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Regulation of synaptic adhesion complexes by alternative splicing Peter Scheiffele, Columbia University, NY.

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1 Regulation of synaptic adhesion complexes by alternative splicing Peter 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 Adhesion molecules represent one of the most important morphogenic factors in all tissues. Adhesive interactions between cells and the extracellular matrix or with other cells are central regulators for cell polarization, migration, and for three-dimensional organization. Moreover 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 Alternative splicing is one of the key processes
that increases the diversity of cell adhesion molecules a promoter b promoter Neurexin-1 genomic locus 1 2 4 5 3 6 9 10 7 b a 8 11 12 13 14 15 16 17 18 21 20 19 22 23 1 2 3 5 7 8 10 12 13 11 14 15 16 18 20 21 19 22 23 24 exon4 exon6 exon9 exon17 TM DSCAM genomic locus Variable (1) Constant (1) Single exon genes 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 Neurexins Neuroligins b-neurexin a-neurexin
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) Neurexins Neuroligins 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)

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 Freeze-fracture replica immuno-EM
Elaine Budreck Ryuichi Shigemoto 5 nm gold: pan-neuroligin 10 nm gold: PSD95 Quick-freezing in helium Freeze-fracture and platinum/carbon shadowing SDS digestion and immuno-gold labeling Kazushi Fujimoto, J. Cell Sci., 108: , 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 Neurexin and neuroligin genes encode large numbers of splice variants
a-NRX b-NRX GPLTKKHTDDLGDNDGAEDE GNRWSNSTK NL1 NL2

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

15 Neuroligin variants generated by alternative splicing
GPLTKKHTDDLGDNDGAEDE GNRWSNSTK Leora Gollan

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

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

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

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

20 Neuroligin-1 splice variants
differ in their interactions with neurexin-1b isoforms Fc-control NRXb4(-) NRXb4(+) 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 NRXa NRXb4(+) ? ? NRXb4(-) a + NL1B NL1A NL2A glutamatergic axon
GABAergic axon NRXa NRXb4(+) ? ? NRXb4(-) a + NL1B NL1A NL2A dendrite vGlut1 VGAT 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 What is the spatial and temporal regulation of Neurexin splicing?
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 Processing of splice reporter RNAs in hippocampal neurons
DNA transfection RFP RNA delivery 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): suggested splicing-like mRNA processing in dendrites

35 Acknowledgements 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 (POSTER: Wednesday /B25)

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