Global, Cell Type Specific Modulation of Selected Glutamate Transporters

选定谷氨酸转运蛋白的全局、细胞类型特异性调节

• 批准号: 7345415
• 负责人: DAVID J POULSEN
• 金额: $ 15.48万
• 依托单位: UNIVERSITY OF MONTANA
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-04-01 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7345415
• 关键词: Amino Acid Transporter; Amyotrophic Lateral Sclerosis; Astrocytes; Biological Models; Biology; Brain; Cell membrane; Cell surface; Cessation of life; Condition; Data; Dependovirus; Development; Disease; Environment; Epilepsy; Excitatory Amino Acids; Future; Gene Delivery; Gene Expression; Genes; Glutamate Transporter; Glutamates; Goals; Individual; Injection of therapeutic agent; Injury; Ischemia; Literature; Mediating; Methods; Nerve Degeneration; Neurons; Numbers; Pathology; Physiological; Play; Proteins; Rattus; Recombinant adeno-associated virus (rAAV); Recombinants; Role; Serotyping; Solid; Stroke; Synaptic Cleft; Testing; Translational Research; Ventricular; Viral; adeno-associated viral vector; analog; base; cell type; extracellular; knock-down; neuropathology; neuroprotection; new technology; novel; promoter; pup; receptor; tool; tool development; trafficking; transmission process; vector

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.

描述（由申请人提供）：在正常的生理条件下，神经元上谷氨酸介导的兴奋受体激活是短暂的。然而，在病理条件下，细胞外谷氨酸水平可以升高，导致兴奋受体的刺激延长，并导致神经元的兴奋性死亡。五个兴奋性氨基酸转运蛋白（EAATS 1-5）在确保兴奋性传播是通过快速清除突触裂口和细胞外环境的瞬时传播方面起着关键作用。但是，在某些病理状况（例如缺血）下，这些转运蛋白的逆转实际上可能导致病理和神经元损害。因此，谷氨酸转运蛋白具有促进神经保护或神经退行性的潜力。目前的文献中存在矛盾，该文献对单个转运蛋白同种型在兴奋性毒性损伤的背景下对神经保护或神经变性的贡献产生了贡献。用于研究谷氨酸转运蛋白的模型系统主要仅限于转运蛋白抑制。药理学研究成功地鉴定了许多全球抑制谷氨酸转运蛋白的谷氨酸类似物。很少有人鉴定出可以增强谷氨酸转运蛋白活性的药理学剂，并且没有能力以细胞类型的特定方式区分选定的转运蛋白。相反，我们的初步数据强烈表明，通过使用重组腺癌相关病毒（AAV）载体（在细胞类型的特定于促销剂的控制下），可以在细胞类型的特定方式抑制或增强单个转运蛋白同种型的表达。但是，我们已经观察到基因表达仅限于AAV载体立体定向递送后中枢神经系统内的焦点区域。我们在该提案中的目标是严格测试该工具，并确定可以调节功能转运蛋白表达的程度，并进一步开发该方法，以通过新型AAV血清型对P0大鼠幼崽对P0大鼠幼崽的脑室室心室注入。相关性，这种基于病毒的基因输送方法的发展将提供一种新型工具，该工具将允许以先前不可能的方式检查谷氨酸转运蛋白功能。这种方法将使我们能够直接检验有关单个谷氨酸转运蛋白同种型如何促进疾病和疾病（例如中风，癫痫和ALS）的神经保护或神经病理学的关键假设。这些工具的开发还将为未来的翻译研究提供扎实的科学基础，涉及所选转运蛋白的细胞类型特定调节，作为涉及兴奋性毒性成分的疾病的潜在治疗方法。