Leucine-rich repeat kinase 2 mutations and Parkinson’s disease: three questions

Mutations in the gene encoding LRRK2 (leucine-rich repeat kinase 2) were first identified in 2004 and have since been shown to be the single most common cause of inherited Parkinson’s disease. The protein is a large GTP-regulated serine/threonine kinase that additionally contains several protein–protein interaction domains. In the present review, we discuss three important, but unresolved, questions concerning LRRK2. We first ask: what is the normal function of LRRK2? Related to this, we discuss the evidence of LRRK2 activity as a GTPase and as a kinase and the available data on protein–protein interactions. Next we raise the question of how mutations affect LRRK2 function, focusing on some slightly controversial results related to the kinase activity of the protein in a variety of in vitro systems. Finally, we discuss what the possible mechanisms are for LRRK2-mediated neurotoxicity, in the context of known activities of the protein

Exclusion of PINK1 as candidate gene for the late-onset form of Parkinson's disease in two European populations

BACKGROUND: Parkinson's disease (PD) is the second most common neurodegenerative disorder. Recently, mutations in the PINK1 (PARK6) gene were shown to rarely cause autosomal-recessively transmitted, early-onset parkinsonism. In order to evaluate whether PINK1 contributes to the risk of common late-onset PD we analysed PINK1 sequence variations. A German (85 patients) and a Norwegian cohort (90 patients) suffering from late-onset PD were screened for mutations and single nucleotide polymorphisms (SNPs) in the PINK1 gene. Both cohorts consist of well-characterized patients presenting a positive family history of PD in ~17%. Investigations were performed by single strand conformation polymorphism (SSCP), denaturating high performance liquid chromatography (DHPLC) and sequencing analyses. SNP frequencies were compared by the χ(2 )test RESULTS: Several common SNPs were identified in our cohorts, including a recently identified coding variant (Q115L) in exon 1. Genotyping of the Q115L variation did not reveal significant frequency differences between patients and controls. Pathogenic mutations in the PINK1 gene were not identified, neither in the German nor in the Norwegian cohort. CONCLUSION: Sequence variation in the PINK1 gene appears to play a marginal quantitative role in the pathogenesis of the late-onset form of PD, in German and Norwegian cohorts, if at all

Neither Replication nor Simulation Supports a Role for the Axon Guidance Pathway in the Genetics of Parkinson's Disease

Susceptibility to sporadic Parkinson's disease (PD) is thought to be influenced by both genetic and environmental factors and their interaction with each other. Statistical models including multiple variants in axon guidance pathway genes have recently been purported to be capable of predicting PD risk, survival free of the disease and age at disease onset; however the specific models have not undergone independent validation. Here we tested the best proposed risk panel of 23 single nucleotide polymorphisms (SNPs) in two PD sample sets, with a total of 525 cases and 518 controls. By single marker analysis, only one marker was significantly associated with PD risk in one of our sample sets (rs6692804: P = 0.03). Multi-marker analysis using the reported model found a mild association in one sample set (two sided P = 0.049, odds ratio for each score change = 1.07) but no significance in the other (two sided P = 0.98, odds ratio = 1), a stark contrast to the reported strong association with PD risk (P = 4.64×10−38, odds ratio as high as 90.8). Following a procedure similar to that used to build the reported model, simulated multi-marker models containing SNPs from randomly chosen genes in a genome wide PD dataset produced P-values that were highly significant and indistinguishable from similar models where disease status was permuted (3.13×10−23 to 4.90×10−64), demonstrating the potential for overfitting in the model building process. Together, these results challenge the robustness of the reported panel of genetic markers to predict PD risk in particular and a role of the axon guidance pathway in PD genetics in general

123I-MIBG cardiac uptake and smell identification in parkinsonian patients with LRRK2 mutations

Reduced uptake of 123I- metaiodobenzylguanidine (MIBG) on cardiac gammagraphy and impaired odor identification are markers of neurodegenerative diseases with Lewy bodies (LB) as a pathological hallmark, such as idiopathic Parkinson’s disease (IPD). LRRK2 patients present with a clinical syndrome indistinguishable from IPD, but LB have not been found in some cases. Patients with such mutations could behave differently than patients with IPD with respect to MIBG cardiac uptake and olfaction. We studied 14 LRRK2 patients, 14 IPD patients matched by age, gender, disease duration and severity, and 13 age and gender matched control subjects. Olfaction was analyzed through the University of Pennsylvania Smell Identification Test (UPSIT). MIBG cardiac uptake was evaluated through the H/M ratio. The late H/M was 1.44 ± 0.31 for LRRK2 patients, 1.19 ± 0.15 for PD patients, and 1.67 ± 0.16 for control subjects. LRRK2 patients presented lower but not statistically significant MIBG cardiac uptake than controls (p = 0.08) and significant higher uptake than PD patients (p = 0.04). UPSIT mean scores were 21.5 ± 7.3 for LRRK2 patients, 18.7 ± 6.2 for IPD patients and 29.7 ± 5.7 for control subjects. UPSIT score was lower in both LRRK2 and PD than in controls. In LRRK2 patients a positive correlation was found between myocardial MIBG uptake and UPSIT scores, (R = 0.801, p < 0.001). In LRRK2 patients, MIBG cardiac uptake was less impaired than in PD; a positive correlation between MIBG cardiac uptake and UPSIT scores was observed. As MIBG cardiac reduced uptake and impaired odor identification are markers of LB pathology, this findings may represent neuropathological heterogeneity among LRRK2 patients

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

A homologue of the Parkinson's disease-associated protein LRRK2 undergoes a monomer-dimer transition during GTP turnover.

Mutations in LRRK2 are a common cause of genetic Parkinson's disease (PD). LRRK2 is a multi-domain Roco protein, harbouring kinase and GTPase activity. In analogy with a bacterial homologue, LRRK2 was proposed to act as a GTPase activated by dimerization (GAD), while recent reports suggest LRRK2 to exist under a monomeric and dimeric form in vivo. It is however unknown how LRRK2 oligomerization is regulated. Here, we show that oligomerization of a homologous bacterial Roco protein depends on the nucleotide load. The protein is mainly dimeric in the nucleotide-free and GDP-bound states, while it forms monomers upon GTP binding, leading to a monomer-dimer cycle during GTP hydrolysis. An analogue of a PD-associated mutation stabilizes the dimer and decreases the GTPase activity. This work thus provides insights into the conformational cycle of Roco proteins and suggests a link between oligomerization and disease-associated mutations in LRRK2

Increasing microtubule acetylation rescues axonal transport and locomotor deficits caused by LRRK2 Roc-COR domain mutations

​Leucine-rich repeat kinase 2 (​LRRK2) mutations are the most common genetic cause of Parkinson’s disease. ​LRRK2 is a multifunctional protein affecting many cellular processes and has been described to bind microtubules. Defective microtubule-based axonal transport is hypothesized to contribute to Parkinson’s disease, but whether ​LRRK2 mutations affect this process to mediate pathogenesis is not known. Here we find that ​LRRK2 containing pathogenic Roc-COR domain mutations (R1441C, Y1699C) preferentially associates with deacetylated microtubules, and inhibits axonal transport in primary neurons and in Drosophila, causing locomotor deficits in vivo. In vitro, increasing microtubule acetylation using deacetylase inhibitors or the tubulin acetylase ​αTAT1 prevents association of mutant ​LRRK2 with microtubules, and the deacetylase inhibitor ​trichostatin A (​TSA) restores axonal transport. In vivo knockdown of the deacetylases ​HDAC6 and ​Sirt2, or administration of ​TSA rescues both axonal transport and locomotor behavior. Thus, this study reveals a pathogenic mechanism and a potential intervention for Parkinson’s disease

GTPase Activity and Neuronal Toxicity of Parkinson's Disease–Associated LRRK2 Is Regulated by ArfGAP1

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common cause of autosomal dominant familial Parkinson's disease (PD) and also contribute to idiopathic PD. LRRK2 encodes a large multi-domain protein with GTPase and kinase activity. Initial data indicates that an intact functional GTPase domain is critically required for LRRK2 kinase activity. PD–associated mutations in LRRK2, including the most common G2019S variant, have variable effects on enzymatic activity but commonly alter neuronal process morphology. The mechanisms underlying the intrinsic and extrinsic regulation of LRRK2 GTPase and kinase activity, and the pathogenic effects of familial mutations, are incompletely understood. Here, we identify a novel functional interaction between LRRK2 and ADP-ribosylation factor GTPase-activating protein 1 (ArfGAP1). LRRK2 and ArfGAP1 interact in vitro in mammalian cells and in vivo in brain, and co-localize in the cytoplasm and at Golgi membranes. PD–associated and functional mutations that alter the GTPase activity of LRRK2 modulate the interaction with ArfGAP1. The GTP hydrolysis activity of LRRK2 is markedly enhanced by ArfGAP1 supporting a role for ArfGAP1 as a GTPase-activating protein for LRRK2. Unexpectedly, ArfGAP1 promotes the kinase activity of LRRK2 suggesting a potential role for GTP hydrolysis in kinase activation. Furthermore, LRRK2 robustly and directly phosphorylates ArfGAP1 in vitro. Silencing of ArfGAP1 expression in primary cortical neurons rescues the neurite shortening phenotype induced by G2019S LRRK2 overexpression, whereas the co-expression of ArfGAP1 and LRRK2 synergistically promotes neurite shortening in a manner dependent upon LRRK2 GTPase activity. Neurite shortening induced by ArfGAP1 overexpression is also attenuated by silencing of LRRK2. Our data reveal a novel role for ArfGAP1 in regulating the GTPase activity and neuronal toxicity of LRRK2; reciprocally, LRRK2 phosphorylates ArfGAP1 and is required for ArfGAP1 neuronal toxicity. ArfGAP1 may represent a promising target for interfering with LRRK2-dependent neurodegeneration in familial and sporadic PD

LRRK2 at the interface of autophagosomes, endosomes and lysosomes

Over the past 20 years, substantial progress has been made in identifying the underlying genetics of Parkinson’s disease (PD). Of the known genes, LRRK2 is a major genetic contributor to PD. However, the exact function of LRRK2 remains to be elucidated. In this review, we discuss how familial forms of PD have led us to hypothesize that alterations in endomembrane trafficking play a role in the pathobiology of PD. We will discuss the major observations that have been made to elucidate the role of LRRK2 in particular, including LRRK2 animal models and high-throughput proteomics approaches. Taken together, these studies strongly support a role of LRRK2 in vesicular dynamics. We also propose that targeting these pathways may not only be beneficial for developing therapeutics for LRRK2-driven PD, but also for other familial and sporadic cases

Dopaminergic Neuronal Loss, Reduced Neurite Complexity and Autophagic Abnormalities in Transgenic Mice Expressing G2019S Mutant LRRK2

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause late-onset, autosomal dominant familial Parkinson's disease (PD) and also contribute to idiopathic PD. LRRK2 mutations represent the most common cause of PD with clinical and neurochemical features that are largely indistinguishable from idiopathic disease. Currently, transgenic mice expressing wild-type or disease-causing mutants of LRRK2 have failed to produce overt neurodegeneration, although abnormalities in nigrostriatal dopaminergic neurotransmission have been observed. Here, we describe the development and characterization of transgenic mice expressing human LRRK2 bearing the familial PD mutations, R1441C and G2019S. Our study demonstrates that expression of G2019S mutant LRRK2 induces the degeneration of nigrostriatal pathway dopaminergic neurons in an age-dependent manner. In addition, we observe autophagic and mitochondrial abnormalities in the brains of aged G2019S LRRK2 mice and markedly reduced neurite complexity of cultured dopaminergic neurons. These new LRRK2 transgenic mice will provide important tools for understanding the mechanism(s) through which familial mutations precipitate neuronal degeneration and PD