Mechanisms underlying glutamate dyshomeostasis in Alzheimer's disease

阿尔茨海默病认知功能下降的关键神经病理基础是 突触。阿尔茨海默病发病机制的一个主要观点是，突触异常是 导致增强突触消除的产物。阿尔茨海默病中的突触被认为主要是由于 Aβ1-42肽(OAβ)的毒性可溶低聚物的生产。可溶性Aβ低聚物，但不是单体， 已被证明导致突触功能障碍，表现为抑制LTP，增强LTD，丢失 树突棘、生化异常和多动症。增强的LTD和多动症似乎 这是由于谷氨酸重摄取受损导致细胞外谷氨酸升高所致。虽然 已有大量证据支持这一假说，在我们的 了解OAβ如何扰乱谷氨酸动态平衡。具体地说，谷氨酸的身份 一个或多个被OAβ靶向产生谷氨酸稳态缺陷的转运体是未知的，就像 OAβ影响谷氨酸转运功能的分子机制。这些差距隐约可见 鉴于最近有证据表明，针对可溶性Aβ的单抗(阿杜卡努单抗；BAN2401) AD患者中的寡聚体可能会减缓认知能力的下降。前脑中主要的谷氨酸转运体是GLT- 1(人类同源基因EAAT2)，占大脑蛋白质的1%。GLT-1在星形胶质细胞和星形胶质细胞均有表达 谷氨酸能轴突终末。申请人最近使用有条件的GLT-1基因敲除(KO)进行的工作 结果表明，轴突末端表达的GLT-1是介导谷氨酸摄取的主要转运体 转化为粗制突触体制剂，也称为质膜囊泡(PMV)。GLT-1 在突触前终末的表达也被证明在突触线粒体中起着重要作用 新陈代谢。几项研究表明，在人类和小鼠模型中，谷氨酸转运体存在缺陷 在AD中出现表达和/或功能。在关键实验中，PMV对谷氨酸的摄取源于 用OAβ处理脑片时，脑片减少，提示有神经元GLT-1的存在。中环 支持这个项目的假设是，GLT-1是OAβ产生谷氨酸的主要机制靶点 稳态失调。鉴于这些发现，确定GLT-1是否是特定的谷氨酸是很重要的 以OAβ为靶向的转运体，OAβ是否影响星形胶质细胞或神经元的GLT-1功能，或两者都影响，以及 β与OA-1相互作用的分子基础。该项目的具体目标是： 目的1：确定OAβ功能受损的谷氨酸转运体。 目的2：用条件性β-1KO法确定OA对GLT-1影响的细胞定位。 对这些目标的追求将导致分子上理解OAβ，AD的主要细胞毒素， 扰乱AD中的谷氨酸平衡，最终导致预防和治疗AD的新方法。

The critical neuropathology underlying the cognitive decline in Alzheimer's disease is the loss of synapses. A leading view of the pathogenesis of Alzheimer's disease is that synaptic abnormalities are produced that lead to enhanced synapse elimination. The synaptopathy in AD is thought to be due largely to the production of toxic soluble oligomers of the Aβ1-42 peptide (oAβ). Soluble Aβ oligomers, but not monomers, have been shown to cause synaptic dysfunction, manifest by inhibition of LTP, enhancement of LTD, loss of dendritic spines, biochemical abnormalities, and hyperactivity. The enhanced LTD and hyperactivity appear to be due to elevation of extracellular glutamate as a consequence of impaired glutamate reuptake. Although substantial evidence has accumulated to support this hypothesis, there are significant gaps in our understanding of how oAβ perturbs glutamate homeostasis. Specifically, the identity of the glutamate transporter or transporters targeted by oAβ to produce the defect in glutamate homeostasis is unknown, as are the molecular mechanisms by which glutamate transport function is compromised by oAβ. These gaps loom greater in light of recent evidence that monoclonal antibodies (aducanumab; BAN2401) targeting soluble Aβ oligomers in AD patients may slow cognitive decline. The major glutamate transporter in the forebrain is GLT- 1 (human homolog EAAT2), which represents 1% of brain protein. GLT-1 is expressed in both astrocytes and glutamatergic axon terminals. Recent work by the applicant using a conditional GLT-1 knockout (KO) has shown that GLT-1 expressed in axon terminals is the dominant transporter mediating glutamate uptake into crude synaptosome preparations, also known as plasma membrane vesicles (PMVs). GLT-1 expressed in presynaptic terminals has also been shown to play an important role in synaptic mitochondrial metabolism. Several studies suggest that in the human and in mouse models deficits in glutamate transporter expression and/or function occur in AD. In critical experiments, glutamate uptake into PMVs derived from brain slices was decreased when the slices were treated with oAβ, implicating neuronal GLT-1. The central hypothesis motivating this project is that GLT-1 is the primary mechanistic target of oAβ causing glutamate dyshomeostasis. Given these findings it is important to ascertain whether GLT-1 is the specific glutamate transporter targeted by oAβ, whether oAβ affects GLT-1 function in astrocytes or neurons, or both, and the molecular basis for the interaction of oAβ with GLT-1. The specific goals of this project are to: Aim 1: Identify the glutamate transporter whose function is impaired by oAβ. Aim 2: Determine the cellular localization of effects of oAβ on GLT-1 using a conditional GLT-1 KO. The pursuit of these goals will lead to a molecular understanding of how oAβ, the salient AD cytotoxins, perturb glutamate homeostasis in AD and ultimately lead to novel approaches to prevent and treat AD.