Regulation of Neuroligins and Effects on Synapse Number and Function

National Institute Of Neurological Disorders And Stroke

Investigators

Linked publications & trials

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. We focus on the role of protein-protein interactions and post-translational modifications in dictating NLGN trafficking and functional regulation. Over the last few years, we have identified several different phosphorylation sites on the different neuroligin isoforms. We have been characterizing the kinases involved and the physiological relevance to synapse formation. 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. Interestingly, all the ASD-associated missense mutations in NLGN3 and NLGN4X reported thus far reside in their extracellular domains except for a single point mutation in the intracellular domain of NLGN4X at arginine (R) 704, which is modified to a cysteine (C). We discovered that endogenous NLGN4X is robustly phosphorylated by protein kinase C (PKC) at T707 in human embryonic neurons. This 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 establish 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. Human NLGN4Y is located on the Y chromosome and is almost identical to NLGN4X. In fact, NLGN4X and NLGN4Y have only eight amino acid differences in the extracellular domain and five in the intracellular domain. We recently reported that NLGN4Y has a trafficking defect. Specifically, we used biochemistry, electrophysiology, and imaging analyses to study NLGN4Y and identified severe deficits in maturation, surface expression, and synaptogenesis compared to NLGN4X. Strikingly, the functional differences were primarily regulated by one amino acid difference with NLGN4X (P93 in NLGN4X, but S93 in NLGN4Y). Furthermore, 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 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. Although this initiative was slowed down due to the pandemic, it is now moving forward, and we have generated stable cell lines expressing the NLGN4X ASD-associated mutations with the appropriate tags to facilitate the high throughput screen. We will further investigate any candidate from the high throughput screen using differentiated neurons from human iPSCs. We will introduce the relevant mutations using CRISPR-edited iPSCs and perform trafficking assays on those cells. 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. To study kalirin-7 and Trio interactors, we used specific antibodies to immunoprecipitate endogenous kalirin-7 or Trio and identify potential interactions using LC MS/MS. For kalirin-7, we identified members of the neuroligin family of cell adhesion molecules, which were of particular interest given that their phenotype and subcellular localization closely resembles that of kalirin-7. Using both in vitro and in vivo techniques we have validated this interaction, showing that kalirin-7 can interact with all members of the neuroligin family, but not all isoforms of kalirin can interact with neuroligins. 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 current experiments, we are examining Trio interactors that we identified via mass spec and their role in axon outgrowth, synapse development and synaptic dysfunction in neurodevelopmental disorders.