Glycolysis and Glutamate Storage into Synaptic Vesicles

描述（申请人提供）：葡萄糖代谢对维持正常的突触传递和大脑功能至关重要。急性血糖引起异常突触传递和脑功能，而不显著降低细胞总ATP水平。这种机制在分子水平上还没有得到很好的理解。我们最近提供的证据表明，甘油醛磷酸脱氢酶和3-磷酸甘油激酶，负责产生ATP的糖酵解酶，与突触囊泡有关，这些酶的激活足以支持谷氨酸摄取到突触囊泡。其他证据表明，在神经末梢的突触囊泡中，谷氨酸的积累是由突触囊泡表面糖酵解和局部产生的ATP而不是线粒体合成的ATP发挥重要作用。最近，我们已经获得了初步证据，表明丙酮酸激酶（PK），另一种能够合成ATP的糖酵解酶，也与突触囊泡结合，并且该酶的激活导致突触囊泡对谷氨酸的显著摄取。谷氨酸是脊椎动物中枢神经系统中一种主要的兴奋性神经递质，谷氨酸的正常传递对神经元的交流、学习和记忆功能至关重要。有趣的是，苯丙酮酸在苯丙酮尿患者的血液中含量很高，被发现可以抑制pk介导的水疱性谷氨酸摄取，这表明这种水疱结合酶具有一定的临床意义。我们假设突触囊泡结合糖酵解酶PK与v型质子泵atp酶在功能上偶联，产生电化学质子梯度（谷氨酸摄取到突触囊泡的驱动力），从而在谷氨酸传递中起关键作用。我们计划提供足够的证据来支持上述假设，目的如下：(1)表征囊泡结合的PK介导的谷氨酸摄取到突触囊泡中；(2)证明PK和苯丙酮酸在突触小体中谷氨酸的囊泡积累中起重要作用。为了实现目标1，我们将使用分离的大鼠脑突触囊泡，从多个方面研究磷酸烯醇丙酮酸和adp依赖性摄取。为了实现2，我们将确定膜渗透的PK抑制剂对突触体中囊泡谷氨酸含量和释放的影响。本研究将有助于更好地理解糖代谢与突触传递/脑功能之间的关系。希望从这项研究中获得的知识将用于创造一种治疗某些神经和精神疾病的新方法。

DESCRIPTION (provided by applicant): Glucose metabolism is of vital importance in maintaining proper synaptic transmission and brain function. Acute glycemia induces aberrant synaptic transmission and brain function without a significant reduction in global cellular ATP levels. The mechanism is not well understood at the molecular level. We have recently provided evidence that glyceraldehyde phosphate dehydrogenase and 3-phosphoglycerate kinase, glycolytic enzymes responsible for production of ATP, are associated with synaptic vesicles and that activation of these enzymes is sufficient to support glutamate uptake into synaptic vesicles. Additional evidence suggests that glycolytically and locally produced ATP on the surface of synaptic vesicles, rather than ATP synthesized in mitochondria, plays an important role in accumulating glutamate into synaptic vesicles in the nerve ending. More recently we have obtained preliminary evidence that pyruvate kinase (PK), another glycolytic enzyme capable of ATP synthesis, is also bound to synaptic vesicles, and that activation of this enzyme leads to marked glutamate uptake into synaptic vesicles. Glutamate is a major excitatory neurotransmitter in the vertebrate central nervous system, and proper glutamate transmission is essential for neuronal communication, learning and memory function. Of interest, phenylpyruvate, whose blood levels are high in phenylketonuria, was found to inhibit PK-mediated vesicular glutamate uptake, suggesting some clinical implication for this vesicle-bound enzyme. We postulate that the synaptic vesicle-bound glycolytic enzyme PK is functionally coupled to V-type proton pump ATPase, which generates an electrochemical proton gradient (the driving force for glutamate uptake into synaptic vesicles) and thereby plays a critical role in setting the stage for glutamate transmission. We plan to provide sufficient evidence to support the hypothesis mentioned above, with the following aims: (1) to characterize vesicle-bound PK-mediated glutamate uptake into synaptic vesicles, and (2) to demonstrate that PK and phenylpyruvate play important roles in vesicular accumulation of glutamate in synaptosomes. To achieve aim 1, we will study phosphoenol pyruvate plus ADP-dependent uptake in a number of respects, using isolated rat brain synaptic vesicles. To achieve 2, we will determine the effect of membrane-permeant PK inhibitors on vesicular glutamate content in and release from synaptosomes. This study is expected to contribute to better understanding of the relation between glucose metabolism and synaptic transmission/brain function. It is hoped that knowledge gained from this research will be of use in creating a new approach to treatment of certain neurological and mental disorders.