Global, Cell Type Specific Modulation of Selected Glutamate Transporters

描述(申请人提供)：在正常的生理条件下，谷氨酸介导的神经元兴奋性受体的激活是短暂的。然而，在病理条件下，细胞外谷氨酸水平会升高，导致兴奋性受体的长时间刺激，并导致神经元的兴奋性死亡。5种兴奋性氨基酸转运体(EAATs 1-5)通过快速清除突触间隙和细胞外环境中的谷氨酸，在确保兴奋性传递是一过性的过程中起着关键作用。然而，在某些病理条件下，如缺血，这些转运蛋白的逆转实际上可能导致病理和神经元损伤。因此，谷氨酸转运体具有促进神经保护或神经变性的潜力。在目前的文献中，关于单个转运体同种类型对兴奋性毒性损伤中的神经保护或神经变性的贡献，存在着矛盾。用于研究谷氨酸转运体的模型系统主要局限于转运体抑制。药理学研究已经成功地确定了一些谷氨酸类似物，它们在全球范围内抑制谷氨酸转运体。目前已确定的增强谷氨酸转运蛋白活性的药物很少，而且没有一种药物能够以细胞类型特异性的方式区分选定的转运蛋白。相反，我们的初步数据强烈表明，在细胞类型特异性启动子的控制下，通过重组腺相关病毒(AAV)载体以正义或反义方向递送选定的谷氨酸转运蛋白基因，可以以细胞类型特异性的方式抑制或增强个别转运体同种类型的表达。然而，我们观察到，在立体定向递送AAV载体后，基因表达仅限于中枢神经系统内的焦点区域。我们在这项建议中的目标是严格测试这一工具，并确定在多大程度上可以调节功能性转运蛋白的表达，并进一步发展这一方法，通过脑室注射具有新AAV血清型的P0大鼠幼鼠，在中枢神经系统提供全球、细胞类型特异性转运蛋白基因的分布和表达。相关性这种基于病毒的基因传递方法的发展将提供一种新的工具，它将允许以一种以前不可能的方式检查谷氨酸转运蛋白的功能。这种方法将使我们能够直接测试关键假设，即单个谷氨酸转运体同种类型如何在中风、癫痫和ALS等疾病和障碍中对神经保护或神经病理做出贡献。这些工具的开发也将为未来的转译研究提供坚实的科学基础，涉及特定细胞类型对选定转运体的调节，作为涉及兴奋毒性成分的疾病的潜在治疗方法。

DESCRIPTION (provided by applicant): Under normal physiological conditions, glutamate-mediated activation of excitatory receptors on neurons is transient. However, under pathological conditions, extracellular glutamate levels can become elevated, resulting in prolonged stimulation of the excitatory receptors, and leading to excitotoxic death of neurons. Five excitatory amino acid transporters (EAATs 1-5) play a key role in insuring that excitatory transmission is transient by rapidly clearing glutamate from synaptic clefts, and the extracellular environment. However, under certain pathological conditions, such as ischemia, reversal of these transporters may actually contribute to pathology and neuronal damage. Thus, glutamate transporters have the potential to promote either neuroprotection or neurodegeneration. Contradictions exist in the current literature in regards to the contribution that individual transporter isotypes provide towards neuroprotection or neurodegeneration in the context of excitotoxic injury. The model systems used to study glutamate transporters have been primarily limited to transporter inhibition. Pharmacological studies have successfully identified a number of glutamate analogs that globally inhibit glutamate transporters. Very few pharmacological agents have been identified which enhance glutamate transporter activity and none have the ability to discriminate between selected transporters in a cell type specific manner. In contrast, our preliminary data strongly suggests that the expression of individual transporter isotypes can be either inhibited or enhanced in a cell type specific manner by using recombinant Adeno-associated virus (AAV) vectors to deliver selected glutamate transporter genes in either the sense or antisense orientation, under the control of cell type specific promoters. However, we have observed that gene expression is limited to focal regions within the CNS following stereotactic delivery of AAV vectors. Our goal in this proposal is to rigorously test this tool and determine to what extent functional transporter expression can be modulated and further develop this method to provide global, cell type specific transporter gene distribution and expression in the CNS through intracerebral ventricular injection of P0 rat pups with novel AAV serotypes. Relevance The development of this viral based gene delivery method will provide a novel tool which will permit the examination of glutamate transporter function in a way that has not been previously possible. This approach will allow us to directly test critical hypotheses regarding how individual glutamate transporter isotypes contribute to neuroprotection or neuropathology in diseases and disorders such as stroke, epilepsy and ALS. The development of these tools will also provide a solid scientific basis for future translational research involving the cell type specific modulation of selected transporters as potential treatments for disorders involving an excitotoxic component.