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。急性血糖可诱导异常的突触传递和脑功能，而不会显著降低整体细胞的ATP水平。其机制在分子水平上还没有被很好地理解。我们最近提供的证据表明，负责产生ATP的糖酵解酶磷酸甘油醛脱氢酶和3-磷酸甘油酸激酶与突触小泡有关，这些酶的激活足以支持谷氨酸摄取到突触小泡。更多的证据表明，糖酵解和局部产生的ATP在突触小泡表面，而不是线粒体合成的ATP，在神经末梢将谷氨酸积累到突触小泡中起着重要作用。最近，我们获得了初步证据，丙酮酸激酶(PK)，另一种能够合成ATP的糖酵解酶，也与突触小泡结合，该酶的激活导致显著的谷氨酸摄取到突触小泡。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，适当的谷氨酸传递对神经元的交流、学习和记忆功能至关重要。值得注意的是，苯丙酮尿症患者的血液中苯丙酮酸水平很高，它被发现抑制PK介导的囊泡谷氨酸摄取，这表明这种囊泡结合酶具有一定的临床意义。我们推测，突触小泡结合的糖酵解酶PK在功能上与V型质子泵ATPase偶联，从而产生电化学质子梯度(谷氨酸摄取到突触小泡的驱动力)，从而在谷氨酸传递过程中发挥关键作用。我们计划提供足够的证据支持上述假设，目的如下：(1)表征囊泡结合的PK介导的谷氨酸在突触小泡中的摄取；(2)证明PK和苯丙酮酸在突触体内谷氨酸的囊泡积累中起重要作用。为了实现目标1，我们将利用分离的大鼠脑突触小泡，在多个方面研究磷酸烯醇丙酮酸加ADP依赖的摄取。为了实现2，我们将确定膜穿透PK抑制剂对突触体内囊泡谷氨酸含量和释放的影响。这项研究有望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有助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.