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，并且在大约三分之一的患者中不被当前的抗癫痫药物（AED）控制。对于第三个没有控制癫痫发作的人和那些发现AED有不可接受的副作用的人，需要新的治疗方法。替代治疗可以是代谢和蛋白质和不含葡萄糖的高脂肪饮食，生酮饮食，可以改善对常规AED无反应的患者的癫痫控制。生酮饮食抑制糖酵解，糖的分解，当摄入葡萄糖时，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）抑制糖酵解，2DG是一种葡萄糖类似物， 糖酵解途径中的异构化步骤。2DG正在被评估为研究性新药，并提供了一种新的治疗选择。2DG在点燃模型中可减少癫痫的发展，在6 Hz和听源性刺激模型中具有急性抗癫痫作用，在海马切片中具有抗化学惊厥作用。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。通过定义2DG抗癫痫特性的机制，我们预计癫痫治疗的新方法将随之而来。2DG似乎也具有疾病缓解作用，本提案将研究2DG效应的使用依赖性作用。我们的基本假设是，抑制糖酵解的2DG的结果在抗癫痫作用，我们将解决可能的机制，因为它们涉及到神经元膜的兴奋性和突触传递的变化。 我们将评估以下2个具体目标：目标1：确定2DG治疗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。这将包括分析可能因钠-钾泵降低而改变的动作电位生成、因ATP水平降低而激活的三磷酸腺苷（ATP）敏感性钾通道增强以及动作电位生成后的后超极化变化。 目标二：评估2DG对突触前机制介导的突触传递的影响，该机制改变海马CA 3区的网络兴奋性。具体来说，我们将看到2DG如何改变异常的突触活动后，发生长时间激活组I代谢型谷氨酸受体激活和产生CA 3癫痫样同步。我们还将使用最小刺激和微型突触后电流的分析来定义突触传递的变化。 所有研究都将使用海马的CA 3区，该区域易受癫痫样同步的影响，部分与CA 3神经元的特性有关 和它们的周期性兴奋性突触网络。我们将使用尖锐的细胞内电极记录来评估CA 3神经元的放电特征，以及它们在2DG后如何变化。我们还将使用膜片钳和全细胞电压钳记录来研究内源性和突触后电流。 我们的方法将测试特定的变化，单神经元和网络突触功能的修改2DG，并将阐明潜在的作用机制，糖酵解抑制的抗癫痫作用。在拟议的研究中，我们将进行实验，增加神经元活动，以用DG加载神经元，并监测可能解释2DG治疗的任何疾病改善特征的使用依赖性效应。明确这些作用将为抗癫痫药物的开发提供新的治疗靶点，并有助于了解2DG的急性抗癫痫作用机制。

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.