Axonal transport, synapse development and mental retardation

1. Axonal transport, synapse development and mental retardation Yong Q. Zhang Institute of Genetics and Developmental Biology Chinese Academy of Sciences

2. Mental retardation (Intellectual Disability, Cognitive Disorder) a generalized disorder, characterized by significantlyimpaired 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

3. 82 MR genes on the X-chrom. Syndromic MR: black Non-syndromic MR: *grey MR with neuromuscular defects: +grey CUL4B Chiurazzi et al., EJHG, 2008

4. Molecular Functions of MR Genes 3 ✓ 2 1 3 5 Chiurazzi et al., EJHG, 2008

5. Nature 2011 Transcriptional/translational Cell-cycle-related Ros/Rho/PSD95

6. Neuronal functions of MR-related genes 1, Neurogenesis (microcephaly) 2, Neuronal migration (Lissencephaly) 3, Synapse formation and plasticity

7. Neuronal functions of MR-related genes 1, Neurogenesis (microcephaly) 2, Neuronal migration (Lissencephaly) 3, Synapse formation and plasticity

8. Synapse dysgenesis in MR patients Dendritic spines Purpura, Science, 1974

9. Mutations in ACSL4 are associated with non-syndromic X-linked mental retardation ACSL: Acyl-CoA Synthetase Long-chain (C12-20) aa Splicing mutant resulting in truncation Meloni et al., Nat Genet, 2002 Longo et al., J Med Genet, 2003

10. ACSL in Lipid Synthesis and ATP Production Lipid biosynthesis Fatty acid degradation Fatty acid Coenzyme A Fatty acid ACSL Coenzyme A Acyl-CoA ACSL Glycerol-3-P Acyl-CoA Phosphatidic acid (PA) Diacylglycerol (DAG) β-oxidation PC, PE, PS Membrane Components ATP Triacylglycerol (TAG) Lipid droplet Endoplasmic Reticulum Mitochondria Coleman et al., Annu Rev Nutri, 2000

11. ACSL Family ACSL1, 5, 6 ACSL3, 4 { subfamilies The role of ACSL4 in neurodevelopment? The mechanism of how the disease develops? no mouse models

12. ACSL Family: 1, 3, 4, 5, and 6 ACSL1, 5, 6 ACSL3, 4 (fly dAcsl) 50% identity and 67% similarity Zhaohui Wang and colleagues, Hum Mol Genet, 2009

13. Larval Neuromuscular Junctions Nerves and NMJ synapses Muscles NMJ synapses Large, simple and accessible Griffth and Budnik, 2006

14. Axonal accumulation of synaptic vesicle protein CSP in dAcsl mutants WT Khc, Dhc Wild type dAcsl Ubi. rescue Hurd and Saxton, Genetics, 1996 Martin et al, Mol Cell Biol, 1999

15. Distally-Biased Axonal Aggregates of SVs Anterior Posterior Motor Neuron Sensory Neuron A’ A Control OK6>Syt-eGFPChAT OK6>Syt-eGFP B1 dAcslKO/05847 B1’ Motor Neu ChAT B2 B2’ Sens. Neu merge B3 B3' 20 μm ChAT: choline acetyltransferase

16. Accumulation of selective axonal cargos SV protein Dynein Active zone t-SNARE Cell adhesion Mitochondria

17. Retrograde cargos accumulated in dAcsl mutants MVB: Multiple vesicle body . . . . . . . . . . PLB: Prelysosomal body . . Lb: Lamellated body

18. Retrograde cargos accumulated in mutants Late endosome/ lysosome Autophago-some MVBs Lysosome+autophagosome Multiple vesicle body

19. Immunostaining and EM analysis Autophagosome Lysosome Synaptic vesicles dAcsl mutations lead to specific accumulation of retrograde cargoes Why did the axonal aggregates of retrograde cargos form in dAcsl mutants?

20. Live imaging of axonal transport of GFP-tagged synaptic vesicles Flux Velocity Processivity

22. Retrograde transport of synaptic vesicles was impaired in dAcsl mutants

23. Axonal transport of mitochondria was normal in dAcsl mutants

24. dfmr1 regulates axonal transport of mitochondria Anterograde Retrograde A B C time A, B, C, 50 s WT dfmr1 dfmr1 overexpres. 10 mm D E * WT dfmr1 NOE *** FR Time percentage Stop * ** * RR Flux (mito. number/min) *** * * AT RT WT dfmr1 NOE WT dfmr1 NOE RT AT Yao et al., Hum Mol Genet, 2011

25. SV transport defects in dAcsl mutants Mitochondria transport defects in dfmr1 mutants Role of axonal transport in neurodegenerative diseases De Vos et al., Annu Rev Neurosci, 2008 Axonal transport and neurodegenerative disease Chevalier-Larsen and Holzbaur Biochim Biophys Acta, 2006

26. Accumulation of retrograde cargoes and transport defects ? Defects in synaptic development and function

27. Larval Neuromuscular Junctions Nerves and NMJ synapses NMJ synapses Muscles Large, simple and accessible Griffth and Budnik, 2006

28. dAcsl mutants show atrophic synaptic terminals Neuronal but not muscular rescue by human ACSL4

29. dAcsl is required for NMJ growth and stability

30. Pre- and post-synaptic components concomitantly reduced in dystrophic NMJs Futsch/MAP1B active zone protein Postsynaptic scaffold No increased apoptosis

31. Will atrophic NMJs show defective transmission? Spontaneous release of single SVs mEJP: miniature EJPs EJP: Excitatory Junction Potentials

32. dAcsl mutations impaired neurotransmission EJP: Excitatory Junction Potentials mEJP: miniature excitatory junction potentials

33. dAcsl mutations impaired neurotransmission Neuronal but not muscular rescue by human ACSL4

34. Axonal jam and retrograde transport defects NMJ synapse atrophy Reduced neurotransmission Can these phenotypes be corrected by induced expression?

35. Drug-induced tissue specific expression Gal4 Osterwalder et al., PNAS, 2001

36. Aggregates were rescued by induced expression Together with NMJ rescue

37. Summary dAcsl mutants show distally-biased axonal aggregates and impaired retrograde transport of synaptic vesicles dAcsl is required for synaptic growth, stability and neurotransmission SV SV SV SV SV SV Liu et al., J Neurosci., 2011

38. Acknowledgments Zhihua Liu Zhihua Liu, PhD Yan Huang Zhaohui Wang (PI） Yi Zhang (mutants & Ab) Di Chen (mutants) Yan Huang Grants: National Science Foundation of China Chinese Academy of Sciences The Ministry of Science and Technology

40. Acyl-CoA is central to diverse processes

41. Conservation is not restricted K294E P375L Q419R R570S V594D W685@ dACSL-8 dACSL-1 mutations in patients