Lysosomal stress drives the release of pathogenic α-synuclein from macrophage lineage cells via the LRRK2-Rab10 pathway
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ABSTRACT:
SUBMITTER: Abe T
PROVIDER: S-EPMC10835446 | biostudies-literature | 2024 Jan
REPOSITORIES: biostudies-literature
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Project description:Genetic variation at the leucine-rich repeat kinase 2 (LRRK2) locus contributes to an enhanced risk of familial and sporadic Parkinson's disease. Previous data have demonstrated that recruitment to various membranes of the endolysosomal system results in LRRK2 activation. However, the mechanism(s) underlying LRRK2 activation at endolysosomal membranes and the cellular consequences of these events are still poorly understood. Here, we directed LRRK2 to lysosomes and early endosomes, triggering both LRRK2 autophosphorylation and phosphorylation of the direct LRRK2 substrates Rab10 and Rab12. However, when directed to the lysosomal membrane, pRab10 was restricted to perinuclear lysosomes, whereas pRab12 was visualized on both peripheral and perinuclear LRRK2+ lysosomes, suggesting that lysosomal positioning provides additional regulation of LRRK2-dependent Rab phosphorylation. Anterograde transport of lysosomes to the cell periphery by increasing the expression of ARL8B and SKIP or by knockdown of JIP4 blocked the recruitment and phosphorylation of Rab10 by LRRK2. The absence of pRab10 from the lysosomal membrane prevented the formation of a lysosomal tubulation and sorting process we previously named LYTL. Conversely, overexpression of RILP resulted in lysosomal clustering within the perinuclear area and increased LRRK2-dependent Rab10 recruitment and phosphorylation. The regulation of Rab10 phosphorylation in the perinuclear area depends on counteracting phosphatases, as the knockdown of phosphatase PPM1H significantly increased pRab10 signal and lysosomal tubulation in the perinuclear region. Our findings suggest that LRRK2 can be activated at multiple cellular membranes, including lysosomes, and that lysosomal positioning further provides the regulation of some Rab substrates likely via differential phosphatase activity or effector protein presence in nearby cellular compartments.
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Project description:Mutations in LRRK2 increase its kinase activity and cause Parkinson's disease. LRRK2 phosphorylates a subset of Rab proteins which allows for their binding to RILPL1. The phospho-Rab/RILPL1 interaction causes deficits in ciliogenesis and interferes with the cohesion of duplicated centrosomes. We show here that centrosomal deficits mediated by pathogenic LRRK2 can also be observed in patient-derived iPS cells, and we have used transiently transfected cell lines to identify the underlying mechanism. The LRRK2-mediated centrosomal cohesion deficits are dependent on both the GTP conformation and phosphorylation status of the Rab proteins. Pathogenic LRRK2 does not displace proteinaceous linker proteins which hold duplicated centrosomes together, but causes the centrosomal displacement of CDK5RAP2, a protein critical for centrosome cohesion. The LRRK2-mediated centrosomal displacement of CDK5RAP2 requires RILPL1 and phospho-Rab proteins, which stably associate with centrosomes. These data provide fundamental information as to how pathogenic LRRK2 alters the normal physiology of a cell.
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Project description:Considerable evidence supports the release of pathogenic aggregates of the neuronal protein α-Synuclein (αSyn) into the extracellular space. While this release is proposed to instigate the neuron-to-neuron transmission and spread of αSyn pathology in synucleinopathies including Parkinson's disease, the molecular-cellular mechanism(s) remain unclear. To study this, we generated a new mouse model to specifically immunoisolate neuronal lysosomes, and established a long-term culture model where αSyn aggregates are produced within neurons without the addition of exogenous fibrils. We show that neuronally generated pathogenic species of αSyn accumulate within neuronal lysosomes in mouse brains and primary neurons. We then find that neurons release these pathogenic αSyn species via SNARE-dependent lysosomal exocytosis. The released aggregates are non-membrane enveloped and seeding-competent. Additionally, we find that this release is dependent on neuronal activity and cytosolic Ca2+. These results propose lysosomal exocytosis as a central mechanism for the release of aggregated and degradation-resistant proteins from neurons.
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Project description:Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) cause familial Parkinson's disease, and sequence variations are associated with the sporadic form of the disease. LRRK2 phosphorylates a subset of RAB proteins implicated in secretory and recycling trafficking pathways, including RAB8A and RAB10. Another RAB protein, RAB29, has been reported to recruit LRRK2 to the Golgi, where it stimulates its kinase activity. Our previous studies revealed that G2019S LRRK2 expression or knockdown of RAB8A deregulate epidermal growth factor receptor (EGFR) trafficking, with a concomitant accumulation of the receptor in a RAB4-positive recycling compartment. Here, we show that the G2019S LRRK2-mediated EGFR deficits are mimicked by knockdown of RAB10 and rescued by expression of active RAB10. By contrast, RAB29 knockdown is without effect, but expression of RAB29 also rescues the pathogenic LRRK2-mediated trafficking deficits independently of Golgi integrity. Our data suggest that G2019S LRRK2 deregulates endolysosomal trafficking by impairing the function of RAB8A and RAB10, while RAB29 positively modulates non-Golgi-related trafficking events impaired by pathogenic LRRK2.
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Project description:Missense mutations in the multi-domain kinase LRRK2 cause late onset familial Parkinson's disease. They most commonly with classic proteinopathy in the form of Lewy bodies and Lewy neurites comprised of insoluble α-synuclein, but in rare cases can also manifest tauopathy. The normal function of LRRK2 has remained elusive, as have the cellular consequences of its mutation. Data from LRRK2 null model organisms and LRRK2-inhibitor treated animals support a physiological role for LRRK2 in regulating lysosome function. Since idiopathic and LRRK2-linked PD are associated with the intraneuronal accumulation of protein aggregates, a series of critical questions emerge. First, how do pathogenic mutations that increase LRRK2 kinase activity affect lysosome biology in neurons? Second, are mutation-induced changes in lysosome function sufficient to alter the metabolism of α-synuclein? Lastly, are changes caused by pathogenic mutation sensitive to reversal with LRRK2 kinase inhibitors? Here, we report that mutation of LRRK2 induces modest but significant changes in lysosomal morphology and acidification, and decreased basal autophagic flux when compared to WT neurons. These changes were associated with an accumulation of detergent-insoluble α-synuclein and increased neuronal release of α-synuclein and were reversed by pharmacologic inhibition of LRRK2 kinase activity. These data demonstrate a critical and disease-relevant influence of native neuronal LRRK2 kinase activity on lysosome function and α-synuclein homeostasis. Furthermore, they also suggest that lysosome dysfunction, altered neuronal α-synuclein metabolism, and the insidious accumulation of aggregated protein over decades may contribute to pathogenesis in this late-onset form of familial PD.
| S-EPMC5803451 | biostudies-literature
Project description:In Parkinson's disease (PD) misfolded alpha-synuclein (aSyn) accumulates in the substantia nigra, where dopaminergic neurons are progressively lost. The mechanisms underlying aSyn pathology are still unclear, but they are hypothesized to involve the autophagy-lysosome pathway (ALP). LRRK2 mutations are a major cause of familial and sporadic PD, and LRRK2 kinase activity has been shown to be involved in pS129-aSyn inclusion modulation. We observed selective downregulation of the novel PD risk factor RIT2 in vitro and in vivo. Rit2 overexpression in G2019S-LRRK2 cells rescued ALP abnormalities and diminished aSyn inclusions. In vivo, viral mediated overexpression of Rit2 operated neuroprotection against AAV-A53T-aSyn. Furthermore, Rit2 overexpression prevented the A53T-aSyn-dependent increase of LRRK2 kinase activity in vivo. On the other hand, reduction of Rit2 levels leads to defects in the ALP, similar to those induced by the G2019S-LRRK2 mutation. Our data indicate that Rit2 is required for correct lysosome function, inhibits overactive LRRK2 to ameliorate ALP impairment, and counteracts aSyn aggregation and related deficits. Targeting Rit2 could represent an effective strategy to combat neuropathology in familial and idiopathic PD.
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Project description:Pathologic accumulation of alpha-synuclein is a feature of human parkinsonism and other neurodegenerative diseases. This accumulation may be counteracted by mechanisms of protein degradation that have been investigated in vitro but remain to be elucidated in animal models. In this study, lysosomal clearance of alpha-synuclein in vivo was indicated by the detection of alpha-synuclein in the lumen of lysosomes isolated from the mouse midbrain. When neuronal alpha-synuclein expression was enhanced as a result of toxic injury (i.e. treatment of mice with the herbicide paraquat) or transgenic protein overexpression, the intralysosomal content of alpha-synuclein was also significantly increased. This effect was paralleled by a marked elevation of the lysosome-associated membrane protein type 2A (LAMP-2A) and the lysosomal heat shock cognate protein of 70 kDa (hsc70), two essential components of chaperone-mediated autophagy (CMA). Immunofluorescence microscopy revealed an increase in punctate (lysosomal) LAMP-2A staining that co-localized with alpha-synuclein within nigral dopaminergic neurons of paraquat-treated and alpha-synuclein-overexpressing animals. The data provide in vivo evidence of lysosomal degradation of alpha-synuclein under normal conditions and, quite importantly, under conditions of enhanced protein burden. In the latter, increased lysosomal clearance of alpha-synuclein was mediated, at least in part, by CMA induction. It is conceivable that these neuronal mechanisms of protein clearance play an important role in neurodegenerative processes characterized by abnormal alpha-synuclein buildup.
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Project description:Mutations that activate LRRK2 protein kinase cause Parkinson's disease. We showed 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 the loss of cilia seen upon serum readdition. 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, and 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; LRRK2 blockade of CP110 uncapping requires Rab10 and RILPL1 proteins and is due to failure to recruit TTBK2, a kinase needed for CP110 release. In contrast, deciliation probability does not change in cells lacking Rab10 or RILPL1 and relies on a distinct LRRK2 pathway. 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.
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Project description:Backgroundα-Synuclein (SNCA) gene hypomethylation was reported in idiopathic Parkinson's disease (iPD). Based on a high clinical resemblance between iPD and leucine-rich repeat kinase 2 (LRRK2)-driven Parkinson's disease (L2PD), we investigated the epigenetic status of SNCA in an extensive LRRK2 clinical cohort from Spain.MethodsWe assessed the methylation levels of 23 CpG sites in the SNCA promoter region using peripheral blood DNA from L2PD patients (n = 151), LRRK2 nonmanifesting carriers (n = 55), iPD patients (n = 115), and healthy control subjects (n = 154) (total: N = 475).ResultsCompared with control subjects, we found significant SNCA hypomethylation in 11 of 23 CpGs in L2PD (48%), whereas 22 CpGs (96%) were hypomethylated in iPD. In line with a healthy status, asymptomatic mutation carriers had similar SNCA methylation profiles to control subjects.ConclusionsThis study shows for the first time that SNCA hypomethylation occurs in patients with L2PD. Further studies addressing SNCA methylation status in additional worldwide LRRK2 cohorts are warranted. © 2024 The Author(s). Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.
| S-EPMC11926512 | biostudies-literature
Project description:Propagation of α-synuclein aggregates has been suggested as a contributing factor in Parkinson's disease (PD) progression. However, the molecular mechanisms underlying α-synuclein aggregation are not fully understood. Here, we demonstrate in cell culture, nematode, and rodent models of PD that leucine-rich repeat kinase 2 (LRRK2), a PD-linked kinase, modulates α-synuclein propagation in a kinase activity-dependent manner. The PD-linked G2019S mutation in LRRK2, which increases kinase activity, enhances propagation efficiency. Furthermore, we show that the role of LRRK2 in α-synuclein propagation is mediated by RAB35 phosphorylation. Constitutive activation of RAB35 overrides the reduced α-synuclein propagation phenotype in lrk-1 mutant C. elegans. Finally, in a mouse model of synucleinopathy, administration of an LRRK2 kinase inhibitor reduced α-synuclein aggregation via enhanced interaction of α-synuclein with the lysosomal degradation pathway. These results suggest that LRRK2-mediated RAB35 phosphorylation is a potential therapeutic target for modifying disease progression.
| S-EPMC6110743 | biostudies-literature