Proteomics of Apopotosis in GFP-Labeled Motor Neurons

这是一个研究肌萎缩侧索硬化症(ALS)的项目，ALS是一种年龄相关的脊髓运动神经元退行性疾病。在大约25%的家族性肌萎缩侧索硬化症(FAL)中，铜/锌超氧化物歧化酶(SOD1)基因的突变会导致运动神经元变性。有证据表明，死亡过程涉及到凋亡基因的激活。这个项目的主要目标是阐明突变的SOD1介导的细胞凋亡死亡的机制(S)。 我们在小鼠神经母细胞瘤N2A细胞和转基因ALS小鼠上的研究表明，多个SOD1突变体启动毒素级联反应，导致caspase-1(细胞死亡的缓慢起始者)和caspase-3(最终效应者)的顺序激活。肌萎缩侧索硬化症小鼠在运动神经元死亡前几个月出现活化的caspase-1，表明caspase-1可能通过激活其他凋亡途径而起到细胞死亡的早期中介作用。这表明，至少在突变的SOD1相关的ALS中，细胞凋亡不是导致运动神经元最终死亡的次要现象，而是一个随着时间推移而发展的缓慢过程。随着时间的推移，运动神经元对原发损伤(SOD1蛋白中的遗传分子缺陷)有逐渐的反应，这种反应增强了运动神经元对 外源性刺激(如氧化应激)或与年龄相关的细胞凋亡途径的变化。与这一观点一致，我们发现在N2A细胞中caspase-1的慢性激活并不会引起细胞的快速死亡，但会使细胞对氧化损伤更加敏感。在级联反应的最后，caspase-3激活与细胞死亡之间的密切时间关系表明，这个执行者caspase直接参与了运动神经元的死亡。对运动神经元相关的细胞凋亡机制进行详细分析，对于理解运动神经元细胞死亡的生物学意义重大。我们现在计划使用高灵敏度的SELDI蛋白质芯片技术在GFP标记的初级运动神经元中剖析ALS小鼠随着时间的推移细胞死亡级联中的个别步骤。为此，我们建议(1) 建立GFP荧光蛋白在运动神经元中特异表达的ALS小鼠，以及(2)使用从这些小鼠中分离的荧光运动神经元进行SELDI分析凋亡蛋白。

This is a project to study Amyotrophic Lateral Sclerosis (ALS), an age-dependent neurodegenerative disease of spinal motor neurons. Mutations in the gene encoding for copper/zinc superoxide dismutase (SOD1) cause motor neuron degeneration in about 25% of familial ALS cases (FALS). Evidence indicates that the death process involves activation of apoptotic genes. The broad goal of this project is to elucidate the mechanism(s) governing mutant SODl-mediated apoptotic death. Our investigations in mouse neuroblastoma N2A cells and transgenic ALS mice show that multiple SOD1 mutants initiate a toxic cascade that entails sequential activation of caspase-1 (a slowly initiator of cell death) and caspase-3 (the final effector). The appearance of activated caspase-1 in the ALS mice months prior to motor neurons death indicates that caspase-1 plays a role as an early mediator of cell death probably by activating other apoptotic pathways. This suggests the hypothesis that at least in mutant SODl-related ALS, apoptosis is not a secondary phenomenon that contributes to the final demise of motor neurons, but rather a slow process that develops over time. There is a gradual response to the primary insult (the inherited molecular defect in the SOD1 protein) that over time enhances motor neurons susceptibility to either an exogenous stimulus (e.g. oxidative stress) or to age-related changes in apoptotic pathways. Consistent with this view, we have found that chronic activation of caspase-1 in the N2A cells does not provoke rapid cell death, but renders the cells more sensitive to an oxidative insult. At the end of the cascade, the close temporal relationship between caspase-3 activation and cell death argues that this executioner caspase directly contributes to motor neuron death. A detailed analysis of motor neuron-related apoptotic mechanisms may have a great importance in understanding the biology of motor neuron cell death. We now plan to dissect the individual steps in the cell death cascade over time in the ALS mice using high sensitive SELDI ProteinChip Technology in GFP-labeled primary motor neurons. To this end we propose to (1) Generate ALS mice in which the GFP fluorescent protein is specifically expressed in motor neurons, and (2) use the fluorescent-sorted motor neurons from these mice for SELDI analysis of apoptotic proteins.