Function and Pathogenic Mechanism of LRRK2 in Parkinson's Disease

1.散发性帕金森病患者亮氨酸重复序列蛋白2的表达上调涉及到特定的microRNA。 LRRK2与散发性帕金森病(PD)的进展有关。然而，散发性帕金森病患者脑内LRRK2蛋白表达和功能的调控机制尚不清楚。在此，我们发现散发性帕金森病患者脑组织中LRRK2蛋白的表达显著增加。此外，我们还发现LRRK2和microRNA-205(miR-205)在帕金森病大鼠脑内的表达呈显著负相关。随着小鼠年龄的增长，LRRK2和miR-205的表达也在脑的多个区域动态调节且呈负相关，提示miR-205在调节LRRK2的表达方面具有潜在的转录后调控作用。事实上，miR-205的过表达抑制了LRRK2在细胞系和原代神经元培养中的表达，并挽救了因过度表达与PD相关的LRRK2R1441G突变而导致的轴突生长缺陷。综上所述，我们证明了散发性帕金森病患者大脑中LRRK2蛋白的上调可能是由于miR-205的下调。我们的研究结果还表明，miR-205的过度表达可能有助于抑制PD患者脑内LRRK2的病理性升高。 *miR-205作为生物标记物和治疗靶点的用途已提交专利申请。 *人类分子遗传学正在修改miR-205数据的手稿。 2.LRRK2在突触形成过程中作为PKA通路的调节器 LRRK2积极参与细胞骨架动力学的研究(Parisiadou和Cai，2010)。因此，LRRK2和肌动蛋白动力学之间的关键联系在神经元发育过程中的轴突生长中被表明(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)。脊椎形态发生包括从最初的细长、高度柔韧的丝状足突向更稳定的脊椎过渡，当它们的尖端(脊椎头)和明显的颈部显示出特征的球状扩大时，被认为是成熟的(Oray等人，2006；Yoshihara等人，2009)。维持脊柱形态和密度的重要性体现在脊柱异常属性与脑功能障碍之间的相关性上(Kasai等人，2003年)。Cofilin是肌动蛋白细丝周转的调节器，对以肌动蛋白为基础的脊柱形成的动力学起着重要的调节作用(Meng等人，2002年)。Cofilin活性被LIM激酶介导的磷酸化抑制，并被Slingshot诱导的高度保守的残基3(S3)上的丝氨酸去磷酸化激活(Bernstein和Bamburg，2010)。然而，已经描述了另一种调节机制，包括依赖蛋白激酶A(PKA)的途径(Paavilainen等人，2004年)。 PKA介导的信号通路对神经元的发育和功能至关重要(Frey等人，1993；Greenberg等人，1987；Choi等人，2002)。PKA是一种全酶，由两个调节亚基和两个催化亚基组成。在静息状态下，每个调节亚基与一个催化单元结合，并使其保持不活跃。当与cAMP结合时，调节亚基与催化单元解离，然后催化单元将其底物磷酸化(Scott，1991；Francis和Corbin，1994)。哺乳动物PKA家族可根据其调节亚基分为I型和II型(Brandon等人，1997年)。II-β；调节(RII-β)亚单位在神经元中高度表达(Ventra等人，1996)(Brandon等人，1998)。PKA在细胞内的空间分布在PKA信号转导中具有关键作用，因为它可能有助于信号的特异性和有效性(Lu等人，2007；Zaccolo等人，2002)。II型PKA的亚细胞定位受A激酶锚定蛋白(AKAPs)控制(Wong和Scott，2004；钟等，2009)。 在这里，我们证明了LRRK2是PKARII-β在神经元中亚细胞分布的关键调节因子，并赋予LRRK2一个新的角色，作为PKA通路的调节器，特别是在出生后2-3周左右，这是小鼠大脑中突触形成的关键时期。LRRK2缺失导致PKA活性增加，表现为Cofilin和AMPA型谷氨酸受体亚单位GR1A1的磷酸化增加。COFILIN和GR1A1磷酸化的改变损害了脊髓的形成和突触传递。总体而言，我们的数据揭示了LRRK2在突触发生过程中的一个新的调节作用，其中LRRK2可能作为施加在PKA相关通路上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.