The leucine-rich repeat kinase 2 (
), the most common causative gene for autosomal-dominant familial Parkinson's disease, encodes a large protein kinase harboring multiple characteristic domains. ...LRRK2 phosphorylates a set of Rab GTPases in cells, which is enhanced by the Parkinson-associated LRRK2 mutations. Accumulating evidence suggests that LRRK2 regulates intracellular vesicle trafficking and organelle maintenance including Golgi, endosomes and lysosomes. Furthermore, genetic knockout or inhibition of LRRK2 cause lysosomal abnormalities in rodents and primates, and cells from Parkinson's patients with LRRK2 mutations also exhibit altered lysosome morphology. Cell biological studies on LRRK2 in a diverse cellular context further strengthen the potential connection between LRRK2 and regulation of the endolysosomal system, part of which is mediated by Rab phosphorylation by LRRK2. We will focus on the latest advances on the role of LRRK2 and Rab in relation to the endolysosomal system, and discuss the possible link to the pathomechanism of Parkinson's disease.
Mutations in leucine-rich repeat kinase 2 (LRRK2) are the major genetic cause of autosomal-dominantly inherited Parkinson's disease. LRRK2 is implicated in the regulation of intracellular ...trafficking, neurite outgrowth and PD risk in connection with Rab7L1, a putative interactor of LRRK2. Recently, a subset of Rab GTPases have been reported as substrates of LRRK2. Here we examine the kinase activity of LRRK2 on Rab7L1 in situ in cells. Phos-tag analyses and metabolic labeling assays revealed that LRRK2 readily phosphorylates Golgi-localized wild-type Rab7L1 but not mutant forms that are distributed in the cytoplasm. In vitro assays demonstrated direct phosphorylation of Rab7L1 by LRRK2. Subsequent screening using Rab7L1 mutants harboring alanine-substitution for every single Ser/Thr residue revealed that Ser72 is a major phosphorylation site, which was confirmed by using a phospho-Ser72-specific antibody. Moreover, LRRK2 pathogenic Parkinson mutants altogether markedly enhanced the phosphorylation at Ser72. The modulation of Ser72 phosphorylation in Rab7L1 resulted in an alteration of the morphology and distribution of the trans-Golgi network. These data collectively support the involvement of Rab7L1 phosphorylation in the LRRK2-mediated cellular and pathogenetic mechanisms.
•Rab7L1 is identified as a bona fide substrate of LRRK2.•Ser72 is a major in-cell phosphorylation site of Rab7L1.•Parkinson's-linked mutations in LRRK2 increase the Ser72 phosphorylation of Rab7L1.•Rab7L1 phosphorylation at Ser72 modifies the trans-Golgi morphology.
Previous genetic studies on hereditary Parkinson's disease (PD) have identified a set of pathogenic gene mutations that have strong impacts on the pathogenicity of PD. In addition, genome-wide ...association studies (GWAS) targeted to sporadic PD have nominated an increasing number of genetic variants that influence PD susceptibility. Although the clinical and pathological characteristics in hereditary PD are not identical to those in sporadic PD, α-synuclein, and LRRK2 are definitely associated with both types of PD, with
LRRK2
mutations being the most frequent cause of autosomal-dominant PD. On the other hand, a significant portion of risk genes identified from GWAS have been associated with lysosomal functions, pointing to a critical role of lysosomes in PD pathogenesis. Experimental studies have suggested that the maintenance or upregulation of lysosomal activity may protect against neuronal dysfunction or degeneration. Here we focus on the roles of representative PD gene products that are implicated in lysosomal pathway, namely LRRK2, VPS35, ATP13A2, and glucocerebrosidase, and provide an overview of their disease-associated functions as well as their cooperative actions in the pathogenesis of PD, based on the evidence from cellular and animal models. We also discuss future perspectives of targeting lysosomal activation as a possible strategy to treat neurodegeneration.
Over the last decades, research on the pathobiology of neurodegenerative diseases has greatly evolved, revealing potential targets and mechanisms linked to their pathogenesis. Parkinson's disease ...(PD) is no exception, and recent studies point to the involvement of endolysosomal defects in PD. The endolysosomal system, which tightly controls a flow of endocytosed vesicles targeted either for degradation or recycling, is regulated by a number of Rab GTPases. Their associations with leucine-rich repeat kinase 2 (LRRK2), a major causative and risk protein of PD, has also been one of the hot topics in the field. Understanding their interactions and functions is critical for unraveling their contribution to PD pathogenesis. In this review, we summarize recent studies on LRRK2 and Rab GTPases and attempt to provide more insight into the interaction of LRRK2 with each Rab and its relationship to PD.
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.
•LRRK2 phosphorylation of Rab10 is stimulated by clinically used lysosomotropic drugs.•Lysosomotropic drug treatment commonly induces LRRK2-mediated lysosomal release.•Lysosomotropic drug treatment increases LRRK2-Rab proximity but not LRRK2 activity.•An upstream activator Rab29 mediates a drug-induced increase in Rab10 phosphorylation
Leucine-rich repeat kinase 2 (LRRK2) has been linked to several clinical disorders including Parkinson's disease (PD), Crohn's disease, and leprosy. Furthermore in rodents, LRRK2 deficiency or ...inhibition leads to lysosomal pathology in kidney and lung. Here we provide evidence that LRRK2 functions together with a second PD-associated gene, RAB7L1, within an evolutionarily conserved genetic module in diverse cellular contexts. In C. elegans neurons, orthologues of LRRK2 and RAB7L1 act coordinately in an ordered genetic pathway to regulate axonal elongation. Further genetic studies implicated the AP-3 complex, which is a known regulator of axonal morphology as well as of intracellular protein trafficking to the lysosome compartment, as a physiological downstream effector of LRRK2 and RAB7L1. Additional cell-based studies implicated LRRK2 in the AP-3 complex-related intracellular trafficking of lysosomal membrane proteins. In mice, deficiency of either RAB7L1 or LRRK2 leads to prominent age-associated lysosomal defects in kidney proximal tubule cells, in the absence of frank CNS pathology. We hypothesize that defects in this evolutionarily conserved genetic pathway underlie the diverse pathologies associated with LRRK2 in humans and in animal models.
Leucine-rich repeat kinase 2 (LRRK2) is extensively phosphorylated in cells within a region amino-terminal to the leucine-rich repeat domain. Since phosphorylation in this region of LRRK2, including ...Ser910, Ser935, Ser955, and Ser973, is significantly downregulated upon treatment with inhibitors of LRRK2, it has been hypothesized that signaling pathways downstream of the kinase activity of LRRK2 are involved in regulating the phosphorylation of LRRK2, although the precise mechanism has remained unknown. Here we examined the effects of LRRK2 inhibitors on the phosphorylation state at Ser910, Ser935, and Ser955 in a series of kinase-inactive mutants of LRRK2. We found that the responses of LRRK2 to the inhibitors varied among mutants, in a manner not consistent with the above-mentioned hypothesis. Notably, one of the kinase-inactive mutants, T2035A LRRK2, underwent phosphorylation, as well as the inhibitor-induced dephosphorylation, at Ser910, Ser935, and Ser955, to a similar extent to those observed with wild-type LRRK2. These results suggest that the kinase activity of LRRK2 is not involved in the common mechanism of inhibitor-induced dephosphorylation of LRRK2.
Mutations in leucine-rich repeat kinase 2 (LRRK2) cause autosomal-dominant familial Parkinson's disease. We generated lines of Caenorhabditis elegans expressing neuronally directed human LRRK2. ...Expressing human LRRK2 increased nematode survival in response to rotenone or paraquat, which are agents that cause mitochondrial dysfunction. Protection by G2019S, R1441C, or kinase-dead LRRK2 was less than protection by wild-type LRRK2. Knockdown of lrk-1, the endogenous ortholog of LRRK2 in C. elegans, reduced survival associated with mitochondrial dysfunction. C. elegans expressing LRRK2 showed rapid loss of dopaminergic markers (DAT::GFP fluorescence and dopamine levels) beginning in early adulthood. Loss of dopaminergic markers was greater for the G2019S LRRK2 line than for the wild-type line. Rotenone treatment induced a larger loss of dopamine markers in C. elegans expressing G2019S LRRK2 than in C. elegans expressing wild-type LRRK2; however, loss of dopaminergic markers in the G2019S LRRK2 nematode lines was not statistically different from that in the control line. These data suggest that LRRK2 plays an important role in modulating the response to mitochondrial inhibition and raises the possibility that mutations in LRRK2 selectively enhance the vulnerability of dopaminergic neurons to a stressor associated with Parkinson's disease.
The pathological aggregation of tau characterizes a set of neurodegenerative diseases collectively referred to as tauopathies. Recent studies using cellular and animal models have suggested that tau ...pathology progresses by
-cellular propagation. The process of propagation is mediated by certain species of extracellular tau, which are taken up by recipient cells and serve as a seed for tau aggregation. Tau propagation is currently one of the most active areas of research in dementia. Previous efforts to identify the specific tau molecules involved in propagation have suggested that multiple forms of tau with different molecular weights derived from recombinant tau or brain lysates exert seeding activity. Nonetheless, the molecular characteristics of the "extracellular" seed-competent tau as well as its release mechanisms remain to be elucidated. Given that tau is physiologically released into the extracellular space, it is critical to distinguish seed-competent tau from normal monomeric tau. Utilizing biosensor cells expressing P301S mutant tau fused to CFP/YFP, here we discriminated between seed-competent tau and inert monomer tau released from HEK293 cells. By analyzing the size-exclusion fractions of the media, we found that seed-competent tau was enriched in high molecular weight fractions of >2,000 kDa, while the majority of soluble tau in the media positively detected by ELISA was in low molecular weight fractions. We also found that lysosomal stress not only increased Ca
-dependent release of seed-competent tau but also altered its molecular size. Inhibiting lysosomal exocytosis specifically decreased release of seed-competent tau without influencing total tau. These data underscore the differential response of seed-competent tau and inert tau to lysosomal stress and indicates the presence of distinct release mechanisms via lysosomes.
α-Synuclein and LRRK2 are associated with both familial and sporadic Parkinson’s disease (PD), although the mechanistic link between these two proteins has remained elusive. Treating cells with ...lysosomotropic drugs causes the recruitment of LRRK2 and its substrate Rab10 onto overloaded lysosomes and induces extracellular release of lysosomal contents. Here we show that lysosomal overload elicits the release of insoluble α-synuclein from macrophages and microglia loaded with α-synuclein fibrils. This release occurred specifically in macrophage lineage cells, was dependent on the LRRK2-Rab10 pathway and involved exosomes. Also, the uptake of α-synuclein fibrils enhanced the LRRK2 phosphorylation of Rab10, which was accompanied by an increased recruitment of LRRK2 and Rab10 onto lysosomal surface. Our data collectively suggest that α-synuclein fibrils taken up in lysosomes activate the LRRK2-Rab10 pathway, which in turn upregulates the extracellular release of α-synuclein aggregates, leading to a vicious cycle that could enhance α-synuclein propagation in PD pathology.
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•Lysosomal stress elicits the exocytic release of pre-loaded insoluble α-synuclein•α-Synuclein release occurs specifically in macrophages and microglial cells•The release is mediated via the LRRK2-Rab10 pathway and involves exosomes•α-Synuclein fibril uptake induces the activation and lysosomal recruitment of LRRK2
Molecular biology; Cell biology
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