Novel Mechanisms of LRRK2-Dependent Neurodegeneration in Parkinson's Disease

 描述(申请人提供)：帕金森病(PD)是一种常见的神经退行性运动障碍，主要由黑质中的多巴胺能神经元退化引起。富含亮氨酸的重复蛋白激酶2(LRRK2)基因突变导致晚发性常染色体显性帕金森病，LRRK2基因变异增加了帕金森病的风险。LRRK2已成为治疗帕金森病的重要靶点，因此了解LRRK2依赖性神经退行性变的分子机制至关重要。LRRK2是一种含有Ras-of-Complex(Ras-of-Complex，Ras-of-Complex我们以前已经证明，家族性LRRK2突变增加了KK活性(G2019S)或削弱了GTP酶活性(R1441C/G/H或Y1699C)，但通常会导致培养细胞的神经元损伤。我们的研究还强调了GTPase结构域在调节LRRK2激酶活性和神经元毒性方面的重要作用，强调了GTPase结构域是一个有希望的抑制LRRK2的靶点。我们还发现，G2019S突变可以通过一种未知的机制通过腺病毒介导的基因转移诱导大鼠多巴胺能神经元变性。在目前的应用中，我们现在建议探索在基于腺病毒的LRRK2大鼠模型(AIM 1)中，由家族性PD突变(R1441C、Y1699C和G2019S)引起的多巴胺能神经变性是否普遍需要激酶活性。我们假设某些家族性突变通过一种依赖于激酶的机制来发挥其有害作用。因此，在这个腺病毒模型中，对LRRK2激酶活性的遗传和药物抑制将被评估以改善疾病的效果。LRRK2激酶活性的真实底物还没有在体内鉴定出来。我们最近发现ArfGAP1是LRRK2的一个强大的激酶底物，这是LRRK2在培养中诱导神经元毒性所必需的。我们现在建议确定ArfGAP1被LRRK2在体外和体内脑组织中磷酸化的位置，并评估ArfGAP1的磷酸化和表达在LRRK2诱导的原代神经元和腺病毒基础上的大鼠模型神经元损伤中的作用(AIM 2)。最后，我们的研究将探索Roc-COR串联结构域在调节LRRK2活性和毒性中的作用(目标3)。我们假设LRRK2的功能是二聚化激活的GTP酶(GAD)，因此我们将探索分子间(即COR结构域介导的二聚化)和分子内(ROC/COR相互作用)相互作用如何影响LRRK2的活性和毒性。我们将确定破坏这些独特的ROC-COR相互作用是否有助于减弱LRRK2介导的神经退行性变。我们的建议是新颖的、创新的和及时的，并将提供关键的机制洞察力，以了解GTP酶和激酶活性在LRRK2介导的神经变性中的相对贡献。我们的研究将对基于减轻LRRK2活性和神经元毒性的帕金森病治疗策略的确定具有重要意义。

 DESCRIPTION (provided by applicant): Parkinson's disease (PD) is a common neurodegenerative movement disorder caused primarily by the degeneration of dopaminergic neurons in the substantia nigra. Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause late-onset, autosomal dominant PD, and LRRK2 genomic variation increases PD risk. LRRK2 has emerged has an important therapeutic target for treating PD and therefore it is critical to understand the molecular mechanisms that lead to LRRK2-dependent neurodegeneration. LRRK2 is a multi- domain protein containing Ras-of-complex (Roc) GTPase and C-terminal-of-Roc (COR) domains, in addition to a protein kinase domain. We have previously shown that familial LRRK2 mutations increase kinase activity (G2019S) or impair GTPase activity (R1441C/G/H or Y1699C) but commonly induce neuronal damage in cultured cells. Our studies have also highlighted an important role for the GTPase domain in regulating LRRK2 kinase activity and neuronal toxicity, highlighting the GTPase domain as a promising target for inhibiting LRRK2. We have also shown that the G2019S mutation, which produces a hyperactive kinase, can induce dopaminergic neuronal degeneration in rats via adenoviral-mediated gene transfer through an unknown mechanism. In the present application, we now propose to explore whether kinase activity is commonly required for dopaminergic neurodegeneration induced by familial PD mutations (R1441C, Y1699C and G2019S) in an adenoviral-based LRRK2 rat model (Aim 1). We hypothesize that certain familial mutations exert their detrimental effects through a kinase-dependent mechanism. Accordingly, genetic and pharmacological inhibition of LRRK2 kinase activity will be evaluated in this adenoviral model for disease- modifying effects. Authentic substrates of LRRK2 kinase activity have not yet been identified in vivo. We recently identified ArfGAP1 as a robust kinase substrate of LRRK2 that is critically required for LRRK2-induced neuronal toxicity in cultures. We now propose to identify the sites of ArfGAP1 phosphorylation by LRRK2 in vitro and in vivo in brain tissue, and evaluate the contribution of ArfGAP1 phosphorylation and expression to LRRK2-induced neuronal damage in primary neuronal and adenoviral-based rat models (Aim 2). Finally, our studies will explore the role of the Roc-COR tandem domain in regulating LRRK2 activity and toxicity (Aim 3). We hypothesize that LRRK2 functions as a GTPase activated by dimerization (GAD) and accordingly we will explore how intermolecular (i.e. COR domain-mediated dimerization) and intramolecular (Roc/COR interactions) interactions contribute to LRRK2 activity and toxicity. We will determine whether disrupting these unique Roc-COR interactions serve to attenuate LRRK2-mediated neurodegeneration. Our proposal is novel, innovative and timely and will provide critical mechanistic insight into the relative contributions of GTPase and kinase activity to LRRK2-mediated neurodegeneration. Our studies will have important implications for the identification of therapeutic strategies for PD based upon attenuating LRRK2 activity and neuronal toxicity.