Mechanisms of 2DGs Antiepileptic Effects

描述(由申请人提供)： 癫痫发生在大约1%的退伍军人患者中，在大约三分之一的患者中，目前的抗癫痫药物(AED)无法控制癫痫。对于没有控制癫痫发作的第三人，以及那些发现AEDs有不可接受的副作用的人，需要新的治疗方法。另一种治疗方法可以是代谢和不含葡萄糖的蛋白质和高脂肪饮食，即生酮饮食，可以改善那些对传统AEDs无效的患者的癫痫控制。生酮饮食抑制糖酵解，糖的分解，当摄取葡萄糖时癫痫发作失去控制。饮食的一种新的替代方法是使用2-脱氧-D-葡萄糖(2DG)来抑制糖酵解，这是一种葡萄糖类似物，会干扰最初的 糖酵解途径中的异构化步骤。2DG正在被评估为研究中的新药，并提供了一种新的治疗选择。2DG可减少点燃模型癫痫的发生，对6 Hz刺激模型和听源性刺激模型均有急性抗癫痫作用，对海马片中的化学惊厥药也有明显的拮抗作用。我们将尝试确定2DG在单个神经元水平和神经元网络水平上对神经元兴奋性的影响。通过明确2DG抗癫痫作用的机制，我们期待着癫痫治疗的新途径随之而来。2DG似乎也有改善疾病的作用，这项提议将研究2DG作用的使用依赖作用。我们的基本假设是，2DG抑制糖酵解可以产生抗癫痫作用，我们将讨论与神经细胞膜兴奋性和突触传递变化有关的可能机制。我们将评估以下两个特定目标：目标1：确定2DG处理产生的膜兴奋性的变化。我们将评估一些可能的作用机制，因为它们与糖酵解的抑制和膜附近能量底物的变化有关。这将包括分析动作电位的产生可能会因钠-钾泵的减少而改变，随着三磷酸腺苷(ATP)水平的降低而激活的腺苷敏感钾通道的增强，以及动作电位产生后超极化的变化。目的：观察2DG对突触前机制介导的突触传递的影响，以改变海马区CA3区的网络兴奋性。具体地说，我们将看到2DG如何改变在I组代谢性谷氨酸受体激活延长激活后发生的异常突触活动，并产生CA3癫痫样同步。我们还将使用最小刺激和分析微小突触后电流来定义突触传递的变化。所有研究都将使用容易出现癫痫样同步的海马区CA3区，这部分与CA3神经元的特性有关 以及它们反复出现的兴奋性突触网络。我们将使用锐利的细胞内电极记录来评估CA3神经元的放电特征，以及它们在2DG后的变化。我们还将使用斑片和全细胞电压钳记录来研究固有电流和突触后电流。我们的方法将测试被2DG修饰的单个神经元和网络突触功能的特定变化，并将阐明糖酵解抑制抗癫痫作用的潜在作用机制。在拟议的研究中，我们将进行实验，以增加神经元活性，使神经元负载DG，并监测使用依赖效应，这些效应可能解释2DG治疗的任何疾病改善特征。明确这些作用将为抗癫痫药物的开发提供新的治疗靶点，并了解2DG急性抗癫痫作用的机制。

DESCRIPTION (provided by applicant): Epilepsy occurs in about 1% of Veteran patients and is not controlled by current anti-epileptic drugs (AEDs) in approximately one third of patients. For the third who do not have their seizures controlled and for those who find AEDs to have unacceptable side effects, new treatment approaches are needed. An alternative treatment can be metabolic and a protein and high fat diet without glucose, the ketogenic diet, can improve seizure control in those patients who do not respond to conventional AEDs. The ketogenic diet inhibits glycolysis, the breakdown of sugar, and seizure control is lost when glucose ingested. A novel alternative to the diet is to inhibit glycolysis using 2-deoxy-D-glucose (2DG), a glucose analog that interferes with the initial isomerization step in the glycolytic pathway. 2DG is being evaluated as investigational new drug and offers a new therapy option. 2DG has been shown to decrease the development of epilepsy in the kindling model as well as having acute antiepileptic effects in the 6 Hz and audiogenic stimulation models, and against chemoconvulsants in hippocampal slices. We will try to determine what effects 2DG has on neuronal excitability at a single neuron level and at the neuronal network level. By defining mechanisms of 2DG's antiepileptic properties, we expect new approaches to therapy of epilepsy will follow. 2DG also appears to have disease-modifying actions, and this proposal will study the use-dependent action of 2DG's effects. Our underlying hypothesis is that inhibition of glycolysis by 2DG results in antiepileptic effects, and we will address possible mechanisms as they relate to neuronal membrane excitability and changes in synaptic transmission. We will assess the following 2 specific aims: Aim 1: To identify changes in membrane excitability produced by 2DG treatment. We will evaluate a number of possible mechanisms of action as they relate to inhibition of glycolysis and a change in energy substrate near the membrane. This will include analysis of action potential generation which may be changed by a decrease in sodium-potassium pump, enhancement in the adenosine triphosphate (ATP) sensitive potassium channel that is activated with a decrease in ATP levels, and changes in the afterhyperpolarization that follows action potential generation. Aim 2: To assess 2DG's effect on synaptic transmission mediated by presynaptic mechanisms that alter the network excitability of the CA3 region of the hippocampus. Specifically we will see how 2DG alters the abnormal synaptic activity that occurs after prolonged activation of group I metabotropic glutamate receptor activation and produces CA3 epileptiform synchronization. We will also define changes in synaptic transmission using minimal stimulation and analysis of miniature post synaptic currents. All studies will use the CA3 region of the hippocampus which is vulnerable to epileptiform synchronization, in part related to the properties of the CA3 neurons and their recurrent excitatory synaptic network. We will assess CA3 neuron firing characteristics using sharp intracellular electrode recordings and how they change after 2DG. We will also use patch and whole-cell voltage clamp recordings to study intrinsic and postsynaptic currents. Our approach will test for specific changes in single neuron and network synaptic function that are modified by 2DG and will elucidate potential mechanisms of action for the antiepileptic effects of glycolytic inhibition. In the proposed study, we will be performing experiments that will increase neuronal activity to load neurons with DG and monitor for use-dependent effects that may explain any disease modifying features of 2DG treatment. Defining these actions will give new therapeutic targets for antiepileptic drug development as well as understanding the mechanism of 2DG's acute antiepileptic action.