Synapse Remodeling and Neuronal MHC Class I

 描述(由申请人提供)：这项研究的长期目标是了解发育关键期活性依赖的电路调节的细胞和分子机制。这些时候需要适当的神经活动和经验模式才能正常形成大脑回路，而不正常的活动或经验可能会扰乱结果。这里的具体假设是，小鼠(MHCI；人类的HLA)中两个特定的主要组织相容性I类基因H2-DB(DB)和H2-KB(KB)在神经元和突触中表达，在发育关键期调节依赖活性的突触修剪和可塑性。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在中枢神经系统神经元中的表达出人意料地被发现是在体内无偏见地筛选由发育中的视觉系统的神经活动调节的基因。下一项研究表明，50多个MHCI基因中只有两个基因Db和/或Kb的功能丧失，阻止了体内突触的修剪，并出人意料地增强了视觉皮质的可塑性和运动学习。此外，仅通过在体内选择性地将DB恢复到神经元，就可以挽救突触的修剪，以及视网膜生成突触的LTD和AMPA型谷氨酸受体亚单位的组成。这些观察表明，DB必须在神经元中发挥作用，并表明DB在神经元和免疫细胞中的作用是分开的和平行的。1)利用体外系统调控皮质2/3层锥体神经元中DB水平的系统，验证和扩展了体内观察到的DB、突触修剪和AMPA型谷氨酸受体(GluA1/2)亚单位组成之间的关系。Db、Kb或shRNAs的表达结构将用于WT与KbDb-/-或Db-/-小鼠的皮质神经元培养中功能的获得或丧失，或抢救实验。将对突触修剪和GluA1/2亚基组成进行生化和电生理学评估。此外，还将研究阻断突触传递的效果。2)用生化方法和质谱学方法鉴定Db(或Kb)与相关分子之间的分子相互作用。MS的初步结果表明，可能与突触成分以及与膜转运相关的蛋白质存在直接相互作用。这种相互作用将通过免疫共沉淀得到验证，并可以阐明DB(或KB)如何直接或间接调节谷氨酸受体。3)通过突变分析，研究Db的短胞内All在调控修剪、AMPA受体亚单位组成和受体转运中的可能作用。总之，这些实验应该有助于在Db、突触修剪、神经活动和AMPA型谷氨酸受体亚单位组成等MHCI分子之间建立强大的联系。这一知识有朝一日可能会纠正神经发育障碍，如精神分裂症或自闭症，其中修剪出现问题，甚至治疗神经退行性疾病，如阿尔茨海默氏症，过度突触修剪会破坏记忆所在的神经回路。

 DESCRIPTION (provided by applicant): The long term goal of this research is to understand cellular and molecular mechanisms of activity dependent circuit tuning during developmental critical periods. These are times when proper patterns of neural activity and experience are required for brain circuits to form normally, and when abnormal activity or experience can perturb the outcome. The specific hypothesis here is that two specific Major Histocompatibility Class I genes in mouse (MHCI; HLA in human) H2-Db (Db) and H2-Kb (Kb) expressed in neurons and at synapses regulate activity-dependent synaptic pruning and plasticity during developmental critical periods. MHCI genes are famous for their roles in cell-mediated immunity, but here we study a novel role in neurons. Neuronal MHCI expression in CNS neurons was discovered unexpectedly in an unbiased in vivo screen for genes regulated by neural activity in the developing visual system. Research next showed that loss of function of just 2 of the 50+ MHCI genes, Db and/or Kb, prevents synaptic pruning in vivo and unexpectedly enhances visual cortical plasticity and motor learning. Moreover, just by restoring Db selectively to neurons in vivo, synapse pruning is rescued, as well as LTD and AMPA type glutamate receptor subunit composition at retinogeniculate synapses. These observations demonstrated that Db must function in neurons, and showed that there are separate and parallel roles for Db in neurons vs in immune cells. Three Specific Aims are proposed here: 1) Validate and extend understanding of the relationship between Db, synapse pruning and AMPA type glutamate receptor (GluA1/2) subunit composition observed in vivo using an in vitro system to manipulate systematically Db levels in cortical Layer 2/3 pyramidal neurons. Expression constructs for Db, Kb, or shRNAs, will be used for gain- or loss-of function, or rescue experiments in cortical neuron cultures from WT vs KbDb-/- or Db-/- mice. Synapse pruning and GluA1/2 subunit composition will be assessed biochemically and electrophysiologically. The effects of blocking synaptic transmission will also be studied. 2) Identify molecular interactions between Db (or Kb) and associated molecules using biochemical methods and Mass Spectrometry (MS). Preliminary results from MS suggest that there may be direct interactions with synaptic components as well as proteins associated with membrane trafficking. Such interactions will be validated using co-immunoprecipitation, and can illuminate how Db (or Kb) might directly or indirectly regulate glutamate receptors. 3) Investigate a possible role of the short intracellular ail of Db in regulating pruning, AMPA receptor subunit composition and receptor trafficking by mutational analysis. Together these experiments should help establish a strong link between MHCI molecules such as Db, synapse pruning, neural activity and AMPA type glutamate receptor subunit composition. This knowledge may make it possible someday to correct neurodevelopmental disorders such as Schizophrenia or Autism in which pruning has gone awry, or even to treat neurodegenerative disorders such as Alzheimer's in which excessive synapse pruning destroys the very neural circuits in which memories reside.