Early motor neuron alteration in a non-cell autonomous ALS model

描述（由申请人提供）：本提案旨在通过寻找突变型超氧化物歧化酶-1 （SOD1）引起的最早细胞扰动来继续我们对肌萎缩性侧索硬化症（ALS）发病机制的研究，SOD1是这种致命疾病的已知原因。该项目的前提是，即使在临床表型出现之前，突变SOD1也会引起运动神经元表达的基因和蛋白质的反应，这是了解运动神经元退行性过程基础和制定有效神经保护策略的关键。我们已经证明，野生型原代或胚胎干细胞衍生的脊髓运动神经元可以被表达突变SOD1的星形胶质细胞杀死，这种神经毒性作用可以在突变预先调节的培养基中重现，而不是野生型星形胶质细胞（Nagai et al., Nat. Neurosci. 2007）。鉴于这些结果，我们假设我们的体外细胞系统应该提供一种独特的工具来识别野生型运动神经元中响应SOD1突变而产生的初始mRNA和蛋白质变化。因此，在Specific Aim (SA)- 1中，我们将使用高度纯化的原代脊髓和胚胎干细胞衍生的运动神经元在层粘胶蛋白上培养，然后在特定时刻暴露于突变型或野生型星形胶质细胞条件培养基，通过基因阵列来确定运动神经元对突变型星形胶质细胞条件培养基的反应。在选定的时间点，收集突变型或野生型星形胶质细胞条件培养基中培养的运动神经元，分离总mRNA并通过基因表达谱定量比较。结果将通过real-time PCR进行确认。在SA-II中，我们将使用与sa - 1中相同的神经元培养和条件培养基，通过蛋白质组学来定义运动神经元对突变星形胶质细胞条件培养基的反应。在选定的时间点，如上所述培养的运动神经元将被收获，蛋白质将被分离并通过差异荧光二维凝胶电泳蛋白质组分析进行定量比较。Western blot检测结果。这个R21项目提供了一组高风险/高收益的研究，由于互补的方法，可能会确定SOD1突变模型中运动神经元变性的分子途径和关键介质。所产生的信息可能对开发有效的神经保护疗法对于与SOD1突变相关的家族型ALS以及可能对其散发性形式也是至关重要的。公共卫生相关性：肌萎缩性侧索硬化症（ALS）是一种无法治愈的致命麻痹性疾病，原因不明。我们在培养皿中发现，从模拟这种疾病的动物身上分离出的特定炎症细胞产生了能够杀死导致ALS瘫痪的神经细胞的因子。在这个项目中，我们建议使用这种独特的疾病模型来寻找神经细胞对邻近炎症细胞介导的损伤的早期病理变化。我们期望这些发现将为开发新的治疗策略打开大门，旨在保护神经细胞免受ALS的侵害，甚至在它们被疾病过程严重破坏之前。

DESCRIPTION (provided by applicant): This proposal seeks to pursue our investigations on the pathogenesis of amyotrophic lateral sclerosis (ALS) by searching for the earliest cellular perturbations provoked by mutant superoxide dismutase-1 (SOD1), a known cause of this fatal disease. This project rests on the premise that mutant SOD1 induces responses in genes and proteins expressed by motor neurons even before the emergence of the clinical phenotype which are key to understanding the basis of motor neuron degenerative process and to developing effective neuroprotective strategies. We have shown that wild-type primary or embryonic stem cell-derived spinal motor neurons can be killed by astrocytes expressing mutant SOD1, a neurotoxic effect that can be recapitulated with medium pre-conditioned by mutant, but not wild-type astrocytes (Nagai et al., Nat. Neurosci. 2007). Given these results, we hypothesize that our in vitro cell system should afford a unique tool to identify those initial mRNA and protein changes that arise in wild-type motor neurons in response to a mutant SOD1. Accordingly, in Specific Aim (SA)-I, we will determine motor neuron response to mutant astrocyte conditioned medium by gene array using highly purified primary spinal cord and embryonic stem cell-derived motor neurons cultured on laminin, and then, at a defined moment, exposed to either mutant or wild-type astrocyte conditioned medium. At selected time points, motor neurons cultured in mutant or wild-type astrocyte conditioned medium will be harvested, and total mRNA will be isolated and quantitatively compared by gene expression profiling. Results will be confirmed by real-time PCR. In SA-II, we will define motor neuron response to mutant astrocyte conditioned medium by proteomics using the same neuronal cultures and conditional media as in SA-I. At selected time points, motor neurons, cultured as above, will be harvested and proteins will be isolated and quantitatively compared by differential fluorescence 2-D gel electrophoresis proteome profiling. Results will be confirmed by Western blot. This R21 project offers a high-risk/high-yield set of studies, which may identify, thanks to complementary approaches, molecular pathways and key mediators of motor neuron degeneration in the mutant SOD1 model. The generated information may be of critical importance for the development of effective neuroprotective therapies for the familial form of ALS linked to mutant SOD1 and perhaps for its sporadic form as well. PUBLIC HEALTH RELEVANCE: Amyotrophic lateral sclerosis (ALS) is an incurable fatal paralytic disorder of uncertain cause. We have found in a dish that specific inflammatory cells isolated from animals modeling this disease produce factors capable of killing the nerve cells responsible for ALS paralysis. In this project, we propose to use this unique disease model to hunt for the earliest pathological changes taking place in nerve cells in response to the insult mediated by neighboring inflammatory cells. We expect that these findings will open the door to the development of new therapeutic strategies aimed at protecting nerve cells against ALS even before they become significantly damaged by the disease process.