Mutations In Leucine-rich Repeat Kinase Research Articles

LRRK2 and the Endolysosomal System in Parkinson's Disease.

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause autosomal dominant familial Parkinson’s disease (PD), with pathogenic mutations enhancing LRRK2 kinase activity. There is a growing body of evidence indicating that LRRK2 contributes to neuronal damage and pathology both in familial and sporadic PD, making it of particular interest for understanding the molecular pathways that underlie PD. Although LRRK2 has been extensively studied to date, our understanding of the seemingly diverse functions of LRRK2 throughout the cell remains incomplete. In this review, we discuss the functions of LRRK2 within the endolysosomal pathway. Endocytosis, vesicle trafficking pathways, and lysosomal degradation are commonly disrupted in many neurodegenerative diseases, including PD. Additionally, many PD-linked gene products function in these intersecting pathways, suggesting an important role for the endolysosomal system in maintaining protein homeostasis and neuronal health in PD. LRRK2 activity can regulate synaptic vesicle endocytosis, lysosomal function, Golgi network maintenance and sorting, vesicular trafficking and autophagy, with alterations in LRRK2 kinase activity serving to disrupt or regulate these pathways depending on the distinct cell type or model system. LRRK2 is critically regulated by at least two proteins in the endolysosomal pathway, Rab29 and VPS35, which may serve as master regulators of LRRK2 kinase activity. Investigating the function and regulation of LRRK2 in the endolysosomal pathway in diverse PD models, especially in vivo models, will provide critical insight into the cellular and molecular pathophysiological mechanisms driving PD and whether LRRK2 represents a viable drug target for disease-modification in familial and sporadic PD.

Chetomin rescues pathogenic phenotype of LRRK2 mutation in drosophila.

Leucine-rich repeat kinase 2 (LRRK2) is a complex protein kinase involved in a diverse set of functions. Mutations in LRRK2 are a common cause of autosomal dominant familial Parkinson’s disease. Peroxiredoxin 2 (PRDX2) belongs to a family of anti-oxidants that protect cells from oxidative stress. Importantly, PRDX2 is a cytoplasmic protein, similar to Leucine-rich repeat kinase 2, which localizes predominantly in the cytosol. Here, we demonstrated that Leurice-rich repeat kinase 2 phosphorylates PRDX2 in Drosophila, leading to a loss of dopaminergic neurons, climbing ability and shortened lifespan. These pathogenic phenotypes in the LRRK2 Drosophila were rescued with transgenic expression of PRDX2. Chetomin, a PRDX2 mimic, belongs to a class of epidithio-diketopiperazine fungal secondary metabolites (containing a dithiol group that has hydrogen peroxide-reducing activity). As proof of principle, we demonstrated that Chetomin recapitulated the rescue in these mutant Drosophila. Our findings suggest that Chetomin can be a potential therapeutic compound in LRRK2 linked Parkinson’s disease.

Dementia with Lewy bodies in first-generation immigrants in a European memory clinic.

We wanted to explore possible differences in disease presentation, frequency, and age of onset of dementia with Lewy bodies (DLB) between first-generation immigrants (FGI) and patients born in Belgium (PBIB). We conducted a retrospective study on all patients of our Memory Clinic between June 1, 2010 and January 31, 2020. A synucleinopathy was diagnosed in 150 of 2702 patients (5.5%): 91 received a diagnosis of DLB (3.4%). FGI were two times more likely to receive a diagnosis of DLB, due to a higher prevalence in North-Africans and Latin-Americans. Visual hallucinations were less frequent in North-Africans than in other immigrants. FGI were younger than PBIB and reported more often parasomnia. Our data suggest a higher risk for DLB in certain immigrant groups. Especially for North-African patients, a genetic factor can be suspected, namely mutations in Leucine-rich repeat kinase 2 (LRRK2). Memory clinics with a high rate of FGI may provide interesting data and insights into the prevalence of DLB, genetic and environmental differences.

Somatic Mutations in LRRK2 Identify a Subset of Invasive Mammary Carcinomas Associated with High Mutation Burden

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common cause of familial Parkinson disease. Although LRRK2-related Parkinson disease patients have a heightened risk of certain nonskin cancers, including breast cancer, it is unknown whether LRRK2 somatic mutations occur and are associated with breast cancer. The objective of this study was to evaluate the occurrence of LRRK2 somatic mutations in breast cancer and the clinicopathologic features associated with LRRK2-mutated tumors. Using The Cancer Genome Atlas Breast Cancer Project, somatic LRRK2 DNA sequence information was obtained for 93 cases, of which 17 cases (18%) with 18 mutations were identified. LRRK2-mutated mammary carcinomas are enriched with stop-gain, truncating mutations predicted to result in loss of function; missense mutations frequently targeted the GTPase and kinase domains. Tumors displayed predominantly high-grade morphology with abundant granular eosinophilic cytoplasm, resembling mitochondria-rich apocrine-like carcinomas. Exploration of the genomic landscape of LRRK2-mutated carcinomas yielded frequent TP53 deactivation and a remarkably high tumor mutation burden. More important, breast cancers with LRRK2 mutations are associated with reduced patient survival compared with The Cancer Genome Atlas Breast Cancer Project cohort. These findings, for the first time, show that somatic LRRK2 mutations occur frequently in breast cancer, and the high mutation burden seen in this subset of tumors suggests that LRRK2 mutations may herald benefit from immune checkpoint inhibition.

Roles of lysosomotropic agents on LRRK2 activation and Rab10 phosphorylation

Leucine-rich repeat kinase 2 (LRRK2), the major causative gene product of autosomal-dominant Parkinson's disease, is a protein kinase that phosphorylates a subset of Rab GTPases. Since pathogenic LRRK2 mutations increase its ability to phosphorylate Rab GTPases, elucidating the mechanisms of how Rab phosphorylation is regulated by LRRK2 is of great importance. We have previously reported that chloroquine-induced lysosomal stress facilitates LRRK2 phosphorylation of Rab10 to maintain lysosomal homeostasis. Here we reveal that Rab10 phosphorylation by LRRK2 is potently stimulated by treatment of cells with a set of lysosome stressors and clinically used lysosomotropic drugs. These agents commonly promoted the formation of LRRK2-coated enlarged lysosomes and extracellular release of lysosomal enzyme cathepsin B, the latter being dependent on LRRK2 kinase activity. In contrast to the increase in Rab10 phosphorylation, treatment with lysosomotropic drugs did not increase the enzymatic activity of LRRK2, as monitored by its autophosphorylation at Ser1292 residue, but rather enhanced the molecular proximity between LRRK2 and its substrate Rab GTPases on the cytosolic surface of lysosomes. Lysosomotropic drug-induced upregulation of Rab10 phosphorylation was likely a downstream event of Rab29 (Rab7L1)-mediated enzymatic activation of LRRK2. These results suggest a regulated process of Rab10 phosphorylation by LRRK2 that is associated with lysosomal overload stress, and provide insights into the novel strategies to halt the aberrant upregulation of LRRK2 kinase activity.

Accurate MS-based Rab10 Phosphorylation Stoichiometry Determination as Readout for LRRK2 Activity in Parkinson's Disease

Pathogenic mutations in the Leucine-rich repeat kinase 2 (LRRK2) are the predominant genetic cause of Parkinson's disease (PD). They increase its activity, resulting in augmented Rab10-Thr73 phosphorylation and conversely, LRRK2 inhibition decreases pRab10 levels. Currently, there is no assay to quantify pRab10 levels for drug target engagement or patient stratification. To meet this challenge, we developed an high accuracy and sensitivity targeted mass spectrometry (MS)-based assay for determining Rab10-Thr73 phosphorylation stoichiometry in human samples. It uses synthetic stable isotope-labeled (SIL) analogues for both phosphorylated and nonphosphorylated tryptic peptides surrounding Rab10-Thr73 to directly derive the percentage of Rab10 phosphorylation from attomole amounts of the endogenous phosphopeptide. The SIL and the endogenous phosphopeptides are separately admitted into an Orbitrap analyzer with the appropriate injection times. We test the reproducibility of our assay by determining Rab10-Thr73 phosphorylation stoichiometry in neutrophils of LRRK2 mutation carriers before and after LRRK2 inhibition. Compared with healthy controls, the PD predisposing mutation carriers LRRK2 G2019S and VPS35 D620N display 1.9-fold and 3.7-fold increased pRab10 levels, respectively. Our generic MS-based assay further establishes the relevance of pRab10 as a prognostic PD marker and is a powerful tool for determining LRRK2 inhibitor efficacy and for stratifying PD patients for LRRK2 inhibitor treatment.

Defects in mRNA Translation in LRRK2-Mutant hiPSC-Derived Dopaminergic Neurons Lead to Dysregulated Calcium Homeostasis.

The G2019S mutation in leucine-rich repeat kinase 2 (LRRK2) is a common cause of familial Parkinson's disease (PD). This mutation results in dopaminergic neurodegeneration via dysregulated protein translation, although how alterations in protein synthesis contribute to neurodegeneration in human neurons is not known. Here we define the translational landscape in LRRK2-mutant dopaminergic neurons derived from human induced pluripotent stem cells (hiPSCs) via ribosome profiling. We found that mRNAs that have complex secondary structure in the 5' untranslated region (UTR) are translated more efficiently in G2019S LRRK2 neurons. This leads to the enhanced translation of multiple genes involved in Ca2+ regulation and to increased Ca2+ influx and elevated intracellular Ca2+ levels, a major contributor to PD pathogenesis. This study reveals a link between dysregulated translation control and Ca2+ homeostasis in G2019S LRRK2 human dopamine neurons, which potentially contributes to the progressive and selective dopaminergic neurotoxicity in PD.

Mitochondrial Mechanisms of LRRK2 G2019S Penetrance.

Several mutations in leucine-rich repeat kinase-2 (LRRK2) have been associated with Parkinson's disease (PD). The most common substitution, G2019S, interferes with LRRK2 kinase activity, which is regulated by autophosphorylation. Yet, the penetrance of this gain-of-function mutation is incomplete, and thus far, few factors have been correlated with disease status in carriers. This includes (i) LRRK2 autophosphorylation in urinary exosomes, (ii) serum levels of the antioxidant urate, and (iii) abundance of mitochondrial DNA (mtDNA) transcription-associated 7S DNA. In light of a mechanistic link between LRRK2 kinase activity and mtDNA lesion formation, we previously investigated mtDNA integrity in fibroblasts from manifesting (LRRK2+/PD+) and non-manifesting carriers (LRRK2+/PD−) of the G2019S mutation as well as from aged-matched controls. In our published study, mtDNA major arc deletions correlated with PD status, with manifesting carriers presenting the highest levels. In keeping with these findings, we now further explored mitochondrial features in fibroblasts derived from LRRK2+/PD+ (n = 10), LRRK2+/PD− (n = 21), and control (n = 10) individuals. In agreement with an accumulation of mtDNA major arc deletions, we also detected reduced NADH dehydrogenase activity in the LRRK2+/PD+ group. Moreover, in affected G2019S carriers, we observed elevated mitochondrial mass and mtDNA copy numbers as well as increased expression of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), which regulates antioxidant signaling. Taken together, these results implicate mtDNA dyshomeostasis—possibly as a consequence of impaired mitophagy—in the penetrance of LRRK2-associated PD. Our findings are a step forward in the pursuit of unveiling markers that will allow monitoring of disease progression of LRRK2 mutation carriers.

LRRK2 kinase inhibitors reduce alpha-synuclein in human neuronal cell lines with the G2019S mutation

Kinase activating missense mutations in leucine-rich repeat kinase 2 (LRRK2) predispose to Parkinson's disease. Consequently, there is much interest in delineating LRRK2 biology, both in terms of gaining further insight into disease causes, and also determining whether or not LRRK2 is a potential Parkinson's disease therapeutic target. Indeed, many potent and selective small molecule inhibitors of LRRK2 have been developed and are currently being used for pre-clinical testing in cell and animal models. In the current study, we have obtained fibroblasts from four subjects with the common LRRK2 mutation, G2019S. Fibroblasts were reprogrammed to induced pluripotent stem cells and then to neural stem cells and ultimately neurons. Two clones for each of the human neural cell lines were then chronically treated with and without either of two distinct inhibitors of LRRK2 and effects on toxicity and Parkinson's disease related phenotypes were assessed. Cells with the G2019S mutation had a propensity to accumulate the pathological Parkinson's disease protein α-synuclein. Moreover, α-synuclein accumulation in the G2019S cells was significantly reduced with both LRRK2 inhibitors in seven of the eight cell lines studied. LRRK2 inhibitors also improved the nuclear morphology of G2019S cells and impacted on measures of autophagy and endoplasmic reticulum stress. Lastly, we did not find evidence of inhibitor toxicity under the chronic treatment conditions. These results add to evidence that LRRK2 inhibitors may have utility in the treatment of Parkinson's disease via reducing α-synuclein.

The In Situ Structure of Parkinson’s Disease-Linked LRRK2

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most frequent cause of familial Parkinson's disease. LRRK2 is a multi-domain protein containing a kinase and GTPase. Using correlative light and electron microscopy, in situ cryo-electron tomography, and subtomogram analysis, we reveal a 14-Å structure of LRRK2 bearing a pathogenic mutation that oligomerizes as a right-handed double helix around microtubules, which are left-handed. Using integrative modeling, we determine the architecture of LRRK2, showing that the GTPase and kinase are in close proximity, with the GTPase closer to the microtubule surface, whereas the kinase is exposed to the cytoplasm. We identify two oligomerization interfaces mediated by non-catalytic domains. Mutation of one of these abolishes LRRK2 microtubule-association. Our work demonstrates the power of cryo-electron tomography to generate models of previously unsolved structures in their cellular environment.

The Role of Human LRRK2 in Methylmercury-Induced Inhibition of Microvesicle Formation of Cephalic Neurons in Caenorhabditis elegans.

In a previous study, we have shown that methylmercury (MeHg) exposure causes focal aggregation of intracellular transgenic mCherry protein in dendrites of cephalic (CEP) neurons in Caenorhabditis elegans (C. elegans). However, the underlying mechanism is unknown. We hypothesized that reduced cellular release of mCherry via extracellular vesicles by MeHg contributes to its accumulation and intracellular aggregation. Thus, we characterized vesicular structures in CEP dendrites, which were 1-3μm in diameter and could readily bud off from the plasma membrane of the dendrites. Chronic treatment of C. elegans with MeHg (5μM, 4-10days) reduced the number of vesicles attached to CEP dendrites (attached vesicles) and vesicles unattached to CEP dendrites (unattached vesicles), as well as the presence of extracellular mCherry, supporting the hypothesis that release of mCherry by microvesicle formation is inhibited by MeHg. Leucine-rich repeat kinase 2 (LRRK2) has an important function in membrane biology. Further investigation showed that the effects of MeHg were modified by human LRRK2. In worms with the wild-type LRRK2, the vesicle numbers were significantly reduced by MeHg (0.5 and 5μM). The effects of MeHg on the presence of extracellular mCherry and attached vesicles were modified by the human wild-type LRRK2. Independent of MeHg treatment, the G2019S mutant LRRK2 showed reduced number of unattached vesicles; however, the levels of extracellular mCherry were increased. Knockdown of C. elegans irk-1, the homolog of human LRRK2, reduced the number of attached vesicles, corroborating that LRRK2 plays an important role in the formation of microvesicles.

From Synaptic Dysfunction to Neuroprotective Strategies in Genetic Parkinson's Disease: Lessons From LRRK2.

The pathogenesis of Parkinson’s disease (PD) is thought to rely on a complex interaction between the patient’s genetic background and a variety of largely unknown environmental factors. In this scenario, the investigation of the genetic bases underlying familial PD could unveil key molecular pathways to be targeted by new disease-modifying therapies, still currently unavailable. Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are responsible for the majority of inherited familial PD cases and can also be found in sporadic PD, but the pathophysiological functions of LRRK2 have not yet been fully elucidated. Here, we will review the evidence obtained in transgenic LRRK2 experimental models, characterized by altered striatal synaptic transmission, mitochondrial dysfunction, and α-synuclein aggregation. Interestingly, the processes triggered by mutant LRRK2 might represent early pathological phenomena in the pathogenesis of PD, anticipating the typical neurodegenerative features characterizing the late phases of the disease. A comprehensive view of LRRK2 neuronal pathophysiology will support the possible clinical application of pharmacological compounds targeting this protein, with potential therapeutic implications for patients suffering from both familial and sporadic PD.

LRRK2 and Protein Aggregation in Parkinson's Disease: Insights From Animal Models.

Mutations in leucine-rich repeat kinase 2 (LRRK2) instigate an autosomal dominant form of Parkinson’s disease (PD). Despite the neuropathological heterogeneity observed in LRRK2-PD, accumulating evidence suggests that alpha-synuclein and tau pathology are observed in a vast majority of cases. Intriguingly, the presence of protein aggregates spans both LRRK2-PD and idiopathic disease, supportive of a common pathologic mechanism. Thus, it is important to consider how LRRK2 mutations give rise to such pathology, and whether targeting LRRK2 might modify the accumulation, transmission, or toxicity of protein aggregates. Likewise, it is not clear how LRRK2 mutations drive PD pathogenesis, and whether protein aggregates are implicated in LRRK2-dependent neurodegeneration. While animal models have been instrumental in furthering our understanding of a potential interaction between LRRK2 and protein aggregation, the biology is far from clear. We aim to provide a thoughtful overview of the evidence linking LRRK2 to protein aggregation in animal models.

Dopaminergic neurodegeneration induced by Parkinson's disease-linked G2019S LRRK2 is dependent on kinase and GTPase activity

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of late-onset, autosomal-dominant familial Parkinson's disease (PD). LRRK2 functions as both a kinase and GTPase, and PD-linked mutations are known to influence both enzymatic activities. While PD-linked LRRK2 mutations can commonly induce neuronal damage in culture models, the mechanisms underlying these pathogenic effects remain uncertain. Rodent models containing familial LRRK2 mutations often lack robust PD-like neurodegenerative phenotypes. Here, we develop a robust preclinical model of PD in adult rats induced by the brain delivery of recombinant adenoviral vectors with neuronal-specific expression of human LRRK2 harboring the most common G2019S mutation. In this model, G2019S LRRK2 induces the robust degeneration of substantia nigra dopaminergic neurons, a pathological hallmark of PD. Introduction of a stable kinase-inactive mutation or administration of the selective kinase inhibitor, PF-360, attenuates neurodegeneration induced by G2019S LRRK2. Neuroprotection provided by pharmacological kinase inhibition is mediated by an unusual mechanism involving the robust destabilization of human LRRK2 protein in the brain relative to endogenous LRRK2. Our study further demonstrates that G2019S LRRK2-induced dopaminergic neurodegeneration critically requires normal GTPase activity, as hypothesis-testing mutations that increase GTP hydrolysis or impair GTP-binding activity provide neuroprotection although via distinct mechanisms. Taken together, our data demonstrate that G2019S LRRK2 induces neurodegeneration in vivo via a mechanism that is dependent on kinase and GTPase activity. Our study provides a robust rodent preclinical model of LRRK2-linked PD and nominates kinase inhibition and modulation of GTPase activity as promising disease-modifying therapeutic targets.

Lrrk2 modulation of Wnt signaling during zebrafish development.

Mutations in leucine-rich repeat kinase 2 (lrrk2) are the most common genetic cause of Parkinson's disease. Difficulty in elucidating the pathogenic mechanisms resulting from disease-associated Lrrk2 variants stems from the complexity of Lrrk2 function and activities. Lrrk2 contains multiple protein-protein interacting domains, a GTPase domain, and a kinase domain. Lrrk2 is implicated in many cellular processes including vesicular trafficking, autophagy, cytoskeleton dynamics, and Wnt signaling. Here, we generated a zebrafish lrrk2 allelic series to study the requirements for Lrrk2 during development and to dissect the importance of its various domains. The alleles are predicted to encode proteins that either lack all functional domains (lrrk2sbu304 ), the GTPase, and kinase domains (lrrk2sbu71 ) or the kinase domain (lrrk2sbu96 ). All three lrrk2 mutants are viable, morphologically normal, and display wild-type-like locomotion. Because Lrrk2 modulates Wnt signaling in some contexts, we assessed Wnt signaling in all three mutant lines. Analysis of Wnt signaling by studying the expression of target genes using whole mount RNA in situ hybridization and a transgenic Wnt reporter revealed wild-type domains of Wnt activity in each of the mutants. However, we found that Wnt pathway activation is attenuated in lrrk2sbu304/sbu304 , which lacks both scaffolding and catalytic domains, but not in the other alleles during late embryogenesis. This supports a model in which Lrrk2 scaffolding functions are key to a context-dependent role in promoting canonical Wnt signaling.

Dietary Amino Acids Impact LRRK2-Induced Neurodegeneration in Parkinson's Disease Models.

The G2019S mutation in leucine-rich repeat kinase 2 (LRRK2) is a common cause of Parkinson's disease (PD) and results in age-related dopamine neuron loss and locomotor dysfunction in Drosophila melanogaster through an aberrant increase in bulk neuronal protein synthesis. Under nonpathologic conditions, protein synthesis is tightly controlled by metabolic regulation. Whether nutritional and metabolic influences on protein synthesis can modulate the pathogenic effect of LRRK2 on protein synthesis and thereby impact neuronal loss is a key unresolved question. Here, we show that LRRK2 G2019S-induced neurodegeneration is critically dependent on dietary amino acid content in Drosophila studies with both sexes. Low dietary amino acid concentration prevents aberrant protein synthesis and blocks LRRK2 G2019S-mediated neurodegeneration in Drosophila and rat primary neurons. Unexpectedly, a moderately high-amino acid diet also blocks dopamine neuron loss and motor deficits in Drosophila through a separate mechanism involving stress-responsive activation of 5'-AMP-activated protein kinase (AMPK) and neuroprotective induction of autophagy, implicating the importance of protein homeostasis to neuronal viability. At the highest amino acid diet of the range tested, PD-related neurodegeneration occurs in an age-related manner, but is also observed in control strains, suggesting that it is independent of mutant LRRK2 expression. We propose that dietary influences on protein synthesis and autophagy are critical determinants of LRRK2 neurodegeneration, opening up possibilities for future therapeutic intervention.SIGNIFICANCE STATEMENT Parkinson's disease (PD) prevalence is projected to rise as populations continue to age, yet there are no current therapeutic approaches that delay or stop disease progression. A broad role for leucine-rich repeat kinase 2 (LRRK2) mutations in familial and idiopathic PD has emerged. Here, we show that dietary amino acids are important determinants of neurodegeneration in a Drosophila model of LRRK2 PD. Restricting all amino acids effectively suppresses dopaminergic neuron loss and locomotor deficits and is associated with reduced protein synthesis, while moderately high amino acids similarly attenuate these PD-related phenotypes through a stress-responsive induction of 5'-AMP-activated protein kinase and autophagy. These studies suggest that diet plays an important role in the development of PD-related phenotypes linked to LRRK2.

LRRK2-Related Parkinson's Disease Due to Altered Endolysosomal Biology With Variable Lewy Body Pathology: A Hypothesis.

Mutations in the gene encoding for leucine-rich repeat kinase 2 (LRRK2) are associated with both familial and sporadic Parkinson’s disease (PD). LRRK2 encodes a large protein comprised of a GTPase and a kinase domain. All pathogenic variants converge on enhancing LRRK2 kinase substrate phosphorylation, and distinct LRRK2 kinase inhibitors are currently in various stages of clinical trials. Although the precise pathophysiological functions of LRRK2 remain largely unknown, PD-associated mutants have been shown to alter various intracellular vesicular trafficking pathways, especially those related to endolysosomal protein degradation events. In addition, biochemical studies have identified a subset of Rab proteins, small GTPases required for all vesicular trafficking steps, as substrate proteins for the LRRK2 kinase activity in vitro and in vivo. Therefore, it is crucial to evaluate the impact of such phosphorylation on neurodegenerative mechanisms underlying LRRK2-related PD, especially with respect to deregulated Rab-mediated endolysosomal membrane trafficking and protein degradation events. Surprisingly, a significant proportion of PD patients due to LRRK2 mutations display neuronal cell loss in the substantia nigra pars compacta in the absence of any apparent α-synuclein-containing Lewy body neuropathology. These findings suggest that endolysosomal alterations mediated by pathogenic LRRK2 per se are not sufficient to cause α-synuclein aggregation. Here, we will review current knowledge about the link between pathogenic LRRK2, Rab protein phosphorylation and endolysosomal trafficking alterations, and we will propose a testable working model whereby LRRK2-related PD may present with variable LB pathology.

Quantitative Measurements of LRRK2 in Human Cerebrospinal Fluid Demonstrates Increased Levels in G2019S Patients.

Leucine-rich repeat kinase 2 (LRRK2) mutations are among the most significant genetic risk factors for developing late onset Parkinson’s disease (PD). To understand whether a therapeutic can modulate LRRK2 levels as a potential disease modifying strategy, it is important to have methods in place to measure the protein with high sensitivity and specificity. To date, LRRK2 measurements in cerebrospinal fluid (CSF) have used extracellular vesicle enrichment via differential ultracentrifugation and western blot detection. Our goal was to develop a methodology which could be deployed in a clinical trial, therefore throughput, robustness and sensitivity were critical. To this end, we developed a Stable Isotope Standard Capture by Anti-peptide Antibody (SISCAPA) assay which is capable of detecting LRRK2 from 1 ml of human CSF. The assay uses a commercially available LRRK2 monoclonal antibody (N241A/34) and does not require extracellular vesicle enrichment steps. The assay includes stable isotope peptide addition which allows for absolute quantitation of LRRK2 protein. We determined that the assay performed adequately for CSF measurements and that blood contamination from traumatic lumbar puncture does not pose a serious analytical challenge. We then applied this technique to 106 CSF samples from the MJFF LRRK2 Cohort Consortium which includes healthy controls, sporadic PD patients and LRRK2 mutation carriers with and without PD. Of the 105 samples that had detectable LRRK2 signal, we found that the PD group with the G2019S LRRK2 mutation had significantly higher CSF LRRK2 levels compared to all other groups. We also found that CSF LRRK2 increased with the age of the participant. Taken together, this work represents a step forward in our ability to measure LRRK2 in a challenging matrix like CSF which has implications for current and future LRRK2 therapeutic clinical trials.

Sequential screening nominates the Parkinson's disease associated kinase LRRK2 as a regulator of Clathrin-mediated endocytosis

Mutations in leucine-rich repeat kinase 2 (LRRK2) are an established cause of inherited Parkinson's disease (PD). LRRK2 is expressed in both neurons and glia in the central nervous system, but its physiological function(s) in each of these cell types is uncertain. Through sequential screens, we report a functional interaction between LRRK2 and Clathrin adaptor protein complex 2 (AP2). Analysis of LRRK2 KO tissue revealed a significant dysregulation of AP2 complex components, suggesting LRRK2 may act upstream of AP2. In line with this hypothesis, expression of LRRK2 was found to modify recruitment and phosphorylation of AP2. Furthermore, expression of LRRK2 containing the R1441C pathogenic mutation resulted in impaired clathrin-mediated endocytosis (CME). A decrease in activity-dependent synaptic vesicle endocytosis was also observed in neurons harboring an endogenous R1441C LRRK2 mutation. Alongside LRRK2, several PD-associated genes intersect with membrane-trafficking pathways. To investigate the genetic association between Clathrin-trafficking and PD, we used polygenetic risk profiling from IPDGC genome wide association studies (GWAS) datasets. Clathrin-dependent endocytosis genes were found to be associated with PD across multiple cohorts, suggesting common variants at these loci represent a cumulative risk factor for disease. Taken together, these findings suggest CME is a LRRK2-mediated, PD relevant pathway.

CSF total and oligomeric \u03b1-Synuclein along with TNF-\u03b1 as risk biomarkers for Parkinson\u2019s disease: a study in LRRK2 mutation carriers

BackgroundAsymptomatic carriers of leucine-rich repeat kinase 2 (LRRK2) gene mutations constitute an ideal population for discovering prodromal biomarkers of Parkinson’s disease (PD). In this study, we aim to identify CSF candidate risk biomarkers of PD in individuals with LRRK2 mutation carriers.MethodsWe measured the levels of CSF total- (t-), oligomeric (o-) and phosphorylated S129 (pS129-) α-syn, total-tau (tTau), phosphorylated threonine 181 tau (pTau), amyloid-beta 40 (Aβ-40), amyloid-beta-42 (Aβ-42) and 40 inflammatory chemokines in symptomatic (n = 23) and asymptomatic (n = 51) LRRK2 mutation carriers, subjects with a clinical diagnosis of PD (n = 60) and age-matched healthy controls (n = 34). General linear models corrected for age and gender were performed to assess differences in CSF biomarkers between the groups. Markers that varied significantly between the groups were then analyzed using backward-elimination logistic regression analysis to identify an ideal biomarkers panel of prodromal PD.ResultsDiscriminant function analysis revealed low levels of CSF t-α-syn, high levels of CSF o-α-syn and TNF-α best discriminated asymptomatic LRRK2 mutation carriers from both symptomatic PD and healthy controls. Assessing the discriminative power using receiver operating curve analysis, an area under the curve > 0.80 was generated.ConclusionsThe current study suggests that CSF t-, o-α-syn and TNF-α are candidate risk biomarkers for the detection of PD at the prodromal stage. Our findings also highlight the dynamic interrelationships between CSF proteins and the importance of using a biomarkers’ panel approach for an accurate and timely diagnosis of PD.