Regulation of Neuroligins and Effects on Synapse Number and Function

National Institute Of Neurological Disorders And Stroke

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Abstract

Neuroligins (NLGNs) are brain-specific cell adhesion molecules. They are expressed on the postsynaptic membrane and bind to presynaptic neurexins (NRXNs) spanning the synaptic cleft. Interestingly, some de novo mutations in both NLGNs and NRXNs have been deemed causative of neurodevelopmental disorders such as autism spectrum disorder (ASD) and intellectual disability (ID). This has led researchers to develop genetically engineered NLGN mouse models to study the etiology of ASD/ID. These studies have shown that NLGN dysfunction can shift the balance of inhibition and excitation in the brain. NLGN isoforms are highly conserved, yet each isoform displays a distinct synaptic localization. However, the molecular mechanisms that regulate isoform-specific targeting and localization are not well understood. Over the last decade, we have focused on the role of protein-protein interactions and post-translational modifications in dictating NLGN trafficking and functional regulation. We have identified isoform-specific phosphorylation sites of the neuroligins. In a recently published study, we found that endogenous NLGN4X is robustly phosphorylated by protein kinase C (PKC) at T707 in human embryonic neurons. The ASD-associated mutation (R704C) eliminates T707 phosphorylation, which is critical for NLGN4X-mediated excitatory enhancement. Interestingly, unlike other NLGN ASD-associated mutations, R704C, did not disrupt the stability or surface expression of NLGN4X, yet still led to synaptic dysfunction. Our results established a potential causality between a genetic mutation, a key posttranslational modification, and robust synaptic changes and will provide insights in elucidating the pathophysiology of ASDs. Recently, we have expanded this work to examine the phosphorylation of NLGN4X by PKA and CDK5 and compare NLGN4X regulation to that of NLGN4Y, revealing differences in these two isoforms. While PKA phosphorylates NLGN4X, but not 4Y on the analogous residue, we find that CDK5 phosphorylates both NLGN4X and NLGN4Y. Our findings reveal that NLGN4X and 4Y share both distinct and overlapping modulation by kinases, which yields more diversity in function. More recently, our studies on neuroligins have shifted to focus on their role in synaptic dysfunction and disease. ASDs are a group of neurodevelopmental disorders that have a high genetic predisposition and higher occurrence rates in males than females. A variety of de novo missense mutations have been identified in the X-linked NLGN3 and 4X genes in patients with intellectual disability and ASD. Most of the ASD-associated missense mutations in NLGN3 and NLGN4X reside in their extracellular domains and many result in functional deficits in binding to neurexins and therefore decreased synaptogenesis. In previous studies, we analyzed ASD-associated mutations in NLGN4X and identified a cluster in the region surrounding S93 in NLGN4X. Importantly, these de novo mutations identified in patients phenocopied the trafficking deficit we had observed for NLGN4Y. Because NLGN4Y cannot compensate for the trafficking and functional deficits observed in ASD-associated NLGN4X mutations, our data reveal a potential pathogenic mechanism for male bias in NLGN4X-associated ASD. We continue to study NLGN4X and 4Y and believe that a better investigation of the sex-linked isoforms of NLGNs will lead to better insight regarding the sex bias associated with some cases of ASD. We are now collaborating with NCATS to screen small molecules for candidates to rescue the trafficking defect in the NLGN4X ASD-associated mutations and in NLGN4Y. We have generated stable cell lines expressing NLGN4Y and the NLGN4X ASD-associated mutations with the appropriate tags to facilitate the high throughput screens. Furthermore, we have identified several candidate molecules from our screens. Currently, we are conducting further analyses on these candidates identified from the high throughput screens. We are using in utero expression to understand the effects on cortical development and studying these variants in differentiated neurons from human iPSCs. These experiments are ongoing. NLGN3 is cleaved in an activity-dependent manner, and the resulting soluble ectodomain has been shown to be a potent mitogen. Several years ago, we published findings on the molecular mechanisms underlying NLGN3 cleavage. We have several ongoing studies examining the mechanisms regulating the specificity of NLGN3 cleavage, the role in neurons, the role in glial biology, and the post cleavage fate of the NLGN3 cleavage fragments. We have also studied synaptic proteins that interact with neuroligins to better understand downstream signaling. The RhoGEF kalirin-7 is a brain-specific kalirin isoform thought to be an important signaling hub at the postsynaptic density. The mechanisms by which kalirin-7 regulates synaptic transmission, particularly which protein-protein interactions are important, remain largely unknown. In addition, kalirin has a paralogue, Trio, which is strongly associated with neurodevelopmental disorders. A few years ago, we found that NLGN-dependent potentiation of synapses and spine growth are mediated, at least in part, by kalirin-7. Thus, we identified the first downstream effector of NLGN1. In a paper recently accepted for publication, we examined Trio interactors that we identified via mass spec including collapsing response mediator protein (CRMP) family members. We investigated their role in axon outgrowth, synapse development and synaptic dysfunction in neurodevelopmental disorders. We find that some CRMP -mediated effects on growth cone collapse are mediated through Trio. In addition, we find that axon guidance cues such as BDNF and Sema3a act via Trio-dependent signaling. These findings reveal a role for Trio early in neuronal development that likely plays a role in the etiology of Trio-related disorders. In another recently published project, we developed a new approach to analyze human variation within NLGN genes to identify sensitive regions that have an increased frequency of neurodevelopmental disorder-associated variants. By carefully comparing genetics’ databases, we can gain insight into NLGN function. Therefor, we developed an algorithm that assesses tolerance to missense mutations in human genetic variation by comparing clinical variants from ClinVar to reference variants from gnomAD. This approach provides tolerance values to subdomains within the protein. This approach helped us identify several critical regions that were conserved across multiple NLGN isoforms.