Mutations in LRRK2 linked to Parkinson’s disease sequester Rab8a to damaged lysosomes and regulate transferrin-mediated iron uptake in microglia
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PROVIDER: PRJNA774526 | ENA |
REPOSITORIES: ENA
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Project description:Mutations in leucine-rich repeat kinase 2 (LRRK2) cause autosomal dominant Parkinson’s disease (PD) while polymorphic LRRK2 variants are associated with sporadic PD. PD-linked mutations increase LRRK2 kinase activity and induce neurotoxicity in vitro and in vivo. The small GTPase Rab8a is a LRRK2 kinase substrate and is involved in receptor-mediated recycling and endocytic trafficking of transferrin, but the effect of PD-linked LRRK2 mutations on the function of Rab8a is poorly understood. Here, we show that gain-of-function mutations in LRRK2 induce sequestration of endogenous Rab8a to lysosomes in over-expression cell models while pharmacological inhibition of LRRK2 kinase activity reverses this phenotype. Furthermore, we show that LRRK2 mutations drive association of endocytosed transferrin with Rab8a-positive lysosomes. LRRK2 has been nominated as an integral part of cellular responses downstream of proinflammatory signals and is activated in microglia in post-mortem PD tissue. Here, we show that iPSC-derived microglia from patients carrying the most common LRRK2 mutation, G2019S, mistraffic transferrin to lysosomes proximal to the nucleus in proinflammatory conditions. Furthermore, G2019S knock-in mice show a significant increase in iron deposition in microglia following intrastriatal LPS injection compared to wild type mice, accompanied by striatal accumulation of ferritin. Conclusion: Our data support a role of LRRK2 in modulating iron uptake and storage in response to proinflammatory stimuli in microglia. Neuroinflammation remodels endolysosomal gene expression in microglia, in vitro and in vivo.
2021-10-29 | GSE186559 | GEO
Project description:Mutations in leucine-rich repeat kinase 2 (LRRK2) cause autosomal dominant Parkinson disease (PD), while polymorphic LRRK2 variants are associated with sporadic PD. PD-linked mutations increase LRRK2 kinase activity and induce neurotoxicity in vitro and in vivo. The small GTPase Rab8a is a LRRK2 kinase substrate and is involved in receptor-mediated recycling and endocytic trafficking of transferrin, but the effect of PD-linked LRRK2 mutations on the function of Rab8a is poorly understood. Here, we show that gain-of-function mutations in LRRK2 induce sequestration of endogenous Rab8a to lysosomes in overexpression cell models, while pharmacological inhibition of LRRK2 kinase activity reverses this phenotype. Furthermore, we show that LRRK2 mutations drive association of endocytosed transferrin with Rab8a-positive lysosomes. LRRK2 has been nominated as an integral part of cellular responses downstream of proinflammatory signals and is activated in microglia in postmortem PD tissue. Here, we show that iPSC-derived microglia from patients carrying the most common LRRK2 mutation, G2019S, mistraffic transferrin to lysosomes proximal to the nucleus in proinflammatory conditions. Furthermore, G2019S knock-in mice show a significant increase in iron deposition in microglia following intrastriatal LPS injection compared to wild-type mice, accompanied by striatal accumulation of ferritin. Our data support a role of LRRK2 in modulating iron uptake and storage in response to proinflammatory stimuli in microglia.
| S-EPMC8675653 | biostudies-literature
Project description:Lysosomal membrane permeabilization (LMP) or lysosomal membrane damage is commonly associated with aging and age-related diseases. In searching for cellular mechanisms in response to LMP, we used a proteomic approach to identify proteins enriched on damaged lysosomes. This unbiased approach identified a new phosphoinositide signaling pathway triggered by LMP to mediate rapid lysosomal repair. Specifically, LMP induces fast lysosomal recruitment of PI4K2A which generates high levels of the lipid messenger phosphatidylinositol-4-phosphate (PtdIns4P) on damaged lysosomes. Lysosomal PtdIns4P in turn recruits multiple oxysterol-binding protein (OSBP)-related protein (ORP) family members, including ORP9, ORP10, and ORP11, to orchestrate extensive membrane contact sites (MCSs) between damaged lysosomes and the endoplasmic reticulum (ER). The ORPs subsequently catalyze robust ER-to-lysosomal transport of phosphatidylserine (PS), which is critical for rapid lysosomal repair. The lipid transporter ATG2 is also recruited to damaged lysosomes, activated by PS, and is essential for rapid lysosomal repair. Our findings identify a phosphoinositide-dependent membrane tethering and lipid transport (PITT) pathway essential for the maintenance of lysosomal membrane integrity, with important implications for a wide range of diseases characterized by impaired lysosomal function.
2022-09-07 | PXD028852 | Pride
Project description:Leucine-rich repeat kinase 2 (LRRK2) variants associated with Parkinson's disease (PD) and Crohn's disease lead to increased phosphorylation of its Rab substrates. While it has been recently shown that perturbations in cellular homeostasis including lysosomal damage can increase LRRK2 activity and localization to lysosomes, the molecular mechanisms by which LRRK2 activity is regulated have remained poorly defined. We performed a targeted siRNA screen to identify regulators of LRRK2 activity and identified Rab12 as a novel modulator of LRRK2-dependent phosphorylation of one of its substrates, Rab10. Using a combination of imaging and immunopurification methods to isolate lysosomes, we demonstrated that Rab12 is actively recruited to damaged lysosomes and leads to a local and LRRK2-dependent increase in Rab10 phosphorylation. PD-linked variants, including LRRK2 R1441G and VPS35 D620N, lead to increased recruitment of LRRK2 to the lysosome and a local elevation in lysosomal levels of pT73 Rab10. Together, these data suggest a conserved mechanism by which Rab12, in response to damage or expression of PD-associated variants, facilitates the recruitment of LRRK2 and phosphorylation of its Rab substrate(s) at the lysosome.
| S-EPMC10708889 | biostudies-literature
Project description:LRRK2-G2019S (LRRK2-GS), a pathogenic mutation in the PD-associated gene LRRK2, promote ER stress-induced cell death by reducing transcriptional activity of X-box-binding protein 1 (XBP1), a key transcription factor of the UPR. We used microarrays to detail the programme of gene expression and identified distinct classes of up-regulated or down-regulated genes during ER stress.
2019-10-31 | GSE139579 | GEO
Project description:G2019S mutaion of LRRK2 is known to increase mRNA translation. We perform ribosome profiling to study defective translation in mammalian LRRK2 models. We used G2019S LRRK2 transgenic mice, G2019S/D1994A LRRK2 transgenic mice, LRRK2 knockout mice.
2021-02-27 | GSE167704 | GEO
Project description:BACKGROUND: Previous data implicates neuroinflammation in the pathogenesis of Parkinson’s disease (PD). Of the genes associated with PD, Leucine rich repeat kinase 2 (LRRK2) is expressed in microglia, resident brain inflammatory cells. However, the extent to which LRRK2 affects neuroinflammatory responses is unclear. OBJECTIVE: To examine whether mutations in Lrrk2 affect responses to neuroinflammation in vivo. METHODS: We injected cohorts of mice carrying mutations in Lrrk2, homologous to those causing human PD, with a single intrastriatal injection of lipopolysaccharide (LPS) or control. We used single cell RNA-Sequencing to examine cell type specific responses to treatment and genotype and validated key results with orthogonal approaches. RESULTS: We found that our chosen paradigm of acute LPS exposure evokes robust transcriptional changes consistent with a multicellular neuroinflammatory response. We also found evidence of peripheral immune cell recruitment into the brain and interaction with brain-resident microglia. However, the transcriptional effects of Lrrk2 mutations were limited to small numbers of genes, including down regulation of gene ontogeny terms related to lysosomes, predominantly in microglia. CONCLUSIONS: Our data clearly demonstrate that many cells in the brain respond to a single inflammatory insult with strong transcriptional responses and that, even in a model focussed on CNS injection, there is interaction between peripheral and central immune cells. In contrast, the quantitative effects of Lrrk2 mutations are modest at least at the level of transcription, demonstrating that additional studies are needed to clarify whether Lrrk2 affects neuroinflammation in an endogenous context.
2024-04-29 | GSE265833 | GEO
Project description:Removal of damaged organelles via the process of selective autophagy constitutes a major form of cellular quality control. Damaged organelles are recognized by a dedicated surveillance machinery, leading to the assembly of an autophagosome around the damaged organelle, prior to fusion with the degradative lysosomal compartment. Lysosomes themselves are also prone to damage and are degraded through the process of lysophagy. While early steps involve recognition of ruptured lysosomal membranes by glycan-binding Galectins and ubiquitylation of transmembrane lysosomal proteins, many steps in the process, and their inter-relationships, remain poorly understood, including the role and identity of cargo receptors required for completion of lysophagy. Here, we employ quantitative organelle capture and proximity biotinylation proteomics of autophagy adaptors, cargo receptors, and Galectins in response to acute lysosomal damage, thereby revealing the landscape of lysosomal proteome remodeling during lysophagy. Among proteins dynamically recruited to damaged lysosomes were ubiquitin-binding autophagic cargo receptors. Using newly developed lysophagic flux reporters including Lyso-Keima, we demonstrate that TAX1BP1, together with its associated kinase TBK1, are both necessary and sufficient to promote lysophagic flux in both Hela cells and induced neurons (iNeurons). While the related receptor OPTN can drive damage-dependent lysophagy when overexpressed, cells lacking either OPTN or CALCOCO2 still maintain significant lysophagic flux in HeLa cells. Mechanistically, TAX1BP1-driven lysophagy requires its N-terminal SKICH domain, which binds both TBK1 and the autophagy regulatory factor RB1CC1, and requires upstream ubiquitylation events for efficient recruitment and lysophagic flux. These results identify TAX1BP1 as a central component in the lysophagy pathway and provide a proteomic resource for future studies of the lysophagy process.
2021-09-23 | PXD027476 | Pride
Project description:Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most frequent cause of familial and sporadic ParkinsonM-bM-^@M-^Ys disease (PD). Here, we investigated in parallel gene and microRNA transcriptome profiles of three different LRRK2 mouse models. Striatal tissue was isolated from adult LRRK2 knockout mice, as well as mice expressinghuman LRRK2 wildtype (hLRRK2-WT) or PD-associated R1441G mutation (hLRRK2-R1441G). We used microarraya to measure gene and microRNA expression levels in various LRRK2 mouse models. LRRK2 wildtype and knockout mice were bred on a C57BL/6 background. LRRK2 non-transgenic and hLRRK2 transgenic mice were bred on a FVB/N background.
2013-11-22 | E-GEOD-52584 | biostudies-arrayexpress
Project description:Recent advances in generating 3 dimensional (3D) organoid systems from stem cells offer new possibilities for disease modeling. In this study, we generate isogenic 3D midbrain organoids with or without a Parkinson’s disease-associated LRRK2 G2019S mutation. LRRK2-G2019S midbrain organoids derived from LRRK2 targeted human iPSCs in vitro have LRRK2-associated sporadic Parkinson's disease phenotypes. Midbrain-like 3D organoids expressing LRRK2-G2019S showed dynamic changes in globle gene expression.
2019-02-02 | GSE125234 | GEO