Regulation of Neuroligins and Effects on Synapse Number and Function

Neuroligins 的调节及其对突触数量和功能的影响

• 批准号: 10915990
• 负责人: Katherine Roche
• 金额: $ 202.39万
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10915990
• 关键词: Amino Acids; Arginine; Axon; Binding; Binding Proteins; Biochemistry; Biological; Biological Assay; Biology; Brain; Brain-Derived Neurotrophic Factor; CDK5 gene; Cell Adhesion Molecules; Cells; Chemosensitization; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Compensation; Cues; Cyclic AMP-Dependent Protein Kinases; Cysteine; DNA Sequence Alteration; Data; Defect; Development; Electrophysiology (science); Embryo; Equilibrium; Etiology; Excitatory Synapse; Extracellular Domain; Female; Functional disorder; Gene Family; Genes; Genetic Engineering; Genetic Predisposition to Disease; Growth; Growth Cones; Human; Image; Inhibitory Synapse; Intellectual functioning disability; Investigation; Link; Maintenance; Mediating; Mediator; Missense Mutation; Molecular; Mutation; NRCAM gene; National Center for Advancing Translational Sciences; Neurodevelopmental Disorder; Neuronal Differentiation; Neurons; Pathogenicity; Patients; Phenocopy; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physiological; Point Mutation; Post-Translational Protein Processing; Postsynaptic Membrane; Protein Family; Protein Isoforms; Protein Kinase C; Proteins; Publishing; Regulation; Reporting; Research Personnel; Rodent; Role; Semaphorin-3A; Sex Bias; Signal Pathway; Signal Transduction; Specific qualifier value; Specificity; Surface; Synapses; Synaptic Cleft; Synaptic Transmission; Vertebral column; Work; Y Chromosome; autism spectrum disorder; axon guidance; de novo mutation; density; experimental study; high throughput screening; induced pluripotent stem cell; insight; interest; male; mouse model; pandemic disease; postsynaptic; presynaptic; protein protein interaction; response; sex; small molecule; stable cell line; synaptogenesis; trafficking

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. 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. 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 the trafficking deficit observed in 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. 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 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. We find that some CRMP protein-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. We are characterizing this signaling pathway in more detail. NLGN3 is cleaved in an activity dependent manner. We published findings on the molecular mechanisms underlying NLGN3 cleavage several years ago. We continue to study 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.

神经素（nlgn）是脑特异性细胞粘附分子。它们在突触后膜上表达，并结合跨越突触间隙的突触前神经素（NRXNs）。有趣的是，nlgn和nrxn的一些新生突变被认为是自闭症谱系障碍（ASD）和智力残疾（ID）等神经发育障碍的病因。这使得研究人员开发了基因工程的NLGN小鼠模型来研究ASD/ID的病因。这些研究表明，NLGN功能障碍可以改变大脑中抑制和兴奋的平衡。NLGN异构体是高度保守的，但每个异构体都显示出不同的突触定位。然而，调控异构体特异性靶向和定位的分子机制尚不清楚。我们专注于蛋白质-蛋白质相互作用和翻译后修饰在NLGN运输和功能调节中的作用。在过去的几年里，我们已经在不同的神经素异构体上发现了几个不同的磷酸化位点。我们一直在描述所涉及的激酶及其与突触形成的生理相关性。

项目成果

Advances in Proteomics Allow Insights Into Neuronal Proteomes.

• DOI: 10.3389/fnmol.2021.647451
• 发表时间: 2021
• 期刊: Frontiers in molecular neuroscience
• 影响因子: 4.8
• 作者: Fingleton E;Li Y;Roche KW
• 通讯作者: Roche KW

Posttranslational modifications of neuroligins regulate neuronal and glial signaling.

• DOI: 10.1016/j.conb.2017.05.017
• 发表时间: 2017-08
• 期刊: Current opinion in neurobiology
• 影响因子: 5.7
• 作者: Jeong J;Paskus JD;Roche KW
• 通讯作者: Roche KW

Modeling human mutations to understand TRIO GEF function during development.

对人类突变进行建模以了解 TRIO GEF 在发育过程中的功能。

• DOI: 10.1016/j.tins.2023.03.004
• 发表时间: 2023
• 期刊: Trends in neurosciences
• 影响因子: 15.9
• 作者: Fingleton,Erin;Roche,KatherineW
• 通讯作者: Roche,KatherineW

CaMKII phosphorylation of neuroligin-1 regulates excitatory synapses.

• DOI: 10.1038/nn.3601
• 发表时间: 2014-01
• 期刊: NATURE NEUROSCIENCE
• 影响因子: 25
• 作者: Bemben, Michael A.;Shipman, Seth L.;Hirai, Takaaki;Herring, Bruce E.;Li, Yan;Badger, John D., II;Nicoll, Roger A.;Diamond, Jeffrey S.;Roche, Katherine W.
• 通讯作者: Roche, Katherine W.