A Novel LRRK2 Variant p.G2294R in the WD40 Domain Identified in Familial Parkinson’s Disease Affects LRRK2 Protein Levels

Leucine-rich repeat kinase 2 (LRRK2) is a major causative gene of late-onset familial Parkinson’s disease (PD). The suppression of kinase activity is believed to confer neuroprotection, as most pathogenic variants of LRRK2 associated with PD exhibit increased kinase activity. We herein report a novel LRRK2 variant—p.G2294R—located in the WD40 domain, detected through targeted gene-panel screening in a patient with familial PD. The proband showed late-onset Parkinsonism with dysautonomia and a good response to levodopa, without cognitive decline or psychosis. Cultured cell experiments revealed that p.G2294R is highly destabilized at the protein level. The LRRK2 p.G2294R protein expression was upregulated in the patient’s peripheral blood lymphocytes. However, macrophages differentiated from the same peripheral blood showed decreased LRRK2 protein levels. Moreover, our experiment indicated reduced phagocytic activity in the pathogenic yeasts and α-synuclein fibrils. This PD case presents an example wherein the decrease in LRRK2 activity did not act in a neuroprotective manner. Further investigations are needed in order to elucidate the relationship between LRRK2 expression in the central nervous system and the pathogenesis caused by altered LRRK2 activity.

Exosome markers of LRRK2 kinase inhibition

Hyper-activated LRRK2 is linked to Parkinson’s disease susceptibility and progression. Quantitative measures of LRRK2 inhibition, especially in the brain, maybe critical in the development of successful LRRK2-targeting therapeutics. In this study, two different brain-penetrant and selective LRRK2 small-molecule kinase inhibitors (PFE-360 and MLi2) were orally administered to groups of cynomolgus macaques. Proposed pharmacodynamic markers in exosomes from urine and cerebrospinal fluid (CSF) were compared to established markers in peripheral blood mononuclear cells (PBMCs). LRRK2 kinase inhibition led to reductions in exosome-LRRK2 protein and the LRRK2-substrate pT73-Rab10 in urine, as well as reduced exosome-LRRK2 and autophosphorylated pS1292-LRRK2 protein in CSF. We propose orthogonal markers for LRRK2 inhibition in urine and CSF can be used in combination with blood markers to non-invasively monitor the potency of LRRK2-targeting therapeutics.

Pathogenic LRRK2 regulates ciliation probability upstream of Tau Tubulin kinase 2

ABSTRACTMutations that activate LRRK2 protein kinase cause Parkinson’s disease. We have shown previously that Rab10 phosphorylation by LRRK2 enhances its binding to RILPL1 and together, these proteins block cilia formation in a variety of cell types including patient derived iPS cells. We have used live cell fluorescence microscopy to identify, more precisely, the effect of LRRK2 kinase activity on both the formation of cilia triggered by serum starvation and loss of cilia seen upon serum re-addition. LRRK2 activity decreases the overall probability of ciliation without changing the rates of cilia formation in R1441C LRRK2 MEF cells. Cilia loss in these cells is accompanied by ciliary decapitation. Kinase activity does not change the timing or frequency of decapitation or the rate of cilia loss, but increases the percent of cilia that are lost upon serum addition. LRRK2 activity, or overexpression of RILPL1 protein, blocks release of CP110 from the mother centriole, a step normally required for early ciliogenesis. In both cases, failure of CP110 uncapping was due to failure to recruit TTBK2, a kinase needed for CP110 release. In contrast, recruitment of EHD1, another step important for ciliogenesis, appears unaltered. These experiments provide critical detail to our understanding of the cellular consequences of pathogenic LRRK2 mutation, and indicate that LRRK2 blocks ciliogenesis upstream of TTBK2 and enhances the deciliation process in response to serum addition.SIGNIFICANCE STATEMENTMutations that activate LRRK2 protein kinase cause Parkinson’s disease. LRRK2 phosphorylates a subset of Rab GTPases, in particular Rab8 and Rab10. Phosphorylated Rabs bind preferentially to a distinct set of effectors and block in primary ciliation in multiple cell types. We show here that the cilia blockade is upstream of the recruitment of TTBK2 kinase to the mother centriole, a step required for the release of CP110 and subsequent cilia formation. This study provides fundamental information related to how pathogenic LRRK2 interferes with normal cell physiology.

Pharmacodynamic Markers of LRRK2 Inhibition in Biofluids

Hyper-activated LRRK2 is linked to Parkinson’s disease susceptibility and progression. Quantitative measures of LRRK2 inhibition, especially in the brain, may be critical in the clinical development of successful LRRK2-targeting therapeutics. In this study, three different brain-penetrant and selective LRRK2 small-molecule kinase inhibitors (PFE-360, MLi2, and RA283) were orally administered to groups of cynomolgus macaques at different doses. Biofluid markers with proposed pharmacodynamic properties for assessing LRRK2 inhibition were measured from samples of blood, urine, and cerebral-spinal fluid (CSF). LRRK2 kinase inhibition led to consistent reduced pS935-LRRK2 and pRab10 proteins in blood mononuclear cells, reduced exosome LRRK2 protein and di-docosahexaenoyl (22:6) bis (monoacylglycerol) phosphate in urine, and reduced exosome LRRK2 and autophosphorylated pS1292-LRRK2 protein in CSF. Incomplete LRRK2 kinase inhibition reduced LRRK2 protein secretion in exosomes whereas high drug exposures may reduce both exosome and tissue levels of LRRK2 protein. These orthogonal markers for LRRK2 inhibition in urine and CSF can be used in combination with blood markers to non-invasively monitor the potency of LRRK2-targeting therapeutics in the brain and periphery.

Caught in the act: LRRK2 in exosomes

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are a frequent genetic cause of late-onset Parkinson's disease (PD) and a target for therapeutic approaches. LRRK2 protein can influence vesicle trafficking events in the cytosol, with action both in endosomal and lysosomal pathways in different types of cells. A subset of late endosomes harbor intraluminal vesicles that can be secreted into the extracellular milieu. These extracellular vesicles, called exosomes, package LRRK2 protein for transport outside the cell into easily accessed biofluids. Both the cytoplasmic complement of LRRK2 as well as the exosome-associated fraction of protein appears regulated in part by interactions with 14-3-3 proteins. LRRK2 inside exosomes have disease-linked post-translational modifications and are relatively stable compared with unprotected proteins in the extracellular space or disrupted cytosolic compartments. Herein, we review the biology of exosome-associated LRRK2 and the potential for utility in diagnosis, prognosis, and theragnosis in PD and other LRRK2-linked diseases.

LRRK2 detection in human biofluids: potential use as a Parkinson's disease biomarker?

Leucine-rich repeat kinase 2 (LRRK2) is a complex signalling protein that is a key therapeutic target, particularly in Parkinson's disease (PD). In addition, there is now evidence showing that LRRK2 expression and phosphorylation levels have potential as markers of disease or target engagement. Indeed, reports show increases in LRRK2 protein levels in the prefrontal cortex of PD patients relative to controls, suggesting that increase in total LRRK2 protein expression is correlated with disease progression. LRRK2 phosphorylation levels are reduced in experimental systems for most disease mutants, and LRRK2 is also rapidly dephosphorylated upon LRRK2 inhibitor treatment, considered potential therapeutics. Recently, the presence of LRRK2 was confirmed in exosomes from human biofluids, including urine and cerebrospinal fluid. Moreover, phosphorylation of LRRK2 at phosphosites S910, S935, S955 and S973, as well as at the autophosphoryation site S1292, was found in urinary exosomes. In this review, we summarize knowledge on detection of LRRK2 in human biofluids and the relevance of these findings for the development of PD-related biomarkers.