LRRK2基因与帕金森病

帕金森病是一种复杂的多因素共同作用的神经变性性疾病,遗传因素和环境因素被认为是发病的重要原因。越来越多的研究发现遗传因素在其发病中起着重要作用,而最近LRRK2基因的发现更加确定了遗传因素在帕金森发病中不可忽视的作用。本文对LRRK2基因以及LRRK2基因在帕金森病中的研究做一综述。     

Genetic Variants of LRRK2 in Taiwanese Parkinson’s Disease

Genetic variants of leucine-rich repeat kinase 2 (LRRK2) were reported to alter the risk for Parkinson’s disease (PD). However, the genetic spectrum of LRRK2 variants has not been clearly disclosed yet in Taiwanese population. Herein, we sequenced LRRK2 coding region in 70 Taiwanese early onset PD patients (age at onset ≤ 50), and found six amino acid-changing single nucleotide polymorphisms (SNPs, N551K, R1398H, R1628P, S1647T, G2385R and M2397T), one reported (R1441H) and 2 novel missense (R767H and S885N) mutations. We examined the frequency of identified LRRK2 variants by genotyping 573 Taiwanese patients with PD and 503 age-matched control subjects. The results showed that PD patients demonstrated a higher frequency of G2385R A allele (4.6%) than control subjects (2.1%; odds ratio = 2.27, 95% confidence interval: 1.38–3.88, P = 0.0017). Fewer PD patients (27.7%) carried the 1647T-2397T haplotype as compared with the control subjects (33.0%; odds ratio = 0.80, 95% confidence interval: 0.65–0.97, P = 0.0215). However, the frequency of 1647T-2385R-2397T haplotype (4.3%) in PD patients was still higher than in control subjects (1.9%, odds ratio: 2.15, 95% confidence interval: 1.27–3.78, P = 0.0058). While no additional subject was found to carry R767H and R1441H, one more patient was observed to carry the S885N variant. Our results indicate a robust risk association regarding G2385R and a new possible protective haplotype (1647T-2397T). Gene-environmental interaction and a larger cohort study are warranted to validate our findings. Additionally, two new missense mutations (R767H and S885N) regarding LRRK2 in PD patients were identified. Functional studies are needed to elucidate the effects of these LRRK2 variants on protein function.     

帕金森病中LRRK2的功能

帕金森病(Parkinson's disease,PD)是一种最常见的神经退行性运动障碍,常染色体显性遗传PD可由LRRK2基因的突变引起．总结了LRRK2功能研究的最新成果,分为分子遗传学、表达分布和亚细胞定位、突变体的功能、蛋白质化学、蛋白质动力学、相互作用蛋白和底物、信号传导途径、与突起和突触囊泡蛋白的关系、结构分析、病理和临床特征等10个方面进行论述．指出已有的研究初步阐明了LRRK2突变导致PD的发病机制,提出了治疗PD的新策略,并对未来研究进行展望．     

Molecular basis of Parkinsons’s disease linked to LRRK2 mutations

Parkinson’s disease (PD) is a common neurodegenerative disorder whose symptoms are consistent with death of dopaminergic neurons in the substantia nigra of the brain. The pathogenesis of PD involves several factors, such as α-synuclein aggregation, oxidative stress, mitochondrial dysfunction, and activation of apoptosis, but the exact molecular mechanism of neurodegeneration remains obscure. PD is usually sporadic, while rare monogenic forms have been identified and described in the past 15 years. Familial Parkinson’s disease is most commonly associated with mutations of the leucine repeat-rich kinase 2 gene (LRRK2). The mechanism of the disease due to LRRK2 mutations is unknown. The signaling cascades regulated by LRRK2 are difficult to study because the physiological substrates of the enzyme are unidentified. The G2019S substitution has been found to be the most common LRRK2 mutation, facilitating a search for patients with LRRK2-associated PD in various populations. The review considers the effects of LRRK2 mutations on protein and, in particular, α-synuclein aggregation, cytoskeletal dynamics, the inflammatory response, and the induction of apoptosis as revealed in both in vitro experiments and studies in PD patients. Investigation of rare hereditary PD forms with known etiology provides for a better understanding of the mechanism of neurodegeneration in more common sporadic PD forms.     

Discovery of potent and selective 5-azaindazole inhibitors of leucine-rich repeat kinase 2 (LRRK2) – Part 1

Parkinson’s disease is a relatively common neurological disorder with incidence increasing with age. Present treatments merely alleviate the symptoms and do not alter the course of the disease, thus identification of disease modifying therapies represents a significant unmet medical need. Mutations in the LRRK2 gene are risk-factors for developing PD and it has been hypothesized that the increased kinase activity of certain LRRK2 mutants are responsible for the damage of the dopaminergic neurons, thus LRRK2 inhibitors offer the potential to target an underlying cause of the disease. In this communication, we describe hit-to-lead medicinal chemistry program on a novel series of 5-azaindazoles. Compound 1, obtained from high-throughput screening was optimized to a highly potent, selective series of molecules with promising DMPK properties. Introduction of heterocycles at the 3-position were found to significantly increase the potency and kinase selectivity, whilst changes to the 4-chlorobenzyl group improved the physicochemical properties. Our series was licensed to a major pharmaceutical company for further development.     

The LRRK2-RAB axis in regulation of vesicle trafficking and α-synuclein propagation

LRRK2 and SNCA, the gene for α-synuclein, are the two of the most important genetic factors of Parkinson's disease (PD). A-synuclein is aggregated and accumulated in neurons and glia in PD and considered the pathogenic culprit of the disease. A-synuclein aggregates spread from a few discrete regions of the brain to larger areas as the disease progresses through cell-to-cell propagation mechanism. LRRK2 is involved in the regulation of vesicle trafficking, in particular in the endolysosomal and autophagic pathways. Studies also suggest that LRRK2 might regulate the pathogenic actions of α-synuclein. However, the relationship between these two proteins in the pathogenesis of PD remains elusive. Here, we review the current literature on the pathophysiological function of LRRK2 with an emphasis on its role in the endolysosomal and autophagic pathways. We also propose a potential mechanism by which LRRK2 is involved in the regulation of aggregation and the propagation of α-synuclein.     

与帕金森病相关的LRRK2-PKA-Cofilin信号通路

<正>"二型富亮氨酸重复激酶"(leucine-rich repeat kinase 2,LRRK2)由帕金森基因家族PARK8基因编码,富含于脑纹状体神经元。最近,NIH研究人员发现了LRRK2参与帕金森症的分子机制。敲除LRRK2分子会导致"纹状体投射神经元"(striatal projection neurons,SPNs)蛋白激酶A(PKA)活性降低、细胞骨架蛋白actin的调节子"Cofilin"过磷酸化、"树突棘"密度降低、突触发育迟滞,呈现帕金森症病理学特征。Parisiadou等人发现:敲除Lrrk2基因会导致SPNs树突棘数量剧减、丝状幼稚树突棘显著多于蘑菇状成熟树突棘、PSD95     

LRRK2 R1628P increases risk of Parkinson’s disease: replication evidence

We showed that the frequency of a LRRK2 variant (c.4883G > C, R1628P) was higher in Parkinson’s disease (PD) compared to controls (8.4 vs. 3.4%, P = 0.046, OR 2.5, 95% CI 1.1–5.6). In the multivariate logistic regression (with adjustments made for the effect of age, age of onset, and gender), the heterozygous R1628P genotype was associated with an increased risk of PD compared to controls (OR 3.3, 95% CI 1.4– 7.9, P = 0.007). We provided an independent confirmation that the R1628P variant increases the risk of PD among Chinese.     

Discovery of potent azaindazole leucine-rich repeat kinase 2 (LRRK2) inhibitors possessing a key intramolecular hydrogen bond – Part 2

The discovery of disease-modifying therapies for Parkinson’s Disease (PD) represents a critical need in neurodegenerative medicine. Genetic mutations in LRRK2 are risk factors for the development of PD, and some of these mutations have been linked to increased LRRK2 kinase activity and neuronal toxicity in cellular and animal models. As such, research towards brain-permeable kinase inhibitors of LRRK2 has received much attention. In the course of a program to identify structurally diverse inhibitors of LRRK2 kinase activity, a 5-azaindazole series was optimized for potency, metabolic stability and brain penetration. A key design element involved the incorporation of an intramolecular hydrogen bond to increase permeability and potency against LRRK2. This communication will outline the structure-activity relationships of this matched pair series including the challenge of obtaining a desirable balance between metabolic stability and brain penetration.     

The role of posttranslational modifications of α-synuclein and LRRK2 in Parkinson's disease: Potential contributions of environmental factors

Parkinson's disease (PD) is the second most common neurodegenerative disease after Alzheimer's disease (AD), and the most prevalent movement disorder. PD is characterized by dopaminergic neurodegeneration in the substantia nigra, but its etiology has yet to be established. Among several genetic variants contributing to PD pathogenesis, α-synuclein and leucine-rich repeat kinase (LRRK2) are widely associated with neuropathological phenotypes in familial and sporadic PD. α-Synuclein and LRRK2 found in Lewy bodies, a pathogenic hallmark of PD, are often posttranslationally modified. As posttranslational modifications (PTMs) are key processes in regulating the stability, localization, and function of proteins, PTMs have emerged as important modulators of α-synuclein and LRRK2 pathology. Aberrant PTMs altering phosphorylation, ubiquitination, nitration and truncation of these proteins promote PD pathogenesis, while other PTMs such as sumoylation may be protective. Although the causes of many aberrant PTMs are unknown, environmental risk factors may contribute to their aberrancy. Environmental toxicants such as rotenone and paraquat have been shown to interact with these proteins and promote their abnormal PTMs. Notably, manganese (Mn) exposure leads to a PD-like neurological disorder referred to as manganism—and induces pathogenic PTMs of α-synuclein and LRRK2. In this review, we highlight the role of PTMs of α-synuclein and LRRK2 in PD pathogenesis and discuss the impact of environmental risk factors on their aberrancy.     

LRRK2特异性调控溶菌酶的运输分泌促进肠道稳态

<正>肠道共生菌在人体健康中发挥重要作用,近年来,有关肠道菌群的研究日益成为科学家们感兴趣的焦点.宿主和肠道菌群共同作用调节肠道内环境稳态,有关这其中复杂机制的探索,是揭示在长期的共进化过程中,肠道菌与宿主如何实现"共生-互益"的关键所在,对研究肠道菌异常引起的疾病的发病机制具有重要意义.中国科学院生物物理研究所刘志华课题组发现了共生菌通过Nod2-LRRK2-Rab2a途径介导潘氏细胞中溶菌酶的分拣来促进共生关系,     

LRRK2 signaling pathways: the key to unlocking neurodegeneration?

Mutations in PARK8, encoding leucine-rich repeat kinase 2 (LRRK2), are a major cause of Parkinson's disease. We contrast data suggesting that changes in LRRK2 activity cause alterations in mitogen-activated protein kinase, translational control, tumor necrosis factor α/Fas ligand and Wnt signaling pathways with the cell biological functions of LRRK2 such as vesicle trafficking. Despite scarce in vivo data on cell signaling, involvement in diverse cell biological functions suggests a role for LRRK2 as an upstream regulator in events leading to neurodegeneration. To stimulate discussion and give direction for future research, we further suggest that despite the importance of the catalytic activity for cytotoxicity, the main cellular function of LRRK2 is linked to assembly of signaling complexes.     

Function and dysfunction of leucine-rich repeat kinase 2 (LRRK2): Parkinson’s disease and beyond

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson’s disease (PD). As such, functions and dysfunctions of LRRK2 in PD have been the subject of extensive investigation. In addition to PD, increasing evidence is suggesting that LRRK2 is associated with a wide range of diseases. Genome-wide association studies have implicated LRRK2 in Crohn’s disease (CD) and leprosy, and the carriers with pathogenic mutations of LRRK2 show increased risk to develop particular types of cancer. LRRK2 mutations are rarely found in Alzheimer’s disease (AD), but LRRK2 might play a part in tauopathies. The association of LRRK2 with the pathogenesis of apparently unrelated diseases remains enigmatic, but it might be related to the yet unknown diverse functions of LRRK2. Here, we reviewed current knowledge on the link between LRRK2 and several diseases, including PD, AD, CD, leprosy, and cancer, and discussed the possibility of targeting LRRK2 in such diseases. [BMB Reports 2015; 48(5): 243-248]     

Leucine-rich Repeat Kinase 2 (LRRK2) Pharmacological Inhibition Abates α-Synuclein Gene-induced Neurodegeneration

Therapeutic approaches to slow or block the progression of Parkinson disease (PD) do not exist. Genetic and biochemical studies implicate α-synuclein and leucine-rich repeat kinase 2 (LRRK2) in late-onset PD. LRRK2 kinase activity has been linked to neurodegenerative pathways. However, the therapeutic potential of LRRK2 kinase inhibitors is not clear because significant toxicities have been associated with one class of LRRK2 kinase inhibitors. Furthermore, LRRK2 kinase inhibitors have not been tested previously for efficacy in models of α-synuclein-induced neurodegeneration. To better understand the therapeutic potential of LRRK2 kinase inhibition in PD, we evaluated the tolerability and efficacy of a LRRK2 kinase inhibitor, PF-06447475, in preventing α-synuclein-induced neurodegeneration in rats. Both wild-type rats as well as transgenic G2019S-LRRK2 rats were injected intracranially with adeno-associated viral vectors expressing human α-synuclein in the substantia nigra. Rats were treated with PF-06447475 or a control compound for 4 weeks post-viral transduction. We found that rats expressing G2019S-LRRK2 have exacerbated dopaminergic neurodegeneration and inflammation in response to the overexpression of α-synuclein. Both neurodegeneration and neuroinflammation associated with G2019S-LRRK2 expression were mitigated by LRRK2 kinase inhibition. Furthermore, PF-06447475 provided neuroprotection in wild-type rats. We could not detect adverse pathological indications in the lung, kidney, or liver of rats treated with PF-06447475. These results demonstrate that pharmacological inhibition of LRRK2 is well tolerated for a 4-week period of time in rats and can counteract dopaminergic neurodegeneration caused by acute α-synuclein overexpression.     

Upregulation of the p53-p21 pathway by G2019S LRRK2 contributes to the cellular senescence and accumulation of α-synuclein

Parkinson’s disease (PD) is characterized by the degeneration of dopaminergic neurons in the substantia nigra and the presence of Lewy bodies (LB) in neurons. α-Synuclein (αSyn) is a major component of LB and promote the PD pathogenesis via its accumulation by the impaired proteasomal or autophagic clearance. Numerous studies have revealed that the reduction of proteasome activity and autophagy is accelerated by cellular senescence. Leucine-rich repeat kinase 2 (LRRK2) contributes to PD progression and its most prevalent mutation, G2019S LRRK2, increases its activity. Our previous report has shown that the G2019S LRRK2 mutant promoted p53-induced p21 expression and neuronal cytotoxicity. The p53-p21 pathway plays a role in cellular senescence. We hypothesized that the loss of dopaminergic neurons by the stimulated p53-p21 pathway via the G2019S LRRK2 mutation might be associated with cellular senescence, thereby promoting the accumulation of αSyn. We confirmed that the ectopic expression of the phosphomimetic p53 mutant, p21, or G2019 in differentiated SH-SY5Y cells increased the following: 1) the expression of β-galactosidase, a marker of cellular senescence, and the activity of senescence-associated β-galactosidase, 2) endogenous αSyn protein level, but not its mRNA level, and 3) αSyn fibril accumulation in dSH-SY5Y via low proteasome and cathepsin D activities. Elevated oligomeric αSyn and the increase in β-galactosidase with induced p21 were observed in brain lysates of G2019S transgenic mice. Our results suggest that cellular senescence is promoted via the p53-p21 pathway due to the G2019S LRRK2 mutation. Eventually, decreased protein degradation by G2019S-mediated senescence could accelerate αSyn aggregate formation.     

Nonmotor Symptoms in LRRK2 G2019S Associated Parkinson’s Disease

Background

Idiopathic Parkinson’s disease (IPD) and LRRK2-associated PD (LRRK2-PD) might be expected to differ clinically since the neuropathological substrate of LRRK2-PD is heterogeneous. The range and severity of extra-nigral nonmotor features associated with LRRK2 mutations is also not well-defined.

Objective

To evaluate the prevalence and time of onset of nonmotor symptoms (NMS) in LRRK2-PD patients.

Methods

The presence of hyposmia and of neuropsychiatric, dysautonomic and sleep disturbances was assessed in 33 LRRK2-G2019S-PD patients by standardized questionnaires and validated scales. Thirty-three IPD patients, matched for age, gender, duration of parkinsonism and disease severity and 33 healthy subjects were also evaluated.

Results

University of Pennsylvania Smell Identification Test (UPSIT) scores in LRRK2-G2019S-PD were higher than those in IPD (23.5±6.8 vs 18.4±6.0; p = 0.002), and hyposmia was less frequent in G2019S carriers than in IPD (39.4% vs 75.8%; p = 0.01). UPSIT scores were significantly higher in females than in males in LRRK2-PD patients (26.9±4.7 vs 19.4±6.8; p<0.01). The frequency of sleep and neuropsychiatric disturbances and of dysautonomic symptoms in LRRK2-G2019S-PD was not significantly different from that in IPD. Hyposmia, depression, constipation and excessive daytime sleepiness, were reported to occur before the onset of classical motor symptoms in more than 40% of LRRK2-PD patients in whom these symptoms were present at the time of examination.

Conclusion

Neuropsychiatric, dysautonomic and sleep disturbances occur as frequently in patients with LRRK2-G2019S-PD as in IPD but smell loss was less frequent in LRRK2-PD. Like in IPD, disturbances such as hyposmia, depression, constipation and excessive daytime sleepiness may antedate the onset of classical motor symptoms in LRRK2-G2019S-PD.     

帕金森病相关蛋白质LRRK2在脑中的表达分布与定位分析

帕金森病（Parkinson’s disease,PD）是一种最常见的神经退行性运动障碍,常染色体显性遗传PD可由LRRK2 (leucine-rich repeat kinase 2)基因突变引起.原核表达和纯化得到LRRK2多肽与GST的融合蛋白,免疫新西兰雄兔,得到了anti-LRRK2兔多克隆抗体. 抗体用途分析实验表明,自制的anti-LRRK2兔多克隆抗体可用于Western 印迹, 免疫组织化学,免疫细胞染色等实验.利用自制的抗体,Western 印迹证明,LRRK2在大鼠脑主要神经解剖学部位均有表达.免疫荧光双染色的结果表明,LRRK2定位于线粒体.本研究对了解LRRK2在PD中的分子生物学功能具有重要的意义.     

LRRK2: a common pathway for parkinsonism, pathogenesis and prevention?

The presence of alpha-synuclein Lewy body pathology is used to distinguish Parkinson's disease from parkinsonism, for which a broader spectrum of neuropathologies, including tau-immunopositive neurofibrillary tangles and ubiquitin inclusions, might accompany nigral neuronal loss. These neuropathologies define the endpoint of many neurodegenerative disorders but might be symptomatic rather than causative. Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) were recently discovered in late-onset parkinsonism, the phenotype of which can be clinically and pathologically indistinguishable from Parkinson's disease. However, in some kindreds with LRRK2- associated disease, pathologically distinct forms of parkinsonism, including nigral neuronal loss with Lewy body disease or tau-immunopositive neurofibrillary tangles, were discovered. Understanding the molecular function of the LRRK2 protein and its associated pathways might elucidate the switch between Lewy body pathology and neurofibrillary tangles, and holds promise for prospective therapeutics that might slow or halt progression of many forms of parkinsonism.     

Conformational heterogeneity of the Roc domains in C. tepidum Roc–COR and implications for human LRRK2 Parkinson mutations

Ras of complex proteins (Roc) is a Ras-like GTP-binding domain that always occurs in tandem with the C-terminal of Roc (COR) domain and is found in bacteria, plants and animals. Recently, it has been shown that Roco proteins belong to the family of G-proteins activated by nucleotide (nt)-dependent dimerization (GADs). We investigated the RocCOR tandem from the bacteria Chlorobium tepidum with site-directed spin labelling and pulse EPR distance measurements to follow conformational changes during the Roco G-protein cycle. Our results confirm that the COR domains are a stable dimerization device serving as a scaffold for the Roc domains that, in contrast, are structurally heterogeneous and dynamic entities. Contrary to other GAD proteins, we observed only minor structural alterations upon binding and hydrolysis of GTP, indicating significant mechanistic variations within this protein class. Mutations in the most prominent member of the Roco family of proteins, leucine-rich repeat (LRR) kinase 2 (LRRK2), are the most frequent cause of late-onset Parkinson''s disease (PD). Using a stable recombinant LRRK2 Roc-COR-kinase fragment we obtained detailed kinetic data for the G-protein cycle. Our data confirmed that dimerization is essential for efficient GTP hydrolysis and PD mutations in the Roc domain result in decreased GTPase activity. Previous data have shown that these LRRK2 PD-mutations are located in the interface between Roc and COR. Importantly, analogous mutations in the conserved C. tepidum Roc/COR interface significantly influence the structure and nt-induced conformational changes of the Roc domains.