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

描述(申请人提供)：线粒体功能障碍和氧化应激在帕金森病(PD)中起着重要作用。PGC-1α是一种转录共激活因子，它上调线粒体的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-1α的小鼠对线粒体毒素MPTP的易感性增加，而过度表达PGC-1α则保护细胞系免受氧化挑战。帕金森病早期黑质神经元中受PGC-1α调控的基因表达水平较低。帕金森病的两个重要遗传原因现在被认为与低PGC-1α有关。首先，最近的一项研究表明，Parkin功能的丧失会导致名为“Paris”的新蛋白水平增加，这种蛋白在转录上抑制PGC-1α的表达。最近发现α-突触核蛋白与PGC-1α启动子结合并抑制其转录。综上所述，这些数据强烈暗示了帕金森病患者PGC-1α活性低的致病作用，并增加了纠正帕金森病患者PGC-1α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，在非常高的水平过度表达PGC-1α会导致BDNF减少，并抑制多巴胺能表型。我们的初步数据表明，这可能是由于对转录因子Pitx3的抑制，该转录因子对维持多巴胺能表型和BDNF的表达至关重要。使用我们的AAV-PGC-1α载体获得的非常高水平的PGC-1α增加了对MPTP的脆弱性。因此，无论是低水平还是非常高的PGC-1α水平都可能是有害的。综上所述，这些数据表明，将PGC-1α的活性水平维持在“治疗”的范围内，对多巴胺能神经元的生存和功能至关重要。我们假设，非常高水平的PGC-1α导致Pitx3的抑制，导致多巴胺能表型的丧失，并由于BDNF的丢失而增强了对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-1α活性增加的神经保护潜力，同时避免与非常高水平的过度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易感性的影响来测试这一点，方法是适度上调PGC-1α，或在共表达PGC-1α以防止更高水平的PGC-1α的有害影响之后。此外，还将研究Pitx3本身的潜在神经保护作用。这些实验将验证我们的假设，即Pitx3的抑制介导了PGC-1α诱导的BDNF和多巴胺能表型的下调。此外，已经在帕金森病患者中进行了多个基因治疗试验，因此拟议的研究也将作为具有翻译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.