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）：在正常生理条件下，谷氨酸介导的神经元兴奋性受体激活是短暂的。然而，在病理条件下，细胞外谷氨酸水平可能升高，导致兴奋性受体的长期刺激，并导致神经元的兴奋性毒性死亡。五种兴奋性氨基酸转运蛋白（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短暂的方面发挥关键作用。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，谷氨酸转运蛋白具有促进神经保护或神经变性的潜力。目前的文献中存在矛盾的贡献，个别转运蛋白同种型提供对神经保护或神经变性的兴奋性毒性损伤的背景下。用于研究谷氨酸转运蛋白的模型系统主要限于转运蛋白抑制。药理学研究已经成功地确定了许多谷氨酸类似物，全面抑制谷氨酸转运蛋白。已经鉴定了非常少的增强谷氨酸转运蛋白活性的药理学试剂，并且没有一种具有以细胞类型特异性方式区分所选转运蛋白的能力。相反，我们的初步数据强烈表明，通过使用重组腺相关病毒（AAV）载体在细胞类型特异性启动子的控制下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方式抑制或增强单个转运蛋白同种型的表达。然而，我们已经观察到，在立体定向递送AAV载体后，基因表达限于CNS内的病灶区域。我们在该提案中的目标是严格测试该工具，并确定在何种程度上可以调节功能性转运蛋白表达，并进一步开发该方法，通过脑室内注射具有新型AAV血清型的P0大鼠幼崽，提供CNS中的全局细胞类型特异性转运蛋白基因分布和表达。这种基于病毒的基因递送方法的发展将提供一种新的工具，这将允许以以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测试关于单个谷氨酸转运蛋白同种型如何有助于中风，癫痫和ALS等疾病和病症的神经保护或神经病理学的关键假设。这些工具的开发也将为未来的转化研究提供坚实的科学基础，这些研究涉及选定转运蛋白的细胞类型特异性调节，作为涉及兴奋性毒性成分的疾病的潜在治疗方法。