Pathological α-synuclein recruits LRRK2 expressing pro-inflammatory monocytes to the brain

Abstract Background Leucine rich repeat kinase 2 (LRRK2) and SNCA are genetically linked to late-onset Parkinson’s disease (PD). Aggregated α-synuclein pathologically defines PD. Recent studies identified elevated LRRK2 expression in pro-inflammatory CD16+ monocytes in idiopathic PD, as well as increased phosphorylation of the LRRK2 kinase substrate Rab10 in monocytes in some LRRK2 mutation carriers. Brain-engrafting pro-inflammatory monocytes have been implicated in dopaminergic neurodegeneration in PD models. Here we examine how α-synuclein and LRRK2 interact in monocytes and subsequent neuroinflammatory responses. Methods Human and mouse monocytes were differentiated to distinct transcriptional states resembling macrophages, dendritic cells, or microglia, and exposed to well-characterized human or mouse α-synuclein fibrils. LRRK2 expression and LRRK2-dependent Rab10 phosphorylation were measured with monoclonal antibodies, and myeloid cell responses to α-synuclein fibrils in R1441C-Lrrk2 knock-in mice or G2019S-Lrrk2 BAC mice were evaluated by flow cytometry. Chemotaxis assays were performed with monocyte-derived macrophages stimulated with α-synuclein fibrils and microglia in Boyden chambers. Results α-synuclein fibrils robustly stimulate LRRK2 and Rab10 phosphorylation in human and mouse macrophages and dendritic-like cells. In these cells, α-synuclein fibrils stimulate LRRK2 through JAK-STAT activation and intrinsic LRRK2 kinase activity in a feed-forward pathway that upregulates phosphorylated Rab10. In contrast, LRRK2 expression and Rab10 phosphorylation are both suppressed in microglia-like cells that are otherwise highly responsive to α-synuclein fibrils. Corroborating these results, LRRK2 expression in the brain parenchyma occurs in pro-inflammatory monocytes infiltrating from the periphery, distinct from brain-resident microglia. Mice expressing pathogenic LRRK2 mutations G2019S or R1441C have increased numbers of infiltrating pro-inflammatory monocytes in acute response to α-synuclein fibrils. In primary cultured macrophages, LRRK2 kinase inhibition dampens α-synuclein fibril and microglia-stimulated chemotaxis. Conclusions Pathologic α-synuclein activates LRRK2 expression and kinase activity in monocytes and induces their recruitment to the brain. These results predict that LRRK2 kinase inhibition may attenuate damaging pro-inflammatory monocyte responses in the brain.

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Dopaminergic neurodegeneration induced by Parkinson's disease-linked G2019S LRRK2 is dependent on kinase and GTPase activity

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1922184117 ◽

2020 ◽

Vol 117 (29) ◽

pp. 17296-17307 ◽

Cited By ~ 2

Author(s):

An Phu Tran Nguyen ◽

Elpida Tsika ◽

Kaela Kelly ◽

Nathan Levine ◽

Xi Chen ◽

...

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Kinase Inhibitor ◽

Late Onset ◽

Neuronal Damage ◽

Preclinical Model ◽

Gtpase Activity ◽

Kinase Inhibition ◽

Dopaminergic Neurodegeneration ◽

The Brain

Mutations inleucine-rich repeat kinase 2(LRRK2) are the most common cause of late-onset, autosomal-dominant familial Parkinson’s disease (PD). LRRK2 functions as both a kinase and GTPase, and PD-linked mutations are known to influence both enzymatic activities. While PD-linked LRRK2 mutations can commonly induce neuronal damage in culture models, the mechanisms underlying these pathogenic effects remain uncertain. Rodent models containing familial LRRK2 mutations often lack robust PD-like neurodegenerative phenotypes. Here, we develop a robust preclinical model of PD in adult rats induced by the brain delivery of recombinant adenoviral vectors with neuronal-specific expression of human LRRK2 harboring the most common G2019S mutation. In this model, G2019S LRRK2 induces the robust degeneration of substantia nigra dopaminergic neurons, a pathological hallmark of PD. Introduction of a stable kinase-inactive mutation or administration of the selective kinase inhibitor, PF-360, attenuates neurodegeneration induced by G2019S LRRK2. Neuroprotection provided by pharmacological kinase inhibition is mediated by an unusual mechanism involving the robust destabilization of human LRRK2 protein in the brain relative to endogenous LRRK2. Our study further demonstrates that G2019S LRRK2-induced dopaminergic neurodegeneration critically requires normal GTPase activity, as hypothesis-testing mutations that increase GTP hydrolysis or impair GTP-binding activity provide neuroprotection although via distinct mechanisms. Taken together, our data demonstrate that G2019S LRRK2 induces neurodegeneration in vivo via a mechanism that is dependent on kinase and GTPase activity. Our study provides a robust rodent preclinical model ofLRRK2-linked PD and nominates kinase inhibition and modulation of GTPase activity as promising disease-modifying therapeutic targets.

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Immune System in the Brain: A Modulatory Role on Dendritic Spine Morphophysiology?

Neural Plasticity ◽

10.1155/2012/348642 ◽

2012 ◽

Vol 2012 ◽

pp. 1-7 ◽

Cited By ~ 31

Author(s):

Oscar Kurt Bitzer-Quintero ◽

Ignacio González-Burgos

Keyword(s):

Central Nervous System ◽

Nervous System ◽

Immune System ◽

Dendritic Spines ◽

Cell Lineage ◽

Myeloid Cell ◽

Brain Parenchyma ◽

Immune Mediators ◽

The Central Nervous System ◽

The Brain

The central nervous system is closely linked to the immune system at several levels. The brain parenchyma is separated from the periphery by the blood brain barrier, which under normal conditions prevents the entry of mediators such as activated leukocytes, antibodies, complement factors, and cytokines. The myeloid cell lineage plays a crucial role in the development of immune responses at the central level, and it comprises two main subtypes: (1) resident microglia, distributed throughout the brain parenchyma; (2) perivascular macrophages located in the brain capillaries of the basal lamina and the choroid plexus. In addition, astrocytes, oligodendrocytes, endothelial cells, and, to a lesser extent, neurons are implicated in the immune response in the central nervous system. By modulating synaptogenesis, microglia are most specifically involved in restoring neuronal connectivity following injury. These cells release immune mediators, such as cytokines, that modulate synaptic transmission and that alter the morphology of dendritic spines during the inflammatory process following injury. Thus, the expression and release of immune mediators in the brain parenchyma are closely linked to plastic morphophysiological changes in neuronal dendritic spines. Based on these observations, it has been proposed that these immune mediators are also implicated in learning and memory processes.

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Microglia-specific overexpression of α-synuclein leads to severe dopaminergic neurodegeneration by phagocytic exhaustion and oxidative toxicity

Nature Communications ◽

10.1038/s41467-021-26519-x ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Simone Bido ◽

Sharon Muggeo ◽

Luca Massimino ◽

Matteo Jacopo Marzi ◽

Serena Gea Giannelli ◽

...

Keyword(s):

Immune Cells ◽

Neuronal Degeneration ◽

Brain Parenchyma ◽

Microglial Cells ◽

Dopaminergic Neurodegeneration ◽

Progressive Degeneration ◽

Toxic Environment ◽

Inflammatory State ◽

Peripheral Immune Cells ◽

The Brain

AbstractRecent findings in human samples and animal models support the involvement of inflammation in the development of Parkinson’s disease. Nevertheless, it is currently unknown whether microglial activation constitutes a primary event in neurodegeneration. We generated a new mouse model by lentiviral-mediated selective α-synuclein (αSYN) accumulation in microglial cells. Surprisingly, these mice developed progressive degeneration of dopaminergic (DA) neurons without endogenous αSYN aggregation. Transcriptomics and functional assessment revealed that αSYN-accumulating microglial cells developed a strong reactive state with phagocytic exhaustion and excessive production of oxidative and proinflammatory molecules. This inflammatory state created a molecular feed-forward vicious cycle between microglia and IFNγ-secreting immune cells infiltrating the brain parenchyma. Pharmacological inhibition of oxidative and nitrosative molecule production was sufficient to attenuate neurodegeneration. These results suggest that αSYN accumulation in microglia induces selective DA neuronal degeneration by promoting phagocytic exhaustion, an excessively toxic environment and the selective recruitment of peripheral immune cells.

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Pathologic α-Synuclein Species Activate LRRK2 in Pro-Inflammatory Monocyte and Macrophage Responses

10.1101/2020.05.04.077065 ◽

2020 ◽

Author(s):

Enquan Xu ◽

Ravindra Boddu ◽

Hisham A. Abdelmotilib ◽

Kaela Kelly ◽

Arpine Sokratian ◽

...

Keyword(s):

Immune Responses ◽

Immune Cells ◽

Kinase Activity ◽

Interferon Γ ◽

Missense Mutations ◽

Inflammatory Monocyte ◽

Lrrk2 Kinase ◽

The Brain ◽

Classical Monocytes ◽

Lrrk2 Kinase Activity

AbstractMissense mutations in the LRRK2 gene that lead to LRRK2 kinase hyperactivity can cause Parkinson’s disease (PD). The link between LRRK2 and α-synuclein aggregation in PD remains enigmatic. Numerous reports suggest critical LRRK2 functions in microglial responses. Herein, we find that LRRK2-positive immune cells in the brain represent CD68-positive pro-inflammatory, monocyte-derived macrophages, distinct from microglia. Rod α-synuclein fibrils stimulate LRRK2 kinase activity in monocyte-derived macrophages, and LRRK2 mutations lead to enhanced recruitment of classical monocytes into the midbrain in response to α-synuclein. LRRK2 kinase inhibition blocks α-synuclein fibril induction of LRRK2 protein in both human and murine macrophages, with human cells demonstrating much higher LRRK2 levels and kinase activity than equivalent murine cells. Further, interferon-γ strongly induces LRRK2 kinase activity in primary human macrophages in comparison to weak effects observed in murine cells. These results highlight peripheral immune responses in LRRK2-linked paradigms that further connect two central proteins in PD.

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Surface GluA1 and glutamatergic transmission are increased in cortical neurons of a VPS35 D620N knock-in mouse model of parkinsonism and altered by LRRK2 kinase inhibition

10.1101/2021.01.18.427223 ◽

2021 ◽

Author(s):

Chelsie A Kadgien ◽

Anusha Kamesh ◽

Jaskaran Khinda ◽

Li Ping Cao ◽

Jesse Fox ◽

...

Keyword(s):

Cortical Neurons ◽

Late Onset ◽

Glutamate Release ◽

Surface Expression ◽

Cellular Functions ◽

Kinase Inhibition ◽

Kinase Substrate ◽

Lrrk2 Kinase ◽

Protein Density ◽

Vacuolar Protein

AbstractVacuolar protein sorting 35 (VPS35) regulates receptor recycling from endosomes. A missense mutation in VPS35 (D620N) leads to autosomal-dominant, late-onset Parkinson’s disease. Here, we use a VPS35 D620N knock-in mouse to study the neurobiology of this mutation. In brain tissue, we confirm previous findings that the mutation results in reduced binding of VPS35 with WASH-complex member FAM21, and robustly elevated phosphorylation of the LRRK2 kinase substrate Rab10. In cultured cortical neurons, the mutation results in increased endosomal recycling protein density (VPS35-FAM21 co-clusters and Rab11 clusters), glutamate release, and GluA1 surface expression. LRRK2 kinase inhibition exerted genotype-specific effects on GluA1 surface expression, but did not impact glutamate release phenotypes. These results improve our understanding of the early effects of the D620N mutation on cellular functions that are specific to neurons. These observations provide candidate pathophysiological pathways that may drive eventual transition to late-stage parkinsonism in VPS35 families, and support a synaptopathy model of neurodegeneration.

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Pharmacodynamic Markers of LRRK2 Inhibition in Biofluids

10.1101/2020.01.28.923557 ◽

2020 ◽

Cited By ~ 1

Author(s):

Shijie Wang ◽

Kaela Kelly ◽

Nathalie Schussler ◽

Sylviane Boularand ◽

Laurent Dubois ◽

...

Keyword(s):

Disease Susceptibility ◽

Kinase Inhibitors ◽

Mononuclear Cells ◽

Kinase Inhibition ◽

Cynomolgus Macaques ◽

Pharmacodynamic Markers ◽

Lrrk2 Kinase ◽

Lrrk2 Protein ◽

The Brain ◽

Different Doses

AbstractHyper-activated LRRK2 is linked to Parkinson’s disease susceptibility and progression. Quantitative measures of LRRK2 inhibition, especially in the brain, may be critical in the clinical development of successful LRRK2-targeting therapeutics. In this study, three different brain-penetrant and selective LRRK2 small-molecule kinase inhibitors (PFE-360, MLi2, and RA283) were orally administered to groups of cynomolgus macaques at different doses. Biofluid markers with proposed pharmacodynamic properties for assessing LRRK2 inhibition were measured from samples of blood, urine, and cerebral-spinal fluid (CSF). LRRK2 kinase inhibition led to consistent reduced pS935-LRRK2 and pRab10 proteins in blood mononuclear cells, reduced exosome LRRK2 protein and di-docosahexaenoyl (22:6) bis (monoacylglycerol) phosphate in urine, and reduced exosome LRRK2 and autophosphorylated pS1292-LRRK2 protein in CSF. Incomplete LRRK2 kinase inhibition reduced LRRK2 protein secretion in exosomes whereas high drug exposures may reduce both exosome and tissue levels of LRRK2 protein. These orthogonal markers for LRRK2 inhibition in urine and CSF can be used in combination with blood markers to non-invasively monitor the potency of LRRK2-targeting therapeutics in the brain and periphery.

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The industrial solvent trichloroethylene induces LRRK2 kinase activity and dopaminergic neurodegeneration in a rat model of Parkinson’s disease

10.1101/2020.11.02.365775 ◽

2020 ◽

Author(s):

Sandra L. Castro ◽

Emily M. Rocha ◽

Christopher R. Bodle ◽

Katrina E. Johnson ◽

J. Timothy Greenamyre ◽

...

Keyword(s):

Parkinson’S Disease ◽

Organic Solvents ◽

Dopaminergic Neurons ◽

Kinase Activity ◽

Environment Interaction ◽

Dopaminergic Neurodegeneration ◽

Gene Environment Interaction ◽

Gene Environment ◽

Lrrk2 Kinase ◽

Lrrk2 Kinase Activity

Gene-environment interaction is implicated in the majority of idiopathic Parkinson’s disease (PD) risk, and some of the most widespread environmental contaminants are selectively toxic to dopaminergic neurons. Pesticides have long been connected to PD incidence, however, it has become increasingly apparent that other industrial byproducts likely influence neurodegeneration. For example, organic solvents, which are used in chemical, machining, and dry-cleaning industries, are of growing concern, as decades of solvent use and their effluence into the environment has contaminated much of the world’s groundwater and soil. Like some pesticides, certain organic solvents, such as the chlorinated halocarbon trichloroethylene (TCE), are mitochondrial toxicants, which are collectively implicated in the pathogenesis of dopaminergic neurodegeneration. Recently, we hypothesized a possible gene-environment interaction may occur between environmental mitochondrial toxicants and the protein kinase LRRK2, mutations of which are the most common genetic cause of familial and sporadic PD. In addition, emerging data suggests that elevated wildtype LRRK2 kinase activity also contributes to the pathogenesis of idiopathic PD. To this end, we investigated whether chronic, systemic TCE exposure (200 mg/kg) in aged rats produced wildtype LRRK2 activation and influenced predegenerative dopaminergic dysfunction. Interestingly, we found that TCE not only induced LRRK2 kinase activity in the brain, but produced a significant dopaminergic lesion in the nigrostriatal tract, elevated oxidative stress, and caused endolysosomal dysfunction and protein accumulation (α-synuclein). Together, these data suggest that TCE-induced LRRK2 kinase activity contributed to the selective toxicity of dopaminergic neurons. We conclude that gene-environment interactions between certain industrial contaminants and LRRK2 likely influence PD risk.

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Dopaminergic Neurodegeneration Induced by Parkinson’s Disease-Linked G2019S LRRK2 is Dependent on Kinase and GTPase Activity

10.1101/2019.12.17.879759 ◽

2019 ◽

Cited By ~ 1

Author(s):

An Phu Tran Nguyen ◽

Elpida Tsika ◽

Kaela Kelly ◽

Nathan Levine ◽

Xi Chen ◽

...

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Kinase Inhibitor ◽

Late Onset ◽

Neuronal Damage ◽

Gtpase Activity ◽

Adult Rats ◽

Kinase Inhibition ◽

Dopaminergic Neurodegeneration ◽

Cellular Models

AbstractMutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause late-onset, autosomal dominant familial Parkinson’s disease (PD) and represent the most common known cause of PD. LRRK2 can function as both a protein kinase and GTPase and PD-linked mutations are known to influence both of these enzymatic activities. While PD-linked LRRK2 mutations can commonly induce neuronal damage and toxicity in cellular models, the mechanisms underlying these pathogenic effects remain uncertain. Rodent models based upon familial LRRK2 mutations often lack the hallmark features of PD and robust neurodegenerative phenotypes in general. Here, we develop a robust pre-clinical model of PD in adult rats induced by the brain delivery of recombinant adenoviral vectors with neuronal-specific expression of full-length human LRRK2 harboring the most common G2019S mutation. In this model, G2019S LRRK2 induces the robust degeneration of substantia nigra dopaminergic neurons, a pathological hallmark of PD. Introduction of a stable kinase-inactive mutation or in-diet dosing with the selective kinase inhibitor, PF-360, attenuates neurodegeneration induced by G2019S LRRK2. Neuroprotection provided by pharmacological kinase inhibition is mediated by an unusual mechanism involving the selective and robust destabilization of human LRRK2 protein in the rat brain relative to endogenous LRRK2. Our study further demonstrates that dopaminergic neurodegeneration induced by G2019S LRRK2 critically requires normal GTPase activity. The introduction of hypothesis-testing mutations that increase GTP hydrolysis or impair GTP binding activity provide neuroprotection against G2019S LRRK2 via distinct mechanisms. Taken together, our data demonstrate that G2019S LRRK2 induces neurodegeneration in vivo via a mechanism that is dependent on kinase and GTPase activity. Our study provides a robust rodent model of LRRK2-linked PD and nominates kinase inhibition and modulation of GTPase activity as promising disease-modifying therapeutic targets.

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Exosome markers of LRRK2 kinase inhibition

npj Parkinson s Disease ◽

10.1038/s41531-020-00138-7 ◽

2020 ◽

Vol 6 (1) ◽

Author(s):

Shijie Wang ◽

Kaela Kelly ◽

Jonathan M. Brotchie ◽

James B. Koprich ◽

Andrew B. West

Keyword(s):

Disease Susceptibility ◽

Kinase Inhibitors ◽

Mononuclear Cells ◽

Peripheral Blood Mononuclear ◽

Kinase Inhibition ◽

Cynomolgus Macaques ◽

Pharmacodynamic Markers ◽

Lrrk2 Kinase ◽

Lrrk2 Protein ◽

The Brain

AbstractHyper-activated LRRK2 is linked to Parkinson’s disease susceptibility and progression. Quantitative measures of LRRK2 inhibition, especially in the brain, maybe critical in the development of successful LRRK2-targeting therapeutics. In this study, two different brain-penetrant and selective LRRK2 small-molecule kinase inhibitors (PFE-360 and MLi2) were orally administered to groups of cynomolgus macaques. Proposed pharmacodynamic markers in exosomes from urine and cerebrospinal fluid (CSF) were compared to established markers in peripheral blood mononuclear cells (PBMCs). LRRK2 kinase inhibition led to reductions in exosome-LRRK2 protein and the LRRK2-substrate pT73-Rab10 in urine, as well as reduced exosome-LRRK2 and autophosphorylated pS1292-LRRK2 protein in CSF. We propose orthogonal markers for LRRK2 inhibition in urine and CSF can be used in combination with blood markers to non-invasively monitor the potency of LRRK2-targeting therapeutics.

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