PGC-1alpha and Pitx3 as individual and combined targets for neuroprotection

描述（由申请人提供）：线粒体功能障碍和氧化应激在帕金森病（PD）中起重要作用。pgc -1 α是一种转录辅助激活因子，上调线粒体生物发生和抗氧化防御，因此是帕金森病神经保护的一个有吸引力的靶点。缺乏pgc -1 α的小鼠对MPTP（一种线粒体毒素）的易感性增加，而过表达pgc -1 α的细胞系可以保护细胞免受氧化挑战。pgc -1 α调控基因在PD早期黑质（SN）神经元中的表达水平较低。目前，PD的两个重要遗传原因与低pgc -1 α有关。首先，最近的一项研究表明，帕金蛋白功能的丧失会导致一种名为“PARIS”的新型蛋白水平升高，这种蛋白通过转录抑制pgc -1 α的表达。最近有研究表明-突触核蛋白与pgc - 1α启动子结合并抑制其转录。总之，这些数据强烈暗示pgc -1 α活性低在PD中的致病作用，并提出了纠正PD中pgc -1 α缺陷将具有神经保护作用的希望。然而，出乎意料的是，我们和其他人发现PGC-1alpha的高水平过表达会导致Bdnf降低和多巴胺能表型抑制。我们的初步数据表明，这可能是由于Pitx3的抑制，Pitx3是维持多巴胺能表型和Bdnf表达的关键转录因子。使用我们的AAV-PGC-1alpha载体获得的非常高水平的PGC-1alpha增加了对MPTP的脆弱性。因此，低或非常高的pgc -1 α水平都可能是有害的。综上所述，这些数据表明，将pgc -1 α活性水平维持在“治疗”范围内对多巴胺能神经元的存活和功能至关重要。我们假设，非常高水平的pgc -1 α导致Pitx3的抑制，导致多巴胺能表型的丧失，并由于Bdnf的丧失而增加对MPTP的易感性。我们进一步假设，有可能利用病毒载体介导的pgc -1 α活性增加的神经保护潜力，同时避免与非常高水平的过表达相关的潜在有害影响。我们建议通过研究多巴胺能神经元对线粒体功能、氧化应激、多巴胺能表型和MPTP易感性的影响来验证这一点，在适度上调pgc -1 α水平或共同表达Pitx3以防止较高pgc -1 α水平的有害影响之后。Pitx3本身的潜在神经保护作用也将被研究。这些实验将验证我们的假设，即抑制Pitx3介导pgc -1 α诱导的Bdnf和多巴胺能表型的下调。此外，已经在PD患者中进行了多项基因治疗试验，因此提出的研究也将作为具有转化潜力的治疗策略的初步测试。

DESCRIPTION (provided by applicant): Mitochondrial dysfunction and oxidative stress play important roles in Parkinson's disease (PD). PGC-1alpha, a transcriptional coactivator, upregulates mitochondrial biogenesis and antioxidant defenses, and thus is an attractive target for neuroprotection in PD. Susceptibility to MPTP, a mitochondrial toxin, is increased in mice lacking PGC-1alpha, whereas overexpressing PGC-1alpha protects against an oxidative challenge in cell lines. Levels of expression of genes regulated by PGC-1alpha are low in substantia nigra (SN) neurons in early PD. Two important genetic causes of PD now have been linked to low PGC-1alpha. First, a recent study showed that loss of Parkin function leads to increased levels of novel protein called "PARIS" which transcriptionally inhibits expression of PGC-1alpha. And recently it was demonstrated that alpha-synuclein binds to the PGC-1alphha promoter and also suppresses its transcription. Together, these data strongly implicate a pathogenic role for low PGC-1alpha activity in PD, and raise the hope that correction of the PGC-1alpha deficit in PD will be neuroprotective. However, unexpectedly, we and others find that overexpressing PGC-1alpha at very high levels leads to reduced Bdnf and suppression of the dopaminergic phenotype. Our preliminary data suggest that this may result from suppression of Pitx3, a transcription factor that is critical for maintaining the dopaminergic phenotype and also for expression of Bdnf. Vulnerability to MPTP is increased by the very high levels of PGC-1alpha achieved using our AAV-PGC-1alpha vector. Thus, either low or very high levels of PGC-1alpha can be deleterious. Together, these data reveal that maintenance of PGC-1alpha activity levels within a "therapeutic" range is critical for the survival and function of dopaminergic neurons. We hypothesize that very high levels of PGC-1alpha lead to suppression of Pitx3, leading to loss of the dopaminergic phenotype and to enhanced vulnerability to MPTP due to loss of Bdnf. We further hypothesize that it will be possible to harness the neuroprotective potential of viral vector- mediated increases in PGC-1alpha activity while avoiding the potentially deleterious effects associated with very high levels of overexpression. We propose to test this by studying the impact in dopaminergic neurons on mitochondrial function, oxidative stress, the dopaminergic phenotype, and susceptibility to MPTP following more modest levels of upregulation of PGC-1alpha, or following co-expression of Pitx3 to prevent the deleterious effects of higher PGC-1alpha levels. The potential neuroprotective effects of Pitx3 on its own also will be studied. These experiments will test our hypothesis that suppression of Pitx3 mediates the PGC-1alpha-induced downregulation of Bdnf and of the dopaminergic phenotype. In addition, multiple gene therapy trials have been conducted in PD patients, and thus the proposed studies also will serve as initial tests of therapeutic strategies with translational potential.