Structural and Catalytic Functions of CaMKII in Neurons

描述(申请人提供)：钙依赖的蛋白激酶II(CaMKII)是学习和记忆形式所必需的。在大脑中，CaMKII的表达水平与结构蛋白相似(占总蛋白的1-2%)，其12聚体全酶结构允许多种蛋白质同时相互作用。然而，CaMKII的结构作用或其与其他蛋白质相互作用的功能充其量也是知之甚少。哺乳动物脑中的主要兴奋性神经递质谷氨酸可诱导海马神经元CaMKII两种类型的移位：短暂刺激时(0.5-2分钟)移位到突触后位置；长时间刺激后(3-5分钟)移位到突触外簇。这两种类型的易位都需要多价CaMKII全酶结构(未发表的观察结果)。短暂的谷氨酸治疗可以诱导各种形式的突触可塑性，被认为是学习和记忆的基础，而长期暴露会导致兴奋性毒性，这是一种缺血性细胞死亡的细胞模型。这一建议的目的是验证我们的假设，即突触易位需要与NMDA型谷氨酸受体亚单位NR2B结合并参与突触可塑性，而突触外簇的形成是由CaMKII自结合介导的，并参与缺血性细胞死亡。阐明CaMKII靶向的机制和功能将有助于我们理解高级脑功能和行为背后的突触原理，并为缺血性损伤提供新的治疗途径。我们将在以下方面实现我们的目标(使用大鼠海马和皮质培养作为神经元研究的主要模型系统)：(1)确定神经元中亚细胞CaMKII定位所需的蛋白质相互作用。我们将在体外鉴定阻止NR2B结合或自结合的CaMKII突变和抑制剂，并确定它们对GFP-CaMKII在神经元中定位的影响。(2)确定CaMKII活性、突触靶向和突触外聚集的神经保护作用与神经毒性作用。我们将利用CaMKII活性和聚集的抑制剂，通过RNAi抑制表达，以及在活性、调节和靶向方面具有特定损伤的CaMKII野生型和突变体的过表达。(3)确定CaMKII在突触数目和强度调节中的作用。我们将使用与(2)中相同的工具，并在已建立的分析中确定对突触数量和强度的影响。

DESCRIPTION (provided by applicant): The Ca2????dependent protein kinase II (CaMKII) is required for forms of learning and memory. In brain, CaMKII is expressed at levels reminiscent of structural proteins (1-2% of total protein), and its 12meric holoenzyme structure allows multiple simultaneous protein interactions. However, structural roles of CaMKII or functions of its interactions with other protein are understood poorly at best. The major excitatory neuro- transmitter in mammalian brain, glutamate, induces two types of CaMKII translocation in hippocampal neurons: To postsynaptic sites upon brief stimulation (0.5-2 min) and to extra-synaptic clusters after extended exposure (3-5 min). Both types of translocation require the multivalent CaMKII holoenzyme structure (unpublished observations). Brief glutamate treatment can induce forms of synaptic plasticity thought to underlie learning and memory, while extended exposure leads to excitotoxicity, a cellular model for ischemic cell death. The goal of this proposal is to test our hypotheses that synaptic translocation requires binding to the NMDA-type glutamate receptor subunit NR2B and is involved in synaptic plasticity, while formation of extrasynaptic clusters is mediated by CaMKII self-association and is involved in ischemic cell death. Elucidating the mechanisms and functions of CaMKII targeting will aid our understanding of synaptic principles underlying higher brain functions and behavior, and provide new therapeutic avenues for ischemic damage. We will accomplish our goal in the following aims (using rat hippocampal and cortical cultures as main model systems for studies in neurons): (1) Determine protein interactions required for subcellular CaMKII localization in neurons. We will identify CaMKII mutations and inhibitors that prevent NR2B-binding or self-association in vitro and determine their effect on GFP-CaMKII localization in neurons. (2) Determine neuroprotective versus neurotoxic effects of CaMKII activity, synaptic targeting, and extrasynaptic clustering. We will make use of an inhibitor of CaMKII activity and clustering, knockdown of expression by RNAi, and overexpression of CaMKII wildtype and mutants with specific impairments in activity, regulation and targeting. (3) Determine functions of CaMKII in regulation of synapse number and strength. We will make use of the same tools as in (2) and determine effects on synapse number and strength in established assays.