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Adhesion complexes at CNS synapses

Regulation of synaptic adhesion complexes by alternative splicing Peter Scheiffele, Columbia University, NY. Adhesion complexes at CNS synapses. adhesion molecules represent one of the most important morphogenic determinants in all tissues

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Adhesion complexes at CNS synapses

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  1. Regulation of synaptic adhesion complexesby alternative splicingPeter Scheiffele, Columbia University, NY

  2. Adhesion complexes at CNS synapses • adhesion molecules represent one of the most important morphogenic determinants in all tissues • central regulators for cell polarization, migration, and for three-dimensional organization • cell adhesion complexes are dynamically regulated to alter cellular architecture and function

  3. Adhesion complexes mediate cell-cell interactions at multiple synaptic sub-domains • peri-active zones • synaptic growth • endocytic zone • active zone / • postsynaptic density • synaptic transmission • alignment & recruitment presynaptic terminal Nectins Cadherins FasII (Still life) Protocadherins Neuroligins • neuron-glia junction • transmitter uptake • signaling • spine morphogenesis EphA4 Glt-1 glia postsynaptic terminal

  4. Potential roles for adhesion molecules in local regulation of connectivity Laminar specificity Cell type specificity Branching, tiling (repulsive self-recognition) Subcellular specificity

  5. 1 2 3 4 5 6 7 a 7 b 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 a 22 b 23 exon4 exon6 exon9 exon17 1 2 3 5 7 8 10 11 12 13 14 15 16 18 19 20 21 22 23 24 TM Variable (1) Constant (1) Single exon genes Variable (1) Constant (1) Alternative splicing is one of the key processes that increases the diversity of cell adhesion molecules apromoter bpromoter Neurexin-1 genomic locus DSCAM genomic locus Protocadherin genomic locus a b g Cadherin-related Neuronal Receptor (CNR) Families of surface molecules encoded by multiple genes: olfactory receptors, classical cadherins, immunoglobulin-domain proteins, leucine-rich repeat proteins

  6. a-neurexin Neurexins Neuroligins b-neurexin • four genes in mouse, five in human • further isoforms are generated by alternative splicing at two sites in the extracellular domain (Ichtchenko et al. 1995, 1996) • neuroligins are postsynaptic adhesion molecules, interact with postsynaptic scaffolding molecules (Irie et al., 1997, Song et al 1999) • inactivating mutations in NL3 and NL4 are associated with autism-spectrum disorders and mental retardation (Jaimain et al. 2003, Laumonnier et al 2004) • three neurexin genes in mice (NRX1,2,3) • alternative promoter choice generates 2 transcripts per gene (a and b NRX) • alternative splicing at 5 sites generates more than 1,000 variants (Ushkaryov et al., 1992)

  7. Adhesion complexes mediate cell-cell interactions at multiple synaptic sub-domains • peri-active zones • synaptic growth • endocytic zone • active zone / • postsynaptic density • synaptic transmission • alignment & recruitment presynaptic terminal Nectins Cadherins FasII (Still life) Neurexins • neuron-glia junction • transmitter uptake • signaling • spine morphogenesis Neuroligins EphA4 Glt-1 glia postsynaptic terminal

  8. Outline • Subcellular localization of neuroligins and neurexins at hippocampal synapses • Analysis of splice isoforms-specific functions • Mechanisms that control alternative splicing

  9. 5 nm gold: pan-neuroligin 10 nm gold: PSD95 Elaine Budreck Ryuichi Shigemoto Freeze-fracture replica immuno-EM Quick-freezing in helium Freeze-fracture and platinum/carbon shadowing SDS digestion and immuno-gold labeling Kazushi Fujimoto, J. Cell Sci., 108:3443-3449, 1995

  10. Neuroligins are closely associated with neurotransmitter receptors in the postsynaptic membrane 10 nm gold: GABA-A R beta 2 subunit 5 nm gold: neuroligin-2 Elaine Budreck, Ryuichi Shigemoto

  11. GFP-beta-Neurexin-1 mice pan-neurexin antibodies Chiye Aoki Francisco G. Scholl

  12. Model of the synaptic Neuroligin-Neurexin adhesion complex presynaptic: Neurexin postsynaptic: Neuroligin

  13. GPLTKKHTDDLGDNDGAEDE GNRWSNSTK Neurexin and neuroligin genes encode large numbers of splice variants a-NRX b-NRX NL1 NL2

  14. Presynaptic Receptors? 2 2 1 1 Selectivity of neuroligins for glutamatergic and GABAergic synapses Glutamatergic Axon GABAergic Axon Dendrite (Song et al. 1999; Prange et al, 2004; Varoqueaux et al., 2004, Graf et al. 2004)

  15. GPLTKKHTDDLGDNDGAEDE GNRWSNSTK Neuroligin variants generated by alternative splicing Leora Gollan

  16. VGAT : VGlut1 % overlap Puncta/10µm Ben Chih

  17. Alternative splicing controls localization of neuroligin-2 VGAT : VGlut1 Ben Chih, Leora Gollan

  18. NL1B VGlut1 :VGAT Presence of the B insertion is sufficient to prevent activity of neuroligin-2 towards GABAergic axons Ben Chih

  19. Specific Neurexin isoforms? Synaptic selectivity of NL splice variants glutamatergic axon GABAergic axon NL1A NL2A NL1B dendrite insertion A: localization and function at GABAergic contactsinsertion B: localization and function at glutamatergic contacts B insertion is dominant

  20. Neuroligin-1 splice variants differ in their interactions with neurexin-1b isoforms NRXb4(-) NRXb4(+) Fc-control NL Fc NL NX-Fc NL NX-Fc NL1A NL1B NL2A similar findings: Boucard et al., 2005; Graf et al. 2006 Ben Chih

  21. Candidate neurexins for interactions at GABAergic synapses glutamatergic axon GABAergic axon NRXa NRXb4(+) ? ? NRXb4(-) a + NL1B NL1A NL2A dendrite

  22. Selective function for neurexin variants in GABAergic postsynaptic differentiation PSD95 gephyrin Ben Chih

  23. vGlut1 VGAT glutamatergic axon GABAergic axon NRXa NRXb4(+) ? ? NRXb4(-) a + NL1B NL1A NL2A dendrite Ben Chih

  24. presynaptic: Neurexin postsynaptic: Neuroligin • alternative splicing of neuroligin-1 and -2 regulates localization and function at GABAergic vs. glutamatergic contacts • neurexin splice variants that interact selectively with the GABAergic neuroligin variants selectively induce GABAergic postsynaptic differentiation • alternative splicing underlies selective trans-synaptic interactions of the neuroligin - neurexin complex

  25. What is the spatial and temporal regulation of Neurexin splicing?

  26. Dynamic alterations in neurexin splicing have been reported, e.g. in response to growth factor signaling, seizure or ischemia (Patzke and Ernsberger, 2000; Gorecki et al. 1999, Sun et al. 2000) What is the spatial and temporal regulation of Neurexin splicing? • Cell-type specific expression of splicing factors • Dynamic regulation by post-translational modifications, e.g. phosphorylation

  27. Leora Gollan

  28. A quantitative assay for neurexin splicing Leora Gollan

  29. Splice reporters with inactivated donor and acceptor sites Leora Gollan

  30. RNA delivery Processing of splice reporter RNAs in hippocampal neurons DNA transfection RFP GFP merge Leora Gollan

  31. specific introns are retained in cytoplasmic neurexin-1 mRNA • unspliced RNA delivered into the cytoplasm of hippocampal neurons can be processed by the cellular machinery • is the neurexin-1 mRNA processed through a cytoplasmic splicing mechanism?

  32. RNA processing in mechanically isolated axons Leora Gollan

  33. Cytoplasmic splicing of synaptic proteins would provide a novel mechanism for local modifications of cell function In the neuroligin-neurexin complex alternative splicing regulates selective adhesive interactions

  34. Candidate mechanisms for cytoplasmic neurexin mRNA processing Non-conventional cytoplasmic mechanism: Ire1p - cleavage tRNA ligase - exon joining Sidrauski, Walter and colleagues Cytoplasmic mechanism using conventional machinery: Glanzer et al. PNAS 102(46):16859-64 suggested splicing-like mRNA processing in dendrites

  35. Acknowledgements (POSTER: Wednesday 715.13/B25) Ben Chih (Genentech) Leora Gollan Elaine Budreck Francisco Gomez-Scholl (Univ. Seville) Collaborators: Ryuichi Shigemoto (NIPS, Okazaki) Chiye Aoki (NYU) Support: NIDA NINDS Irma T. Hirschl Fund The John Merck Fund The Simons Foundation Searle Scholar Program Alfred P. Sloan Foundation Joseph and Esther Klingenstein Fund National Alliance for Autism Research

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