Synapse Remodeling and Neuronal MHC Class I

描述（由申请人提供）：本研究的长期目标是了解在大脑发育的关键时期，由神经回路功能介导的经验如何转化为突触连通性的持久结构变化。本研究的具体假设是，在神经元和突触中表达的MHCI类基因（MHCI；人类HLA）作为负调节因子限制活动依赖性突触可塑性。有分子和信号通路正常工作反对突触可塑性的想法是新颖的，具有重要的治疗意义。MHCI基因以其在细胞介导免疫中的作用而闻名，但在这里我们研究了神经元中的新作用。中枢神经系统中神经元MHCI的表达是在对发育过程中受神经活动调节的基因进行无偏筛选时意外发现的。对小鼠的初步研究为这些分子在突触可塑性中的作用提供了间接证据（Huh et al, 2000）。在过去的资助期内的研究表明，在60多个MHCI基因中，只有2个基因（H2-Kb和/或H2-Db）的功能丧失会改变突触可塑性规则，出乎意料地经常增强可塑性和学习能力（McConnell et al, 2009）。计划有三个具体目标：1)证明H2- Db， H2- kb在突触可塑性中的需求：双突变小鼠（KbDB-/-）将被研究，以确定这两个基因是否可以解释在缺乏大多数MHCI蛋白表面表达的小鼠的初步研究中观察到的突触可塑性的许多变化。过表达H2-Db的小鼠也将进行检测。将进行拯救实验：产生双转基因小鼠（NSE-Db+/+; KbDb-/-），其中Db功能仅在神经元中被拯救。gfp标记的H2-Kb或H2-Db全长cdna将使用慢病毒载体表达。2)确定MHCI蛋白是否位于突触：神经元MHCI的一个工作模型表明，位于突触后的MHCI蛋白通过突触与突触前受体（如PirB）结合。MHCI抗体免疫染色将用于检查突触分布。阵列断层扫描（AT）将用于与多种突触标记物以及PirB直接相关的MHCI蛋白的更高分辨率定位。生化分离和Western Blotting也将用于评估MHCI的亚细胞定位和潜在的相互作用伙伴。3)生成用于神经元功能研究的H2-Db条件等位基因：生成转基因小鼠，获得脑和神经元细胞类型特异性敲除，进一步研究神经元中H2-D的需求和特异性。所有实验将利用小鼠体内和体外视觉系统的电生理、解剖和成像研究来评估活动依赖性突触的发育和可塑性。通过研究MHCI功能增加或减少的小鼠，这些实验应该有助于确定神经元是否以及在何处正常需要H2-Kb和H2-Db，并且应该允许对大脑中特定MHCI分子的功能进行更系统的研究。7.

DESCRIPTION (provided by applicant): The long-term goal of this research is to understand how experience during critical periods of brain development, mediated by the functioning of neural circuits, is translated into lasting structural change in synaptic connectivity. The specific hypothesis under study here is that MHC Class I genes (MHCI; HLA in human) expressed in neurons and at synapses act as a negative regulators to limit activity-dependent synaptic plasticity. The idea that there are molecules and signaling pathways normally working to oppose synaptic plasticity is novel and has significant therapeutic implications. MHCI genes are famous for their role in cell-mediated immunity, but here we study a novel role in neurons. Neuronal MHCI expression in the CNS was discovered unexpectedly in an unbiased screen for genes regulated by neural activity in development. Initial studies in mice provided indirect evidence for a role for these molecules in synaptic plasticity (Huh et al, 2000). Research during the past funding period has revealed that loss of function of just 2 of the 60+ MHCI genes, H2-Kb and/or H2-Db, alters synaptic plasticity rules, unexpectedly often enhancing plasticity and learning (McConnell et al, 2009). Three specific aims are planned: 1) Demonstrate requirement for H2- Db, H2-Kb in synaptic plasticity: Double mutant mice (KbDB-/-) will be studied to determine if these 2 genes can account for many of the changes in synaptic plasticity observed in initial studies of mice lacking surface expression of the majority of MHCI proteins. Mice overexpressing H2-Db will also be examined. Rescue experiments will be performed: Double transgenic mice (NSE-Db+/+; KbDb-/-) have been generated in which Db function is rescued only in neurons. GFP-tagged full-length cDNAs for H2-Kb or H2-Db will be expressed using Lentiviral vectors. 2) Determine if MHCI protein is located at synapses: A working model for neuronal MHCI suggests that MHCI protein located postsynaptically binds across the synapse to presynaptic receptors such as PirB. Immunostaining with MHCI antibodies will be used to examine synaptic distribution. Array Tomography (AT) will be used for higher resolution localization of MHCI protein in direct relation to multiple synaptic markers, as well as to PirB. Biochemical fractionation and Western Blotting will also be used to assess subcellular localization of MHCI and potential interacting partners. 3) Generate a conditional allele of H2-Db for studies of neuronal function: A transgenic mouse will be generated to obtain brain and neuronal cell-type specific knockouts for further study of requirement and specificity of H2-D in neurons. All experiments will make use of electrophysiological, anatomical and imaging studies of mouse visual system in vivo and in vitro to assess activity-dependent synapse development and plasticity. By studying mice with gain- or loss- of MHCI function, these experiments should help to establish whether and where H2-Kb and H2-Db are required normally in neurons and should permit a more systematic investigation of the function of specific MHCI molecules in the brain. 7.