Function and Pathogenic Mechanism of LRRK2 in Parkinson's Disease

1.LRRK2是一种新的NFAT调节因子，调节炎症性肠病的严重程度。 炎症性肠病(IBD)通常被认为是由于对肠腔生物群的免疫反应失调而发展起来的，包括两种主要形式：克罗恩病(CD)和溃疡性结肠炎。CD1的发生发展受遗传因素和环境因素共同作用。克隆氏病的全基因组关联研究已确定了40多个易感基因2-4。易感基因的大量存在可能反映了IBD胃肠道炎症过程的复杂性。胃肠道内的淋巴组织经常遇到正常的共生微生物区系以及潜在的致病菌；因此，在这个位置保持免疫反应性和耐受性之间的微妙平衡是至关重要的。对GWAS确定的易感基因的研究证明了这一概念，并对IBD的发病机制产生了重要的见解。例如，Treg细胞的调节功能、自噬的参与以及IL23/IL23R信号事件都与IBD5有关。尽管取得了这一进展，但大多数易感基因还没有已知的机制来解释它们与CD的关系。我们研究了LRRK2在IBD中的作用及其潜在的分子机制。为了研究先天性免疫系统和获得性免疫系统在IBD发病机制中的作用，已经建立了各种实验小鼠模型。鉴于LRRK2在先天免疫细胞中的优势表达，而在T细胞21中不表达，本研究采用葡聚糖硫酸钠(DSS)诱导的结肠炎。DSS引起的急性结肠炎依赖于先天免疫系统，因为它发生在缺乏B和T淋巴细胞的严重联合免疫缺陷(SCID)小鼠22。在这里，我们表明，LRRK2缺乏加剧了使用DSS治疗的小鼠的结肠炎。我们利用黑腹隐翅虫RNAi屏幕上的数据寻找潜在的调节因子，发现LRRK2与NFAT相互作用。通过生化检测，我们发现LRRK2抑制了NRON复合体内细胞质中的NFAT1，并抑制了NFAT1的激活。我们还确定LRRK2不改变NFAT1的磷酸化，而是有效地调节细胞质保留和NFAT1与NRON复合体之间的相互作用 诱导剂，如脂多糖(LPS)。最后，人类中与CD相关的LRRK2风险等位基因导致LRRK2蛋白水平微妙地降低，NFAT活性微妙地增强，这与其作为人类IBD多因素易感性的一部分的作用一致。总体而言，我们的数据表明，LRRK2负向调节NFAT驱动的髓系细胞的先天性免疫反应，并且人类中较低水平的LRRK2为该蛋白如何导致CD风险增加提供了分子洞察力。未来的研究将探讨LRRK2/NFAT通路在脑内潜在的小胶质细胞增多症和神经变性中的作用。 *上述数据稿件已被《自然免疫学》接受 2.散发性帕金森病中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。 *miR-205数据手稿正在由Brain进行最终修订 3.将LRRK2 WT和G2019S转基因小鼠和LRRK2基因敲除小鼠放置在JAX中供公众使用。 JAX小鼠数据库-012441 C57BL/6J-TG(TETO-LRRK2*G2019S)E3Cai/J JAX小鼠数据库-012449 TC57BL/6J-TG(teto-LRRK2)C7874Cai/J JAX小鼠数据库-012453 B6.129X1(FVB)-Lrrk2tm1.1Cai/J

1. LRRK2 is a novel regulator of NFAT that modulates the severity of inflammatory bowel disease. Inflammatory bowel disease (IBD), which is generally thought to develop from a dysregulated immune response to the gut luminal biota, includes two major forms: Crohns disease (CD) and ulcerative colitis. Both genetic and environmental factors contribute to the development of CD1. Genome-wide association studies (GWAS) for Crohns disease have identified over 40 susceptibility loci2-4. The large number of the susceptibility genes likely reflects the complexity of the inflammatory process taking place along the gastrointestinal tract in IBD. The lymphoid tissue within the gastrointestinal tract constantly encounters normal commensal microbiota as well as potentially pathogenic bacteria; hence it is critical in this location to maintain a delicate balance between immune responsiveness and tolerance. Research on the susceptibility genes identified by GWAS has exemplified this concept, and yielded important insights into the pathogenesis of IBD. For example, the regulatory function of Treg cells, the involvement of autophagy, and IL23/IL23R signaling events have been implicated in IBD5. Despite this progress, the majority of susceptibility genes have no known mechanism to explain their involvement with CD. We investigated the role of LRRK2 in IBD and the underlying molecular mechanism. Various experimental murine models have been established to study the roles of innate and adaptive immune systems in the pathogenesis of IBD. Given the predominant expression of LRRK2 in innate immune cells but not in T cells21, dextran sulfate sodium (DSS) induced colitis was used in our study. Acute colitis induced by DSS depends on the innate immune system, because it occurs in severe combined immunodeficiency (SCID) mice which lack B and T lymphocytes22. Here we show that LRRK2 deficiency exacerbated colitis in mice treated with DSS. We searched for potential mediators of the effect using data from a RNAi screen in D. Melanogaster and found that LRRK2 was an interactor with NFAT. Examining this connection biochemically, we found that LRRK2 restrained NFAT1 in the cytoplasm within the NRON complex and inhibited NFAT1 activation. We also determined that LRRK2 did not alter NFAT1 phosphorylation but instead potently modulated cytoplasmic retention and the interaction between NFAT1 and the NRON complex in response to selected inducers such as lipopolysaccharide (LPS). Finally, a LRRK2 risk allele associated with CD in humans resulted in subtly reduced LRRK2 protein levels and delicately enhanced NFAT activity consistent with its role as part of a multifactorial susceptibility to human IBD. Collectively, our data demonstrate that LRRK2 negatively regulates NFAT-driven innate immune responses in myeloid cells and that lower levels of LRRK2 in humans provides a molecular insight into how this protein can contribute to an increased risk of CD. Future studies will address the involvement of LRRK2/NFAT pathway in the potential microgliosis and neurodegeneration in the brain. * The manuscript of the above data has been accepted by Nature Immunology. 2. 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 the final revision by Brain. 3. Deposition of LRRK2 WT and G2019S transgenic and LRRK2 knockout mice in the JAX for public access. JAX Mice Database - 012441 C57BL/6J-Tg(tetO-LRRK2*G2019S)E3Cai/J JAX Mice Database - 012449 TC57BL/6J-Tg(teto-LRRK2)C7874Cai/J JAX Mice Database - 012453 B6.129X1(FVB)-Lrrk2tm1.1Cai/J