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大脑或视网膜的谷氨酸能系统的紊乱。这项建议的目标是了解神经系统中谷氨酸水平的调节机制。大量证据表明，在神经传递过程中，神经胶质细胞迅速内化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，现在很清楚的是，胶质细胞摄取的谷氨酸中，可能有多达50%实际上转化为丙酮酸和乳酸。因此，神经系统必须通过丙酮酸羧化酶(PC)催化丙酮酸的羧化来再生丢失的碳，PC被发现是星形胶质细胞。其次，现在看来，必需的支链氨基酸(BCAA)不是氨，而是提供充足的α-氨基氮，以实现谷氨酸从头合成的最佳速率。事实上，我们的假设是支链氨基酸和支链氨基转移酶同功酶(胞质BCATm，线粒体BCATm)参与了氮穿梭，神经胶质BCATm和神经元特异性BCATm串联作用，将氮从神经元转移到星形胶质细胞。这项提议的目的是为了了解调节神经系统中谷氨酸水平的机制，并测试支链氨基酸穿梭假说。1)我们将建立一种方法来同时测定体外视网膜和体内大脑中谷氨酸合成速率和谷氨酰胺周期蛋白的速率，并将神经元活动的影响与流量相关联。H14CO3和13C核磁共振波谱将用于体内动力学分析。2)BCAT同工酶在调节谷氨酸合成和脑神经递质池大小方面的功能将在体内大脑和体外视网膜量化。神经活性药物加巴喷丁通过特异性抑制胞浆BCATC发挥作用的假设将得到验证。3)将评估逆转录酶丙酮酸羧基酶(PC)相对于BCAT同工酶的控制程度。反义技术将被用来改变体内的酶水平。数据将使用控制强度理论进行分析，以确定BCAT和PC在从头合成谷氨酸的途径上的相对控制强度。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，并可能为未来产生新的治疗化合物奠定基础。

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.