Exploring synaptic abnormalities that underlie hyperactivity in a stem-cell derived model of amyotrophic lateral sclerosis

 描述(申请人提供)：该研究项目的长期目标是确定神经退行性疾病肌萎缩侧索硬化症(ALS)的病理生理学基础，并确定导致有效治疗ALS的药物靶点。肌萎缩侧索硬化症是一种影响上、下运动神经元的衰弱和致命的神经退行性疾病，在美国的患病率为每25,000人中就有1人，每年给经济造成2.56亿至4.33亿美元的赤字。虽然自第一例ALS导致基因突变被发现以来，我们已经研究了20多年，但我们还没有发现导致ALS运动神经元进行性退化的病理过程，也没有开发出有效的治疗方法。然而，多年来的研究表明，兴奋性毒性可能在ALS的发病机制中发挥作用，最近的研究表明，在活体ALS模型运动神经元中观察到的自发动作电位放电(活动)增加是脊髓环路异常的结果，尽管其潜在的通路尚未阐明。在这里，我们展示了由小鼠胚胎干细胞产生的运动神经元表现出类似的过度活跃的表型，并且，模仿体内模型系统，潜在的原因是由于突触异常。有趣的是，尽管高度活跃，我们发现ALS模型运动神经元本质上是低兴奋性的，可能是由于代偿性内稳态机制。初步数据表明，在我们的体外ALS模型系统中，改变突触连接的细胞自主过程调节了这些观察到的异常。因此，我们假设要么是兴奋性突触与抑制性突触比率的改变，要么是兴奋性突触后反应的增加，要么是两者的结合，都是在ALS模型运动神经元中观察到的自发活动增加的基础。发现这些途径对未来基于机制的药物发现至关重要，这些药物将使运动神经元生理学正常化。这项建议的目的是利用已建立的干细胞模型，通过检测突触的类型、相对患病率和有效性，阐明ALS运动神经元中驱动多动的机制。我将通过以下两个目的来实现这一目标：1)用全细胞膜片钳检测星形胶质细胞微岛上的野生型和ALS模型运动神经元的突触后电流；2)用免疫细胞化学和电生理学分析比较ALS模型运动神经元和WT模型运动神经元中离子型突触输入的密度。这些分析的结果将揭示ALS兴奋毒性病理生理学的可能基础，并将进一步努力实现我们有效治疗ALS的长期目标。

 DESCRIPTION (provided by applicant): The long-term goal of this research project is to determine the pathophysiology underlying the neurodegenerative disease Amyotrophic Lateral Sclerosis (ALS) and to identify drug targets that lead to effective ALS treatments. ALS is a debilitating and fatal neurodegenerative disease affecting upper and lower motor neurons, with a prevalence in the United States of 1 in 25,000 persons and an annual deficit to the economy of $256 to $433 million. Though studied for over two decades since the first ALS causing genetic mutation was discovered, we have yet to uncover the pathological processes that lead to progressive degeneration of motor neurons in ALS, or to develop effective treatments. However, years of research have revealed that excitotoxicity likely plays a role in ALS pathogenesis, and recent studies have shown that increased spontaneous action potential firing (activity) observed in in vivo ALS-model motor neurons is a consequence of abnormalities in spinal circuitry, though the underlying pathways have yet to be elucidated. Here, we show that motor neurons generated from mouse embryonic stem cells exhibit a similar phenotype of hyperactivity, and, mimicking the in vivo model system, the underlying cause is due to synaptic aberrations. Intriguingly, despite being hyperactive, we find the ALS-model motor neurons to be intrinsically hypoexcitable, likely due to compensatory homeostatic mechanisms. Preliminary data suggest that cell-autonomous processes that alter synaptic connectivity mediate these observed irregularities in our in vitro ALS-model system. Therefore, we hypothesize that either altered ratios of excitatory to inhibitory synapses, increased excitatory post-synaptic response, or a combination of both underlie the increase in spontaneous activity observed in ALS-model motor neurons. Uncovering these pathways is paramount to future mechanism-based drug discoveries that normalize motor neuron physiology. The objective of this proposal is to use an established stem cell model to elucidate mechanisms in ALS motor neurons that drive hyperactivity, by examining the types, relative prevalence, and efficacy of synapses. I will accomplish this objective with the following two aims: 1) Examine post-synaptic currents in wild type and ALS-model motor neurons plated on astrocyte microislands using whole-cell patch clamp and 2) Compare the density of ionotropic synaptic input in ALS model motor neurons to WT using immunocytochemical and electrophysiological analyses. The results of these analyses will uncover pathways that may underlie excitotoxic pathophysiology in ALS, and will further efforts toward our long-term goal of effectively treating ALS.