Kynurenic Acid Synthesis in Mouse Brain

描述（由申请人提供）：目前估计，到2050年，治疗各种神经退行性疾病引起的老年痴呆症的费用将消耗工业化国家的全部国民生产总值。因此，必须紧急开发有效的预防措施和治疗神经退行性疾病。在哺乳动物脑中，谷氨酸介导的神经传递负责基础兴奋性突触传递和许多形式的神经可塑性反应。然而，大幅增加谷氨酸介导的兴奋活性水平的事件通常诱导兴奋毒性神经元损伤，导致神经退行性疾病的发展。犬尿烯酸（KYNA，色氨酸代谢物）作为谷氨酸受体拮抗剂，被认为在调节谷氨酸介导的神经传递中发挥作用。脑KYNA的缺乏或过度积累与多种神经退行性疾病如阿尔茨海默病和帕金森病有关，这表明未能维持脑KYNA的生理水平可能是神经退行性疾病的致病因素之一。KYNA是由犬尿氨酸氨基转移酶（KAT）催化的犬尿氨酸转氨反应产生的。由于KYNA不能通过血脑屏障，它必须在大脑中产生。基于脑KYNA水平异常与神经退行性疾病之间的密切关系，脑KAT已被认为是调节人脑KYNA合成和预防神经退行性疾病的潜在靶点。然而，为了靶向KAT用于脑KYNA调节，有必要确定存在的KAT的数量，每个对脑KYNA产生的具体贡献以及它们的结构特征。我们假设脑KYNA合成是许多氨基转移酶的集体贡献，并且各个KAT的具体贡献取决于它们在脑中的相对含量和定位以及它们对犬尿氨酸和其他底物的总体催化效率而不同。这项研究建议的具体目标是：（1）通过其重组蛋白的表达和广泛的生物化学表征来分析目前鉴定的KAT对犬尿氨酸和其它底物的底物特异性和催化效率，（2）通过基于抗原-抗体的技术使用小鼠作为模型物种来确定脑KAT的身份、相对量和细胞定位，（3）通过大分子晶体学方法建立KATs的结构/功能关系。实验室小鼠被广泛认为是研究人类疾病的模型物种，因为小鼠和人类共享其绝大多数生理和病理特征。预计计划研究的结果将有助于我们了解人脑KYNA生物合成和调控，这可能会提供有关维持人脑正常KYNA水平的策略的见解。 公共卫生相关性：犬尿烯酸（KYNA）是一种非选择性的兴奋性氨基酸受体拮抗剂，参与调节哺乳动物脑内的多巴胺能神经传递。脑KYNA的异常浓度与许多神经退行性疾病如阿尔茨海默病、帕金森病和亨廷顿病有关。本提案中描述的研究调查了负责使用小鼠作为模型物种产生脑KYNA的酶的生理学和生物化学。

DESCRIPTION (provided by applicant): It is currently estimated that in 2050, the cost of treating the aging population for dementia caused by various neurodegenerative diseases will consume the entire gross national product of the industrialized countries. Consequently, effective preventive measures and treatments for neurodegenerative diseases must be urgently developed. In mammalian brains, glutamate-mediated neurotransmission is responsible for basal excitatory synaptic transmission and many forms of neuroplastic responses. However, events that substantially increase the level of glutamate-mediated excitatory activity often induce excitotoxic neuronal injury, leading to the development of neurodegenerative diseases. Kynurenic acid (KYNA, a tryptophan metabolite) acts as a glutamate receptor antagonist and is believed to play a role in modulating glutamate-mediated neurotransmission. Deficiency or overaccumulation of brain KYNA has been linked to a variety of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases, suggesting that failure to maintain physiological levels of brain KYNA could be one of the causative factors of neurodegenerative diseases. KYNA is produced by the kynurenine aminotransferase (KAT)-catalyzed transamination of kynurenine. Because KYNA cannot pass through the blood brain barrier, it must be produced in the brain. Based on the intimate relationship between abnormal brain KYNA levels and neurodegenerative diseases, brain KATs have been considered potential targets for regulating human brain KYNA synthesis and preventing neurodegenerative diseases. To target KATs for brain KYNA regulation, however, it is necessary to determine the number of KATs present, the specific contribution of each to brain KYNA production, and their structural characteristics. We hypothesize that brain KYNA synthesis is the collective contribution of a number of aminotransferases and that the specific contribution of individual KATs differs depending on their relative content and localization in the brain as well as their overall catalytic efficiency to kynurenine and other substrates. The specific aims of this research proposal are: (1) to analyze the substrate specificity and catalytic efficiency of the currently identified KATs to kynurenine and other substrates through the expression and extensive biochemical characterization of their recombinant proteins, (2) to determine the identity, relative amount and cellular localization of brain KATs through antigen- antibody based techniques using mice as the model species, and (3) to establish the structure/function relationship of KATs through macromolecular crystallography. Laboratory mice are widely considered the model species for studying human diseases because mice and humans share the vast majority of their physiological and pathological features. It is anticipated that the results from the planned research will contribute to our understanding of the human brain KYNA biosynthesis and regulation, which may provide insight regarding strategies towards maintaining normal KYNA levels in the human brain. PUBLIC HEALTH RELEVANCE: Kynurenic acid (KYNA) is a nonselective antagonist of excitatory amino acid receptors and involved in modulating glutamatergic neurotransmission in mammalian brains. Abnormal concentrations of brain KYNA have been linked to a number of neurodegenerative diseases such as Alzheimer's, Parkinson's and Huntington's diseases. The research described in this proposal investigates physiology and biochemistry of enzymes responsible for the production of brain KYNA using mice as model species.