LRRK2 and its substrate Rab GTPases are sequentially targeted onto stressed lysosomes and maintain their homeostasis

Leucine-rich repeat kinase 2 (LRRK2) has been associated with a variety of human diseases, including Parkinson’s disease and Crohn’s disease, whereas LRRK2 deficiency leads to accumulation of abnormal lysosomes in aged animals. However, the cellular roles and mechanisms of LRRK2-mediated lysosomal regulation have remained elusive. Here, we reveal a mechanism of stress-induced lysosomal response by LRRK2 and its target Rab GTPases. Lysosomal overload stress induced the recruitment of endogenous LRRK2 onto lysosomal membranes and activated LRRK2. An upstream adaptor Rab7L1 (Rab29) promoted the lysosomal recruitment of LRRK2. Subsequent family-wide screening of Rab GTPases that may act downstream of LRRK2 translocation revealed that Rab8a and Rab10 were specifically accumulated on overloaded lysosomes dependent on their phosphorylation by LRRK2. Rab7L1-mediated lysosomal targeting of LRRK2 attenuated the stress-induced lysosomal enlargement and promoted lysosomal secretion, whereas Rab8 stabilized by LRRK2 on stressed lysosomes suppressed lysosomal enlargement and Rab10 promoted lysosomal secretion, respectively. These effects were mediated by the recruitment of Rab8/10 effectors EHBP1 and EHBP1L1. LRRK2 deficiency augmented the chloroquine-induced lysosomal vacuolation of renal tubules in vivo. These results implicate the stress-responsive machinery composed of Rab7L1, LRRK2, phosphorylated Rab8/10, and their downstream effectors in the maintenance of lysosomal homeostasis.

Download Full-text

Neuron-autonomous susceptibility to induced synuclein aggregation is exacerbated by endogenous Lrrk2 mutations and ameliorated by Lrrk2 genetic knock-out

Brain Communications ◽

10.1093/braincomms/fcz052 ◽

2020 ◽

Vol 2 (1) ◽

Cited By ~ 3

Author(s):

Sarah MacIsaac ◽

Thaiany Quevedo Melo ◽

Yuting Zhang ◽

Mattia Volta ◽

Matthew J Farrer ◽

...

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Therapeutic Strategy ◽

Germ Line ◽

Lewy Bodies ◽

Neuronal Cultures ◽

Leucine Rich Repeat ◽

Kinase Inhibition ◽

Knock Out

Abstract Neuronal aggregates containing α-synuclein are a pathological hallmark of several degenerative diseases; including Parkinson’s disease, Parkinson’s disease with dementia and dementia with Lewy bodies. Understanding the process of α-synuclein aggregation, and discovering means of preventing it, may help guide therapeutic strategy and drug design. Recent advances provide tools to induce α-synuclein aggregation in neuronal cultures. Application of exogenous pre-formed fibrillar α-synuclein induces pathological phosphorylation and accumulation of endogenous α-synuclein, typical of that seen in disease. Genomic variability and mutations in α-synuclein and leucine-rich repeat kinase 2 proteins are the major genetic risk factors for Parkinson’s disease. Reports demonstrate fibril-induced α-synuclein aggregation is increased in cells from leucine-rich repeat kinase 2 pathogenic mutant (G2019S) overexpressing mice, and variously decreased by leucine-rich repeat kinase 2 inhibitors. Elsewhere in vivo antisense knock-down of leucine-rich repeat kinase 2 protein has been shown to protect mice from fibril-induced α-synuclein aggregation, whereas kinase inhibition did not. To help bring clarity to this issue, we took a purely genetic approach in a standardized neuron-enriched culture, lacking glia. We compared fibril treatment of leucine-rich repeat kinase 2 germ-line knock-out, and G2019S germ-line knock-in, mouse cortical neuron cultures with those from littermates. We found leucine-rich repeat kinase 2 knock-out neurons are resistant to α-synuclein aggregation, which predominantly forms within axons, and may cause axonal fragmentation. Conversely, leucine-rich repeat kinase 2 knock-in neurons are more vulnerable to fibril-induced α-synuclein accumulation. Protection and resistance correlated with basal increases in a lysosome marker in knock-out, and an autophagy marker in knock-in cultures. The data add to a growing number of studies that argue leucine-rich repeat kinase 2 silencing, and potentially kinase inhibition, may be a useful therapeutic strategy against synucleinopathy.

Download Full-text

Genetic analysis of Parkinson's disease-linked leucine-rich repeat kinase 2

Biochemical Society Transactions ◽

10.1042/bst20120112 ◽

2012 ◽

Vol 40 (5) ◽

pp. 1042-1046 ◽

Cited By ~ 11

Author(s):

Youren Tong ◽

Jie Shen

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Oxidative Damage ◽

Inflammatory Responses ◽

Dopamine D2 Receptor ◽

Apoptotic Cell ◽

Protein Carbonyls ◽

Mutant Form ◽

Leucine Rich Repeat

Mutations in LRRK2 (leucine-rich repeat kinase 2) are the most common genetic cause of PD (Parkinson's disease). To investigate how mutations in LRRK2 cause PD, we generated LRRK2 mutant mice either lacking its expression or expressing the R1441C mutant form. Homozygous R1441C knockin mice exhibit no dopaminergic neurodegeneration or alterations in steady-state levels of striatal dopamine, but they show impaired dopamine neurotransmission, as was evident from reductions in amphetamine-induced locomotor activity and stimulated catecholamine release in cultured chromaffin cells as well as impaired dopamine D2 receptor-mediated functions. Whereas LRRK2−/− brains are normal, LRRK2−/− kidneys at 20 months of age develop striking accumulation and aggregation of α-synuclein and ubiquitinated proteins, impairment of the autophagy–lysosomal pathway, and increases in apoptotic cell death, inflammatory responses and oxidative damage. Our further analysis of LRRK2−/− kidneys at multiple ages revealed unique age-dependent biphasic alterations of the autophagic activity, which is unchanged at 1 month of age, enhanced at 7 months, but reduced at 20 months. Levels of α-synuclein and protein carbonyls, a general oxidative damage marker, are also decreased in LRRK2−/− kidneys at 7 months of age. Interestingly, this biphasic alteration is associated with increased levels of lysosomal proteins and proteases as well as progressive accumulation of autolysosomes and lipofuscin granules. We conclude that pathogenic mutations in LRRK2 impair the nigrostriatal dopaminergic pathway, and LRRK2 plays an essential role in the dynamic regulation of autophagy function in vivo.

Download Full-text

Deciphering the function of leucine-rich repeat kinase 2 and targeting its dysfunction in disease1

Biochemical Society Transactions ◽

10.1042/bst20120178 ◽

2012 ◽

Vol 40 (5) ◽

pp. 1039-1041 ◽

Cited By ~ 3

Author(s):

Patrick A. Lewis ◽

Dario R. Alessi

Keyword(s):

Parkinson’S Disease ◽

Inflammatory Bowel Disease ◽

Parkinson's Disease ◽

Bowel Disease ◽

Therapeutic Target ◽

Unknown Function ◽

Normal Function ◽

Human Diseases ◽

Leucine Rich Repeat ◽

Inflammatory Bowel

LRRK2 (leucine-rich repeat kinase 2) is a gene of unknown function that has been linked to a number a human diseases, including PD (Parkinson's disease), IBD (inflammatory bowel disease), leprosy and cancer. The papers from the LRRK2: Function and Dysfunction meeting in this issue of Biochemical Society Transactions explore our growing knowledge of LRRK2's normal function, the role that it plays in disease and emerging strategies to exploit LRRK2 as a therapeutic target.

Download Full-text

Rab27 GTPases regulate alpha-synuclein uptake, cell-to-cell transmission, and toxicity

10.1101/2020.11.17.387449 ◽

2020 ◽

Author(s):

Rachel Underwood ◽

Bing Wang ◽

Aneesh Pathak ◽

Laura Volpicelli-Daley ◽

Talene A. Yacoubian

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Lewy Bodies ◽

Neuronal Loss ◽

Alpha Synuclein ◽

Rab Gtpases ◽

Double Knockout ◽

The Impact

SUMMARYParkinson’s disease and Dementia with Lewy Bodies are two common neurodegenerative disorders marked by proteinaceous aggregates composed primarily of the protein α-synuclein. α-Synuclein is hypothesized to have prion-like properties, by which misfolded α-synuclein induces the pathological aggregation of endogenous α-synuclein and neuronal loss. Rab27a and Rab27b are two highly homologous Rab GTPases that regulate α-synuclein secretion, clearance, and toxicity in vitro. In this study, we tested the impact of Rab27a/b on the transmission of pathogenic α-synuclein. Double knockout of both Rab27 isoforms eliminated α-synuclein aggregation and neuronal toxicity in primary cultured neurons exposed to fibrillary α-synuclein. In vivo, Rab27 double knockout mice lacked fibril-induced α-synuclein inclusions, dopaminergic neuron loss, and behavioral deficits seen in wildtype mice with fibril-induced inclusions. Studies using AlexaFluor488-labeled α-synuclein fibrils revealed that Rab27a/b knockout prevented α-synuclein internalization without affecting bulk endocytosis. Rab27a/b knockout also blocked the cell-to-cell spread of α-synuclein pathology in multifluidic, multichambered devices. This study provides critical insight into the role of Rab GTPases in Parkinson’s disease and identifies Rab27s as key players in the progression of synucleinopathies.

Download Full-text

Membrane targeting activates Leucine-rich repeat kinase 2 with differential effects on downstream Rab activation

10.1101/2020.12.01.406223 ◽

2020 ◽

Author(s):

Jillian H. Kluss ◽

Alexandra Beilina ◽

Patrick A. Lewis ◽

Mark R. Cookson ◽

Luis Bonet-Ponce

Keyword(s):

Parkinson’S Disease ◽

Genetic Variation ◽

Membrane Targeting ◽

Potential Mechanism ◽

Leucine Rich Repeat ◽

Differential Effects ◽

Lysosomal Membranes ◽

Golgi Membranes ◽

Sporadic Parkinson’S Disease ◽

Perinuclear Area

ABSTRACTGenetic variation at the Leucine-rich repeat kinase 2 (LRRK2) locus contributes to risk of familial and sporadic Parkinson’s disease. Recent data have shown a robust association between localization to various membranes of the endolysosomal system and LRRK2 activation. However, the mechanism(s) underlying LRRK2 activation at endolysosomal membranes are still poorly understood. Here we artificially direct LRRK2 to six different membranes within the endolysosomal system. We demonstrate that LRRK2 is activated and able to phosphorylate three of its Rab substrates (Rab10, Rab12 and Rab29) at each compartment. However, we report differing localization of pRab10 and pRab12 at the lysosomal and Golgi membranes. Specifically, we found that pRab10 colocalizes with a sub-population of perinuclear LRRK2-positive Golgi/lysosomal compartments whereas pRab12 localized to all LRRK2-positive Golgi/lysosomal membranes across the cell. When organelle positioning is manipulated by sequestering lysosomes to the perinuclear area, pRab10 colocalization with LRRK2 significantly increases. We also show recruitment of JIP4, a pRab10 effector that we have recently linked to LYTL, after trapping LRRK2 at various membranes. Taken together, we demonstrate that the association of LRRK2 to membranous compartments is sufficient for its activation and Rab phosphorylation independent of membrane identity. Our system also identifies a potential mechanism underlying the distinct relationships between LRRK2 and its substrates Rab10 and Rab12.

Download Full-text

Preclinical modeling of chronic inhibition of the Parkinson’s disease associated kinase LRRK2 reveals altered function of the endolysosomal system in vivo

Molecular Neurodegeneration ◽

10.1186/s13024-021-00441-8 ◽

2021 ◽

Vol 16 (1) ◽

Author(s):

Jillian H. Kluss ◽

Melissa Conti Mazza ◽

Yan Li ◽

Claudia Manzoni ◽

Patrick A. Lewis ◽

...

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Physiological Activity ◽

Response To Treatment ◽

Common Mutation ◽

Rab Gtpases ◽

Major Pathway ◽

Cellular Processes ◽

Downstream Effects

AbstractThe most common mutation in the Leucine-rich repeat kinase 2 gene (LRRK2), G2019S, causes familial Parkinson’s Disease (PD) and renders the encoded protein kinase hyperactive. While targeting LRRK2 activity is currently being tested in clinical trials as a therapeutic avenue for PD, to date, the molecular effects of chronic LRRK2 inhibition have not yet been examined in vivo. We evaluated the utility of newly available phospho-antibodies for Rab substrates and LRRK2 autophosphorylation to examine the pharmacodynamic response to treatment with the potent and specific LRRK2 inhibitor, MLi-2, in brain and peripheral tissue in G2019S LRRK2 knock-in mice. We report higher sensitivity of LRRK2 autophosphorylation to MLi-2 treatment and slower recovery in washout conditions compared to Rab GTPases phosphorylation, and we identify pS106 Rab12 as a robust readout of downstream LRRK2 activity across tissues. The downstream effects of long-term chronic LRRK2 inhibition in vivo were evaluated in G2019S LRRK2 knock-in mice by phospho- and total proteomic analyses following an in-diet administration of MLi-2 for 10 weeks. We observed significant alterations in endolysosomal and trafficking pathways in the kidney that were sensitive to MLi-2 treatment and were validated biochemically. Furthermore, a subtle but distinct biochemical signature affecting mitochondrial proteins was observed in brain tissue in the same animals that, again, was reverted by kinase inhibition. Proteomic analysis in the lung did not detect any major pathway of dysregulation that would be indicative of pulmonary impairment. This is the first study to examine the molecular underpinnings of chronic LRRK2 inhibition in a preclinical in vivo PD model and highlights cellular processes that may be influenced by therapeutic strategies aimed at restoring LRRK2 physiological activity in PD patients.

Download Full-text

Manganese is a Physiologically Relevant TORC1 Activator in Yeast and Mammals.

10.1101/2021.12.09.471923 ◽

2021 ◽

Author(s):

Raffaele Nicastro ◽

Helene Gaillard ◽

Laura Zarzuela ◽

Elisabet Fernandez-Garcia ◽

Mercedes Tome ◽

...

Keyword(s):

Metal Ion ◽

Human Diseases ◽

Natural Resistance ◽

Homeostatic Process ◽

Downstream Effectors ◽

Mtorc1 Signaling ◽

The Family ◽

Macrophage Protein

The essential biometal manganese (Mn) functions as a cofactor for several enzymatic activities that are critical for the prevention of human diseases. Whether intracellular Mn levels may also modulate signaling events has so far remained largely unexplored. The target of rapamycin complex 1 (TORC1, mTORC1 in mammals) is a conserved protein kinase complex that requires metal co-factors to phosphorylate its downstream effectors as part of a central, homeostatic process that coordinates cell growth and metabolism in response to nutrient and/or growth factor availability. Using genetic and biochemical approaches, we show here that TORC1 activity is exquisitely sensitive to stimulation by Mn both in vivo and in vitro. Mn-mediated control of TORC1 depends on Smf1 and Smf2, two members of the family of natural resistance-associated macrophage protein (NRAMP) metal ion transporters, the turnover of which is subjected to feedback control by TORC1 activity. Notably, increased Mn levels and consequent activation of TORC1 cause retrograde dysregulation and antagonize the rapamycin-induced gene expression and autophagy programs in yeast. Because Mn also activates mTORC1 signaling in aminoacid starved human cells, our data indicate that intracellular Mn levels may constitute an evolutionary conserved physiological cue that modulates eukaryotic TORC1/mTORC1 signaling. Our findings therefore reveal a hitherto elusive connection between intracellular Mn levels, mTORC1 activity, and human diseases.

Download Full-text

Preclinical Modeling of Chronic Inhibition of the Parkinson’s Disease Associated Kinase LRRK2 Reveals Altered Function of the Endolysosomal System in Vivo

10.21203/rs.3.rs-78203/v1 ◽

2020 ◽

Cited By ~ 3

Author(s):

JILLIAN H. KLUSS ◽

Melissa Conti Mazza ◽

Yan Li ◽

Claudia Manzoni ◽

Patrick A. Lewis ◽

...

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Physiological Activity ◽

Common Mutation ◽

Rab Gtpases ◽

Peripheral Tissues ◽

Cellular Processes ◽

Downstream Effects ◽

Molecular Effects

Abstract BackgroundThe most common mutation in the Leucine-rich repeat kinase 2 gene (LRRK2), G2019S, causes familial Parkinson’s Disease (PD) and renders the encoded protein kinase hyperactive. To date, the molecular effects of chronic LRRK2 inhibition have not yet been examined in vivo. MethodsWe evaluated the utility of newly available phospho-antibodies for Rab substrates pT73 Rab10, pS106 Rab12, pT71 Rab29 and LRRK2 autophosphorylation to examine the pharmacodynamic response to acute treatment paradigms with the potent and specific LRRK2 inhibitor, MLi-2, in brain and peripheral tissue in G2019S LRRK2 knock-in mice to define the relative target engagement between brain and LRRK2-enriched peripheral tissues. The molecular effects of 10 days and 10 weeks of chronic in-diet dosing were also evaluated using TMTpro reagents for LC-MS/MS total and phospho- proteomics in brain and kidney tissues. ResultsWe report higher sensitivity of LRRK2 autophosphorylation to MLi-2 treatment and slower recovery in washout conditions compared to Rab GTPases phosphorylation, and we identify pS106 Rab12 as a robust readout of downstream LRRK2 activity across tissues. The downstream effects of long-term chronic LRRK2 inhibition in vivo were evaluated in G2019S LRRK2 knock-in mice by phospho- and total proteomic analyses following an in-diet administration of MLi-2 for 10 weeks. We observed alterations in endolysosomal and trafficking pathways in the kidney that were sensitive to MLi-2 treatment and that we validated biochemically. Furthermore, a subtle but distinct biochemical signature affecting mitochondrial proteins was observed in brain tissue in the same animals that was reverted by kinase inhibition. ConclusionsThis is the first study to examine the molecular underpinnings of chronic LRRK2 inhibition in a preclinical in vivo PD model and highlights cellular processes that may be influenced by therapeutic strategies aimed at restoring LRRK2 physiological activity in PD patients.

Download Full-text

Phosphoproteomics reveals that Parkinson's disease kinase LRRK2 regulates a subset of Rab GTPases

eLife ◽

10.7554/elife.12813 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 383

Author(s):

Martin Steger ◽

Francesca Tonelli ◽

Genta Ito ◽

Paul Davies ◽

Matthias Trost ◽

...

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Kinase Inhibitors ◽

Rab Gtpases ◽

Common Amino Acid ◽

Bona Fide ◽

Lrrk2 Kinase ◽

Conserved Residue

Mutations in Park8, encoding for the multidomain Leucine-rich repeat kinase 2 (LRRK2) protein, comprise the predominant genetic cause of Parkinson's disease (PD). G2019S, the most common amino acid substitution activates the kinase two- to threefold. This has motivated the development of LRRK2 kinase inhibitors; however, poor consensus on physiological LRRK2 substrates has hampered clinical development of such therapeutics. We employ a combination of phosphoproteomics, genetics, and pharmacology to unambiguously identify a subset of Rab GTPases as key LRRK2 substrates. LRRK2 directly phosphorylates these both in vivo and in vitro on an evolutionary conserved residue in the switch II domain. Pathogenic LRRK2 variants mapping to different functional domains increase phosphorylation of Rabs and this strongly decreases their affinity to regulatory proteins including Rab GDP dissociation inhibitors (GDIs). Our findings uncover a key class of bona-fide LRRK2 substrates and a novel regulatory mechanism of Rabs that connects them to PD.

Download Full-text