Metabolic control of neuronal activity by fuel substrate switching

描述（由申请人提供）：通过燃料底物转换对神经元活动的代谢控制。燃料代谢不仅对大脑神经元和星形胶质细胞的细胞生死决定至关重要，而且是神经元活动的关键调节因子。代谢如何改变大脑活动的一个显著例子是BAD缺失或改变小鼠对癫痫发作的强烈抵抗，BAD是一种BCL-2家族蛋白，对葡萄糖和酮体消耗具有相互作用，独立于其调节细胞凋亡的能力。我们发现BAD代谢功能的改变可导致神经元和星形胶质细胞中葡萄糖的线粒体氧化减少和酮体氧化增强。与用生酮饮食治疗的动物类似，具有这些代谢变化的BAD突变小鼠对化学前惊厥药引起的行为和电痉挛表现出惊人的抵抗力。遗传和电生理研究也表明，BAD中这些相同的突变增加了atp敏感钾（KATP）通道的打开概率，这些通道的激活是代谢诱导癫痫抵抗的必要中间过程。我们提出的研究解决了关于BAD改变代谢和神经元兴奋性的机制的两个关键问题。第一个问题是在燃料利用的代谢途径的水平上：当BAD被修饰时，葡萄糖和酮体的代谢发生变化的精确步骤是什么？靶向代谢组学和13C追踪将与BAD的遗传操作结合使用，以具体了解葡萄糖和酮体如何通过不同的代谢途径，以及在培养的神经元和星形胶质细胞以及脑切片中，这种途径如何受到BAD的影响。其次，BAD突变神经元中KATP通道激活的可能原因和后果是什么？我们将使用荧光生物传感器来评估培养或切片中单个神经元和星形胶质细胞中关键代谢信号（ATP， ROS/谷胱甘肽氧化还原和NADH氧化还原）的水平，以了解KATP通道激活的原因。补充的电生理学研究将解决齿状颗粒神经元的兴奋性和信号在BAD突变的脑切片中是如何改变的。这些问题的答案应该提供一个连接BAD和燃料代谢与改变的神经元兴奋性的途径的综合图片。了解这一途径将提供有价值的线索，将代谢的这种有效的抗惊厥作用转化为对许多难治性癫痫患者的改进治疗。

DESCRIPTION (provided by applicant): Metabolic control of neuronal activity by fuel substrate switching. Fuel metabolism is important not only for cellular life-and-death decisions of neurons and astrocytes in the brain, but also as a key regulator of neuronal activity. A remarkable example of how metabolism can alter brain activity is the strong resistance to epileptic seizures exhibited by mice with deletion or alteration of BAD, a BCL-2 family protein that imparts reciprocal effects on glucose and ketone body consumption independent of its ability to regulate apoptosis. We have found that alterations in BAD's metabolic function can lead to reduced mitochondrial oxidation of glucose and enhanced oxidation of ketone bodies, in both neurons and astrocytes. Similar to animals treated with ketogenic diet, BAD mutant mice with these metabolic changes show a striking resistance to behavioral and electrographic seizures induced by chemical proconvulsants. Genetic and electrophysiologic studies also show that these same mutations in BAD increase the open probability of the ATP-sensitive potassium (KATP) channels and that activation of these channels is a necessary intermediate in this metabolically induced seizure resistance. Our proposed studies address two key questions about the mechanism by which BAD alters metabolism and neuronal excitability. The first question is at the level of the metabolic pathways for fuel utilization: What are the precise steps at which the metabolism of glucose and ketone bodies is changed when BAD is modified? Targeted metabolomics and 13C tracing will be used in conjunction with genetic manipulation of BAD to learn specifically how glucose and ketone bodies are routed through different metabolic pathways, and how this routing is affected by BAD, in cultured neurons and astrocytes as well as in brain slices. Second, what are the possible causes and consequences of KATP channel activation in BAD mutant neurons? We will employ fluorescent biosensors to assess the levels of key metabolic signals - ATP, ROS/glutathione redox, and NADH redox - in individual neurons and astrocytes in culture or slices, to learn the causes of KATP channel activation. Complementary electrophysiological studies will address how excitability and signaling in dentate granule neurons are altered in BAD mutant brain slices. The answer to these questions should give an integrated picture of the pathway connecting BAD and fuel metabolism with altered neuronal excitability. Understanding this pathway should yield valuable clues for translating this potent anticonvulsant effect of metabolism into improved therapies for the many individuals with intractable epilepsy.