Cellular mechanisms of dietary therapy for epilepsy

描述（由申请人提供）：我们的目标是了解代谢诱导的抗癫痫发作机制。癫痫极为常见，约占人口的1%，约三分之一的癫痫患者无法通过现有药物得到很好的治疗。饮食疗法，如生酮饮食可以非常有效的治疗这种药物抗性癫痫，但饮食往往是困难的病人和提供者。然而，如果我们能够了解这些饮食产生的癫痫抵抗机制，就有可能找到新的药物来治疗目前治疗不良的癫痫。在之前的资助期内，我们发现ATP敏感钾通道（KATP通道）对代谢性癫痫抵抗至关重要。我们观察到神经元中的KATP通道可以被神经元活动和酮体（酮体是大脑在生酮饮食中使用的替代燃料）协同激活。我们发现了一种不需要改变饮食的小鼠代谢癫痫抵抗遗传模型，并了解到在该模型中，KATP通道是癫痫抵抗所必需的。我们还设计了新的荧光生物传感器来检测细胞内关键代谢辅助因子ATP和NADH的变化，以便更好地了解细胞代谢。我们的第一个目标是了解脑细胞代谢如何对刺激的能量需求做出反应，以及这些反应如何依赖于不同的细胞燃料（例如在生酮饮食中大脑可用的酮体）。我们自己的生物传感器和其他传感器将用于监测单个细胞中特定关键代谢信号的反应：ATP， NADH，谷胱甘肽氧化还原和AMP激活的蛋白激酶活性。这些信号不仅与代谢有关，而且对影响神经元兴奋性的下游机制也有影响。我们将在培养的海马神经元中研究这些反应，因为我们对细胞外燃料有最佳的控制。我们还将研究急性脑切片，这将使我们能够检查兴奋性神经元，抑制性中间神经元和附近星形胶质细胞在突触或电刺激下的独特行为。这些研究应该提供一个前所未有的代谢观点

DESCRIPTION (provided by applicant): Our goal is to understand the mechanism of metabolically induced resistance to epileptic seizures. Epilepsy is extremely common, affecting on the order of 1% of the population, and about a third of people with epilesy are not well treated by existing medications. Dietary therapies such as the ketogenic diet can be very effective for such pharmacoresistant epilepsy, but the diets are often difficul for patients and providers. However, if we can understand the mechanism of the seizure resistance produced by these diets, it may be possible to find new medications to treat a wide range of currently poorly treated epilepsies. In the previous grant period, we found suppor for the hypothesis that ATP--sensitive potassium channels (KATP channels) are critical for metabolic seizure resistance. We observed that KATP channels in neurons could be activated synergistically by neuronal activity and by ketone bodies (which are an alternative fuel used by the brain on the ketogenic diet). We discovered a mouse genetic model for metabolic seizure resistance that does not require a change in diet and learned that in this model, KATP channels are required for the seizure resistance. We also engineered new fluorescent biosensors to detect intracellular changes of the key metabolic cofactors ATP and NADH, to enable a better understanding of cellular metabolism. Our first aim for this grant renewal is to learn how brain cell metabolism responds o the energy demands of stimulation and how those responses depend on different cellular fuels (such as the ketone bodies that become available to the brain on a ketogenic diet). Our own biosensors and others will be used to monitor the responses, in individual cells, of specific key metabolic signals: ATP, NADH, glutathione redox, and AMP-- activated protein kinase activity. Each of these signals not only reports on metabolism but also has effects on downstream mechanisms that affect neuronal excitability. We will study these responses in cultured hippocampal neurons, for which we have optimal control of extracellular fuels. We will also study acute brain slices, which will allow us to examine distinctive behavior i excitatory neurons, inhibitory interneurons, and nearby astrocytes, in response to either synaptic or electrical stimulation. These studies should provide an unprecedented view of metabolic responses in brain cells, and give fundamental insights into how neurons and astrocytes respond to energy demands. These insights will be valuable not only for understanding the basis of metabolic seizure resistance, but also for how brain cells respond to metabolic challenges in disorders ranging from traumatic brain injury to neurodegeneration. Our second aim is to link metabolic changes to KATP channel activity by learning how KATP channels respond to energy levels in intact neurons. We will record the ope probability of KATP channels simultaneously with biosensor measurements of [ATP] or ATP:ADP ratio, to learn the actual dose--response relation for intact neurons, and the conditions for engaging these channels to produce seizure resistance.