Reversible and Temporally Inducible LRRK2 Knockout Mice

描述（由申请方提供）：LRRK 2/Dardarin基因突变是常染色体显性帕金森病（PD）的常见原因。已经在许多家族中鉴定出几种显性遗传的错义突变，其表现出广泛的神经病理学特征，包括α-突触核蛋白和tau蛋白的沉积。LRRK 2（Leucine-rich repeat kinase 2）是一种多功能蛋白。它属于ROCO蛋白家族，包括MAPKKK类的蛋白激酶结构域和几个其他主要功能结构域，包括Ras/GTK和WD 40结构域。目前很难完全理解LRRK 2基因突变是如何导致PD的，因为它的功能在很大程度上是未知的。我们建议产生和表征LRRK 2敲除小鼠，以更好地了解LRRK 2功能，并确定LRRK 2功能的缺失是否是由于LRRK 2突变导致PD的原因。我们计划利用最近的技术进步，在抑制剂敏化突变的产生，引入突变到LRRK 2激酶结构域，这将允许LRRK 2信号级联的特异性抑制。在饮用水中或通过注射引入药物将允许在发育或衰老期间的任何时间特异性和可逆地抑制LRRK 2激酶活性。这种创新的方法提供了一种非常有价值的方法来剖析LRRK 2信号在PD中的作用。此外，它将使LRRK 2激酶活性的作用，在维持一个功能性的黑质纹状体多巴胺系统在发展过程中，早期出生后的发展和成熟和患病的大脑中的分子解剖。我们的靶向策略还将使我们能够使用Cre-Lox系统在LRRK 2中产生无效突变以及LRRK 2的组织特异性缺失。因此，提出了进一步表征LRRK 2在PD发病机制中的作用的实验。在具体目标#1中，我们将使用可逆和时间特异性中断细胞和动物中激酶活性的新方法开发和表征可逆和时间诱导的LRRK 2敲除小鼠。在具体目标#2中，我们将评估LRRK 2基因靶向小鼠对环境毒素（包括MPTP诱导的多巴胺能细胞死亡）的敏感性。在具体目标#3中，我们将通过将LRRK 2基因靶向小鼠与α-突触核蛋白和tau转基因小鼠杂交来确定LRRK 2、α-突触核蛋白和/或tau是否参与共同的致病途径。可逆的和暂时诱导的LRRK 2基因敲除小鼠的开发和表征，将增加我们对LRRK 2激酶活性在PD发病机制中的关系的理解。此外，它可以提供深入了解该基因突变诱导神经元损伤的分子机制，并可以提供新的治疗靶点，以防止该家族相关基因在PD退行性过程中的毒性作用。

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.