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发育关键期活动依赖性电路调谐的细胞和分子机制。在这些时候，大脑回路的正常形成需要适当的神经活动和经验模式，而异常的活动或经验可能会扰乱结果。这里的具体假设是，在小鼠中的两个特定的主要组织相容性I类基因（MHCI;人类中的HLA）H2-Db（Db）和H2-Kb（Kb）在神经元和突触中表达，在发育关键期调节活性依赖性突触修剪和可塑性。MHCI基因以其在细胞介导的免疫中的作用而闻名，但在这里，我们研究了神经元中的新作用。神经元MHCI在CNS神经元中的表达在发育中的视觉系统中由神经活动调节的基因的无偏体内筛选中意外地被发现。接下来的研究表明，50多个MHCI基因中只有2个基因（Db和/或Kb）的功能丧失会阻止体内突触修剪，并出乎意料地增强视觉皮层可塑性和运动学习。此外，仅仅通过在体内将Db选择性地恢复到神经元，突触修剪被拯救，以及在retinogeniculate突触处的LTD和AMPA型谷氨酸受体亚基组成。这些观察结果表明，Db必须在神经元中发挥作用，并表明Db在神经元中与免疫细胞中具有独立和平行的作用。本研究的目的有三：1）通过体外系统调控皮层2/3层锥体神经元Db水平，进一步了解Db、突触修剪和AMPA型谷氨酸受体（GluA 1/2）亚基组成之间的关系。Db、Kb或shRNA的表达构建体将用于来自WT与KbDb-/-或Db-/-小鼠的皮质神经元培养物中的功能获得或丧失或拯救实验。突触修剪和GluA 1/2亚基组成将进行生化和电生理评估。还将研究阻断突触传递的影响。2)使用生物化学方法和质谱法（MS）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，可能存在与突触成分以及与膜运输相关的蛋白质的直接相互作用。这种相互作用将使用免疫共沉淀进行验证，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ail在调节修剪、AMPA受体亚基组成和受体运输中的可能作用。总之，这些实验应该有助于建立一个强大的联系之间的MHCI分子，如Db，突触修剪，神经活动和AMPA型谷氨酸受体亚基组成。这一知识可能使我们有可能在某一天纠正神经发育障碍，如精神分裂症或自闭症，其中修剪已经出错，甚至治疗神经退行性疾病，如阿尔茨海默氏症，其中过度的突触修剪破坏了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.