Leucine-rich Repeat Kinase 2 Variants Research Articles

Left corticospinal tract could be a biomarker to identify the dual prodromal LRRK2/GBA mutated Parkinson's disease.

Prodromal Parkinson's disease (PD) carriers of dual leucine-rich repeat kinase 2 (LRRK2) and glucosylceramidase β (GBA) variants are rare, and their biomarkers are less well developed. This study aimed to investigate the biomarkers for diagnosing the prodromal phase of LRRK2-GBA-PD (LRRK2-GBA-prodromal). We assessed the clinical and whole-brain white matter microstructural characteristics of 54 prodromal PD carriers of dual LRRK2 (100% M239T) and GBA (95% N409S) variants, along with 76 healthy controls (HCs) from the Parkinson's Progression Markers Initiative (PPMI) cohort. By analyzing the four values of 100 nodes on 20 fiber bundles, totaling 8000 data points, we identified the smallest p value in the fractional anisotropy (FA) value of the 38th segment of left corticospinal tract (L-CST) with differences between LRRK2-GBA-prodromal and HCs (p = 8.94 × 10-9). The FA value of the 38th node of the L-CST was significantly lower in LRRK2-GBA-prodromal (FA value, 0.65) compared with HCs (FA value, 0.71). The receiver-operating characteristic curve showed a cut-off value of 0.218 for the FA value of L-CST, providing sufficient sensitivity (79.2%) and specificity (72.2%) to distinguish double mutation prodromal PD from the healthy population. L-CST, especially the 38th node, may potentially serve as a biomarker for distinguishing individuals with double mutation prodromal PD from the healthy population.

CSF proteo‐genomic studies identify an interaction between LRRK2 genetic variants and GRN, GPNMB, CTSB, and ENTPD1

AbstractBackgroundParkinson’s disease (PD) is a movement disorder characterized by dopaminergic neurodegeneration in the substantia nigra of the brain. Another neurodegenerative disorder, Alzheimer’s disease (AD), is a progressive memory loss disorder with pathological changes mainly at hippocampus and entorhinal cortex. As dementia is also present in late stage of PD, there might be common molecular mechanism between the pathogenesis of AD and PD. Evidence has nominated Leucine Rich Repeat Kinase 2 (LRRK2), Progranulin (GRN), Glycoprotein nonmetastatic melanoma protein B () since these proteins were linked to AD and PD. Identifying genetic variants of 5 proteins will be critical to shed lights on common disrupted pathways shared by AD and PD.MethodTo identify genetic variants associated with these 5 proteins, we performed protein quantitative trait loci (pQTL) analyses of log10 transformed CSF GRN, GPNMB, CTSB, ENTPD1 and LRRK2 assayed in SomaScan platform. We considered ∼11 million sequenced variants from 787 individuals recruited at PPMI in the discovery and ∼4.3 million imputed variants from 799 Knight ADRC samples in replication. Age, sex and 10 principal components were included as covariates. Genome‐wide threshold (5.0×10−8) was used in the meta‐analyses of discovery and replication for significance.ResultOn chromosome 12, exonic missense LRRK2 variant rs33939927 (MAF = 1.4%) was identified as shared pQTL for GRN (P = 7.87×10−10), CTSB (P = 8.99×10−9), GPNMB (P = 3.59×10−9), and ENTPD1 (P = 1.24×10−13). The second LRRK2 exonic missense variant rs34637584 (MAF = 11%) was pQTL consistent for 3 proteins (GRN: P = 2.34×10−8, GPNMB: P = 8.55×10−13, and ENTPD1: P = 1.75×10−13). The third LRRK2 exonic missense variant rs35303786 (MAF = 3.4%) was identified for GPNMB (P = 1.53×10−8) and ENTPD1 (P = 2.31×10−8). The risk allele of 3 SNPs were associated with higher amount of corresponding CSF proteins. No significant LRRK2 pQTL was identified. Additional analyses including colocalisation with AD and PD GWAS results are ongoing.ConclusionTwo low frequency exonic missense LRRK2 variants (rs33939927 and rs35303786) and one common exonic missense LRRK2 variant (rs34637584) were pQTL shared by GRN, CTSB, GPNMB, and ENTPD1. Our findings indicate that the LRRK2 exonic variants are the key genetic regulators of LRRK2 activity and CSF level of GRN, CTSB, GPNMB, and ENTPD1.

Impact of 100 LRRK2 variants linked to Parkinson's disease on kinase activity and microtubule binding.

Mutations enhancing the kinase activity of leucine-rich repeat kinase-2 (LRRK2) cause Parkinson's disease (PD) and therapies that reduce LRRK2 kinase activity are being tested in clinical trials. Numerous rare variants of unknown clinical significance have been reported, but how the vast majority impact on LRRK2 function is unknown. Here, we investigate 100 LRRK2 variants linked to PD, including previously described pathogenic mutations. We identify 23 LRRK2 variants that robustly stimulate kinase activity, including variants within the N-terminal non-catalytic regions (ARM (E334K, A419V), ANK (R767H), LRR (R1067Q, R1325Q)), as well as variants predicted to destabilize the ROC:CORB interface (ROC (A1442P, V1447M), CORA (R1628P) CORB (S1761R, L1795F)) and COR:COR dimer interface (CORB (R1728H/L)). Most activating variants decrease LRRK2 biomarker site phosphorylation (pSer935/pSer955/pSer973), consistent with the notion that the active kinase conformation blocks their phosphorylation. We conclude that the impact of variants on kinase activity is best evaluated by deploying a cellular assay of LRRK2-dependent Rab10 substrate phosphorylation, compared with a biochemical kinase assay, as only a minority of activating variants (CORB (Y1699C, R1728H/L, S1761R) and kinase (G2019S, I2020T, T2031S)), enhance in vitro kinase activity of immunoprecipitated LRRK2. Twelve variants including several that activate LRRK2 and have been linked to PD, suppress microtubule association in the presence of a Type I kinase inhibitor (ARM (M712V), LRR (R1320S), ROC (A1442P, K1468E, S1508R), CORA (A1589S), CORB (Y1699C, R1728H/L) and WD40 (R2143M, S2350I, G2385R)). Our findings will stimulate work to better understand the mechanisms by which variants impact biology and provide rationale for variant carrier inclusion or exclusion in ongoing and future LRRK2 inhibitor clinical trials.

Pathophysiological evaluation of the LRRK2 G2385R risk variant for Parkinson’s disease

Missense variants in leucine-rich repeat kinase 2 (LRRK2) lead to familial and sporadic Parkinson’s disease (PD). The pathological features of PD patients with LRRK2 variants differ. Here, we report an autopsy case harboring the LRRK2 G2385R, a risk variant for PD occurring mainly in Asian populations. The patient exhibited levodopa-responsive parkinsonism at the early stage and visual hallucinations at the advanced stage. The pathological study revealed diffuse Lewy bodies with neurofibrillary tangles, amyloid plaques, and mild signs of neuroinflammation. Biochemically, detergent-insoluble phospho-α-synuclein was accumulated in the frontal, temporal, entorhinal cortexes, and putamen, consistent with the pathological observations. Elevated phosphorylation of Rab10, a substrate of LRRK2, was also prominent in various brain regions. In conclusion, G2385R appears to increase LRRK2 kinase activity in the human brain, inducing a deleterious brain environment that causes Lewy body pathology.

Penetrance of Parkinson disease LRRK2 G2385R-associated variant in the Chinese population.

Penetrance estimates of the leucine-rich repeat kinase 2 (LRRK2) variants for Parkinson disease (PD) vary widely. G2385R is one of the most common LRRK2 variants in Asian populations, and its penetrance is currently unknown. We aimed to estimate the penetrance of G2385R in the Chinese population. The G2385R variant was tested by Sanger sequencing in 6386 participants older than 50 years, all from the community cohort established by Shanghai Ruijin Hospital in 2009-2011. G2385R carriers and matched noncarriers underwent a brief questionnaire survey (including sex, current age, PD diagnosis, and age at onset) and face-to-face PD assessment during 2020-2021. The penetrance of PD was estimated by the Kaplan-Meier method. A total of 396 G2385R carriers and 415 noncarriers were included, after excluding those with a baseline diagnosis of PD or unwilling to participate. In G2385R carriers, the penetrance of PD was 1.64% at 70 years, 10.26% at 80 years, and 18.49% at 90 years, and reached 25.90% at 95 years. The penetrance of PD in G2385R carriers was higher than in noncarriers (p= 0.0071). In noncarriers, only 0%, 3.72%, and 9.66% developed parkinsonism by 70, 80, and 90 years of age. Among carriers and noncarriers, there were no statistically significant differences in penetrance comparisons between males and females, or between urban and rural. The lifetime penetrance of LRRK2 G2385R in the Chinese population was 25.9%. The penetrance modifier of G2385R in our study was age-related. Further investigation of genetic and environmental modifiers affecting G2385R penetrance is warranted.

Mutations in LRRK2 linked to Parkinson disease sequester Rab8a to damaged lysosomes and regulate transferrin-mediated iron uptake in microglia.

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause autosomal dominant Parkinson disease (PD), while polymorphic LRRK2 variants are associated with sporadic PD. PD-linked mutations increase LRRK2 kinase activity and induce neurotoxicity in vitro and in vivo. The small GTPase Rab8a is a LRRK2 kinase substrate and is involved in receptor-mediated recycling and endocytic trafficking of transferrin, but the effect of PD-linked LRRK2 mutations on the function of Rab8a is poorly understood. Here, we show that gain-of-function mutations in LRRK2 induce sequestration of endogenous Rab8a to lysosomes in overexpression cell models, while pharmacological inhibition of LRRK2 kinase activity reverses this phenotype. Furthermore, we show that LRRK2 mutations drive association of endocytosed transferrin with Rab8a-positive lysosomes. LRRK2 has been nominated as an integral part of cellular responses downstream of proinflammatory signals and is activated in microglia in postmortem PD tissue. Here, we show that iPSC-derived microglia from patients carrying the most common LRRK2 mutation, G2019S, mistraffic transferrin to lysosomes proximal to the nucleus in proinflammatory conditions. Furthermore, G2019S knock-in mice show a significant increase in iron deposition in microglia following intrastriatal LPS injection compared to wild-type mice, accompanied by striatal accumulation of ferritin. Our data support a role of LRRK2 in modulating iron uptake and storage in response to proinflammatory stimuli in microglia.

Parkinson\u2019s Disease\u2013Associated LRRK2 Interferes with Astrocyte-Mediated Alpha-Synuclein Clearance

Parkinson’s disease (PD) is a neurodegenerative, progressive disease without a cure. To prevent PD onset or at least limit neurodegeneration, a better understanding of the underlying cellular and molecular disease mechanisms is crucial. Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene represent one of the most common causes of familial PD. In addition, LRRK2 variants are risk factors for sporadic PD, making LRRK2 an attractive therapeutic target. Mutations in LRRK2 have been linked to impaired alpha-synuclein (α-syn) degradation in neurons. However, in which way pathogenic LRRK2 affects α-syn clearance by astrocytes, the major glial cell type of the brain, remains unclear. The impact of astrocytes on PD progression has received more attention and recent data indicate that astrocytes play a key role in α-syn-mediated pathology. In the present study, we aimed to compare the capacity of wild-type astrocytes and astrocytes carrying the PD-linked G2019S mutation in Lrrk2 to ingest and degrade fibrillary α-syn. For this purpose, we used two different astrocyte culture systems that were exposed to sonicated α-syn for 24 h and analyzed directly after the α-syn pulse or 6 days later. To elucidate the impact of LRRK2 on α-syn clearance, we performed various analyses, including complementary imaging, transmission electron microscopy, and proteomic approaches. Our results show that astrocytes carrying the G2019S mutation in Lrrk2 exhibit a decreased capacity to internalize and degrade fibrillar α-syn via the endo-lysosomal pathway. In addition, we demonstrate that the reduction of α-syn internalization in the Lrrk2 G2019S astrocytes is linked to annexin A2 (AnxA2) loss of function. Together, our findings reveal that astrocytic LRRK2 contributes to the clearance of extracellular α-syn aggregates through an AnxA2-dependent mechanism.

Multiple genetic pathways regulating lifespan extension are neuroprotective in a G2019S LRRK2 nematode model of Parkinson's disease

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most frequent cause of late-onset, familial Parkinson's disease (PD), and LRRK2 variants are associated with increased risk for sporadic PD. While advanced age represents the strongest risk factor for disease development, it remains unclear how different age-related pathways interact to regulate LRRK2-driven late-onset PD. In this study, we employ a C. elegans model expressing PD-linked G2019S LRRK2 to examine the interplay between age-related pathways and LRRK2-induced dopaminergic neurodegeneration. We find that multiple genetic pathways that regulate lifespan extension can provide robust neuroprotection against mutant LRRK2. However, the level of neuroprotection does not strictly correlate with the magnitude of lifespan extension, suggesting that lifespan can be experimentally dissociated from neuroprotection. Using tissue-specific RNAi, we demonstrate that lifespan-regulating pathways, including insulin/insulin-like growth factor-1 (IGF-1) signaling, target of rapamycin (TOR), and mitochondrial respiration, can be directly manipulated in neurons to mediate neuroprotection. We extend this finding for AGE-1/PI3K, where pan-neuronal versus dopaminergic neuronal restoration of AGE-1 reveals both cell-autonomous and non-cell-autonomous neuroprotective mechanisms downstream of insulin signaling. Our data demonstrate the importance of distinct lifespan-regulating pathways in the pathogenesis of LRRK2-linked PD, and suggest that extended longevity is broadly neuroprotective via the actions of these pathways at least in part within neurons. This study further highlights the complex interplay that occurs between cells and tissues during organismal aging and disease manifestation.

Fine-mapping of the non-coding variation driving the Caucasian LRRK2 GWAS signal in Parkinson's disease

IntroductionGenome-wide association studies (GWAS) have confirmed the leucine-rich repeat kinase 2 (LRRK2) gene as a susceptibility locus for idiopathic Parkinson's disease (PD) in Caucasians. Though the rs1491942 and rs76904798 variants have shown the strongest associations, the causal variant(s) remains unresolved. Therefore, the aim of this study was to identify variants that may be driving the LRRK2 GWAS signal by sequencing the entire LRRK2 gene in Caucasian PD patients and controls. MethodsA discovery series (287 PD patients, 294 controls) and replication series (362 PD patients, 168 controls) were included. The entire LRRK2 gene as well as 10 Kb upstream/downstream was sequenced. Candidate potential causal variants were considered to be those that (a) were in at least weak linkage disequilibrium with the two GWAS-nominated variants (rs1491942 and rs76904798), and (b) displayed an association odds ratio (OR) that is stronger than the two GWAS variants. ResultsThirty-four candidate variants (all intronic/intergenic) that may drive the LRRK2 PD GWAS signal were identified in the discovery series. However, examination of the replication series for these variants did not reveal any with a consistently stronger OR than both PD GWAS variants. Evaluation of public databases to determine which candidate variants are most likely to have a direct functional effect on LRRK2 expression was inconclusive. ConclusionThough our findings provide novel insights into the LRRK2 GWAS association, a clear causal variant was not identified. The identified candidate variants can form the basis for future experiments and functional studies that can more definitively assess causal LRRK2 variants.

Identification of Targets from LRRK2 Rescue Phenotypes.

Parkinson’s disease (PD) is an age-dependent neurodegenerative condition. Leucine-rich repeat kinase 2 (LRRK2) mutations are the most frequent cause of sporadic and autosomal dominant PD. The exact role of LRRK2 protective variants (R1398H, N551K) together with a pathogenic mutant (G2019S) in aging and neurodegeneration is unknown. We generated the following myc-tagged UAS-LRRK2 transgenic Drosophila: LRRK2 (WT), N551K, R1398H, G2019S single allele, and double-mutants (N551K/G2019S or R1398H/G2019S). The protective variants alone were able to suppress the phenotypic effects caused by the pathogenic LRRK2 mutation. Next, we conducted RNA-sequencing using mRNA isolated from dopaminergic neurons of these different groups of transgenic Drosophila. Using pathway enrichment analysis, we identified the top 10 modules (p < 0.05), with “LRRK2 in neurons in Parkinson’s disease” among the candidates. Further dissection of this pathway identified the most significantly modulated gene nodes such as eEF1A2, ACTB, eEF1A, and actin cytoskeleton reorganization. The induction of the pathway was successfully restored by the R1398H protective variant and R1398H-G2019S or N551K-G2019S rescue experiments. The oxidoreductase family of genes was also active in the pathogenic mutant and restored in protective and rescue variants. In summary, we provide in vivo evidence supporting the neuroprotective effects of LRRK2 variants. RNA sequencing of dopaminergic neurons identified upregulation of specific gene pathways in the Drosophila carrying the pathogenic variant, and this was restored in the rescue phenotypes. Using protective gene variants, our study identifies potential new targets and provides proof of principle of a new therapeutic approach that will further our understanding of aging and neurodegeneration in PD.

Negative screening for 12 rare LRRK2 pathogenic variants in a cohort of Nigerians with Parkinson's disease

Leucine-rich repeat kinase 2 (LRRK2) gene mutations are the most common genetic cause of Parkinson's disease (PD). More than 300 rare LRRK2 variants have been described, with approximately 17 having confirmed or probable pathogenic role in PD. The distribution differs across ethnic groups, but no PD-related LRRK2 pathogenic variant has been described in persons of Black African ancestry within or outside Africa. We previously reported the absence of LRRK2 p.Gly2019Ser mutation in 126 PD and 55 controls from Nigeria. Using Kompetitive Allele Specific Polymerase Chain Reaction, we screened a new cohort of 92 Nigerians with PD and 210 ethnically matched controls for 12 rare LRRK2 variants shown to be pathogenic in other ethnic populations, including p.Gly2019Ser, p.Arg1441His, p.Gly2385Arg, p.Ala419Val, p.Arg1628Pro, p.Pro755Leu, p.Ile2020Thr, and Tyr1699Cys. All were absent in PD and controls, endorsing our previous findings and confirming that rare LRRK2 pathogenic variants reported in Caucasians, Asians, and persons of mixed ancestry are absent in West Africans. Future studies applying next generation sequencing are necessary to explore novel LRRK2 variants indigenous to Black Africans.

Identification & characterization of leucine-rich repeat kinase 2 & parkin RBR E3 ubiquitin protein ligase variants in patients with Parkinson's disease.

Background & objectives:Parkinson's disease (PD) is a motor disorder that affects movement. More than 24 loci and 28 associated genes have been identified to be associated with this disease. The present study accounts for the contribution of two candidates, leucine-rich repeat kinase 2 (LRRK2) and parkin RBR E3 ubiquitin protein ligase (PRKN) in the PD patients, and their characterization in silico and in vitro.Methods:A total of 145 sporadic PD cases and 120 ethnically matched healthy controls were enrolled with their informed consent. Mutation screening was performed by direct DNA sequencing of the targeted exons of LRRK2 and all exons flanking introns of PRKN. The effect of the pathogenic PRKN variants on a drug (MG-132) induced loss of mitochondrial membrane potential (△ΨM) was measured by a fluorescent dye tetramethylrhodamine methyl ester (TMRM).Results:Twelve and 20 genetic variants were identified in LRRK2 and PRKN, respectively. Interestingly, five out of seven exonic LRRK2 variants were synonymous. Further assessment in controls confirmed the rarity of two such p.Y1527 and p.V1615. Among the pathogenic missense variations (as predicted in silico) in PRKN, two were selected (p.R42H and p.A82E) for their functional study in vitro, which revealed the reduced fluorescence intensity of TMRM as compared to wild type, in case of p.R42H but not the other.Interpretation & conclusions:About 6.2 per cent of the cases (9/145) in the studied patient cohort were found to carry pathogenic (as predicted in silico) missense variations in PRKN in heterozygous condition but not in case of LRRK2 which was rare. The presence of two rare synonymous variants of LRRK2 (p.Y1527 and p.V1615) may support the phenomenon of codon bias. Functional characterization of selected PRKN variations revealed p.R42H to cause disruption of mitochondrial membrane potential (△ΨM) rendering cells more susceptible to cellular stress.

Ameta-analysis of leucine-rich repeat kinase 2 (LRRK2) polymorphisms in Alzheimer's disease.

Pathogenesis and the development of Alzheimer's disease (AD) are subject to several environmental and genetic factors. This study was aimed to estimate the frequency of mutations in leucine-rich repeat kinase 2 (LRRK2) gene to examine the association between these mutations and risk of AD. For finding the articles, four databases including PubMed, Web of Science, Scopus, and Cochrane Library were checked up to August 2018. An analysis was done by RevMan 5.3 using crude odds ratio (OR) and 95% confidence intervals (CIs) to determine the association between LRRK2 polymorphisms and the risk of AD. Of 359 articles identified in the databases, 13 studies were included and analysed in the meta-analysis. There was no significant risk of AD related to five LRRK2 polymorphisms (rs33949390, rs34778348, rs7308720, rs34637584, and rs35870237). The results showed that LRRK2 variants (p.R1628P, p.G2385R, p.N551K, p.G2019S, and p.I2020T) were not associated with the risk of AD and were not acommon cause of AD in populations. Nevertheless, p.R1628P can be examined in patients with AD in other populations in the future studies.

Inhibition of LRRK2 or Casein Kinase 1 Results in LRRK2 Protein Destabilization

Mutations and variations in the leucine-rich repeat kinase 2 (LRRK2) gene are strongly associated with an increased risk to develop Parkinson’s disease (PD). Most pathogenic LRRK2 mutations display increased kinase activity, which is believed to underlie LRRK2-mediated toxicity. Therefore, major efforts have been invested in the development of potent and selective LRRK2 kinase inhibitors. Several of these compounds have proven beneficial in cells and in vivo, even in a LRRK2 wild-type background. Therefore, LRRK2 kinase inhibition holds great promise as disease-modifying PD therapy, and is currently tested in preclinical and early clinical studies. One of the safety concerns is the development of lung pathology in mice and non-human primates, which is most likely related to the strongly reduced LRRK2 protein levels after LRRK2 kinase inhibition. In this study, we aimed to better understand the molecular consequences of chronic LRRK2 kinase inhibition, which may be pivotal in the further development of a LRRK2 kinase inhibitor-based PD therapy. We found that LRRK2 protein levels are not restored during long-term LRRK2 kinase inhibition, but are recovered upon inhibitor withdrawal. Interestingly, LRRK2 kinase inhibitor-induced destabilization does not occur in all pathogenic LRRK2 variants and the N-terminal part of LRRK2 appears to play a crucial role in this process. In addition, we identified CK1, an upstream kinase of LRRK2, as a regulator of LRRK2 protein stability in cell culture and in vivo. We propose that pharmacological LRRK2 kinase inhibition triggers a cascade that results in reduced CK1-mediated phosphorylation of yet unidentified LRRK2 phosphorylation sites. This process involves the N-terminus of LRRK2 and ultimately leads to LRRK2 protein degradation.

Genetic analysis of LRRK2 in Parkinson's disease in Han Chinese population.

Mutations in Leucine-rich repeat kinase 2 (LRRK2) are recognized as the most frequent genetic factors contributing to Parkinson's disease (PD). The aim of our study was to explore LRRK2 variants in PD patients within the mainland Han Chinese population. The whole coding regions of LRRK2 from 296 PD patients were sequenced by targeted regions sequencing and exome sequencing. Eighteen rare variants were identified in 27 PD patients, and 13 of them (M100T, L153W, A459S, S722N, R792K, C925Y, R981K, S1007T, V1447M, R1677S, N2308D, N2313S, and S2350I) were firstly reported in PD. We also tried to explore the genotype-phenotype associations of LRRK2 variants in our data and found that PD with common and rare LRRK2 variants was more likely to have motor fluctuation and nonmotor symptoms. The identification of novel variants in LRRK2 suggests that this gene plays an important role in the pathogenesis and phenotype of PD in Han Chinese population, and our data also rang the alarm bell-more attention should be paid to the whole coding regions of LRRK2.

Case-control analysis of LRRK2 protective variants in Essential Tremor

Co-existence of Parkinson’s disease (PD) and essential tremor (ET) may reflect overlapping pathophysiology underlying both conditions. Furthermore, PD patients with leucine-rich repeat kinase-2 (LRRK2) mutations may present with ET-like features, suggesting the possibility of common genetic underpinnings. Two common LRRK2 variants, R1398H and N551K, have been shown to be protective in multiple PD cohorts. We hypothesized that R1398H and N551K may show a similar effect in ET. In a case-control study involving 3198 subjects (2680 controls and 518 ET cases), R1398H was detected in 16.6% of ET cases compared to 18.0% in controls (OR = 0.91, 95% CI = 0.71–1.17, p = 0.46); while N551K was detected in 16.5% of ET cases compared to 18.0% of controls (OR = 0.89, 95% CI = 0.69–1.15, p = 0.37). While these results suggest that LRRK2 R1398H or N551K do not appear to modulate the risk of ET, it remains possible that a protective trend for both variants may be present in ET and a much larger sample size is required to identify this.

Case-control analysis of leucine-rich repeat kinase 2 protective variants in Alzheimer's disease

Amyloid is the main pathological substrate of Alzheimer's disease (AD) and has been described in leucine-rich repeat kinase 2 (LRRK2) carriers with Parkinson's disease. LRRK2 has been linked with amyloid precursor protein pathways in neurodegeneration. Two common LRRK2 variants, R1398H and N551K, have been shown to be protective in multiple Parkinson's disease cohorts. We hypothesized that R1398H and N551K may be protective in AD. In a case-control study involving 1390 subjects (719 controls and 671 AD cases), R1398H was demonstrated in 16.8% of AD cases compared to 16.7% in controls (odds ratio = 1.01, 95% confidence interval = 0.76–1.34, p = 0.94), whereas N551K was demonstrated in 17.3% of AD cases compared to 17.2% of controls (odds ratio = 1.00, 95% confidence interval = 0.76–1.32, p = 0.98). Overall, these results suggest that LRRK2 R1398H or N551K variants do not appear to modulate the risk of AD.

Lack of Evidence for Decreased Protein Stability in the 2397 (Met) Haplotype of the Leucine Rich Repeat Kinase 2 Protein Implicated in Parkinson’s Disease

Missense mutations in the leucine rich repeat kinase 2 (LRRK2) gene are the leading genetic cause of autosomal dominant familial Parkinson’s disease. We previously reported that two mutations within the ROC domain, namely R1441C and A1442P, exhibit increased protein degradation leading to lowered steady state LRRK2 protein levels in HEK293 cells. More recently, the common WD40 domain LRRK2 haplotype, Met2397, which is a risk factor for Crohn’s disease, has been shown to lower steady state protein levels in HEK293 cells. In view of recent evidence implicating LRRK2 and inflamemation in PD, we investigated the effects of Met2397 on LRRK2 expression, and compared them to the Thr2397 variant and other LRRK2 mutants. In this study, we transfected HEK293 cells with plasmid constructs encoding the different LRRK2 variants, and analyzed the resulting protein levels by Western blot and flow cytometry. Here we found that both the Met2397 and Thr2397 haplotypes yield similar levels of LRRK2 protein expression and do not appear to impact cell viability in HEK293 cells, compared to other LRRK mutants. Thus, we have concluded that the Met2397 haplotype is unlikely to play a role in LRRK2 mediated or idiopathic PD.

Human R1441C LRRK2 regulates the synaptic vesicle proteome and phosphoproteome in a Drosophila model of Parkinson's disease.

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause late-onset, autosomal dominant familial Parkinson`s disease (PD) and variation at the LRRK2 locus contributes to the risk for idiopathic PD. LRRK2 can function as a protein kinase and mutations lead to increased kinase activity. To elucidate the pathophysiological mechanism of the R1441C mutation in the GTPase domain of LRRK2, we expressed human wild-type or R1441C LRRK2 in dopaminergic neurons of Drosophila and observe reduced locomotor activity, impaired survival and an age-dependent degeneration of dopaminergic neurons thereby creating a new PD-like model. To explore the function of LRRK2 variants in vivo, we performed mass spectrometry and quantified 3,616 proteins in the fly brain. We identify several differentially-expressed cytoskeletal, mitochondrial and synaptic vesicle proteins (SV), including synaptotagmin-1, syntaxin-1A and Rab3, in the brain of this LRRK2 fly model. In addition, a global phosphoproteome analysis reveals the enhanced phosphorylation of several SV proteins, including synaptojanin-1 (pThr1131) and the microtubule-associated protein futsch (pSer4106) in the brain of R1441C hLRRK2 flies. The direct phosphorylation of human synaptojanin-1 by R1441C hLRRK2 could further be confirmed by in vitro kinase assays. A protein-protein interaction screen in the fly brain confirms that LRRK2 robustly interacts with numerous SV proteins, including synaptojanin-1 and EndophilinA. Our proteomic, phosphoproteomic and interactome study in the Drosophila brain provides a systematic analyses of R1441C hLRRK2-induced pathobiological mechanisms in this model. We demonstrate for the first time that the R1441C mutation located within the LRRK2 GTPase domain induces the enhanced phosphorylation of SV proteins in the brain.

LRRK2 variation and dementia with Lewy bodies

IntroductionThe leucine-rich repeat kinase 2 (LRRK2) gene contains several variants that cause Parkinson's disease (PD) and others that modify PD risk. However, little is known about the role of LRRK2 in dementia with Lewy bodies (DLB). Aims of this study were to screen DLB patients for pathogenic LRRK2 variants and to evaluate associations between common LRRK2 variants and risk of DLB. Methods417 clinical DLB patients and 1790 controls were included in the primary analysis. Additionally, 355 Lewy body disease patients assessed as having a high likelihood of clinical DLB based on neuropathological findings were included in secondary analysis. Seven pathogenic LRRK2 variants were assessed in patients, while 17 common LRRK2 exonic variants and 1 GWAS-nominated common LRRK2 PD-risk variant were evaluated for association with DLB. ResultsWe identified carriers of 2 different pathogenic LRRK2 variants. One clinical DLB patient was a p.G2019S carrier, while in the pathological high likelihood DLB series there was one carrier of the p.R1441C mutation. However, examination of clinical records revealed the p.R1441C carrier to have PD with dementia. Evaluation of common variants did not reveal any associations with DLB risk after multiple testing adjustment. However, a non-significant trend similar to that previously reported for PD was observed for the protective p.N551K-R1398H-K1423K haplotype in the clinical DLB series (OR: 0.76, P = 0.061). ConclusionLRRK2 does not appear to play a major role in DLB, however further study of p.G2019S and the p.N551K-R1398H-K1423K haplotype is warranted to better understand their involvement in determining DLB risk.