Determining the cellular and molecular basis of mitochondrial encephalomyopathy seizures

 描述(申请人提供)：线粒体编码的ATP合成酶蛋白复合体ATP6亚单位的突变导致人类线粒体脑肌病，包括母系遗传性Leigh综合征(MILS)、神经病变、共济失调和视网膜色素变性(NARP)，以及家族性双侧纹状体坏死(FBSN)。这些破坏性进行性疾病的症状包括寿命缩短、神经退化、器官系统衰竭和癫痫发作。目前还没有找到治愈的方法，而且这些症状的有效治疗也被证明是困难的。例如，这些疾病中出现的癫痫发作通常对常见的抗癫痫药物(AEDs)无效。最近，在果蝇中发现了一个影响ATP6亚单位的点突变，并对其进行了鉴定。该突变系称为ATP6[1]，具有上述疾病的进行性病理特征，因此提供了仅有的可在动物一生中研究的线粒体疾病的稳定动物模型之一。用膜片钳技术对成年ATP6[1]果蝇全脑外植体进行电生理学研究，我们发现果蝇昼夜节律和睡眠回路的大的外侧腹侧神经元(L-LNV)表现出高度的兴奋性，包括膜电位和放电频率的升高，以及对包括直接光暴露和去极化电流注入在内的兴奋性刺激的反应增强。这使它们成为研究与线粒体疾病相关的癫痫发作的极佳模型神经元。我们的初步数据表明，兴奋性的改变可能是由多种因素引起的，其中包括L-LNV中存在的膜通道和突触输入的变化。我们建议使用这些特征良好和电生理上可达的神经元来识别这些通道亚单位和突触贡献，以便在完成这项工作后，我们将知道相关的分子贡献者，最终可能成为线粒体疾病发作的治疗靶点。

 DESCRIPTION (provided by applicant): Mutations in the mitochondrially-encoded ATP6 subunit of the ATP synthase protein complex cause human mitochondrial encephalomyopathies including maternally inherited Leigh Syndrome (MILS), neuropathy, ataxia, and retinitis pigmentosa (NARP), and familial bilateral striatal necrosis (FBSN). Symptoms of these devastating progressive disorders include shortened lifespan, neurodegeneration, organ system failure, and seizures. No cures have been found, and effective treatment of the symptoms has proven difficult. For example, the seizures present in these diseases are often refractory to common anti-epileptic drugs (AEDs). Recently, a point mutation affecting the ATP6 subunit has been identified and characterized in Drosophila melanogaster. This mutant line, termed ATP6[1], shares the progressive pathological features of the above disorders, thus providing one of the only stable animal models of mitochondrial disease that can be studied throughout the animal's lifetime. Using patch clamp electrophysiology on whole-brain explants from adult ATP6[1] flies, we have found that the well-characterized large lateral ventral neurons (l-LNv) of the Drosophila circadian and sleep circuits exhibit hyperexcitability, including elevated interspik membrane potential and firing frequency, and increased responses to excitatory stimuli including direct light exposure and depolarizing current injections. This makes them an excellent model neuron for the study of the seizures associated with mitochondrial diseases. Our preliminary data suggests the altered excitability may be due to multiple factors, which include changes in both membrane channels present in the l-LNv and in synaptic inputs. We propose to use these well- characterized and electrophysiologically-accessible neurons to identify these channel subunits and synaptic contributions, so that upon completion of this work we will know the relevant molecular contributors which may ultimately become therapeutic targets for the seizures of mitochondrial disease.