
1984, Barr et al., 1988).
Our in vivo studies demonstrated that AAV-mediated striatal expression of
CaM-fragment improved motor function, weight loss, and neuropathology in HD
mice. In the long run, to extend the application of CaM-fragment to HD patients, a
less invasive method has to be developed since AAV-mediated delivery of
CaM-fragment by stereotaxic surgery could cause brain injury. The less invasive
approaches include intravenous administration or systemic injection. The delivery of
CaM-fragment to the brain by the intravenous route could be efficient and widespread
to all parts of the brain once the blood-brain barrier (BBB) is traversed. Certain
endogenous large-molecule neuropeptides such as insulin, transferrin, or leptin are
transported into the brain from blood via receptor mediated transport (RMT) across
the BBB (Pardridge, 2003). These transporters are regulated by ligand-specific
receptors such as insulin receptors and transferring receptors, which are highly
expressed on the capillary endothelium of brain (Rip et al., 2009). Molecular Trojan
horses (MTH) are certain monoclonal antibodies (MAbs) or peptide ligands that target
RMT systems (e.g., receptor-binding sequences of insulin) that bind to BBB receptors
and induce receptor mediated transcytosis through the BBB (Pardridge, 2002).
Therefore, the construction of fusion proteins between the CaM-fragment and the
MTH may enable the CaM-fragment to cross the BBB in vivo. It has been reported
that a fusion protein between the insulin receptor mAb and brain-derived neurotrophic
151
factor (BDNF) enables rapid brain penetration of the neurotrophin in animal models,
including nonhuman primates and is intended for treatment of stoke and
neurodegenerative diseases (Boado et al., 2007, Boado et al., 2008, Boado and
Pardridge, 2009).
On the other hand, the identification of the sites on mutant htt that binds to
CaM may also provide an alternative approach to disrupt CaM-htt interaction. A yeast
two-hybrid assay or affinity purification could be used to rapidly screen the sequential
deletions of htt to identify binding domain, and then test their ability to disrupt the
binding of CaM to mutant huntingtin. Next, similar to CaM-fragment (Dudek et al.,
2008, 2009), we could test the ability of these fragments from mutant htt for their
ability to inhibit TGase-catalyzed modifications to mutant htt and for their ability to
increase cell viability in HD cell models. Once the beneficial effects were observed,
we could deliver these fragments to the animal models of HD via different approaches
depending on their molecular size and charge character. The binding domain in
mutant htt could be located at the amino acid sequences upstream or downstream of
the expanded polyglutamine tract or in the middle of the polyglutamine tract. If the
polyglutamine domain is responsible for binding to CaM, the therapeutic strategy that
targets CaM-htt interaction could be applied to the other eight polyglutamine repeat
diseases as well.
CONCLUSIONS
TGase-catalyzed transamidation and activation of the small G protein Rac1 in
152
A1A1v cells is regulated by 5-HT2A receptor signaling and Ca2+/CaM (Figure 25).
Active Rac1 has various physiological functions in neuronal cells, including neurite
outgrowth and neuronal differentiation, via modulating the activity of diverse effector
proteins (Leeuwen et al., 1997, Pan et al., 2005). Pathologically, TGase-modification
of htt is regulated by the association of htt with CaM, which may be involved in the
formation and stabilization of htt-aggregates and play a role in the pathogenesis of
HD. Direct inhibition of TGase or CaM may be able to suppress TGase- and
htt-mediated neuropathological perturbations, but also will interrupt the normal
physiological functions of TGase and CaM because both enzymes regulate a variety
of effectors including kinases and cytoskeletal proteins. Alternatively, specific
disruption of the interaction of CaM with mutant htt, by a competitive
CaM-fragement, is a useful tool to determine the pathogenic mechanisms involved in
HD and can be used to develop new therapeutic strategies for HD (Figure 25). Our in
vivo study demonstrated that striatal expression of the CaM-fragment significantly
improved motor function and neuropathological perturbations in HD mice. Moreover,
TGase and CaM activity were not altered by the CaM-fragment in either HD or WT
mice, suggesting that disrupting CaM-htt interaction is an important new therapeutic
strategy for HD.
153
Figure 25. Regulation of TGase by 5-HT2A receptor signaling and CaM. In A1A1v
cells, upon stimulation of Gq/11 protein-coupled 5-HT2A receptor, the phosphatidyl
inositol pathway was activated, resulting in PLC-mediated hydrolysis of PIP2 to IP3,
which in turn releases Ca2+ from ER stores. A rise of cytoplasmic Ca2+ is sufficient to
activate TGase, which catalyzed the transamidation of Rac1 to bioamines, such as
serotonin transported into the cytoplasm by SERT. CaM, another Ca2+-dependent
enzyme, also contributed to Rac1 transamidation by positive regulation of TGase. In
HD cells, mutant htt associated with CaM at a specific binding domain, and htt were
modified by TGase. This modification may stabilize htt monomers or polymers and
contribute to cytotoxicity and formation of htt-containing aggregates. CaM-fragment
may disrupt CaM-htt interaction by competing with endogenous CaM, resulting in
154
decreased TGase-modified htt and aggregates. Abbreviations: 5-HT2AR, 5-HT2A
receptor; CaM, calmodulin; ER, endoplasmic reticulum; PLC, phospholipase C; PIP2,
phophatidyl inositol-1,4-bisphosphate; IP3, inositol-1,4,5-triphosphate; SERT,
serotonin transporter; TGase, transglutaminase; αq/11, , Gq/11 protein α subunits
155
155
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VITA
Ying Dai was born on July 3, 1978 in Hunan, China to Yiquan Dai and
Zhiyang Song. He attended Nanhua University, China, where he earned his
Bachelor of Medicine degree, with highest distinction in 2001. After graduation, Ying
entered the Master’s Program in the Department of Anatomy and Histology at Jinan
University, China, and joined the laboratory of Dr. Yunquan Liu. His project focused
on cerebral ischemia and reperfusion injury. He completed his Master of Medicine in
July, 2004.
In August of 2004, Ying began to pursue a doctorate of philosophy in
Neuroscience Program at Loyola University Chicago. In 2005, Ying joined the
laboratory of Dr. Nancy A. Muma in the Department of Pharmacology and
Experimental Therapeutics, to study the regulation of transglutaminases by 5-HT2A
receptor signaling and calmodulin. In January of 2007, Ying moved to University of
Kansas with his advisor Dr. Muma, and continued his research at the Department of
Pharmacology and Toxicology.
Following the completion of the doctoral program in Neuroscience at Loyola
University Chicago, Ying will continue to pursue his interest in development of new
therapeutic strategies for neurodegenerative diseases.
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DISSERTATION APPROVAL SHEET
The dissertation submitted by Ying Dai has been read and approved by the following committee: Nancy A. Muma, Ph.D., Director Professor and Chair Department of Pharmacology and Toxicology, University of Kansas George Battaglia, Ph.D. Professor of Pharmacology Department of Pharmacology and Experimental Therapeutics, Loyola University Chicago Thackery Gray, Ph.D. Professor of Anatomy and Associate Dean School of Medicine, American University of the Caribbean Edward J. Neafsey, Ph.D. Professor and Program Director, Neuroscience Program, Loyola University Chicago Karie Scrogin Associate Professor of Pharmacology Department of Pharmacology and Experimental Therapeutics, Loyola University Chicago
The final copies have been examined by the director of the dissertation and the signature which appears below verifies the fact that any necessary changes have been incorporated and that the dissertation is now given final approval by the committee with reference to content and form.
The dissertation is therefore accepted in partial fulfillment of the requirements for the degree of Doctor of Philosophy.
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Date Director’s Signatur