LRRK2 activity does not dramatically alter \u03b1-synuclein pathology in primary neurons

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.

Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) are common genetic risk factors for both familial and sporadic Parkinson’s disease (PD). Pathogenic mutations in LRRK2 have been shown to induce changes in its activity, and abnormal increase in LRRK2 kinase activity is thought to contribute to PD pathology. The precise molecular mechanisms underlying LRRK2-associated PD pathology are far from clear, however the identification of LRRK2 substrates and the elucidation of cellular pathways involved suggest a role of LRRK2 in microtubule dynamics, vesicular trafficking, and synaptic transmission. Moreover, LRRK2 is associated with pathologies of α-synuclein, a major component of Lewy bodies (LBs). Evidence from various cellular and animal models supports a role of LRRK2 in the regulation of aggregation and propagation of α-synuclein. Here, we summarize our current understanding of how pathogenic mutations dysregulate LRRK2 and discuss the possible mechanisms leading to neurodegeneration.

Dominant missense mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common known genetic cause of Parkinson disease. LRRK2 encodes a serine/threonine protein kinase, and pathogenic mutations may increase kinase activity. Intrinsic GTP binding in the GTPase domain may govern kinase activity through an internal signal transduction cascade. As with many protein kinases, LRRK2 self-interacts through mechanisms that may regulate enzymatic activity. We find that the disruption of either GTPase or kinase activity enhances the formation of high molecular weight oligomers and prevents the formation of LRRK2 dimer structures. In addition, brief application of the broad spectrum kinase inhibitor staurosporine ablates LRRK2 dimers and promotes LRRK2 high molecular weight oligomers. LRRK2 interactions with other proteins in cell lines are kinase-independent and include chaperones and cell cytoskeleton components, suggesting that LRRK2 self-assembly principally dictates complex size. To further explore the mechanics of kinase activation, we separate soluble LRRK2 protein that encodes the pathogenic G2019S mutation into high molecular weight oligomers, dimers, and monomers and find that kinase activity resides with dimeric LRRK2. Some PD-associated mutations that increase kinase activity in vitro significantly increase the proportion of dimer structures relative to total LRRK2 protein, providing additional insight into how pathogenic mutations may alter normal enzymatic regulation. Targeting and tracking LRRK2 dimerization may provide a clear way to observe LRRK2 kinase activity in living cells, and disruption of dimeric LRRK2 through kinase inhibition or other means may attenuate pathogenic increases in LRRK2 enzymatic output.

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.

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.

Parkinson’s disease (PD) pathogenic mutations in leucine-rich repeat kinase 2 (LRRK2) are associated with endolysosomal dysfunction across cell types, and carriers of LRRK2 mutations variably present with phosphorylated tau and α-synuclein deposits in post-mortem analysis. LRRK2 mutations increase the phosphorylation of Rab substrates including Rab12 and Rab10. Rab12 and Rab10 are expressed in neuronal and non-neuronal cells with localization to membranes in the endolysosomal compartment, and lysosomal stress activates LRRK2 phosphorylation of Rabs. In this study, using antibodies directed to the LRRK2-mediated phosphorylation sites on Rab12 at amino acid Ser106 (pS106-Rab12) and Rab10 at amino acid Thr73 (pT73-Rab10), we test whether aberrant LRRK2 phosphorylation is associated with tau and/or α-synuclein pathology across clinically distinct neurodegenerative diseases. Analysis of brain tissue lysates and immunohistochemistry of pathology-susceptible brain regions demonstrate that pS106-Rab12 levels are increased in Alzheimer’s disease (AD) and Lewy body disease (LBD), including PD with and without G2019S LRRK2 mutation. At early pathological stages, phosphorylated Rab12 localizes to granulovacuolar degeneration bodies (GVBs), which are thought to be active lysosomal-like structures, in neurons. pS106-Rab12-positive GVBs accumulate with pathological tau across brain tissues in AD and LBD, and in G2019S LRRK2 mutation carriers. In a mouse model of tauopathy, pS106-Rab12 localizes to GVBs during early tau deposition in an age-dependent manner. While GVBs are largely absent in neurons with mature protein pathology, subsets of both tau and α-synuclein inclusions appear to incorporate pS106-Rab12 at later pathological stages. Further, pS106-Rab12 labels GVBs in neurons and shows co-pathology with tau inclusions in primary tauopathies including Pick’s disease, progressive supranuclear palsy, and corticobasal degeneration. Finally, pT73-Rab10 is elevated and localizes to GVBs, but not tau and α-synuclein inclusions, in AD and LBD, including G2019S LRRK2 mutation carriers. These results implicate LRRK2 kinase activity and Rab phosphorylation in endolysosomal dysfunction in tau- and α-synuclein-associated neurodegenerative diseases.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00401-025-02951-x.

Leucine-rich repeat kinase 2 (LRRK2) variants are the most common cause of inherited Parkinson’s disease (PD), and the hyperactivity of the LRRK2 variants represent a validated drug target for PD. The penetration of common LRRK2 variants is incomplete, underscoring the need for molecular biomarkers that predict disease onset and guide therapeutics development. Here, we analyzed large datasets of cerebrospinal fluid (CSF) and urinary proteomics from the Parkinson’s Progression Markers Initiative (PPMI) and identified distinct lysosomal and immune protein signatures as potential biomarkers for LRRK2-linked PD (LRRK2 PD). Longitudinal analysis revealed that levels of specific lysosomal and immune proteins remained elevated in CSF during the prodromal phase but declined following clinical symptom onset. Furthermore, examination of multiple brain cell types from Lrrk2 mutant mice carrying disease variant (G2019S) showed heightened secretion of lysosomal proteins in microglia and astrocytes, but not neurons, supporting a glial origin and intrinsic LRRK2 mutant activity responsible for the elevated CSF lysosomal proteins. Furthermore, proteomics analysis of urine from humanized LRRK2G2019S transgenic mice identified lysosome and glycosphingolipid protein signatures shared with human LRRK2 PD patients. Collectively, our integrated proteomics reveals dynamic changes of functional biofluid signatures for LRRK2 PD, which enables the determination of biomarkers for early disease onset. The humanized LRRK2G2019S mice provide a valuable platform for biomarker refinement and therapeutic development.

Formation of templated inclusions in a forebrain α-synuclein mouse model is independent of LRRK2

Lysosomal dysfunction plays a central role in the pathogenesis of several neurodegenerative disorders, including Parkinson's disease (PD). Several genes linked to genetic forms of PD, including leucine-rich repeat kinase 2 (LRRK2), functionally converge on the lysosomal system. While mutations in LRRK2 are commonly associated with autosomal-dominant PD, the physiological and pathological functions of this kinase remain poorly understood. Here, we demonstrate that LRRK2 regulates lysosome size, number and function in astrocytes, which endogenously express high levels of LRRK2. Expression of LRRK2 G2019S, the most common pathological mutation, produces enlarged lysosomes and diminishes the lysosomal capacity of these cells. Enlarged lysosomes appears to be a common phenotype associated with pathogenic LRRK2 mutations, as we also observed this effect in cells expressing other LRRK2 mutations; R1441C or Y1699C. The lysosomal defects associated with these mutations are dependent on both the catalytic activity of the kinase and autophosphorylation of LRRK2 at serine 1292. Further, we demonstrate that blocking LRRK2's kinase activity, with the potent and selective inhibitor PF-06447475, rescues the observed defects in lysosomal morphology and function. The present study also establishes that G2019S mutation leads to a reduction in lysosomal pH and increased expression of the lysosomal ATPase ATP13A2, a gene linked to a parkinsonian syndrome (Kufor-Rakeb syndrome), in brain samples from mouse and human LRRK2 G2019S carriers. Together, these results demonstrate that PD-associated LRRK2 mutations perturb lysosome function in a kinase-dependent manner, highlighting the therapeutic promise of LRRK2 kinase inhibitors in the treatment of PD.

Parkinson's disease (PD) is characterized by the irreversible loss of dopaminergic neurons and the accumulation of α-synuclein in Lewy bodies. The oligomeric α-synuclein (O-αS) is the most toxic form of α-synuclein species, and it has been reported to be a robust inflammatory mediator. Mutations in Leucine-Rich Repeat Kinase 2 (LRRK2) are also genetically linked to PD and neuroinflammation. However, how O-αS and LRRK2 interact in glial cells remains unclear. Here, we reported that LRRK2 G2019S mutation, which is one of the most frequent causes of familial PD, enhanced the effects of O-αS on astrocytes both invivo and invitro. Meanwhile, inhibition of LRRK2 kinase activity could relieve the inflammatory effects of both LRRK2 G2019S and O-αS. We also demonstrated that nuclear factor κB (NF-κB) pathway might be involved in the neuroinflammatory responses. These findings revealed that inhibition of LRRK2 kinase activity may be a viable strategy for suppressing neuroinflammation in PD.

Low frequency of common LRRK2 mutations in Mexican patients with Parkinson's disease

Development of inducible leucine-rich repeat kinase 2 (LRRK2) cell lines for therapeutics development in Parkinson's disease.

Bacosides, constituents of Bacopa monnieri (Linn.), are reported to be potential therapeutic saponins in the cure of Parkinson’s disease (PD). However, detailed mechanism for control of PD by bacosides is not well documented. PD has been reported to be caused by genetic mutations in leucine-rich repeat kinase 2 (LRRK2) leading to higher kinase activity that has been identified as a major cause of familial PD. The LRRK2 was thus proposed as an important marker in the pathogenesis of PD. This suggests that inhibition of LRRK2 holds promise as a potential treatment for PD. Our study focuses on the possible application of bacoside A constituents as potential inhibitors of LRRK2. In this work, we have carried out the in silico molecular docking studies of bacoside A constituents with LRRK2, proposing their role as an inhibitor in PD. The study has revealed the significant interactions between bacosaponin and LRRK2 having ten H-bonds at receptor-ligand site with binding affinity −7.5 kcal/mol. Hence, amongst the studied triglycosidic saponins, bacosaponin was analyzed to be a better ligand, proposing it to be a major constituent in inhibiting enzymatic activities of mutated LRRK2.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene have emerged as a potential common cause for both sporadic and familial Parkinson’s Disease (PD) in different populations. The pleomorphic features exhibited by LRRK2 mutation carriers and the central role of Lrrk2 protein in the proper functioning of central nervous system suggest that mutations in this protein might be involved in multiple cellular processes leading to other neurodegenerative disorders than PD. The location of LRRK2 gene on chromosome 12, close to a linkage peak for familial late-onset Alzheimer’s Disease (AD), highlights that LRRK2 mutations might be involved in AD pathogenesis. We screened the most common LRRK2 mutation (p.G2019S) in a series of 180 consecutive patients clinically diagnosed with Alzheimer Disease (AD). We identified the p.G2019S in one AD patient with no PD signs, indicating that this mutation is not a common etiological factor for AD in our population (0.5%), corroborating recent data found in Norwegian, North American, Chinese and Italian populations. Nevertheless, these observations together with new information about the Lrrk2 critical multifunctionality do not rule out the possible influence of other variants within LRRK2 in AD, so that other screenings focusing in the whole extension of the LRRK2 using larger sized confirmed AD sample are urgently needed.

Rapid eye movement sleep behavior disorder and sleepiness precede or accompany idiopathic Parkinson's disease (PD), but their presence in subjects with leucine-rich repeat kinase 2 mutations is unknown. Ten patients with leucine-rich repeat kinase 2-associated PD, four healthy leucine-rich repeat kinase 2 mutation carriers, 20 patients with idiopathic PD, and 12 healthy controls underwent clinical assessments and a nighttime video-polysomnography. No sleep changes, no rapid eye movement sleep behavior disorder, or rapid eye movement sleep without atonia was found in the 14 subjects with leucine-rich repeat kinase 2mutations compared with controls, whereas 41% of patients with idiopathic PD had rapid eye movement sleep behavior disorder. Eventually, 20% of patients with leucine-rich repeat kinase 2-associated PD had abnormal periodic leg movements, a frequency similar to the idiopathic PD group frequency. The sleep phenotype in leucine-rich repeat kinase 2 mutations parallels that of idiopathic PD, except for absent rapid eye movement sleep behavior disorder here in the presymptomatic and symptomatic stages.