Impact of Patient-Specific Perturbations in the NRXN1α Isoform Repertoire on Synaptic Composition

Aim 1. Evaluate the impact of patient-specific perturbations in the NRXN1α isoform repertoire on synaptic composition. Rationale: Exclusion of canonical splice site SS4 in mice increased binding affinity to NLGN1β and LRRTMs but decreased binding to CBLNs. Forced Nrxn3 SS4 inclusion in mouse hippocampal neurons decreased post-synaptic AMPAR levels and LTP, but the impact of directly manipulating NRXN1 SS4 is unknown. Hypothesis: Overexpression of each NRXN1α isoform will result in SS-inclusion, genotype-dependent and cell-type specific trans-synaptic alterations of post-synaptic composition in neurons. Research plan. Explore how patient-specific NRXN1 isoforms alter NRXN1 protein-protein interactions and synaptic composition. A. Query the protein-binding partners of FLAG-tagged wildtype and mutant NRXN1 isoforms by Flag-IP and mass spectrometry. B. Apply BioID to specifically resolve the cell-type-specific changes in synaptic composition following overexpression of wildtype and mutant NRXN1 isoforms in human glutamatergic and GABAergic neurons.

Aim 2. Compare cell type-specific protein-interaction-networks of wildtype, loss-of-function (LOF), and gain-of-function (GOF) variants for neurodevelopmental risk genes. Rationale: Up to one third of rare neurodevelopmental disorder genes harbor both LOF and GOF variants. Hypothesis: NDD-associated mutations remodel the PINs of the respective gene in distinct ways that are unique to each cell type. Research plan. We will compare the PINs of WT, LOF, and GOF variants to reveal variant-specific effects on PINs in both iGLUTs and iGABAs. Understanding how these variants differentially impact PINs has important implications for precision medicine, as the phenotypic impact of diverse patient-specific genetic variants may vary, requiring stratification of patients not only by impacted gene, but also by type of mutation. A. Query the protein-binding partners of FLAG-tagged wildtype, GOF, and LOF NDD gene isoforms by Flag-IP and mass spectrometry. B. Apply BioID to resolve the changes in synaptic composition following overexpression of wildtype, GOF, and LOF NDD isoforms in human neurons. C. Functionally validate LOF and GOF mechanisms of proteins in neurodevelopment and/or synaptic function.

Relevance to Addiction: We propose to demonstrate the impact of patient-specific NRXN1+/- mutations on synaptic composition. Given the established links between Neurexins and endocannabinoid signaling (1), cannabis use and psychiatric disease (2,3), and recent trends in legalization and increasing cannabis consumption (4), it is critical to understand the synaptic impact of alternative splicing of neurexin.

Literature Cited

1. Anderson, G. R. et al. (2015) beta-Neurexins Control Neural Circuits by Regulating Synaptic Endocannabinoid Signaling. Cell 162, 593-606, doi:10.1016/j.cell.2015.06.056 (2015).

2. Burns, J. K. (2013) Pathways from cannabis to psychosis: a review of the evidence. Front Psychiatry 4, 128, doi:10.3389/fpsyt.2013.00128.

3. Weiser, M. & Noy, S. (2005) Interpreting the association between cannabis use and increased risk for schizophrenia. Dialogues Clin Neurosci 7, 81-85.

4. Warner, T. D., Roussos-Ross, D. & Behnke, M. (2014) It's not your mother's marijuana: effects on maternal-fetal health and the developing child. Clin Perinatol 41, 877-894, doi:10.1016/j.clp.2014.08.009.