Function and Pathogenic Mechanism of LRRK2 in Parkinson's Disease

1. LRRK 2是一种新型NFAT调节剂，可调节炎症性肠病的严重程度。 炎症性肠病（IBD）通常被认为是由对肠腔生物群的免疫应答失调发展而来，包括两种主要形式：克罗恩病（CD）和溃疡性结肠炎。遗传和环境因素都有助于CD 1的发展。克罗恩病的全基因组关联研究（GWAS）已经确定了40多个易感基因座2 -4。大量的易感基因可能反映了IBD中沿着胃肠道发生的炎症过程的复杂性。胃肠道内的淋巴组织不断遇到正常的肠道微生物群以及潜在的致病细菌;因此，在该位置保持免疫应答和耐受性之间的微妙平衡至关重要。对GWAS鉴定的易感基因的研究例证了这一概念，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、自噬的参与和IL 23/IL 23 R信号传导事件已经涉及IBD 5。尽管取得了这一进展，但大多数易感基因没有已知的机制来解释它们与CD的关系。我们研究了LRRK 2在IBD中的作用及其分子机制。已经建立了各种实验小鼠模型来研究先天性和适应性免疫系统在IBD发病机制中的作用。鉴于LRRK 2在先天免疫细胞中而不是在T细胞中的主要表达21，在我们的研究中使用葡聚糖硫酸钠（DSS）诱导的结肠炎。DSS诱导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）小鼠中22。在这里，我们表明LRRK 2缺乏加剧了DSS治疗小鼠的结肠炎。我们使用来自D.发现LRRK 2与NFAT相互作用。通过生物化学方法研究这种联系，我们发现LRRK 2抑制了NRON复合物内细胞质中的NFAT 1，并抑制了NFAT 1的活化。我们还确定LRRK 2不改变NFAT 1磷酸化，而是有效地调节细胞质滞留以及NFAT 1和NRON复合物之间的相互作用，以响应选择的细胞因子。 诱导剂如脂多糖（LPS）。最后，与人类CD相关的LRRK 2风险等位基因导致LRRK 2蛋白水平轻微降低，NFAT活性轻微增强，这与其作为人类IBD多因素易感性的一部分的作用一致。总的来说，我们的数据表明，LRRK 2负调控骨髓细胞中NFAT驱动的先天免疫反应，并且人体中LRRK 2水平较低，为这种蛋白质如何导致CD风险增加提供了分子见解。未来的研究将解决LRRK 2/NFAT通路参与大脑中潜在的小胶质细胞增生和神经变性。 * 以上数据的稿件已被《自然免疫学》杂志接受。 2.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Brain进行最终修订。 3.将LRRK 2 WT和G2019 S转基因和LRRK 2敲除小鼠存放在JAX中供公众访问。 JAX小鼠数据库- 012441 C57 BL/6 J-Tg（tetO-LRRK 2 * G2019 S）E3 Cai/J JAX小鼠数据库- 012449 TC 57 BL/6 J-Tg（teto-LRRK 2）C7874 Cai/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