Synapse Remodeling and Neuronal MHC Class I

描述(申请人提供)：这项研究的长期目标是了解大脑发育关键时期的经验是如何通过神经回路的功能转化为突触连接的持久结构变化的。这里研究的具体假设是，在神经元和突触表达的MHC Class I基因(在人类中为MHCI；在人类中为HLA)作为负调控因子，限制活性依赖的突触可塑性。有分子和信号通路正常工作来对抗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的免疫中的作用而闻名，但在这里我们研究一个在神经元中的新作用。神经MHCI在中枢神经系统中的表达出人意料地被发现是在对发育中神经活动调节的基因进行无偏见筛选时发现的。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等人，2000年)。在过去的资助期内的研究表明，在60多个MHCI基因中，只有2个基因的功能丧失，即H2-KB和/或H2-DB，改变了突触可塑性规则，出人意料地经常增强可塑性和学习(McConnell等人，2009年)。计划有三个具体目标：1)证明突触可塑性中对H2-DB、H2-KB的需求：将对双突变小鼠(KbDB-/-)进行研究，以确定这两个基因是否可以解释在最初研究中观察到的突触可塑性的许多变化，这些变化是在缺乏大多数MHCI蛋白表面表达的小鼠身上观察到的。过度表达H2-DB的小鼠也将被检查。将进行拯救实验：已经产生了双转基因小鼠(NSE-DB+/+；KbDb-/-)，其中DB功能仅在神经元中被拯救。针对H2-KB或H2-DB的GFP标记的全长cDNA将使用慢病毒载体表达。2)确定MHCI蛋白是否位于突触：一个神经性MHCI的工作模型表明，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的分布。阵列断层成像(AT)将用于MHCI蛋白与多个突触标志物以及PirB直接相关的更高分辨率的定位。生化分级和免疫印迹也将被用来评估MHCI和潜在的相互作用伙伴的亚细胞定位。3)产生H2-DB的条件等位基因用于神经元功能的研究：将产生一只转基因小鼠，以获得脑细胞和神经细胞类型的特异性基因敲除，为进一步研究H2-D在神经元中的需求和特异性奠定基础。所有实验将利用体内和体外小鼠视觉系统的电生理学、解剖学和成像研究，以评估活动依赖型突触的发育和可塑性。通过研究MHCI功能增强或丧失的小鼠，这些实验应该有助于确定神经元是否以及在哪里正常需要H2-KB和H2-DB，并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.项目叙述/关联性2-3句从这里提出的实验结果将扩大对突触的经验依赖的变化的理解，无论是在童年学习的关键时期还是在一生的记忆形成中，最终是如何在神经回路的结构中编码的。了解相关的分子和机制对于解决并最终治愈学习和记忆障碍至关重要，从阅读障碍、自闭症和其他儿童神经疾病，到阿尔茨海默病和老化大脑中的其他记忆功能障碍。此外，已知神经元MHCI受炎症调节，并可被免疫系统的细胞识别，在健康和疾病中提供神经和免疫系统之间的直接沟通手段。 与公共健康相关：这里提出的实验结果将扩大对突触经验依赖性变化的理解，无论是在童年学习的关键时期还是在一生的记忆形成过程中，突触的经验依赖变化最终是如何编码在神经回路结构中的。了解相关的分子和机制对于解决并最终治愈学习和记忆障碍至关重要，从阅读障碍、自闭症和其他儿童神经疾病，到阿尔茨海默病和老化大脑中的其他记忆功能障碍。此外，已知神经元MHCI受炎症调节，并可被免疫系统的细胞识别，在健康和疾病中提供神经和免疫系统之间的直接沟通手段。

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. Project Narrative/Relevance 2-3 sentences Results from experiments proposed here will broaden understanding of how experience-dependent alterations at synapses, both during critical periods of learning in childhood and in memory formation throughout life, are ultimately encoded in the structure of neural circuits. Understanding molecules and mechanisms involved is crucial for addressing and ultimately curing disorders of learning and memory, from Dyslexia, Autism and other childhood neurological disorders, to Alzheimers' and other memory dysfunction in the aging brain. Moreover, neuronal MHCI is known to be modulated by inflammation and can be recognized by cells of the immune system, providing a direct means of communication between the nervous and the immune systems in both health and disease. PUBLIC HEALTH RELEVANCE: Results from experiments proposed here will broaden understanding of how experience- dependent alterations at synapses, both during critical periods of learning in childhood and in memory formation throughout life, are ultimately encoded in the structure of neural circuits. Understanding molecules and mechanisms involved is crucial for addressing and ultimately curing disorders of learning and memory, from Dyslexia, Autism and other childhood neurological disorders, to Alzheimers' and other memory dysfunction in the aging brain. Moreover, neuronal MHCI is known to be modulated by inflammation and can be recognized by cells of the immune system, providing a direct means of communication between the nervous and the immune systems in both health and disease.