Cell-specific expression of AAV-Nurr1 in a model of Parkinson's Disease

 描述（由申请人提供）：迄今为止，没有治疗方法可以减缓帕金森病（PD）的进展。PD症状是由腹侧中脑中多巴胺能（DA）神经元的缓慢变性引起的，其起源于黑质腹侧部（SNpc）。DA神经元的特征在于多巴胺产生所必需的特定基因，其由孤儿核转录因子Nurr 1（NR 4A 1）调节。Nurr 1的表达减少导致体内和体外多巴胺能表型的丧失，因此是正常神经系统健康和DA维持所需的。由胶质细胞激活引发的神经炎症已被证明是PD中DA变性的主要原因。有趣的是，Nurr 1还通过组成性抑制神经胶质细胞中的神经炎性基因表达来提供细胞特异性作用。这表明，如果在整个PD进展过程中在星形胶质细胞和神经元中维持强制表达，Nurr 1可以为SNpc的神经元提供DA表型支持和神经保护。Nurr 1的激活也是核转录所必需的，但尚未鉴定出内源性配体。然而，我们已经证明了小的亲脂性分子化合物C-DIM 12是Nurr 1的有效激活剂，并且在MPTP小鼠PD模型中具有神经保护作用。在没有脱靶副作用的情况下，对大脑中特定细胞类型进行遗传修饰仍然是一个挑战。然而，我们已经测试了特异性非致病性腺相关病毒（AAV）载体，其能够将Nurr 1转基因传递到SNpc中并感染DA神经元和星形胶质细胞。因此，该提议的中心假设是，使用基于AAV的基因递送在DA神经元和星形胶质细胞中选择性过表达Nurr 1将提供免受MPTP诱导的神经毒性的神经保护，并增强C-DIM 12在体外和体内的神经保护功效。以下具体目标将检验这一假设：具体目标1-优化AAV-Nurr 1在星形胶质细胞和神经元中的表达，并确定细胞特异性Nurr 1表达对体外神经毒性损伤的影响;具体目标2-确定细胞特异性AAV-Nurr 1过表达在PD小鼠模型中的神经保护功效。这些目标的完成将增加我们对Nurr 1在PD模型中调节神经元存活和胶质细胞活化的机制的理解。此外，我们预计这些研究将扩大我们对PD治疗靶向候选基因的了解。

 DESCRIPTION (provided by applicant): To date, there is no treatment to slow the progression of Parkinson's disease (PD). PD symptoms are caused by the slow degeneration of dopaminergic (DA) neurons in the ventral midbrain, which originate in the substantia nigra pars compacta (SNpc). DA neurons are characterized by specific genes necessary for dopamine production, which are regulated by orphan nuclear transcription factor Nurr1 (NR4A1). Decreased expression of Nurr1 causes loss of the dopaminergic phenotype in vivo and in vitro, thus is required for normal neurologic health and DA maintenance. Neuroinflammation triggered by glial cell activation has been proven to be a major causing factor of DA degeneration in PD. Interestingly, Nurr1 also provides a cell-specific role by constitutive suppression of neuroinflammatory gene expression in glial cells. This suggests Nurr1 could provide DA phenotypic support and neuroprotection to neurons of the SNpc if forced expression was maintained in astrocytes and neurons throughout PD progression. Nurr1 activation is also necessary for nuclear transcription, yet no endogenous ligands have been identified. However, we have demonstrated small lipophilic molecular compound, C-DIM12, to be a potent activator of Nurr1 and possess neuroprotection in a MPTP mouse model of PD. Challenges remain on genetically modifying specific cell types in the brain without off- target side effects. Yet, we hae tested specific non-pathogenic adeno-associated virus (AAV) vectors capable of transmitting a Nurr1 transgene into the SNpc and infecting both DA neurons and astrocytes. Thus, it is the Central Hypothesis of this proposal that selective overexpression of Nurr1 in DA neurons and astrocytes using AAV-based gene delivery will provide neuroprotection from MPTP-induced neurotoxicity and enhance the neuroprotective efficacy of C-DIM12 both in vitro and in vivo. The following Specific Aims will test this hypothesis: Specific Aim 1- Optimize the expression of AAV-Nurr1 in astrocytes and neurons and determine the effect of cell-specific Nurr1 expression against neurotoxic insult in vitro; Specific Aim 2- Determine the neuroprotective efficacy of cell specific AAV-Nurr1 overexpression in a mouse model of PD. Completion of these aims will increase our understanding of mechanisms by which Nurr1 regulates neuronal survival and glial activation in models of PD. Additionally, we anticipate these studies will expand our knowledge of candidate genes targeted for PD therapeutics.