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是一种年龄依赖性脊髓运动神经元的神经退行性疾病。编码铜/锌超氧化物歧化酶（SOD 1）的基因突变导致约25%的家族性ALS病例（FALS）发生运动神经元变性。有证据表明，死亡过程涉及凋亡基因的激活。该项目的广泛目标是阐明控制突变SOD 1介导的凋亡性死亡的机制。 我们在小鼠神经母细胞瘤N2 A细胞和转基因ALS小鼠中的研究表明，多个SOD 1突变体启动了一个毒性级联反应，该级联反应需要caspase-1（细胞死亡的缓慢启动子）和caspase-3（最终效应子）的顺序激活。在ALS小鼠中，在运动神经元死亡前数月出现活化的半胱天冬酶-1，表明半胱天冬酶-1可能通过活化其他凋亡途径作为细胞死亡的早期介导物发挥作用。这表明了这样的假设，即至少在突变型SOD 1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，而是随着时间发展的缓慢过程。对原发性损伤（SOD 1蛋白中遗传的分子缺陷）有一个逐渐的反应，随着时间的推移，运动神经元对运动神经元的敏感性增加， 外源性刺激（如氧化应激）或凋亡途径中与年龄相关的变化。与这一观点一致，我们发现N2 A细胞中caspase-1的慢性激活不会引起快速细胞死亡，但会使细胞对氧化损伤更敏感。在级联反应的最后，caspase-3激活和细胞死亡之间的密切时间关系表明，这个刽子手caspase直接导致运动神经元死亡。运动神经元相关的凋亡机制的详细分析可能有很大的重要性，在了解运动神经元细胞死亡的生物学。我们现在计划在GFP标记的初级运动神经元中使用高灵敏度的SELDI蛋白芯片技术来剖析ALS小鼠中细胞死亡级联随时间推移的各个步骤。为此，我们建议（1） 产生GFP荧光蛋白在运动神经元中特异性表达的ALS小鼠，和（2）使用来自这些小鼠的荧光分选的运动神经元进行凋亡蛋白的SELDI分析。

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.