Subunit-Specific Regulation Of Glutamate Receptors

我的实验室使用生化和分子技术相结合的方法研究谷氨酸受体的运输和定位的调节。谷氨酸受体是哺乳动物大脑中主要的兴奋性神经递质受体，是一个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和海人藻酸受体亚型，每种亚型都由不同的亚基组成。代谢型谷氨酸受体(mGluR1-8)是G蛋白偶联受体，以同源二聚体的形式组装而成。我们专注于确定调节谷氨酸受体和突触支架蛋白突触定位和功能调节的亚基特异性机制。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，我的研究项目主要集中在调节NMDA受体的分子机制上。总体目标是更好地了解NMDA受体在正常情况下的功能，以及一些神经发育障碍背后的特定功能障碍。NMDA受体是由同源亚基(GluN1、GluN2A-D、GluN3A-B)组成的多亚单位复合体(四聚体)。我们在NMDAR突触表达的详细特征以及GluN2A和GluN2B在受体运输和突触表达中的作用方面取得了重大进展。我们主要关注GluN2A和GluN2B，因为这些亚基在海马体和皮质中高度表达，并已知经历活动和发育调节的贩运事件。 在过去的十年里，我们已经转向了一种新的方法来研究NMDAR的结构/功能，使用人类遗传学来指导我们的研究。我们从这种“床边到长凳”的策略开始，以帮助指导我们测试对突触功能重要的受体区域。我们使用了已发表的论文和公共数据库中的信息，这些信息报告了通过对神经或精神障碍患者进行深度测序而确定的变异。然后，我们开始对在GRIN基因(编码NMDA受体亚基的基因)中识别的错义变体进行实验。具体地说，我们正在检查导致GluN2NMDAR亚单位(GluN2A和GluN2B)细胞内C-末端区域突变的变异。随着人类遗传学数据的积累，NMDA受体亚单位的许多从头突变与包括自闭症谱系障碍、智能障碍和癫痫在内的神经发育障碍高度相关。因此，我们的目标是更好地了解这些与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，我们主要专注于在NMDA受体亚单位的细胞内C末端发现的罕见变异，尽管我们也开始使用GRIN2B单倍体不足的小鼠进行研究。 在最近发表的一项研究中，我们描述了在癫痫队列中发现的一种罕见的GluN2A(S1459G)变异(Mota Vieira等人，2020)。这名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-末端区域内。我们发现这个丝氨酸是一个CaMKII位点，这个残基的磷酸化决定了受体与PSD-95和分类连接蛋白27(SNX27)的相互作用。因此，我们确定了一个调控位点，它决定了含有GluN2A的NMDA受体的运输和突触表达。在一项相关的研究中，我们鉴定了一种罕见的GRIN2A变异体(在神经发育障碍患者中发现)，它编码一种移码，导致C末端结构域的一半截断，并编码一段独特的氨基酸。我们发现，该受体定位错误，表面表达增加，但在突触外部位特异性丰富。在蛋白质相互作用、棘突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，更好地了解临床表现，以便我们可以比较基因和突触功能。我们的方法再次揭示了NMDA受体结构/功能的新发现。我们希望利用从不同致病罕见变异的分析中获得的信息来更好地了解NMDA受体的运输和定位，并帮助测试更精确的治疗方法。 此外，我们正在对GluN2B中导致亚基延长的罕见变异进行研究。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，减少了受体表面的表达。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IPSCs。这需要一个多步骤的方案，因为最常见的单步分化方法不能产生NMDAR可靠而稳健的表达的神经元。我们已经取得了重大进展，目前正在合作使用CRISPR来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.