Dysregulation of the AP2M1 phosphorylation cycle by LRRK2 impairs endocytosis and leads to dopaminergic neurodegeneration

Mutations in the kinase LRRK2 and impaired endocytic trafficking are both implicated in the pathogenesis of Parkinson’s disease (PD). Expression of the PD-associated LRRK2 mutant in mouse dopaminergic neurons was shown to disrupt clathrin-mediated endocytic trafficking. Here, we explored the molecular mechanism linking LRRK2 to endocytosis and found that LRRK2 bound to and phosphorylated the μ2 subunit of the adaptor protein AP2 (AP2M1), a core component of the clathrin-mediated endocytic machinery. Analysis of human SH-SY5Y cells and mouse neurons and tissues revealed that loss of LRRK2 abundance or kinase function resulted in decreased phosphorylation of AP2M1, which is required for the initial formation of clathrin-coated vesicles (CCVs). In contrast, overexpression of LRRK2 or expression of a Parkinson’s disease–associated gain-of-function mutant LRRK2 (G2019S) inhibited the uncoating of AP2M1 from CCVs at later stages and prevented new cycles of CCV formation. Thus, the abundance and activity of LRRK2 must be calibrated to ensure proper endocytosis. Dysregulated phosphorylation of AP2M1 from the brain but not thyroid tissues of LRRK2 knockout and G2019S-knockin mice suggests a tissue-specific regulatory mechanism of endocytosis. Furthermore, we found that LRRK2-dependent phosphorylation of AP2M1 mediated dopaminergic neurodegeneration in a Drosophila model of PD. Together, our findings provide a mechanistic link between LRRK2, AP2, and endocytosis in the pathogenesis of PD.

Small heterodimer partner (SHP) aggravates ER stress in Parkinson’s disease-linked LRRK2 mutant astrocyte by regulating XBP1 SUMOylation

Background Endoplasmic reticulum (ER) stress is a common feature of Parkinson’s disease (PD), and several PD-related genes are responsible for ER dysfunction. Recent studies suggested LRRK2-G2019S, a pathogenic mutation in the PD-associated gene LRRK2, cause ER dysfunction, and could thereby contribute to the development of PD. It remains unclear, however, how mutant LRRK2 influence ER stress to control cellular outcome. In this study, we identified the mechanism by which LRRK2-G2019S accelerates ER stress and cell death in astrocytes. Methods To investigate changes in ER stress response genes, we treated LRRK2-wild type and LRRK2-G2019S astrocytes with tunicamycin, an ER stress-inducing agent, and performed gene expression profiling with microarrays. The XBP1 SUMOylation and PIAS1 ubiquitination were performed using immunoprecipitation assay. The effect of astrocyte to neuronal survival were assessed by astrocytes-neuron coculture and slice culture systems. To provide in vivo proof-of-concept of our approach, we measured ER stress response in mouse brain. Results Microarray gene expression profiling revealed that LRRK2-G2019S decreased signaling through XBP1, a key transcription factor of the ER stress response, while increasing the apoptotic ER stress response typified by PERK signaling. In LRRK2-G2019S astrocytes, the transcriptional activity of XBP1 was decreased by PIAS1-mediated SUMOylation. Intriguingly, LRRK2-GS stabilized PIAS1 by increasing the level of small heterodimer partner (SHP), a negative regulator of PIAS1 degradation, thereby promoting XBP1 SUMOylation. When SHP was depleted, XBP1 SUMOylation and cell death were reduced. In addition, we identified agents that can disrupt SHP-mediated XBP1 SUMOylation and may therefore have therapeutic activity in PD caused by the LRRK2-G2019S mutation. Conclusion Our findings reveal a novel regulatory mechanism involving XBP1 in LRRK2-G2019S mutant astrocytes, and highlight the importance of the SHP/PIAS1/XBP1 axis in PD models. These findings provide important insight into the basis of the correlation between mutant LRRK2 and pathophysiological ER stress in PD, and suggest a plausible model that explains this connection.

The E3 ligase TRIM1 ubiquitinates LRRK2 and controls its localization, degradation, and toxicity

Missense mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson’s Disease (PD); however, pathways regulating LRRK2 subcellular localization, function, and turnover are not fully defined. We performed quantitative mass spectrometry-based interactome studies to identify 48 novel LRRK2 interactors, including the microtubule-associated E3 ubiquitin ligase TRIM1 (Tripartite Motif Family 1). TRIM1 recruits LRRK2 to the microtubule cytoskeleton for ubiquitination and proteasomal degradation by binding LRRK2822-982, a flexible interdomain region we designate the “Regulatory Loop” (RL). Phosphorylation of LRRK2 Ser910/935 within LRRK2 RL serves as a molecular switch controlling LRRK2’s association with cytoplasmic 14-3-3 versus microtubule-bound TRIM1. Association with TRIM1 prevents upregulation of LRRK2 kinase activity by Rab29 and also rescues neurite outgrowth deficits caused by PD-driving mutant LRRK2 G2019S. Our data suggest that TRIM1 is a critical regulator of LRRK2, modulating its cytoskeletal recruitment, turnover, kinase activity, and cytotoxicity.

LRRK2 kinase activity regulates lysosomal glucocerebrosidase in neurons derived from Parkinson’s disease patients

Mutations in LRRK2 and GBA1 are common genetic risk factors for Parkinson’s disease (PD) and major efforts are underway to develop new therapeutics that target LRRK2 or glucocerebrosidase (GCase). Here we describe a mechanistic and therapeutic convergence of LRRK2 and GCase in neurons derived from patients with PD. We find that GCase activity was reduced in dopaminergic (DA) neurons derived from PD patients with LRRK2 mutations. Inhibition of LRRK2 kinase activity results in increased GCase activity in DA neurons with either LRRK2 or GBA1 mutations. This increase is sufficient to partially rescue accumulation of oxidized dopamine and alpha-synuclein in PD patient neurons. We have identified the LRRK2 substrate Rab10 as a key mediator of LRRK2 regulation of GCase activity. Together, these results suggest an important role of mutant LRRK2 as a negative regulator of lysosomal GCase activity.

Lrrk2 alleles modulate inflammation during microbial infection of mice in a sex-dependent manner

Variants in the leucine-rich repeat kinase-2 (LRRK2) gene are associated with Parkinson’s disease, leprosy, and Crohn’s disease, three disorders with inflammation as an important component. Because of its high expression in granulocytes and CD68-positive cells, LRRK2 may have a function in innate immunity. We tested this hypothesis in two ways. First, adult mice were intravenously inoculated with Salmonella typhimurium, resulting in sepsis. Second, newborn mouse pups were intranasally infected with reovirus (serotype 3 Dearing), which induced encephalitis. In both mouse models, wild-type Lrrk2 expression was protective and showed a sex effect, with female Lrrk2-deficient animals not controlling infection as well as males. Mice expressing Lrrk2 carrying the Parkinson’s disease–linked p.G2019S mutation controlled infection better, with reduced bacterial growth and longer animal survival during sepsis. This gain-of-function effect conferred by the p.G2019S mutation was mediated by myeloid cells and was abolished in animals expressing a kinase-dead Lrrk2 variant, p.D1994S. Mouse pups with reovirus-induced encephalitis that expressed the p.G2019S Lrrk2 mutation showed increased mortality despite lower viral titers. The p.G2019S mutant Lrrk2 augmented immune cell chemotaxis and generated more reactive oxygen species during virulent infection. Reovirus-infected brains from mice expressing the p.G2019S mutant Lrrk2 contained higher concentrations of α-synuclein. Animals expressing one or two p.D1994S Lrrk2 alleles showed lower mortality from reovirus-induced encephalitis. Thus, Lrrk2 alleles may alter the course of microbial infections by modulating inflammation, and this may be dependent on the sex and genotype of the host as well as the type of pathogen.

RAB8, RAB10 and RILPL1 contribute to both LRRK2 kinase–mediated centrosomal cohesion and ciliogenesis deficits

Mutations in the LRRK2 kinase are the most common cause of familial Parkinson’s disease, and variants increase risk for the sporadic form of the disease. LRRK2 phosphorylates multiple RAB GTPases including RAB8A and RAB10. Phosphorylated RAB10 is recruited to centrosome-localized RILPL1, which may interfere with ciliogenesis in a disease-relevant context. Our previous studies indicate that the centrosomal accumulation of phosphorylated RAB8A causes centrosomal cohesion deficits in dividing cells, including in peripheral patient-derived cells. Here, we show that both RAB8 and RAB10 contribute to the centrosomal cohesion deficits. Pathogenic LRRK2 causes the centrosomal accumulation not only of phosho-RAB8 but also of phospho-RAB10, and the effects on centrosomal cohesion are dependent on RAB8, RAB10 and RILPL1. Conversely, the pathogenic LRRK2-mediated ciliogenesis defects correlate with the centrosomal accumulation of both phospho-RAB8 and phospho-RAB10. LRRK2-mediated centrosomal cohesion and ciliogenesis alterations are observed in patient-derived peripheral cells, as well as in primary astrocytes from mutant LRRK2 mice, and are reverted upon LRRK2 kinase inhibition. These data suggest that the LRRK2-mediated centrosomal cohesion and ciliogenesis defects are distinct cellular readouts of the same underlying phospho-RAB8/RAB10/RILPL1 nexus and highlight the possibility that either centrosomal cohesion and/or ciliogenesis alterations may serve as cellular biomarkers for LRRK2-related PD.

Novel Cell-Based Assay for Identification of LRRK2 Inhibitors Using Its Aberrant Regulation of a Pluripotency Gene

Mutations in the gene encoding leucine-rich repeat kinase 2 ( LRRK2), such as the G2019S mutation, are the most common cause of familial Parkinson’s disease (PD). The G2019S mutation impairs neurite outgrowth. We hypothesized that those effects could be related to an altered expression of pluripotency genes, which may provide a readout for a screening assay based on LRRK2 function. Here, we show that the G2019S mutation mediates a sustained aberrant upregulation of the transcription factors Nanog and Oct4 that in wild-type are downregulated after differentiation. The aberrant regulation of Nanog can be concentration dependently reversed by LRRK2 tool inhibitors. Building on this knowledge, we developed an assay for the identification and assessment of compounds that inhibit the aberrant pathophysiological activity of mutant LRRK2. Furthermore, the aberrant neural pluripotency is consistent with Parkinson’s pathophysiology and with the epidemiological association between the G2019S genotype and cancer risk.

Mitochondrial clearance and maturation of autophagosomes are compromised in LRRK2 G2019S familial Parkinson’s disease patient fibroblasts

This study utilized human fibroblasts as a preclinical discovery and diagnostic platform for identification of cell biological signatures specific for the LRRK2 G2019S mutation producing Parkinson’s disease (PD). Using live cell imaging with a pH-sensitive Rosella biosensor probe reflecting lysosomal breakdown of mitochondria, mitophagy rates were found to be decreased in fibroblasts carrying the LRRK2 G2019S mutation compared to cells isolated from healthy subject (HS) controls. The mutant LRRK2 increased kinase activity was reduced by pharmacological inhibition and targeted antisense oligonucleotide treatment, which normalized mitophagy rates in the G2019S cells and also increased mitophagy levels in HS cells. Detailed mechanistic analysis showed a reduction of mature autophagosomes in LRRK2 G2019S fibroblasts, which was rescued by LRRK2 specific kinase inhibition. These findings demonstrate an important role for LRRK2 protein in regulation of mitochondrial clearance by the lysosomes, which is hampered in PD with the G2019S mutation. The current results are relevant for cell phenotypic diagnostic approaches and potentially for stratification of PD patients for targeted therapy.