RNA editing of AMPA receptor subunit GluR2 in ischemia

描述（由申请人提供）：缺血性中风是发达国家第三大死亡原因。该疾病的一个关键特征是神经元丧失的高度选择性模式；某些可识别的神经元亚群，特别是海马中的CA1锥体神经元，严重受损，而其他神经元则完好无损。这种选择性神经元损伤的一个步骤涉及Ca2+通过Ca2+可渗透的AMPA受体通道进入。AMPA受体是谷氨酸受体（GluRs）的一个主要亚型，由GluR1-4亚基组装而成。通道的Ca2+通透性主要由Q/R位点的GluR2 RNA编辑控制；编辑的GluR2(R)亚基形成Ca2+不渗透通道，而未编辑的GluR2(Q)通道允许Ca2+进入。在大多数CA1神经元中，AMPA受体通道含有GluR2(R)，因此对Ca2+流动不渗透。最近，我们发现短暂性前脑缺血选择性地破坏GluR2 Q/R位点编辑，从而诱导损伤的Ca2+通过AMPA受体通道进入易感的CA1神经元。我们还发现，GluR2 Q/R位点编辑受损与ADAR2（腺苷脱氨酶作用于RNA的缩写）基因表达减少密切相关，ADAR2是负责GluR2 Q/R位点编辑的核酶。因此，我们假设ADAR2基因的表达减少是GluR2 Q/R位点编辑受损的原因。为了直接解决这一假设，我们将确定ADAR2基因表达的恢复是否可以挽救GluR2 Q/R位点编辑，进而阻断AMPA受体通道的Ca2+通透性，从而导致缺血后大鼠易损神经元的存活。总体而言，本项目将有两个具体目的：具体目的1：确定ADAR2基因表达的恢复是否阻断Ca2+通过AMPA受体通道进入，并拯救缺血后大鼠的易感神经元。特异性目的2：确定稳定ADAR2基因沉默的产生是否诱导缺血不敏感神经元的变性，以及ADAR2缺陷神经元的变性是否由一个或多个谷氨酸受体亚基的RNA编辑缺陷引起。总之，该项目将确定adar2依赖性GluR2 Q/R位点编辑决定神经元对缺血的易感性。因此，这项工作将为脑卒中治疗确定一个有希望的靶点。

DESCRIPTION (provided by applicant): Ischemic stroke is the third leading cause of death in developed countries. A critical feature of the disease is a highly selective pattern of neuronal loss; certain identifiable subsets of neurons, particularly CA1 pyramidal neurons in the hippocampus, are severely damaged while others remain intact. A step in this selective neuronal injury involves Ca2+ entry through Ca2+-permeable AMPA receptor channels. AMPA receptors are a major subtype of glutamate receptors (GluRs) that are assembled from GluR1-4 subunits. Ca2+ permeability of the channels is dominated by GluR2 RNA editing at the Q/R site; edited GluR2(R) subunits form Ca2+-impermeable channels, whereas unedited GluR2(Q) channels allow Ca2+ entry. In most CA1 neurons, AMPA receptor channels contain GluR2(R), and thus are impermeable to Ca2+ flow. Recently, we have identified that transient forebrain ischemia selectively disrupts GluR2 Q/R site editing and hence induces injurious Ca2+ entry through AMPA receptor channels into vulnerable CA1 neurons. We have also shown that impaired GluR2 Q/R site editing is closely correlated with reduced expression of ADAR2 (short for adenosine deaminase acting on RNA) gene, a nuclear enzyme responsible for GluR2 Q/R site editing. We thus hypothesize that reduced expression of ADAR2 gene is responsible for the impaired GluR2 Q/R site editing. To address this hypothesis directly, we will determine if restoration of ADAR2 gene expression rescues GluR2 Q/R site editing and in turn blocks Ca2+ permeability of AMPA receptor channels, leading to the survival of vulnerable neurons in the post-ischemic rats. Overall, this project will have two specific aims: Specific Aim 1: To determine whether restoration of ADAR2 gene expression blocks Ca2+ entry through AMPA receptor channels and rescues vulnerable neurons in the post-ischemic rats. Specific Aim 2: To determine if generation of stable ADAR2 gene silencing induces degeneration of ischemia-insensitive neurons, and if degeneration of ADAR2-deficient neurons results from RNA editing deficits of one or more glutamate receptor subunits. Together, this project will identify that ADAR2-dependent GluR2 Q/R site editing determines vulnerability of neurons to ischemia. Thus, this work will define a promising target for stoke therapy.