LRRK2 Enzymatic Mechanisms of Neurodegeneration in Parkinson's Disease

项目摘要 帕金森氏病（PD）是一种进行性神经退行性运动障碍，主要由帕金森氏病引起。 黑质多巴胺能神经元变性。富含亮氨酸重复序列激酶2的突变 （LRRK 2）基因导致晚发型常染色体显性PD，LRRK 2编码和非编码变体是 与散发性PD的风险相关。LRRK 2已成为治疗PD的重要治疗靶点 因此，了解疾病相关突变的关键机制至关重要。LRRK 2是一个 含有Ras-of-Complex（Roc）GTP酶和蛋白激酶酶结构域的大的多结构域蛋白 由C-末端-of-Roc（COR）结构域分隔。通过Roc结构域的GTP结合对正常激酶至关重要 活动家族性LRRK 2突变在Roc（R1441 C/G/H）、COR（Y1699 C）和激酶（G2019 S， I2020 T）结构域，其中它们通常增强Rab GT3底物亚组的磷酸化。 细胞Roc-COR结构域突变通过削弱GTP水解并促进磷酸三酯酶的活性来间接影响激酶活性 GTP绑定的“on”状态。GT3和激酶结构域代表了抑制LRRK 2的有希望的靶标。 虽然家族性LRRK 2突变具有诱导培养细胞中神经元损伤的能力，但它们对神经元损伤的影响可能与LRRK 2突变有关。 由于缺乏稳健的神经变性表型，动物模型不太确定。此外，两人如何 在由家族性LRRK 2突变诱导的体内神经元损伤中， 明白我们最近在成年大鼠中开发了腺病毒介导的基因转移模型， G2019 S LRRK 2通过激酶依赖性的多巴胺能神经元变性诱导黑质纹状体途径多巴胺能神经元变性 机制在本申请中，我们提出利用这种鲁棒且快速的啮齿动物模型来确定 激酶活性是否是由家族性突变诱导的神经变性所通常需要的（R1441 C， Y1699 C和G2019 S）或PD风险变异体（G2385 R），通过遗传和药理学激酶抑制 (Aim 1.1）。Rab磷酸化对突变型LRRK 2诱导的神经元损伤的作用尚不清楚。 我们将确定在突变型LRRK 2中全面降低Rab磷酸化的神经保护作用。 神经元和腺病毒大鼠模型通过过表达一种新的Rab-specific磷酸酶，PPM 1H（目的1.2）。 PPM 1H的敲除将探索增加Rab磷酸化是否足以复制PPM 1H的表型。 突变LRRK 2的神经毒性作用。我们的研究将进一步探讨基因调节GT3是否 活性可以提供针对啮齿动物中不同家族性LRRK 2突变的共同神经保护机制 (Aim 2.1）。特别是，我们将评估假设检验突变，增加GTP水解和促进 LRRK 2的GDP绑定“关闭”状态。最后，我们将探讨LRRK 2在神经元中的天然相互作用组是如何被激活的。 受GTP酶循环调节，以鉴定与LRRK 2在其GDP-或GTP- 约束状态（目标2.2）。我们的研究将提供关键的机制，深入了解如何谷氨酰胺和激酶活性 调节LRRK 2介导的神经变性，并且对于PD的治疗发现工作将是重要的。

Project Summary Parkinson's disease (PD) is a progressive 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 coding and non-coding variants are associated with risk of sporadic PD. LRRK2 has emerged has an important therapeutic target for treating PD and therefore it is critical to understand the key mechanisms underlying disease-linked mutations. LRRK2 is a large multi-domain protein containing Ras-of-Complex (Roc) GTPase and protein kinase enzymatic domains separated by a C-terminal-of-Roc (COR) domain. GTP-binding via the Roc domain is critical for normal kinase activity. Familial LRRK2 mutations cluster within the Roc (R1441C/G/H), COR (Y1699C) and kinase (G2019S, I2020T) domains where they commonly enhance the phosphorylation of a subset of Rab GTPase substrates in cells. Roc-COR domain mutations act indirectly on kinase activity by impairing GTP hydrolysis and promoting the GTP-bound `on' state. The GTPase and kinase domains represent promising targets for inhibiting LRRK2. While familial LRRK2 mutations share the capacity to induce neuronal damage in cultured cells, their effects in animal models are less certain due to a lack of robust neurodegenerative phenotypes. In addition, how the two enzymatic activities contribute to neuronal damage in vivo induced by familial LRRK2 mutations is poorly understood. We have recently developed an adenoviral-mediated gene transfer model in adult rats where G2019S LRRK2 induces nigrostriatal pathway dopaminergic neurodegeneration through a kinase-dependent mechanism. In the present application, we propose to exploit this robust and rapid rodent model to determine whether kinase activity is commonly required for neurodegeneration induced by familial mutations (R1441C, Y1699C and G2019S) or PD risk variants (G2385R) in LRRK2 by genetic and pharmacological kinase inhibition (Aim 1.1). The contribution of Rab phosphorylation to neuronal damage induced by mutant LRRK2 is not known. We will determine the neuroprotective effects of globally reducing Rab phosphorylation in mutant LRRK2 neuronal and adenoviral rat models by overexpressing a novel Rab-specific phosphatase, PPM1H (Aim 1.2). Knockdown of PPM1H will explore whether increasing Rab phosphorylation is sufficient to phenocopy the neurotoxic effects of mutant LRRK2. Our studies will further explore whether genetically modulating GTPase activity can provide a common neuroprotective mechanism against different familial LRRK2 mutations in rodents (Aim 2.1). In particular, we will evaluate hypothesis-testing mutations that increase GTP hydrolysis and promote the GDP-bound `off' state of LRRK2. Finally, we will explore how the native interactome of LRRK2 in neurons is regulated by the GTPase cycle to identify novel protein targets that interact with LRRK2 in its GDP- or GTP- bound states (Aim 2.2). Our studies will provide critical mechanistic insight into how GTPase and kinase activity regulate LRRK2-mediated neurodegeneration, and will be important for therapeutic discovery efforts for PD.