Motor Neuron Protection through Glutamate Degradation

通过谷氨酸降解保护运动神经元

• 批准号: 6335964
• 负责人: PAUL S FISHMAN
• 金额: $ 14.85万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-01 至 2003-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6335964
• 关键词: alanine transaminase; amyotrophic lateral sclerosis; chimeric proteins; confocal scanning microscopy; cytoprotection; disease /disorder model; enzyme activity; glutamates; high performance liquid chromatography; histology; laboratory mouse; laboratory rat; motor neurons; neuronal transport; neuroprotectants; neurotoxins; neurotransmitter metabolism; neurotransmitter transport; newborn animals; protein localization; spinal cord; tetanus toxin; tissue /cell culture

DESCRIPTION (provided by applicant): Glutamate-provoked excitotoxicity may contribute to the pathogenesis of both acute and degenerative neurological disease. Although glutamate re-uptake is the normal manner through which the action of the neurotransmitter is terminated, glutamate can also be eliminated through enzymatic degradation. We have recently created a functionally active hybrid protein that links a glutamate-degrading enzyme (glutamate-pyruvate-transaminase, GPT-also known as ALT) with the non-toxic neuronal binding domain of tetanus toxin (tetanus toxin fragment C or TTC). The rationale for producing this molecule is to deliver an enhanced capacity to enzymatically-degrade glutamate to synaptic regions surrounding motor neurons, and thereby protect them from toxic levels of glutamate. Our previous studies have demonstrated: 1) TTC can dramatically enhance (100 to 1,000 fold) the delivery of active enzyme to neurons in vitro and motor neurons in vivo. 2) TTC-linked enzymes are targeted to synaptic regions surrounding neurons in vitro and motor neurons in vivo. 3) GPT is the most effective enzyme to rapidly (within minutes) reduce neurotoxic levels of glutamate. 4) GPT can protect neurons in vitro from both direct exposure to toxic levels of glutamate and the toxic effect of inhibition of glutamate re-uptake. We propose to assess TTC-GPT in models of chronic glutamate excitotoxicity and motor neuron degeneration. Initially, we will quantitate the uptake and persistence of active enzymes to explant cultures of neo-natal rat spinal cord with both native GPT and TTC-GPT. We will then assess the capacity of TTC-GPT to prevent progressive motor neuronal death in these spinal cord cultures exposed to inhibitors of glutamate re-uptake, or after reduction of synthesis of high-affinity glutamate transporters using antisense oligonucleotides. We will assess the capacity of TTC-GPT to deliver active enzymes to motor neurons from an intramuscular injection, via retrograde axonal transport, in both normal mice and in a murine model of familial ALS. This proposal will determine the potential of not only glutamate degradation as an anti-excitotoxic strategy, but of TTC as a vector to deliver therapeutic proteins with synaptic sites of action. If successful, TTC-GPT can be assessed in animal models of motor neuron disease, such as the FALS mouse, in the future. These studies will advance understanding of the potential of a novel means of glutamate elimination with therapeutic implications of diseases for not only ALS, but also other neurologic diseases such as stroke, Alzheimer's Disease and Parkinson's Disease.

描述（由申请人提供）： 谷氨酸引起的兴奋性毒性可能有助于两者的发病机制 急性和退行性神经系统疾病。虽然谷氨酸再摄取是 神经递质的正常作用方式是 终止，谷氨酸也可以通过酶促降解消除。我们 最近创造了一种功能活性的杂交蛋白， 谷氨酸降解酶（谷氨酸-丙酮酸-转氨酶，GPT-也称为 ALT）与破伤风毒素（破伤风毒素）的无毒神经元结合结构域 片段C或TTC）。生产这种分子的基本原理是 增强酶促降解谷氨酸至突触区域的能力 周围的运动神经元，从而保护它们免受毒性水平的 谷氨酸盐。我们以前的研究表明：1）TTC可以显着地 增强（100至1，000倍）活性酶向体外神经元的递送 和运动神经元。2)TTC连接的酶靶向突触 体外的神经元周围区域和体内的运动神经元。3)GPT是 最有效的酶，以迅速（在几分钟内）减少神经毒性水平的 谷氨酸盐。4)GPT可以保护体外神经元免受直接暴露于 谷氨酸的毒性水平和抑制谷氨酸的毒性作用 再摄取。我们建议在慢性谷氨酸盐模型中评估TTC-GPT 兴奋性毒性和运动神经元变性。首先，我们将量化 新生大鼠组织块培养中活性酶摄取和持久性 用天然GPT和TTC-GPT两者的脊髓。然后我们将评估 TTC-GPT预防这些脊髓中进行性运动神经元死亡 暴露于谷氨酸再摄取抑制剂的培养物，或在谷氨酸再摄取抑制剂减少后， 使用反义核酸合成高亲和力谷氨酸转运蛋白 寡核苷酸我们将评估TTC-GPT提供活性 酶从肌内注射，通过逆行轴突运动神经元 运输，在正常小鼠和家族性ALS的小鼠模型中。这 该提案将确定不仅谷氨酸降解作为一种 抗兴奋性毒性策略，但TTC作为提供治疗的载体 具有突触作用位点的蛋白质。如果成功，可以评估TTC-GPT 在运动神经元疾病的动物模型中，例如在FALS小鼠中， 未来这些研究将促进对小说潜力的理解 谷氨酸消除的方法与疾病的治疗意义， 不仅是肌萎缩性侧索硬化症，还有其他神经系统疾病， 帕金森氏病和帕金森氏病