Function and Pathogenic Mechanism of LRRK2 in Parkinson's Disease

1.散发性帕金森病中富含亮氨酸重复序列激酶2表达的上调涉及特异性microRNA LRRK 2与散发性帕金森病（PD）的进展有关。然而，调节散发性PD患者脑中LRRK 2蛋白表达和功能的机制仍有待确定。在这里，我们发现LRRK 2蛋白的表达在散发性PD患者的大脑中显著增加。此外，我们发现LRRK 2和microRNA-205（miR-205）在PD脑中的表达之间存在显著的负相关性。随着小鼠年龄的增长，LRRK 2和miR-205的表达也在大脑的多个区域中动态调节并呈负相关，这表明miR-205在调节LRRK 2表达中具有潜在的转录后调节作用。事实上，miR-205的过表达抑制了细胞系和原代神经元培养物中LRRK 2的表达，并挽救了由PD相关LRRK 2 R1441 G突变过表达诱导的神经突生长缺陷。总之，我们证明了LRRK 2蛋白在散发性PD患者的脑中上调，可能是由于miR-205的下调。我们的研究结果还表明，miR-205的过表达可能有助于抑制PD患者脑中LRRK 2的致病性升高。 * miR-205作为生物标志物和治疗靶点的效用已提交专利申请。 * miR-205数据的手稿正在由人类分子遗传学进行修订。 2. LRRK 2作为突触发生过程中PKA通路的调节剂 LRRK 2积极参与细胞骨架动力学以供综述（Parisiadou和Cai，2010）。因此，在神经元发育期间的神经突生长中表明LRRK 2和肌动蛋白动力学之间的关键联系（Parisiadou等人，2009年）。树突棘的形成是大脑中神经元连接和可塑性的基础，也是由肌动蛋白细胞骨架决定的（Ethell和Pasquale，2005; Tada和Sheng，2006; Schubert和Dotti，2007）。棘形态发生包括从最初的长、细、高度柔性的丝状伪足到更稳定的棘的过渡，当在其尖端（棘头）和明显的颈部显示出特征性的球根状扩大时，所述棘被认为是成熟的（Oray等人，2006; Yoshihara等人，2009年）。脊柱形态和密度的维持的重要性由异常脊柱特性和脑功能障碍之间的相关性反映（加塞等人，2003年）。Cofilin是肌动蛋白丝周转的调节剂，严格地调节棘形成的基于肌动蛋白的动力学（Meng等人，2002年）。Cofilin活性被LIM激酶介导的磷酸化抑制，并被弹弓诱导的高度保守的丝氨酸残基3（S3）上的去磷酸化激活（伯恩斯坦and Bamburg，2010）。然而，已经描述了替代的调节机制，包括蛋白激酶A（PKA）依赖性途径（Paavilainen等人，2004年）。 PKA介导的信号传导途径对于神经元发育和功能至关重要（Frey等人，1993; Greenberg等人，1987; Choi等人，2002年）。PKA是由两个调节亚基和两个催化亚基组成的全酶。在静息状态下，每个调节亚基与催化单元结合并使其保持不活动。在与cAMP结合后，调节亚基与催化单元解离，然后催化单元用于磷酸化其底物（Scott，1991;弗朗西斯and Corbin，1994）。哺乳动物PKA家族可基于其调节亚基细分为I型和II型（布兰登等人，1997年）。II-β调节（RII-β）亚基在神经元中高度表达（RII-β等，1996）（布兰登等人，1998年）。PKA空间细胞内分布在PKA信号传导中具有关键作用，因为它可能有助于信号传导特异性和功效（Lu等人，2007; Zaccolo等人，2002年）。II型PKA的亚细胞定位由A激酶锚定蛋白（AKAP）控制（Wong和Scott，2004; Zhong等人，2009年）。 在这里，我们表明，LRRK 2是PKARII-β在神经元中亚细胞分布的关键调节因子，并指定LRRK 2作为PKA通路的调节剂的新角色，特别是在出生后两到三周左右，这是小鼠大脑中突触形成的关键时期。LRRK 2缺失导致PKA活性增加，如通过cofilin和AMPA型谷氨酸受体亚基GR 1A 1的磷酸化增加所证明的。cofilin和GR 1A 1磷酸化的改变损害了棘的形成和突触传递。总的来说，我们的数据揭示了LRRK 2在突触发生过程中的一个新的调节作用，其中LRRK 2可能作为PKA相关通路上施加的发育开关的一部分发挥作用。

1. Upregulation of Leucine-rich Repeat Kinase 2 Expression in Sporadic Parkinson Disease involves Specific MicroRNA. LRRK2 has been implicated in the progression of sporadic Parkinson Disease (PD). However, the mechanisms regulating LRRK2 protein expression and function in the brains of patients with sporadic PD remain to be determined. Here we show that the expression of LRRK2 protein is significantly increased in the brains of patients with sporadic PD. Moreover, we found a significant inverse-correlation between the expression of LRRK2 and microRNA-205 (miR-205) in the PD brains. The expression of LRRK2 and miR-205 were also dynamically regulated and inversely correlated in multiple regions of the brain as mice aged, suggesting a potential post-transcriptional regulatory role of miR-205 in modulating LRRK2 expression. Indeed over-expression of miR-205 suppressed the expression of LRRK2 in both cell lines and primary neuronal cultures, as well as rescued the neurite growth defects induced by over-expressing the PD-related LRRK2 R1441G mutation. In summary, we demonstrate that LRRK2 protein is up-regulated in the brains of patients with sporadic PD possibly due to down-regulation of miR-205. Our findings also suggest that over-expression of miR-205 may help to suppress the pathogenic elevation of LRRK2 in the brains of patients with PD. * The utility of miR-205 as a biomarker and therapeutic target has been submitted for patent application. * The manuscript of miR-205 data is under revision by Human Molecular Genetics. 2. LRRK2 as a Modulator of PKA Pathway during Synaptogenesis LRRK2 is actively involved in cytoskeletal dynamics for review (Parisiadou and Cai, 2010). In accordance, a critical connection between LRRK2 and actin dynamics is indicated in the neurite outgrowth during neuron development (Parisiadou et al., 2009). Dendritic spine formation that underlies the basis for neuron connectivity and plasticity in the brain is also critically determined by the actin cytoskeleton (Ethell and Pasquale, 2005; Tada and Sheng, 2006; Schubert and Dotti, 2007). Spine morphogenesis include the transition from initial long, thin, highly flexible filopodia to more stable spines that are considered mature when show characteristic bulbous enlargements at their tips (spine head) and distinct neck (Oray et al., 2006; Yoshihara et al., 2009). The importance of the maintenance of spine morphology and density is reflected by the correlation between abnormal spine properties and brain dysfunction (Kasai et al., 2003). Cofilin, a modulator of actin filament turnover, critically regulates the actin-based dynamics of spine formation (Meng et al., 2002). Cofilin activities are inhibited by LIM kinase-mediated phosphorylation and activated by Slingshot-induced de-phosphorylation on a highly conserved Serine at residue three (S3) (Bernstein and Bamburg, 2010). However, alternative regulatory mechanisms have been described, including a protein kinase A (PKA)-dependent pathway (Paavilainen et al., 2004). PKA-mediated signaling pathways are critical for neuron development and function (Frey et al., 1993; Greenberg et al., 1987; Choi et al., 2002). PKA is a holoenzyme that consists of two regulatory and two catalytic subunits. At resting state, each regulatory subunit binds to a catalytic unit and keeps it inactive. Upon binding with cAMP, the regulatory subunit dissociates with catalytic unit, which then acts to phosphorylate its substrates (Scott, 1991; Francis and Corbin, 1994). The mammalian PKA family could be subdivided into types I and II based on their regulatory subunits (Brandon et al., 1997). The II-beta; regulatory (RII-beta) subunit is highly expressed in neurons (Ventra et al., 1996) (Brandon et al., 1998). PKA spatial intracellular distribution possesses a critical role in PKA signaling since it might contribute to signaling specificity and efficacy (Lu et al., 2007; Zaccolo et al., 2002). The subcellular localization of type II PKA is controlled by the A kinase anchoring proteins (AKAPs) (Wong and Scott, 2004; Zhong et al., 2009). Here we show that LRRK2 is a critical regulator of PKARII-beta's subcellular distribution in neurons, and assign a new role on LRRK2 as a modulator of PKA pathway particularly around postnatal two to three weeks, a critical period for the synapse formation in the mouse brain. LRRK2-absence resulted to increased PKA activity as evidenced by increased phosphorylation of cofilin and AMPA type glutamate receptor subunit GR1A1. The alterations of cofilin and GR1A1 phosphorylation impaired the spine formation and synaptic transmission. Overall, our data reveal a new regulatory role of LRRK2 during synaptogenesis, in which LRRK2 might function as part of the developmental switch imposed onPKA-related pathways.