REGULATION OF BRAIN NEUROTRANSMITTER SYNTHESIS

谷氨酸是神经系统中应用最广泛的兴奋性神经递质。 某些疾病和神经退行性疾病被认为涉及脑或视网膜的神经递质系统的紊乱。 该提案的目标是了解调节神经系统中谷氨酸水平的机制。 大量证据表明，胶质细胞迅速内化神经传递过程中神经元释放的谷氨酸，将其转化为谷氨酰胺，然后在谷氨酸/谷氨酰胺循环中将其释放回神经元。 然而，现在很清楚，可能多达50%的谷氨酸被神经胶质细胞吸收，实际上转化为丙酮酸和乳酸。 因此，神经系统必须通过在星形胶质细胞中发现的回补酶丙酮酸羧化酶（PC）催化的丙酮酸羧化来再生失去的碳。 其次，现在看来，需要必需支链氨基酸（BCAA）而不是氨来提供足够的α-氨基氮，以获得谷氨酸从头合成的最佳速率。 事实上，我们的假设是BCAA和支链氨基转移酶同工酶（胞质BCATc、线粒体BCATm）参与氮穿梭，由此神经胶质BCATm和神经元特异性BCATc串联作用以将氮从神经元转移到星形胶质细胞。 该提案的目的是了解调节神经系统中谷氨酸水平的机制，并测试BCAA穿梭假说。 1)我们将开发的方法，同时测量从头谷氨酸合成的速率和谷氨酸谷氨酰胺细胞周期蛋白的速率在离体视网膜和在体内的大脑和相关的神经元活动与流量的影响。 H14 CO 3和13 C NMR光谱将在体内用于动力学分析。 2)BCAT同工酶在调节谷氨酸合成和脑神经递质池大小中的功能将在脑中体内和视网膜中离体定量。 将检验神经活性药物加巴喷丁通过特异性抑制胞质BCATc起作用的假设。 3)将评估回补酶丙酮酸羧化酶（PC）相对于BCAT同工酶的控制程度。反义技术将用于改变体内酶水平。将使用控制强度理论分析数据，以确定BCAT和PC对谷氨酸从头合成途径的相对控制强度。 最后，了解调节谷氨酸合成的机制将增加我们对某些疾病过程的了解，并可能允许未来产生新的治疗化合物。

Glutamate is the most widely used excitatory neurotransmitter in the nervous system. Certain diseases and neurodegenerative disorders are thought to involve disturbances of the glutamatergic systems of the brain or retina. The goal of this proposal is to understand the mechanisms that regulate glutamate levels in the nervous system. Considerable evidence indicates that glial cells rapidly internalize glutamate released by neurons during neurotransmission converting it to glutamine before releasing it back to neurons in the glutamate/glutamine cycle. However, it is now clear that possibly as much as 50 percent of the glutamate taken up by the glia is actually converted to pyruvate and lactate. Therefore, the nervous system must regenerate the lost carbon by carboxylation of pyruvate catalyzed by the anaplerotic enzyme pyruvate carboxylase (PC) which is found is astroglia. Second, it now appears that rather than ammonia the essential branched chain amino acids (BCAA) are needed to provide sufficient alpha-amino nitrogen for optimal rates of de novo glutamate synthesis. Indeed, it is our hypothesis that the BCAA and branched chain aminotransferase isoenzymes (cytosolic BCATc, mitochondrial BCATm) participate in a nitrogen shuttle whereby the glial BCATm and neuron specific BCATc act in series to transfer nitrogen from neurons to astrocytes. The aims of this proposal are designed to understand the mechanisms that regulate glutamate levels in the nervous system and test the BCAA shuttle hypothesis. 1) We will develop methods to simultaneously measure the rate of de novo glutamate synthesis and the rate of glutamate glutamine cyclin in the ex vivo retina and in brain in vivo and correlate the influence of neuronal activity with flux. Both H14CO3 and 13C NMR spectroscopy will be used in vivo for the kinetic analysis. 2) The function of the BCAT isoenzymes in regulating glutamate synthesis and brain neurotransmitter pool sizes will be quantified in vivo in brain and ex vivo in the retina. The hypothesis that the neuroactive drug gabapentin acts via specific inhibition of cytosolic BCATc will be tested. 3) The degree of control exerted by the anaplerotic enzyme pyruvate carboxylase (PC) relative to that of the BCAT isoenzymes will be assessed. Antisense technology will be used to vary enzyme levels in vivo. Data will be analyzed using control strength theory to determine the relative control strength of the BCAT and PC on the pathways of de novo glutamate synthesis. Finally, understanding the mechanisms that regulate glutamate synthesis will increase our knowledge of certain disease processes and may allow for future generation of novel therapeutic compounds.