LRRK2 modulates the progression of neuropathology in a mouse model of tauopathy

描述(申请人提供)：tau，一种功能受磷酸化调节的微管结合蛋白，与帕金森病(PD)的发病机制之间的联系尚不清楚。有鉴于此，有趣的是，编码富含亮氨酸重复蛋白激酶2(LRRK2)的基因突变是帕金森病的主要危险因素，与包括tau缠结和tau异常磷酸化在内的病理相关。最常见的突变是G2019S，它位于LRRK2的激活域内，并在体外显示出高活性的激酶活性，这被认为是其细胞毒性的中介。LRRK2的正常功能 而突变如何影响这一功能从而影响疾病的发病机制尚不清楚。最近发现Tau被LRRK2磷酸化，但目前尚不清楚LRRK2和tau在体内是否相互作用，以及这种相互作用如何被LRRK2的突变改变而影响聚集和导致疾病。本申请的总体目标是确定LRRK2是否调节tau病理，并确定其发生的机制(S)。这项建议将使用体外和体内方法相结合的方法来确定野生型(WT)和突变型LRRK2改变tau磷酸化和聚集的机制，并确定这如何影响神经毒性。目标1下的实验将确定WT和突变体LRRK2在体外和细胞培养系统中如何改变tau的磷酸化和聚集。初步数据已经证实，tau是LRRK2激酶活性的良好底物，并在体外发现了多个被tau磷酸化的位点。LRRK2在这些位点上的磷酸化对tau聚集体发育的影响将通过体外和培养细胞中tau包涵体的形成来检验。在目标1中产生的数据的生物学相关性将通过该提案的目标2下的研究来测试，该研究将确定WT和突变的LRRK2如何在新的小鼠模型中调节互变。WT和G2019S LRRK2小鼠将与rTg4510小鼠进行杂交，rTg4510小鼠具有良好的直立性疾病的病程特征。将在这些小鼠的组织中测量tau的磷酸化、聚集和神经毒性。初步数据显示，WT LRRK2/rTg4510在特定位点增加了tau的磷酸化，并增加了tau的聚集，支持在这一提议中使用这些模型。这些研究的结果对于理解帕金森病的病因和指导未来的研究以确定LRRK2介导的tau发病机制是至关重要的。

DESCRIPTION (provided by applicant): The link between tau, a microtubule binding protein whose function is regulated by phosphorylation, and the pathogenesis of Parkinson's disease (PD) remains unclear. In view of this, it is interesting that mutations in the gene encoding Leucine-rich repeat kinase 2 (LRRK2), which are a major risk factor for PD, have been associated with pathologies that include tau tangles and aberrant tau phosphorylation. The most common mutation, G2019S, is within the kinase domain of LRRK2 and shows increased kinase activity in vitro, which is proposed to mediate its cellular toxicity. The normal function of LRRK2 and how mutations affect this function to affect disease pathogenesis is unknown. Tau has recently been shown to be phosphorylated by LRRK2, but it is still not known if LRRK2 and tau interact in vivo and how this interaction is altered by mutations in LRRK2 to affect aggregation and cause disease. The overall objectives of this application are to determine if LRRK2 modulates tau pathology and to identify the mechanism(s) by which this occurs. This proposal will use a combination of in vitro and in vivo approaches to identify mechanisms by which tau phosphorylation and aggregation are altered by wild-type (WT) and mutant LRRK2 and determine how this affects neurotoxicity. Experiments under Aim 1 will determine how tau phosphorylation and aggregation are altered by WT and mutant LRRK2 in vitro and in cell culture systems. Preliminary data has confirmed that tau is a good substrate for LRRK2 kinase activity and has identified multiple sites that are phosphorylated by tau in vitro. The effects of LRRK2 phosphorylation at these sites on the development of tau aggregates will be examined using tau inclusion formation in vitro and in cultured cells. The biological relevance of data generated in Aim 1 will be tested by studies under Aim 2 of this proposal that will determine how WT and mutant LRRK2 modulate tauopathy in novel mouse models. WT and G2019S LRRK2 mice will be crossed with the rTg4510 mouse model of tauopathy, which has a well-characterized disease course of tauopathy. Tau phosphorylation, aggregation and neurotoxicity will be measured in tissue from these mice. Preliminary data show that WT LRRK2/rTg4510 have increased tau phosphorylation at specific sites and increased tau aggregation, supporting the use of these models in this proposal. Results from these studies are essential to understanding PD etiology and directing future research to identify mechanisms of LRRK2-mediated tau pathogenesis.