# Presentation: 157377

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

**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
-  central regulators for cell polarization, migration, and for three-dimensional organization
-  cell adhesion complexes are dynamically regulated to alter cellular architecture and function

**Notes:**
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

## 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**

- Nectins
- Cadherins
- FasII
- (Still life)

- EphA4
- Glt-1

- Protocadherins
- Neuroligins

*presynaptic terminal*

*glia*

*postsynaptic terminal*

**neuron-glia junction**
** ****transmitter uptake**
** ****signaling**
** ****spine morphogenesis**

## Potential roles for adhesion molecules 

- (repulsive self-recognition)

- Laminar
- specificity

- Potential roles for adhesion molecules
- in local regulation of connectivity

- Subcellular
- specificity

- Cell type
- specificity

## Alternative splicing is one of the key processes 

******  ****promoter**

****** ****promoter**

- Neurexin-1
- genomic locus

- DSCAM
- genomic locus

- Protocadherin
- genomic locus

- 

- 

- 

- Cadherin-related
- Neuronal Receptor (CNR)

- Alternative splicing is one of the key processes
- that increases the diversity of cell adhesion molecules

- Families of surface molecules encoded by multiple genes:
*olfactory receptors, classical cadherins, immunoglobulin-domain proteins, leucine-rich repeat proteins*

## Slide 6

## 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**

- Nectins
- Cadherins
- FasII
- (Still life)

- EphA4
- Glt-1

- Neuroligins

*presynaptic terminal*

*glia*

*postsynaptic terminal*

**neuron-glia junction**
** ****transmitter uptake**
** ****signaling**
** ****spine morphogenesis**

- Neurexins

## Outline

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

## 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 nm gold: GABA-A R beta 2 subunit**
**  ****5 nm gold: neuroligin-2 **

- Neuroligins are closely associated with neurotransmitter receptors in the postsynaptic membrane

- Elaine Budreck, Ryuichi Shigemoto

## GFP-beta-Neurexin-1 mice

**pan-neurexin**
**antibodies**

- Chiye Aoki
- Francisco G. Scholl

## Model of the synaptic Neuroligin-Neurexin adhesion complex

- postsynaptic:
**Neuroligin**

- presynaptic:
**Neurexin**

## Neurexin and neuroligin genes encode large numbers of splice variants

- -NRX

- -NRX

- NL1

- NL2

## 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)

## Neuroligin variants generated by alternative splicing

- Leora Gollan

## VGAT : VGlut1

- % overlap

- Puncta/10μm

- VGAT : VGlut1

## Alternative splicing controls localization of neuroligin-2

- VGAT : VGlut1

- Ben Chih, Leora Gollan

## Presence of the B insertion is sufficient to prevent

- Ben Chih

- activity of neuroligin-2 towards GABAergic axons

## Synaptic selectivity of NL splice variants

- GABAergic
- axon

***dendrite***

- glutamatergic
- axon

**insertion A**: localization and function at GABAergic contacts**insertion B**: localization and function at glutamatergic contacts 		B insertion is dominant

- NL1B

- NL1A
- NL2A

## Neuroligin-1 splice variants 

- NL1A

- NL1B

- NL2A

- NRX4(-)

- NRX4(+)

*NL*

*NL*

*NL*

- Fc-control

*NX-Fc*

*NX-Fc*

*Fc*

- Neuroligin-1 splice variants
- differ in their interactions with neurexin-1 isoforms

- Ben Chih

- similar findings: Boucard et al., 2005; Graf et al. 2006

## Candidate neurexins for interactions at GABAergic synapses

- GABAergic
- axon

***dendrite***

- glutamatergic
- axon

- NRX
- NRX4(+)

- NRX4(-)

- NL1B

- NL1A
- NL2A

- 

- +

- ?

- ?

## Selective function for neurexin variants 

- Selective function for neurexin variants
- in GABAergic postsynaptic differentiation

**PSD95**

**gephyrin**

- Ben Chih

## NRX

- GABAergic
- axon

***dendrite***

- glutamatergic
- axon

- NRX
- NRX4(+)

- NRX4(-)

- NL1B

- NL1A
- NL2A

- 

- +

- ?

- ?

- Ben Chih

## postsynaptic:

**Neuroligin**

- presynaptic:
**Neurexin**

- 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

## What is the spatial and temporal regulation of Neurexin splicing?

## 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

## Leora Gollan 

- Leora Gollan

## A quantitative assay for neurexin splicing

- Leora Gollan

## Splice reporters with inactivated donor and acceptor sites

- Leora Gollan

## Processing of splice reporter RNAs in hippocampal neurons

**RFP**

**GFP**

**merge**

**DNA transfection**

- Leora Gollan

- 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?*

## RNA processing in mechanically isolated axons

- Leora Gollan

## 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

## Candidate mechanisms for cytoplasmic neurexin mRNA processing

- 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

## Acknowledgements

**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 715.13/B25)**