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

 描述(由申请人提供)：到目前为止，还没有治疗方法来减缓帕金森病(PD)的进展。帕金森病的症状是由腹侧中脑中多巴胺(DA)能神经元的缓慢变性引起的，该神经元起源于黑质致密部(SNPC)。DA神经元的特征是产生多巴胺所必需的特定基因，该基因受孤儿核转录因子Nurr1(NR4A1)调控。在体内和体外，Nurr1的表达减少会导致多巴胺能表型的丧失，这是正常的神经健康和DA维持所必需的。神经胶质细胞激活引发的神经炎症已被证明是帕金森病患者DA变性的主要原因。有趣的是，Nurr1还通过结构性抑制神经胶质细胞中神经炎性基因的表达，提供了细胞特异性的作用。这表明，如果在帕金森病进展过程中星形胶质细胞和神经元保持强迫表达，Nurr1可以为SNPC的神经元提供DA表型支持和神经保护。Nurr1的激活也是核转录所必需的，但目前还没有鉴定出内源性配体。然而，我们已经证明了亲脂性小分子化合物C-DIM12是Nurr1的有效激活剂，并在MPTP小鼠PD模型中具有神经保护作用。在不产生偏离目标的副作用的情况下，对大脑中特定细胞类型进行基因改造仍然是一个挑战。然而，我们已经测试了特定的非致病性腺相关病毒(AAV)载体，能够将Nurr1转基因转移到SNPC并感染DA神经元和星形胶质细胞。因此，本研究的中心假设是，通过基于AAV的基因传递，选择性地在DA神经元和星形胶质细胞中过表达Nurr1，将在体外和体内提供针对MPTP诱导的神经毒性的神经保护作用，并增强C-DIM12的神经保护作用。以下特定目的将验证这一假设：特定目的1-优化AAV-Nurr1在星形胶质细胞和神经元中的表达，并确定细胞特异性Nurr1表达对体外神经毒性损伤的影响；特定目的2-确定细胞特异性AAV-Nurr1过表达对帕金森病小鼠模型的神经保护作用。这些目标的完成将增加我们对Nurr1调节帕金森病模型中神经元存活和胶质细胞激活的机制的理解。此外，我们预计这些研究将扩大我们对帕金森病治疗目标候选基因的了解。

 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.