Axonal transport, synapse developmentand mental retardation

Yong Q. Zhang

Institute of Genetics and Developmental Biology Chinese Academy of Sciences

Mental retardation(Intellectual Disability, Cognitive Disorder)

a generalized disorder, characterized by significantly impaired cognitive functioning and deficits in two or more adaptive behaviors with onset before the age of 18. 1-3% of general population is mentally retarded

from wikipedia.com

82 MR genes on the X-chrom.

Chiurazzi et al., EJHG, 2008

Syndromic MR: black Non-syndromic MR: *greyMR with neuromuscular defects: +grey

CUL4B

Chiurazzi et al., EJHG, 2008

Molecular Functions of MR Genes

1

2

3

35

✓

Transcriptional/translational Cell-cycle-related Ros/Rho/PSD95

Nature 2011

Neuronal functions of MR-related genes

1, Neurogenesis (microcephaly)

2, Neuronal migration (Lissencephaly)

3, Synapse formation and plasticity

Neuronal functions of MR-related genes

1, Neurogenesis (microcephaly)

2, Neuronal migration (Lissencephaly)

3, Synapse formation and plasticity

Synapse dysgenesis in MR patients

Purpura, Science, 1974Dendritic spines

Mutations in ACSL4 are associated with non-syndromic X-linked mental retardation

Meloni et al., Nat Genet, 2002Longo et al., J Med Genet, 2003

aa

Splicing mutant resulting in truncation

ACSL: Acyl-CoA Synthetase Long-chain (C12-20)

ACSL in Lipid Synthesis and ATP Production

Fatty acid

Acyl-CoA

Coenzyme A

Glycerol-3-P

Diacylglycerol (DAG)

Triacylglycerol (TAG)

PC, PE, PS

Lipid droplet

MembraneComponents

Fatty acid

Acyl-CoA

Coenzyme A

β-oxidation

ATP

ACSL

ACSL

Endoplasmic Reticulum Mitochondria

Lipid biosynthesis Fatty acid degradation

Phosphatidic acid (PA)

Coleman et al., Annu Rev Nutri, 2000

The role of ACSL4 in neurodevelopment?

The mechanism of how the disease develops?

ACSL Family

ACSL1, 5, 6 ACSL3, 4

{subfamilies

no mouse models

50% identity and 67% similarity

Zhaohui Wang and colleagues, Hum Mol Genet, 2009

ACSL Family: 1, 3, 4, 5, and 6

ACSL1, 5, 6 ACSL3, 4 (fly dAcsl)

Larval Neuromuscular Junctions

NMJ synapses

Muscles

Large, simple and accessible

Griffth and Budnik, 2006

Nerves and NMJ synapses

Axonal accumulation of synaptic vesicle protein CSP in dAcsl mutants

Wild type

dAcsl

Ubi. rescue

Khc, DhcWT

Hurd and Saxton, Genetics, 1996Martin et al, Mol Cell Biol, 1999

A Control

B1 dAcslKO/05847

B2

OK6>Syt-eGFP ChAT

B3

B2’

B3'

B1’

A’

OK6>Syt-eGFP

ChAT

merge

20 μm

Distally-Biased Axonal Aggregates of SVs

ChAT: choline acetyltransferase

Motor Neu

Sens. Neu

Anterior Posterior

Sensory NeuronMotor Neuron

SV protein

Dynein

Active zone

t-SNARE

Mitochondria

Cell adhesion

Accumulation of selective axonal cargos

Retrograde cargos accumulated in dAcsl mutants

MVB:Multiple vesicle body

PLB:Prelysosomal body

Lb:Lamellated body

.

..

..

..

..

.

..

Late endosome/lysosome

Autophago-some

MVBs

Retrograde cargos accumulated in mutants

Lysosome+autophagosome Multiple vesicle body

dAcsl mutations lead to specific accumulation of retrograde cargoes

Synaptic vesiclesAutophagosomeLysosome

Immunostaining and EM analysis

Why did the axonal aggregates of retrograde cargos form in dAcsl mutants?

Live imaging of axonal transport of GFP-tagged synaptic vesicles

Flux

Velocity

Processivity

Retrograde transport of synaptic vesicles was impaired in dAcsl mutants

Axonal transport of mitochondria was normal in dAcsl mutants

FR

WT dfmr1 NOE WT dfmr1 NOE

Tim

e p

erce

nta

ge

*

**

*

*

AT RT

Stop

RR

E

10 m

CA B

time

Anterograde Retrograde

50 s

C,A, B,

AT RT

***

***

Flu

x (m

ito.

nu

mb

er/m

in)

*

*

WT

dfmr1

NOE

D

WT dfmr1 dfmr1 overexpres.

Yao et al., Hum Mol Genet, 2011

dfmr1 regulates axonal transport of mitochondria

Role of axonal transport in neurodegenerative diseasesDe Vos et al., Annu Rev Neurosci, 2008

Axonal transport and neurodegenerative diseaseChevalier-Larsen and HolzbaurBiochim Biophys Acta, 2006

SV transport defects in dAcsl mutants

Mitochondria transport defects in dfmr1 mutants

Accumulation of retrograde cargoesand transport defects

?

Defects in synaptic development and function

Larval Neuromuscular Junctions

NMJ synapses

MusclesLarge, simple and accessible

Griffth and Budnik, 2006

Nerves and NMJ synapses

dAcsl mutants show atrophic synaptic terminals

Neuronal but not muscular rescue by human ACSL4

dAcsl is required for NMJ growth and stability

Pre- and post-synaptic components concomitantly reduced in dystrophic NMJs

No increased apoptosis

Futsch/MAP1B active zone protein Postsynaptic scaffold

CNS

7 6

NMJ

Nerve stimulation

Vm

I Postsynaptic current (EJC)

Nerve

Will atrophic NMJs show defective transmission?

mEJP: miniature EJPs

Spontaneous release of single SVs

EJP:Excitatory Junction Potentials

dAcsl mutations impaired neurotransmissionEJP: Excitatory Junction Potentials

mEJP: miniature excitatory junction potentials

dAcsl mutations impaired neurotransmission

Neuronal but not muscular rescue by human ACSL4

Axonal jam and retrograde transport defects

NMJ synapse atrophy

Reduced neurotransmission

Can these phenotypes be corrected by induced expression?

Drug-induced tissue specific expression

Osterwalder et al., PNAS, 2001

Gal4

Aggregates were rescued by induced expression

Together with NMJ rescue

dAcsl mutants show distally-biased axonal aggregates and impaired retrograde transport of synaptic vesicles

dAcsl is required for synaptic growth, stability

and neurotransmission

Summary

Liu et al., J Neurosci., 2011

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AcknowledgmentsZhihua Liu, PhD

Yan Huang

Zhaohui Wang (PI）

Yi Zhang (mutants & Ab)

Di Chen (mutants)

Zhihua Liu

Yan Huang

Grants:

National Science Foundation of China

Chinese Academy of Sciences

The Ministry of Science and Technology

Acyl-CoA is central to diverse processes

K294E

Q419R

W685@

R570S

P375L

V594D

dACSL-8 dACSL-1 mutations in patients

Conservation is not restricted