Leucine-rich Repeat Kinase 2 Levels Research Articles

Cell-autonomous role of leucine-rich repeat kinase in the protection of dopaminergic neuron survival.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of Parkinson's disease (PD). However, whether LRRK2 mutations cause PD and degeneration of dopaminergic (DA) neurons via a toxic gain-of-function or a loss-of-function mechanism is unresolved and has pivotal implications for LRRK2-based PD therapies. In this study, we investigate whether Lrrk2 and its functional homolog Lrrk1 play a cell-intrinsic role in DA neuron survival through the development of DA neuron-specific Lrrk conditional double knockout (cDKO) mice. Unlike Lrrk germline DKO mice, DA neuron-restricted Lrrk cDKO mice exhibit normal mortality but develop age-dependent loss of DA neurons, as shown by the progressive reduction of DA neurons in the substantia nigra pars compacta (SNpc) at the ages of 20 and 24 months. Moreover, DA neurodegeneration is accompanied with increases in apoptosis and elevated microgliosis in the SNpc as well as decreases in DA terminals in the striatum, and is preceded by impaired motor coordination. Taken together, these findings provide the unequivocal evidence for the cell-intrinsic requirement of LRRK in DA neurons and raise the possibility that LRRK2 mutations may impair its protection of DA neurons, leading to DA neurodegeneration in PD.

LRRK2 Quantification in Cerebrospinal Fluid of Patients with Parkinson's Disease and Atypical Parkinsonian Syndromes.

The alteration of leucine-rich repeat kinase 2 (LRRK2) kinase activity is thought to be involved in Parkinson's disease (PD) pathogenesis beyond familiar cases, and LRRK2 inhibitors are currently under investigation. Preliminary data suggest a relationship between LRRK2 alteration and cognitive impairment in PD. To investigate cerebrospinal fluid (CSF) LRRK2 levels in PD and other parkinsonian disorders, also correlating them with cognitive impairment. In this study, we retrospectively investigated by means of a novel highly sensitive immunoassay the levels of total and phosphorylated (pS1292) LRRK2 in CSF of cognitively unimpaired PD (n = 55), PD with mild cognitive impairment (n = 49), PD with dementia (n = 18), dementia with Lewy bodies (n = 12), atypical parkinsonian syndromes (n = 35), and neurological controls (n = 30). Total and pS1292 LRRK2 levels were significantly higher in PD with dementia with respect to PD with mild cognitive impairment and PD, and also showed a correlation with cognitive performances. The tested immunoassay may represent a reliable method for assessing CSF LRRK2 levels. The results appear to confirm an association of LRRK2 alteration with cognitive impairment in PD. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

WHOPPA Enables Parallel Assessment of Leucine-Rich Repeat Kinase 2 and Glucocerebrosidase Enzymatic Activity in Parkinson’s Disease Monocytes

Both leucine-rich repeat kinase 2 (LRRK2) and glucocerebrosidase (GCase) are promising targets for the treatment of Parkinson’s disease (PD). Evidence suggests that both proteins are involved in biological pathways involving the lysosome. However, studies to date have largely investigated the enzymes in isolation and any relationship between LRRK2 and GCase remains unclear. Both enzymes are highly expressed in peripheral blood monocytes and have been implicated in immune function and inflammation. To facilitate the standardized measurement of these readouts in large cohorts of samples collected from persons with PD across the globe, we developed and optimized a sample collection and processing protocol with parallel flow cytometry assays. Assay parameters were first optimized using healthy control peripheral blood mononuclear cells (PBMCs), and then LRRK2 and GCase activities were measured in immune cells from persons with idiopathic PD (iPD). We tested the ability of this protocol to deliver similar results across institutes across the globe, and named this protocol the Wallings-Hughes Optimized Protocol for PBMC Assessment (WHOPPA). In the application of this protocol, we found increased LRRK2 levels and stimulation-dependent enzymatic activity, and decreased GBA index in classical iPD monocytes, as well as increased cytokine release in PD PBMCs. WHOPPA also demonstrated a strong positive correlation between LRRK2 levels, pRab10 and HLA-DR in classical monocytes from subjects with iPD. These data support a role for the global use of WHOPPA and expression levels of these two PD-associated proteins in immune responses, and provide a robust assay to determine if LRRK2 and GCase activities in monocytes have potential utility as reliable and reproducible biomarkers of disease in larger cohorts of subjects with PD.

Effect of LRRK2 protein and activity on stimulated cytokines in human monocytes and macrophages

Leucine-rich-repeat kinase 2 (LRRK2), a potential therapeutic target for the treatment of Parkinson’s disease (PD), is highly expressed in monocytes and macrophages and may play a role in the regulation of inflammatory pathways. To determine how LRRK2 protein levels and/or its activity modulate inflammatory cytokine/chemokine levels in human immune cells, isogenic human induced pluripotent stem cells (iPSC) with the LRRK2-activating G2019S mutation, wild-type LRRK2, and iPSC deficient in LRRK2 were differentiated to monocytes and macrophages and stimulated with inflammatory toll-like receptor (TLR) agonists in the presence and absence of LRRK2 kinase inhibitors. The effect of LRRK2 inhibitors and the effect of increasing LRRK2 levels with interferon gamma on TLR-stimulated cytokines were also assessed in primary peripheral blood-derived monocytes. Monocytes and macrophages with the LRRK2 G2019S mutation had significantly higher levels of cytokines and chemokines in tissue culture media following stimulation with TLR agonists compared to isogenic controls. Knockout of LRRK2 impaired phagocytosis but did not significantly affect TLR-mediated cytokine levels. Interferon gamma significantly increased the levels of LRRK2 and phosphorylation of its downstream Rab10 substrate, and potentiated TLR-mediated cytokine levels. LRRK2 kinase inhibitors did not have a major effect on TLR-stimulated cytokine levels. Results suggest that the LRRK2 G2019S mutation may potentiate inflammation following activation of TLRs. However, this was not dependent on LRRK2 kinase activity. Indeed, LRRK2 kinase inhibitors had little effect on TLR-mediated inflammation under the conditions employed in this study.

Understanding LRRK2 kinase activity in preclinical models and human subjects through quantitative analysis of LRRK2 and pT73\xa0Rab10

Variants in the leucine-rich repeat kinase 2 (LRRK2) gene are associated with increased risk for familial and sporadic Parkinson’s disease (PD). Pathogenic variants in LRRK2, including the common variant G2019S, result in increased LRRK2 kinase activity, supporting the therapeutic potential of LRRK2 kinase inhibitors for PD. To better understand the role of LRRK2 in disease and to support the clinical development of LRRK2 inhibitors, quantitative and high-throughput assays to measure LRRK2 levels and activity are needed. We developed and applied such assays to measure the levels of LRRK2 as well as the phosphorylation of LRRK2 itself or one of its substrates, Rab10 (pT73 Rab10). We observed increased LRRK2 activity in various cellular models of disease, including iPSC-derived microglia, as well as in human subjects carrying the disease-linked variant LRRK2 G2019S. Capitalizing on the high-throughput and sensitive nature of these assays, we detected a significant reduction in LRRK2 activity in subjects carrying missense variants in LRRK2 associated with reduced disease risk. Finally, we optimized these assays to enable analysis of LRRK2 activity following inhibition in human peripheral blood mononuclear cells (PBMCs) and whole blood, demonstrating their potential utility as biomarkers to assess changes in LRRK2 expression and activity in the clinic.

LRRK2 inhibition potentiates PARP inhibitor cytotoxicity through inhibiting homologous recombination-mediated DNA double strand break repair.

PARP inhibitors induce DNA lesions, the repair of which are highly dependent on homologous recombination (HR), and preferentially kill HR‐ deficient cancers. However, cancer cells have developed several mechanisms to transform HR and confer drug resistance to PARP inhibition. Therefore, there is a great clinical interest in exploring new therapies that induce HR deficiency (HRD), thereby sensitizing cancer cells to PARP inhibitors. Here, we found that GSK2578215A, a high‐selective and effective leucine‐rich repeat kinase 2 (LRRK2) inhibitor, or LRRK2 depletion suppresses HR preventing the recruitment of RAD51 to DNA damage sites through disruption of the interaction of RAD51 and BRCA2. Moreover, LRRK2 inhibition or depletion increases the susceptibility of ovarian cancer cells to Olaparib in vitro and in vivo. In clinical specimens, LRRK2 high expression is high related with advanced clinical characteristics and poor survival of ovarian cancer patients. All these findings indicate ovarian cancers expressing high levels of LRRK2 are more resistant to treatment potentially through promoting HR. Furthermore, combination treatment with an LRRK2 and PARP inhibitor may be a novel strategy to improve the effectiveness of LRRK2 expression ovarian cancers.

LRRK2 Ablation Attenuates Αlpha-Synuclein-Induced Neuroinflammation Without Affecting Neurodegeneration or Neuropathology In Vivo.

The development of disease-modifying therapies for Parkinson's disease is a major challenge which would be facilitated by a better understanding of the pathogenesis. Leucine-rich repeat kinase 2 (LRRK2) and α-synuclein are key players in Parkinson's disease, but their relationship remains incompletely resolved. Previous studies investigating the effect of LRRK2 on α-synuclein-induced neurotoxicity and neuroinflammation in preclinical Parkinson's disease models have reported conflicting results. Here, we aimed to further explore the functional interaction between α-synuclein and LRRK2 and to evaluate the therapeutic potential of targeting physiological LRRK2 levels. We studied the effects of total LRRK2 protein loss as well as pharmacological LRRK2 kinase inhibition in viral vector-mediated α-synuclein-based Parkinson's disease models developing early- and late-stage neurodegeneration. Surprisingly, total LRRK2 ablation or in-diet treatment with the LRRK2 kinase inhibitor MLi-2 did not significantly modify α-synuclein-induced motor deficits, dopaminergic cell loss, or α-synuclein pathology. Interestingly, we found a significant effect on α-synuclein-induced neuroinflammatory changes in the absence of LRRK2, with a reduced microglial activation and CD4+ and CD8+ T cell infiltration. This observed lack of protection against α-synuclein-induced toxicity should be well considered in light of the ongoing therapeutic development of LRRK2 kinase inhibitors for idiopathic Parkinson's disease. Future studies will be crucial to understand the link between these neuroinflammatory processes and disease progression as well as the role of α-synuclein and LRRK2 in these pathological events.

LRRK2 Loss-of-Function Variants: When Less Is More.

Gain-of-function variants in leucine-rich repeat kinase 2 (LRRK2) are the most common monogenic cause of Parkinson's disease (PD), albeit with incomplete penetrance, indicating that reduction of LRRK2 kinase activity is a reasonable therapeutic strategy for LRRK2-related PD.1 However, LRRK2 inhibition in animal models was associated with abnormal phenotypes in the lung, kidney, and liver, thus raising safety concerns.2 Resources such as the Genome Aggregation Database or UK Biobank, built on large-scale sequencing projects, represent compelling tools for assessing genetic variant frequency. These were employed by Whiffin and colleagues3 to investigate the frequency of LRRK2 loss-of-function (LoF) variants in humans and their association with specific phenotypes. Among 187,518 individuals in the 2 aforementioned cohorts, they identified 352 carriers of heterozygous LRRK2 LoF variants, implying that approximately 1 in 500 humans harbors such a variant.3 Using genotyping data from the personal genetics company 23andMe, Whiffin and colleagues3 identified an additional 1,103 individuals with LRRK2 LoF variants.3 No reduction in lifespan or overrepresentation of any phenotype affecting the lung, liver, kidney, cardiovascular system, nervous system, immunity, or cancer in carriers of LRRK2 LoF variants was observed.3 In an earlier study, heterozygous LRRK2 LoF variants were identified at comparable percentages of clinically diagnosed patients with PD and controls, suggesting a lack of association between the partial loss of LRRK2 and PD.4 These results collectively suggest that an endogenous decrease of LRRK2 levels and consequently of its activity does not seem to elicit highly penetrant phenotypes, although to date LRRK2 LoF variants have never been investigated specifically in cohorts of patients with unexplained severe manifestations (including those seen in animal models upon LRRK2 inhibition). Thus, the therapeutic administration of compounds intended to decrease LRRK2 enzymatic activity (kinase inhibitors) or levels (antisense oligonucleotides) is not expected to cause harmful effects. Currently, 3 LRRK2-targeted compounds are being tested in phase 1 or 1b clinical trials: the LRRK2 kinase inhibitors DNL151 and DNL201 from Denali Therapeutics Inc., (San Francisco, CA, USA) and the antisense oligonucleotide BIIB094 from Biogen.3 As a caveat, a coinciding loss of both LRRK2 and its functional homolog LRRK1 results in earlier mortality, a loss of dopaminergic neurons, and an increase in α-synuclein in a double-knockout mouse model.5 Hence, targeting LRRK2 may not be safe in individuals with LRRK1 homozygous or heterozygous LoF variants. While it may thus be advisable to exclude the presence of such variants by genetic testing of LRRK1 in individuals prior to the administration of any LRRK2-directed treatment, LRRK2 inhibition is a promising and potentially safe new therapeutic strategy. (1) Research project: A. Conception, B. Organization, C. Execution; (2) Manuscript: A. Writing of the first draft, B. Review and Critique. T.U.: 1A, 1B,1C,2A,2B A.W.:1A,1B,1C,2A,2B T.U. reports the following: employment with University Medical Center Schleswig-Holstein, Campus Lübeck, Germany. A.W. is an advisor for medical writing at CENTOGENE GmbH and reports employment with University Medical Center Schleswig-Holstein, Campus Lübeck, Germany; honoraria from CENTOGENE GmbH; and a grant from the German Research Foundation (Deutsche Forschungsgemeinschaft) Research Unit (FOR2488).

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.

Human Peripheral Blood Neutrophil Isolation for Interrogating the Parkinson's Associated LRRK2 Kinase Pathway by Assessing Rab10 Phosphorylation.

The leucine rich repeat kinase 2 (LRRK2) is the most frequently mutated gene in hereditary Parkinson' disease (PD) and all pathogenic LRRK2 mutations result in hyperactivation of its kinase function. Here, we describe an easy and robust assay to quantify LRRK2 kinase pathway activity in human peripheral blood neutrophils by measuring LRRK2-controlled phosphorylation of one of its physiological substrates, Rab10 at threonine 73. The immunoblotting analysis described requires a fully selective and phosphospecific antibody that recognizes the Rab10 Thr73 epitope phosphorylated by LRRK2, such as the MJFF-pRab10 rabbit monoclonal antibody. It uses human peripheral blood neutrophils, because peripheral blood is easily accessible and neutrophils are an abundant and homogenous constituent. Importantly, neutrophils express relatively high levels of both LRRK2 and Rab10. A potential drawback of neutrophils is their high intrinsic serine protease activity, which necessitates the use of very potent protease inhibitors such as the organophosphorus neurotoxin diisopropylfluorophosphate (DIFP) as part of the lysis buffer. Nevertheless, neutrophils are a valuable resource for research into LRRK2 kinase pathway activity in vivo and should be considered for inclusion into PD biorepository collections.

Leucine-rich repeat kinase 2 (LRRK2) inhibitors differentially modulate glutamate release and Serine935 LRRK2 phosphorylation in striatal and cerebrocortical synaptosomes.

Mutations in leucine‐rich repeat kinase 2 (LRRK2) gene have been pathogenically linked to Parkinson's disease, and pharmacological inhibition of LRRK2 is being pursued to tackle nigro‐striatal dopaminergic neurodegeneration. However, LRRK2 kinase inhibitors may have manifold actions, affecting not only pathological mechanisms in dopaminergic neurons but also physiological functions in nondopaminergic neurons. Therefore, we investigated whether LRRK2 kinase inhibitors differentially modulate dopamine and glutamate release from the mouse striatum and cerebral cortex. Spontaneous and KCl‐evoked [3H]‐dopamine and glutamate release from superfused synaptosomes obtained from wild‐type and LRRK2 knock‐out, kinase‐dead or G2019S knock‐in mice was measured. Two structurally unrelated inhibitors, LRRK2‐IN‐1 and GSK2578215A, were tested. LRRK2, phosphoSerine1292 and phosphoSerine935 LRRK2 levels were measured in all genotypes, and target engagement was evaluated by monitoring phosphoSerine935 LRRK2. LRRK2‐IN‐1 inhibited striatal glutamate but not dopamine release; GSK2578215A inhibited striatal dopamine and cortical glutamate but enhanced striatal glutamate release. LRRK2‐IN‐1 reduced striatal and cortical phosphoSerine935 levels whereas GSK2578215A inhibited only the former. Neither LRRK2 inhibitor affected neurotransmitter release in LRRK2 knock‐out and kinase‐dead mice; however, they facilitated dopamine without affecting striatal glutamate in G2019S knock‐in mice. GSK2578215A inhibited cortical glutamate release in G2019S knock‐in mice. We conclude that LRRK2‐IN‐1 and GSK2578215A modulate exocytosis by blocking LRRK2 kinase activity, although their effects vary depending on the nerve terminal examined. The G2019S mutation unravels a dopamine‐promoting action of LRRK2 inhibitors while blunting their effects on glutamate release, which highlights their positive potential for the treatment of PD, especially of LRRK2 mutation carriers.

LRRK2 is involved in the pathogenesis of system lupus erythematosus through promoting pathogenic antibody production

BackgroundSystemic lupus erythematosus (SLE) is a prototypic autoimmune disease characterized by the presence of pathogenic autoantibodies associated with polyclonal B cell hyperreactivity. Previous study reported that autophagy-related gene Leucine-rich repeat kinase 2 (LRRK2) was likely a susceptible gene for SLE. However, the pathogenic function of LRRK2 in SLE is undefined.MethodsUsing quantitative PCR, we compared the expression levels of LRRK2 in B cells between SLE patients and healthy controls. The expression levels of LRRK2 in in vitro induced CD19hi B cells and naïve B cells were compared as well based on RNA-seq assay. A pristane-induced lupus-like mouse model was used to explore the effects of LRRK2 on the development of SLE. IgG level, B cell subsets in the spleens and bone marrows and pathological features in the kidneys were compared between wildtype (WT) and Lrrk2−/− littermates.ResultsIt was obvious that LRRK2 expression was dramatically up-regulated in primary B cells from SLE patients compared to those from healthy controls, as well as in activated CD19hi B cells. More significantly, LRRK2 expression in B cells was positively correlated with system lupus erythematosus disease activity index (SLEDAI), an indicator for disease severity, and serum IgG levels in SLE patients. Negative correlations were observed between LRRK2 expression and serum C3 or C4 levels, two clinical features associated with SLE-related nephritis. LRRK2 deficiency reduced the death rate of pristane treated mice. Decreased levels of total IgG and autoantibody were detected in the serum with less deposition of immune complexes and attenuated pathological symptoms in the kidneys of Lrrk2−/− mice. Consistent with the reduction in IgG production, the percentages of germinal center B cells and plasma cells decreased significantly as well with LRRK2 deficiency.ConclusionsOur study demonstrates that LRRK2 expression is upregulated in B cells from SLE patients with strong correlations to disease severity. LRRK2 deficiency largely attenuates the pathogenic progress of lupus-like features in pristane-induced mice. This is probably achieved through affecting B cell terminal differentiation and subsequent immunoglobulin production.

Leucine-rich repeat kinase 2 aggravates secondary brain injury induced by intracerebral hemorrhage in rats by regulating the P38 MAPK/Drosha pathway

Leucine-rich repeat kinase 2 (LRRK2) is the genetic cause of both familial and idiopathic Parkinson's disease (PD), and it is associated with neuronal death, vesicle trafficking, mitochondrial dysfunction, and inflammation. However, its role in secondary brain injury (SBI) induced by intracerebral hemorrhage (ICH) has not been evaluated. In this study, an ICH model was induced by injecting autologous whole blood into the right basal ganglia of adult rats. Meanwhile, primary rat cortical neurons treated with Oxyhemoglobin (OxyHb) were used as an in vitro ICH model. Protein levels of LRRK2 increased significantly in brain tissues after ICH. Upregulation of LRRK2 by genetic overexpression augmented inflammatory responses, behavioral and cognitive dysfunction, brain edema, blood-brain barrier (BBB) injury, and cell death involved in SBI following ICH. Downregulation of LRRK2 by GNE7915 (a specific chemical inhibitor of LRRK2) and genetic knockdown yielded opposite effects. Additionally, inhibiting LRRK2 by GNE7915 obviously reduced OxyHb-induced neuronal apoptosis in vitro and attenuated phosphorylation of p38 MAPK and Drosha both in vivo and in vitro. Therefore, we concluded that LRRK2 participated in ICH-induced SBI by mediating inflammatory responses, behavioral and cognitive dysfunction, brain edema, and BBB injury and by modulating neuronal death and dysfunction and regulating the p38 MAPK/Drosha pathway.

The kinase LRRK2 is differently expressed in chronic rhinosinusitis with and without nasal polyps

BackgroundChronic rhinosinusitis (CRS), commonly divided into CRS with nasal polyps (CRSwNP) and without nasal polyps (CRSsNP) is an inflammatory disease which mechanism remain unclear. Leucine-rich repeat kinase 2 (LRRK2) has been proved to be a negative regulator of inflammation response while its role in pathogenesis of CRS has yet to be revealed. This research study was designed to investigate the relationship between the expression level and biologic role of LRRK2 in CRS.MethodsExpression of LRRK2 mRNA and noncoding repressor of NFAT (NRON) were examined by qRT-PCR. Protein levels of LRRK2 were performed by western blot and immunohistochemistry. Nuclear factor of activated T cells (NFAT) nuclear translocation was analyzed by immunohistochemistry. Additionally, LRRK2 mRNA and NRON expression in response to specific inflammatory stimulation was measured in human nasal epithelia cells (HNECs).ResultsThe expression of LRRK2 was increased in CRSsNP patients (p < 0.05) and positively correlated with the expression levels of CD3 and Charot-Leyden crystal. Meanwhile, the NRON expression level is much lower in CRSsNP patients compared to both the control group and CRSwNP group (p < 0.05). Marked enhanced NFAT nuclear localization was observed in CRSwNP groups compared with the CRSsNP and control group (p < 0.0001). And the over-expression of LRRK2 was significantly regulated by lipopolysaccharide (LPS) in HNECs (p < 0.05). Moreover, IL-17A can increase LRRK2 expression and suppress NRON expression in vitro and dexamethasone can rescue the NRON inhibition.ConclusionLRRK2 and NRON may play different role in CRSsNP and CRSwNP. The molecular mechanisms identified here may aid in the design of novel therapeutic strategies to improve clinical outcomes.

Reduced LRRK2 in association with retromer dysfunction in post-mortem brain tissue from LRRK2 mutation carriers.

Missense mutations in leucine-rich repeat kinase 2 (LRRK2) are pathogenic for familial Parkinson's disease. However, it is unknown whether levels of LRRK2 protein in the brain are altered in patients with LRRK2-associated Parkinson's disease. Because LRRK2 mutations are relatively rare, accounting for approximately 1% of all Parkinson's disease, we accessioned cases from five international brain banks to investigate levels of the LRRK2 protein, and other genetically associated Parkinson's disease proteins. Brain tissue was obtained from 17 LRRK2 mutation carriers (12 with the G2019S mutation and five with the I2020T mutation) and assayed by immunoblot. Compared to matched controls and idiopathic Parkinson's disease cases, we found levels of LRRK2 protein were reduced in the LRRK2 mutation cases. We also measured a decrease in two other proteins genetically implicated in Parkinson's disease, the core retromer component, vacuolar protein sorting associated protein 35 (VPS35), and the lysosomal hydrolase, glucocerebrosidase (GBA). Moreover, the classical retromer cargo protein, cation-independent mannose-6-phosphate receptor (MPR300, encoded by IGF2R), was also reduced in the LRRK2 mutation cohort and protein levels of the receptor were correlated to levels of LRRK2. These results provide new data on LRRK2 protein expression in brain tissue from LRRK2 mutation carriers and support a relationship between LRRK2 and retromer dysfunction in LRRK2-associated Parkinson's disease brain.

Elevated LRRK2 autophosphorylation in brain-derived and peripheral exosomes in LRRK2 mutation carriers

Missense mutations in the leucine-rich repeat kinase 2 (LRRK2) gene can cause late-onset Parkinson disease (PD). LRRK2 mutations increase LRRK2 kinase activities that may increase levels of LRRK2 autophosphorylation at serine 1292 (pS1292) and neurotoxicity in model systems. pS1292-LRRK2 protein can be packaged into exosomes and measured in biobanked urine. Herein we provide evidence that pS1292-LRRK2 protein is robustly expressed in cerebral spinal fluid (CSF) exosomes. In a novel cohort of Norwegian subjects with and without the G2019S-LRRK2 mutation, with and without PD, we quantified levels of pS1292-LRRK2, total LRRK2, and other exosome proteins in urine from 132 subjects and in CSF from 82 subjects. CSF and urine were collected from the same morning clinic visit in 55 of the participants. We found that total LRRK2 protein concentration was similar in exosomes purified from either CSF or urine but the levels did not correlate. pS1292-LRRK2 levels were higher in urinary exosomes from male and female subjects with a LRRK2 mutation. Male LRRK2 mutation carriers without PD had intermediate pS1292-LRRK2 levels compared to male carriers with PD and controls. However, female LRRK2 mutation carriers without PD had the same pS1292-LRRK2 levels compared to female carriers with PD. pS1292-LRRK2 levels in CSF exosomes were near saturated in most subjects, ten-fold higher on average than pS1292-LRRK2 levels in urinary exosomes, irrespective of LRRK2 mutation status or PD diagnosis. These results provide insights into the effects of LRRK2 mutations in both the periphery and brain in a well-characterized clinical population and show that LRRK2 protein in brain exosomes may be much more active than in the periphery in most subjects.

Interrogating Parkinson's disease LRRK2 kinase pathway activity by assessing Rab10 phosphorylation in human neutrophils.

There is compelling evidence for the role of the leucine-rich repeat kinase 2 (LRRK2) and in particular its kinase function in Parkinson's disease. Orally bioavailable, brain penetrant and potent LRRK2 kinase inhibitors are in the later stages of clinical development. Here, we describe a facile and robust assay to quantify LRRK2 kinase pathway activity by measuring LRRK2-mediated phosphorylation of Rab10 in human peripheral blood neutrophils. We use the selective MJFF-pRab10 monoclonal antibody recognising the Rab10 Thr73 phospho-epitope that is phosphorylated by LRRK2. We highlight the feasibility and practicability of using our assay in the clinical setting by studying a few patients with G2019S LRRK2 associated and sporadic Parkinson's as well as healthy controls. We suggest that peripheral blood neutrophils are a valuable resource for LRRK2 research and should be considered for inclusion in Parkinson's bio-repository collections as they are abundant, homogenous and express relatively high levels of LRRK2 as well as Rab10. In contrast, the widely used peripheral blood mononuclear cells are heterogeneous and only a minority of cells (monocytes and contaminating neutrophils) express LRRK2. While our LRRK2 kinase pathway assay could assist in patient stratification based on LRRK2 kinase activity, we envision that it may find greater utility in pharmacodynamic and target engagement studies in future LRRK2 inhibitor trials.

LRRK2 Antisense Oligonucleotides Ameliorate α-Synuclein Inclusion Formation in a Parkinson's Disease Mouse Model.

No treatments exist to slow or halt Parkinson’s disease (PD) progression; however, inhibition of leucine-rich repeat kinase 2 (LRRK2) activity represents one of the most promising therapeutic strategies. Genetic ablation and pharmacological LRRK2 inhibition have demonstrated promise in blocking α-synuclein (α-syn) pathology. However, LRRK2 kinase inhibitors may reduce LRRK2 activity in several tissues and induce systemic phenotypes in the kidney and lung that are undesirable. Here, we test whether antisense oligonucleotides (ASOs) provide an alternative therapeutic strategy, as they can be restricted to the CNS and provide a stable, long-lasting reduction of protein throughout the brain. Administration of LRRK2 ASOs to the brain reduces LRRK2 protein levels and fibril-induced α-syn inclusions. Mice exposed to α-syn fibrils treated with LRRK2 ASOs show more tyrosine hydroxylase (TH)-positive neurons compared to control mice. Furthermore, intracerebral injection of LRRK2 ASOs avoids unwanted phenotypes associated with loss of LRRK2 expression in the periphery. This study further demonstrates that a reduction of endogenous levels of normal LRRK2 reduces the formation of α-syn inclusions. Importantly, this study points toward LRRK2 ASOs as a potential therapeutic strategy for preventing PD-associated pathology and phenotypes without causing potential adverse side effects in peripheral tissues associated with LRRK2 inhibition.

CADPS2 gene expression is oppositely regulated by LRRK2 and alpha-synuclein

The Ca2+-dependent activator protein for secretion 2 (CADPS2) is a member of the CAPS/CADPS protein family that plays crucial roles in synaptic vesicle dynamics. Genomic variability in the CADPS2 gene has been associated to autism spectrum disorders and Alzheimer's disease, both characterized by altered neurotransmission. Biological evidence also linked CADPS2 to Parkinson's disease (PD), as a disease-causing mutation in leucine-rich repeat kinase 2 (LRRK2) was reported to increase CADPS2 gene and protein expression. Furthermore, restoration of CADPS2 physiologic levels was able to provide neuroprotection in patient-derived neurons, consistent with the synaptic dysfunction postulated to underlie PD. However, little is known about the influence of PD-related proteins on transcriptional regulation of critical synaptic genes such as CADPS2. Here we aimed at investigating the transcriptional effects of LRRK2 and alpha-synuclein (aSyn) on CADPS2 gene expression, using a combination of in silico analyses and cell biology techniques. First, we identified a predicted promoter in the human CADPS2 genomic sequence, which we then utilized in a luciferase-based gene reporter assay. This approach enabled us to disclose a differential effect of high levels of LRRK2 and aSyn on CADPS2 promoter activity. Specifically, CADPS2 transcriptional activity was enhanced by high cellular levels of LRRK2 and reduced by overexpression of aSyn. Consistently, CADPS2 mRNA levels were diminished in aSyn overexpressing cells.Our results indicate that LRRK2 and aSyn participate in the dysregulation of CADPS2 by altering transcription and support the hypothesis that synaptic dysfunctions, through different mechanisms, might contribute to the neuronal defects of diseases such as PD.

LRRK2 kinase inhibition, a potential Parkinson’s disease therapy, induces LRRK2 protein destabilization and proteasomal degradation

Event Abstract Back to Event LRRK2 kinase inhibition, a potential Parkinson’s disease therapy, induces LRRK2 protein destabilization and proteasomal degradation Evy Lobbestael1*, Laura Civiero2, Tina De Wit1, Jean-Marc Taymans3, Elisa Greggio2 and Veerle Baekelandt1 1 KU Leuven, Neurosciences, Belgium 2 University of Padova, Italy 3 INSERM-CHRU de Lille-Université de Lille, France Leucine-rich repeat kinase 2 (LRRK2) kinase activity is increased in several pathogenic mutations, including the most common mutation, G2019S, and plays a role in Parkinson’s disease (PD) pathobiology. LRRK2 kinase inhibitors are currently one of the most prevailing disease-modifying therapeutic PD strategies and although several lines of evidence support beneficial effects, many questions need to be answered before clinical applications can be envisaged. We aimed to gain insight in the LRRK2 kinase inhibitor-induced reduction in LRRK2 protein levels. We used six different LRRK2 kinase inhibitors administered to mice, primary cultures and cell lines and monitored LRRK2 (phosphorylation) levels via immunoblotting at different time points. LRRK2 kinase inhibition induces LRRK2 dephosphorylation and can reduce LRRK2 protein levels of overexpressed wild-type and G2019S, but not A2016T or K1906M, LRRK2 as well as endogenous LRRK2 in mouse brain and primary astrocytes. This effect could be reversed by proteasomal inhibition, but not by lysosomal inhibition, while mRNA levels remained unaffected. Using LRRK2 S910A and S935A phosphorylation mutants, we show that dephosphorylation of these sites is not required for LRRK2 degradation. Interestingly, a truncated form of LRRK2, detected in mouse kidney, is dephosphorylated upon LRRK2 kinase inhibition, but not destabilized. In conclusion, we show that LRRK2 kinase inhibitor-induced LRRK2 destabilization is mediated by proteasomal degradation. This effect appears to be cell type specific and is not (only) dependent on dephosphorylation at LRRK2 S910 and S935. Increasing our insight in the molecular consequences of LRRK2 kinase inhibition will be crucial in the development of LRRK2-based PD therapies. Keywords: LRRK2, Kinase inhibition, Parkinson's disease, therapy, proteasomal degradation Conference: 12th National Congress of the Belgian Society for Neuroscience, Gent, Belgium, 22 May - 22 May, 2017. Presentation Type: Oral Presentation Topic: Disorders of the Nervous System Citation: Lobbestael E, Civiero L, De Wit T, Taymans J, Greggio E and Baekelandt V (2019). LRRK2 kinase inhibition, a potential Parkinson’s disease therapy, induces LRRK2 protein destabilization and proteasomal degradation. Front. Neurosci. Conference Abstract: 12th National Congress of the Belgian Society for Neuroscience. doi: 10.3389/conf.fnins.2017.94.00100 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 21 Apr 2017; Published Online: 25 Jan 2019. * Correspondence: Dr. Evy Lobbestael, KU Leuven, Neurosciences, Leuven, Belgium, evy.lobbestael@kuleuven.be Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Evy Lobbestael Laura Civiero Tina De Wit Jean-Marc Taymans Elisa Greggio Veerle Baekelandt Google Evy Lobbestael Laura Civiero Tina De Wit Jean-Marc Taymans Elisa Greggio Veerle Baekelandt Google Scholar Evy Lobbestael Laura Civiero Tina De Wit Jean-Marc Taymans Elisa Greggio Veerle Baekelandt PubMed Evy Lobbestael Laura Civiero Tina De Wit Jean-Marc Taymans Elisa Greggio Veerle Baekelandt Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.