Subunit-Specific Regulation Of Glutamate Receptors

我的实验室研究谷氨酸受体运输和本地化的生物化学和分子技术相结合的调节。谷氨酸受体是哺乳动物脑中主要的兴奋性神经递质受体，并且是具有许多不同亚型的多样性家族。离子型谷氨酸受体包括AMPA、NMDA和红藻氨酸受体亚型，它们中的每一个都由多种亚基形成。代谢型谷氨酸受体（mGluR 1 -8）是G蛋白偶联受体（GPCR），组装为同源二聚体。我们专注于定义亚单位的具体机制，调节突触定位和功能调节谷氨酸受体以及突触支架蛋白。这些机制包括翻译后修饰，如磷酸化，以及蛋白质-蛋白质相互作用。 目前，我的研究计划特别集中在调节NMDA受体的分子机制。总体目标是更好地了解正常情况下NMDA受体的功能和某些神经发育障碍的具体功能障碍。NMDA受体是由同源亚基（GluN 1; GluN 2A-D; GluN 3A-B）组成的多亚基复合物（四聚体）。我们已经取得了显着的进展，在详细描述的NMDARs的突触表达和GluN 2A和GluN 2B在受体运输和突触表达的作用。我们主要关注GluN 2A和GluN 2B，因为这些亚基在海马和皮质中高度表达，并且已知它们会经历活动和发育调节的运输事件。 在过去的十年中，我们已经转向一种新的方法来研究NMDAR的结构/功能，使用人类遗传学来为我们的研究提供信息。我们从这个“从床边到实验台”的策略开始，帮助指导我们测试对突触功能很重要的受体结构域。我们使用了来自已发表论文和公共数据库的信息，这些数据库报告了通过对神经系统或精神疾病患者进行深度测序确定的变异。然后，我们开始对GRIN基因（编码NMDA受体亚基）中鉴定的错义变体进行实验。具体来说，我们正在研究导致GluN 2 NMDAR亚基（GluN 2A和GluN 2B）细胞内C-末端结构域突变的变体。随着人类遗传学数据的积累，已经清楚的是，NMDA受体亚基中的许多从头突变与神经发育障碍（包括自闭症谱系障碍、智力残疾和癫痫）高度相关。因此，我们的目标是更好地了解这些疾病相关的罕见变异引起的突触功能障碍，着眼于开发治疗方法。由于我们在研究受体运输和蛋白质相互作用方面的专业知识，我们主要关注NMDA受体亚基细胞内C-末端中鉴定的罕见变体，尽管我们也在着手使用GRIN 2B单倍不足小鼠进行研究。 在最近发表的研究中，我们表征了在癫痫组群中鉴定的GluN 2A（S1459 G）的罕见变体（Mota Vieira等人，2020年）。患者还被诊断为智力残疾。这种从头突变位于PDZ配体附近的极端C-末端结构域内。我们发现该丝氨酸是CaMKII位点，并且该残基的磷酸化决定了与PSD-95和分选连接蛋白27（SNX 27）的受体相互作用。因此，我们确定了一个监管网站，决定了运输和突触表达的GluN 2A-含有NMDA受体。在一项相关的研究中，我们已经表征了GRIN 2A罕见变体（在神经发育障碍患者中鉴定），其编码导致C末端结构域一半截短的移码，以及编码独特的氨基酸片段。我们发现，该受体是错误的，并显示增加表面表达，但特别是丰富的突触外网站。在蛋白质相互作用、棘密度和突触数量方面存在缺陷。我们与临床医生（NINDS和NIMH）合作，收集患者数据并更好地了解临床表现，以便我们可以比较基因型和突触功能。再次，我们的方法揭示了NMDA受体结构/功能的新发现。我们希望利用来自不同致病性罕见变异分析的信息，更好地了解NMDA受体的运输和定位，并帮助测试更精确的治疗方法。 此外，我们正在进行GluN 2B中导致亚基延伸的罕见变体的研究。到目前为止，我们发现这些从头突变破坏了与支架蛋白的结合，减少了受体表面表达。这些研究正在进行中。 在过去的一年里，我们投入了大量的精力来开发可靠的方案，以区分表达NMDA受体的兴奋性神经元和iPSC。这需要多步方案，因为最常见的单步分化方法不能产生具有可靠的NMDAR稳健表达的神经元。我们已经取得了重大进展，目前正在合作使用CRISPR引入患者识别的罕见变异。

My laboratory studies the regulation of glutamate receptor trafficking and localization using a combination of biochemical and molecular techniques. Glutamate receptors are the major excitatory neurotransmitter receptors in the mammalian brain and are a diverse family with many different subtypes. The ionotropic glutamate receptors include AMPA, NMDA, and kainate receptor subtypes, each of which are formed from a variety of subunits. The metabotropic glutamate receptors (mGluR1-8) are G protein-coupled receptors (GPCRs), which are assembled as homodimers. We focus on defining subunit-specific mechanisms that regulate the synaptic localization and functional regulation of glutamate receptors as well as synaptic scaffolding proteins. These mechanisms include posttranslational modifications such as phosphorylation, as well as protein-protein interactions. Currently, my research program is focused specifically of the molecular mechanisms regulating NMDA receptors. The overall goal is to better understand NMDA receptor function under normal circumstances and the specific dysfunction underlying some neurodevelopmental disorders. NMDA receptors are multi-subunit complexes (tetramers) composed of homologous subunits (GluN1; GluN2A-D; GluN3A-B). We have made significant progress in the detailed characterization of the synaptic expression of NMDARs and the role of GluN2A and GluN2B in receptor trafficking and synaptic expression. We primarily focus on GluN2A and GluN2B because these subunits are highly expressed in hippocampus and cortex and are known to undergo activity- and developmentally-regulated trafficking events. Over the last decade, we have shifted to a new approach to studying structure/function of NMDARs using human genetics to inform our research. We began with this "bedside-to-bench" strategy to help guide us in testing receptor domains that are important for synaptic function. We used information from published papers and public databases that report variants identified by deep sequencing of patients with neurological or psychiatric disorders. We then began conducting experiments on missense variants identified in GRIN genes (that encode NMDA receptor subunits. Specifically, we are examining variants causing mutations in the intracellular C-terminal domain of the GluN2 NMDAR subunits (GluN2A and GluN2B). As the human genetics data have accumulated, it has become clear that many de novo mutations in NMDA receptor subunits are highly associated with neurodevelopmental disorders including autism spectrum disorder, intellectual disability and epilepsy. Therefore, our goal is to better understand the synaptic dysfunction caused by these disease-associated rare variants with an eye towards developing therapeutics. Because of our expertise in studying receptor trafficking and protein interactions, we primarily focus on rare variants identified in the intracellular C-termini of NMDA receptor subunits, although we are also embarking on studies using mice with GRIN2B haploinsufficiency. In a recently published study, we characterized a rare variant in GluN2A (S1459G) identified in an epilepsy cohort (Mota Vieira et al., 2020). The patient also was diagnosed with intellectual disability. This de novo mutation is within the extreme C-terminal domain near the PDZ ligand. We found that this serine is a CaMKII site and phosphorylation of this residue dictates the receptor interactions with PSD-95 and sorting nexin 27 (SNX27). Thus, we identified a regulatory site that determines the trafficking and synaptic expression of GluN2A-containing NMDA receptors. In a related study, we have characterized a GRIN2A rare variant (identified in a patient with neurodevelopmental disorders) encoding a frameshift that results in a truncation of half of the C-terminal domain as well as encoding a unique stretch of amino acids. We find that the receptor is mistargeted and displays increased surface expression, but specifically enriched at extrasynaptic sites. There are defects in protein interactions, spine density, and synapse number. We collaborate with clinicians (NINDS and NIMH) to collect patient data and better understand the clinical presentation so we can compare with genotype and synaptic function. Again, our approach reveals new findings in NMDA receptor structure/function. We hope to use the information from the analyses of different pathogenic rare variants to better understand NMDA receptor trafficking and localization and help test for more precise therapeutics. In addition, we have ongoing studies of rare variants in GluN2B that result in extensions of the subunit. Thus far we find that these de novo mutations disrupt binding to scaffolding proteins, reduce receptor surface expression. These studies are ongoing. Over the last year, we have put intense effort into the development of reliable protocol to differentiate excitatory neurons expressing NMDA receptors from iPSCs. This requires a multi-step protocol because the most common single step differentiation method does not yield neurons with reliable robust expression of NMDARs. We have made significant progress and are currently collaborating to use CRISPR to introduce patient identified rare variants.