Reversible and Temporally Inducible LRRK2 Knockout Mice

描述(申请人提供)：LRRK2/Dardarin基因突变是常染色体显性遗传性帕金森病(PD)的常见原因。在一些表现出广泛神经病理特征的家族中发现了几个主要的遗传性错义突变，包括α-突触核蛋白和tau蛋白的沉积。LRRK2(富含亮氨酸的重复蛋白2)编码一种大的多功能蛋白质。它属于Roco蛋白家族，包括一个MAPKKK类蛋白激酶结构域和其他几个主要功能结构域，包括Ras/GTPase和WD40结构域。在这一点上，很难完全了解LRRK2基因突变是如何导致帕金森病的，因为它的功能在很大程度上是未知的。我们建议建立和鉴定LRRK2基因敲除小鼠，以更好地了解LRRK2功能，并确定LRRK2功能缺失是否是LRRK2突变导致的帕金森病的原因。我们计划利用在产生抑制物敏化突变方面的最新技术进展，将突变引入LRRK2激酶结构域，从而允许特异性地抑制LRRK2信号级联反应。将药物引入饮用水或注射将允许在发育或衰老过程中的任何时候特异性和可逆地抑制LRRK2激酶的活性。这种创新的方法为分析LRRK2信号在帕金森病中的作用提供了一个非常有价值的方法。此外，它还将使分子解剖LRRK2激酶活性在发育期间、出生后早期发育以及成熟和患病大脑中维持功能正常的黑质纹状体多巴胺系统中的作用。我们的靶向策略还将使我们能够使用CRE-Lox系统在LRRK2中创建零突变以及组织特异性删除LRRK2。因此，有必要通过实验进一步研究LRRK2在帕金森病发病机制中的作用。在特定的目标#1中，我们将使用一种可逆的和时间特异性的阻断细胞和动物中的激酶活性的新方法来发展和表征可逆的和时间上可诱导的LRRK2基因敲除小鼠。在特定的目标#2中，我们将评估LRRK2基因靶向的小鼠对环境毒素的敏感性，包括MPTP诱导的多巴胺能细胞死亡。在特定的目标#3中，我们将通过将LRRK2基因靶向的小鼠与a-突触核蛋白和tau转基因小鼠杂交，来确定LRRK.2、a-突触核蛋白和/或tau是否参与了常见的致病途径。LRRK2基因敲除小鼠的建立和鉴定将加深我们对LRRK2激酶活性在帕金森病发病机制中的关系的理解。此外，它可能为深入了解该基因突变导致神经元损伤的分子机制提供新的治疗靶点，并可能为防止该家族相关基因在帕金森病退变过程中的毒性作用提供新的治疗靶点。

DESCRIPTION (provided by applicant): Mutations in the LRRK2/Dardarin gene are a common cause of autosomal dominant Parkinson's disease (PD). Several dominantly inherited missense mutations have been identified in a number of families that exhibit a broad spectrum of neuropathological features, including deposition of alpha-synuclein and tau proteins. LRRK2 (leucine-rich repeat kinase 2) encodes a large, multifunctional protein. It belongs to the ROCO protein family and includes a protein kinase domain of the MAPKKK class and several other major functional domains including Ras/GTPase and WD40 domains. It is difficult at this juncture to fully appreciate how mutations in the LRRK2 gene cause PD, as its function is largely unknown. We propose to generate and characterize LRRK2 knockout mice to gain a better understanding of LRRK2 function and to determine whether the absence of LRRK2 function is the cause of PD due to LRRK2 mutations. We plan to utilize the recent technological advances in the generation of inhibitor sensitizing mutations to introduce a mutation into the LRRK2 kinase domain that will allow the specific inhibition of the LRRK2 signaling cascade. Introduction of the drug in drinking water or by injection will permit the specific and reversible inhibition of LRRK2 kinase activity at any time during development or aging. Such an innovative approach provides an extremely valuable method to dissect the role of LRRK2 signaling in PD. Moreover, it will enable a molecular dissection of the role of LRRK2 kinase activity in maintaining a functional nigrostriatal dopamine system during development, early postnatal development and within the mature and diseased brain. Our targeting strategy will also enable us to create a null mutation in LRRK2 as well as tissue specific deletion of LRRK2 using the Cre-Lox system. Accordingly experiments are proposed to further characterize the role of LRRK2 in the pathogenesis of PD. In Specific Aim #1 we will develop and characterize a reversible and temporally inducible LRRK2 knockout mouse using a novel method of reversible and temporally specific interruption of kinase activity in cells and animals. In Specific Aim #2 we will we will evaluate the sensitivity of LRRK2 gene targeted mice to environmental toxins including MPTP-induced dopaminergic cell death. In Specific Aim #3 we will determine whether LRRK.2, a-synuclein and/or tau participate in a common pathogenic pathway by crossing LRRK2 gene-targeted mice with a-synuclein and tau transgenic mice. Development and characterization of a reversible and temporally inducible LRRK2 knockout mouse, will increase our understanding of the relationship of LRRK2 kinase activity in the pathogenesis of PD. Moreover, it may provide insight into the molecular mechanisms by which mutations in this gene induce neuronal damage and may provide novel therapeutics targets to prevent the toxic effects of this familial associated gene in the degenerative process of PD.