Contribution of microglia to the pathogenic mechanisms behind LRRK2-associated Parkinson's disease

Microglia are immune cells of the brain playing critical roles during the inflammatory response. Among the genes mutated in familial Parkinson’s disease, those encoding LRRK2 and α-synuclein have been associated with neuroinflammatory processes. Previous work showed that LRRK2 is a positive regulator of inflammation, while aggregated α-synuclein released by dying neurons can activate microglia triggering the neuroinflammatory process. The main goal of this project was to gain novel insights into the contribution of microglial cells to the pathogenic mechanisms of LRRK2-associated Parkinson’s disease. To this aim, we initially performed RNA-sequencing (RNA-seq) of Lrrk2 wild-type (WT) and knockout (KO) primary microglia treated with α-synuclein pre-formed fibrils (PFFs) or lipopolysaccharide (LPS). We found that LPS and α-synuclein triggered in part overlapping but also different responses, while loss of Lrrk2 had a subtle effect in attenuating the pro-inflammatory response. Moreover, treatment with α-synuclein PFFs caused a significant induction of the antioxidant superoxide dismutase-2 (SOD2) enzyme in WT cells, with the effect attenuated in Lrrk2 KO microglia cells. Since loss of Lrrk2 in primary microglia revealed only small differences in gene expression both under resting or pro-inflammatory conditions, we next moved to an experimental condition that more closely resembles the physiological situation, e.g. acutely isolated microglia cells from LPS-injected adult mouse brains. Using single cell RNA sequencing (scRNA-seq), we confirmed a subset of the genes nominated in primary culture experiments, including IL-1, SOD2 and TXNIP as differentially expressed upon inflammatory stimulation with LPS. However, the overall effect on gene transcription due to loss of Lrrk2 remained subtle. To further explore the impact of LRRK2 on microglia function, we performed ex vivo phagocytic assays. Lrrk2 KO microglia displayed the highest phagocytic activity compared to microglia isolated from WT and pathogenic mutants G2019S and R1441C mice upon treatment with α-synuclein PFFs, suggesting that LRRK2 negatively regulates phagocytosis or delays lysosomal degradation. Additionally, we performed intrastriatal brain injections with lipopolysaccharide (LPS)-inflammatory agent or with PBS as a control, that demonstrated a significant morphological changes in microglial shape (increase in circularity and soma area) in LPS-stimulated animals compare to PBS-treated and a modest differences between LRRK2 genotypes. Additionally, the branch complexity was reduced in LPS-injected animals with stronger effect in Lrrk2-KO and R1441C genotypes and a less response in G2019S animals. Next, we evaluated LRRK2 mRNA and protein expression at the single cell level. We found that LRRK2 has a low and sparse pattern of expression in unstimulated resident microglia. However, upon stimulation with LPS, the proportion of microglia cells expressing LRRK2 increased, while LRRK2 levels remained unchanged per cell. This is, to date, the first attempt to analyze LRRK2 protein expression at the single cell level in brain cells. In summary, the present work provides new insights into the biology of LRRK2 in the brain resident microglia and shows that inflammatory stimulations with α-synuclein PFFs cause a significant induction of pro-inflammatory and anti-oxidant responses, which are attenuated in LRRK2-KO microglia

Convergence of signalling pathways in innate immune responses and genetic forms of Parkinson's disease

In recent years progress in molecular biology and genetics have advanced our understanding of neurological disorders and highlighted synergistic relationships with inflammatory and age-related processes. Parkinson's disease (PD) is a common neurodegenerative disorder that is characterized by loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). Increasing extensive evidence supports the contribution of genetic risk variants and inflammation in the pathobiology of this disease. Functional and genetic studies demonstrate an overlap between genes linked to increased risk for PD and autoimmune diseases. Variants identified in loci adjacent to LRRK2, GBA, and HLA establish a crosstalk between the pathobiologies of the two disease spectra. Furthermore, common signalling pathways associated with the pathogenesis of genetic PD are also relevant to inflammatory signaling include MAPK, NF-κB, Wnt and inflammasome signaling. Importantly, post-mortem analyses of brain and cerebrospinal fluid from PD patients show the accumulation of proinflammatory cytokines. In this review we will focus on the principal mechanisms of genetic, inflammatory and age-related risk that intersect in the pathogenesis of PD

The Parkinson's Disease Associated LRRK2 Exhibits Weaker In Vitro Phosphorylation of 4E-BP Compared to Autophosphorylation

Mutations in the gene encoding Leucine-rich repeat kinase 2 (LRRK2) are the most common cause of inherited Parkinson's disease (PD). LRRK2 is a multi-domain protein kinase containing a central catalytic core and a number of protein-protein interaction domains. An important step forward in the understanding of both the biology and the pathology of LRRK2 would be achieved by identification of its authentic physiological substrates. In the present study we examined phosphorylation of 4E-BP (eukaryotic initiation factor 4E (eIF4E)-binding protein), a recently proposed substrate for LRRKs. We found that LRRK2 is capable of phosphorylating 4E-BP in vitro. The PD related LRRK2-G2019S mutant was ∼2 fold more active than wild type protein. However, LRRK2 autophosphorylation was stronger than 4E-BP phosphorylation under conditions of molar excess of 4E-BP to LRRK2. We also tested three other kinases (STK3, MAPK14/p38α and DAPK2) and found that MAPK14/p38α could efficiently phosphorylate 4E-BP at the same site as LRRK2 in vitro. Finally, we did not see changes in 4E-BP phosphorylation levels using inducible expression of LRRK2 in HEK cell lines. We also found that MAPK14/p38α phosphorylates 4E-BP in transient overexpression experiments whereas LRRK2 did not. We suggest that increased 4E-BP phosphorylation reported in some systems may be related to p38-mediated cell stress rather than direct LRRK2 activity. Overall, our results suggest that 4E-BP is a relatively poor direct substrate for LRRK2

Differential LRRK2 signalling and gene expression in WT-LRRK2 and G2019S- LRRK2 mouse microglia treated with zymosan and MLi2

INTRODUCTION: Mutations in the Leucine Rich Repeat Kinase 2 (LRRK2) gene cause autosomal dominant Parkinson’s disease (PD) with the most common causative mutation being the LRRK2 p.G2019S within the kinase domain. LRRK2 protein is highly expressed in the human brain and also in the periphery, and high expression of dominant PD genes in immune cells suggest involvement of microglia and macrophages in inflammation related to PD. LRRK2 is known to respond to extracellular signalling including TLR4 resulting in alterations in gene expression, with the response to TLR2 signalling through zymosan being less known. METHODS: Here, we investigated the effects of zymosan, a TLR2 agonist and the potent and specific LRRK2 kinase inhibitor MLi-2 on gene expression in microglia from LRRK2-WT and LRRK2 p.G2019S knock-in mice by RNA-Sequencing analysis. RESULTS: We observed both overlapping and distinct zymosan and MLi-2 mediated gene expression profiles in microglia. At least two candidate Genome-Wide Association (GWAS) hits for PD, CathepsinB (Ctsb) and Glycoprotein-nmb (Gpnmb), were notably downregulated by zymosan treatment. Genes involved in inflammatory response and nervous system development were up and downregulated respectively with zymosan treatment while MLi-2 treatment particularly exhibited upregulated genes for ion transmembrane transport regulation. Furthermore, we observed the top twenty most significantly differentially expressed genes in LRRK2 p.G2019S microglia show enriched biological processes in iron transport and response to oxidative stress. DISCUSSION: Overall, these results suggest that microglial LRRK2 may contribute to PD pathogenesis through altered inflammatory pathways. Our findings should encourage future investigations of these putative avenues in the context of PD pathogenesis

Correction: Mutations in LRRK2 linked to Parkinson disease sequester Rab8a to damaged lysosomes and regulate transferrin-mediated iron uptake in microglia

[This corrects the article DOI: 10.1371/journal.pbio.3001480.]

LAG3 is not expressed in human and murine neurons and does not modulate α-synucleinopathies.

While the initial pathology of Parkinson's disease and other α-synucleinopathies is often confined to circumscribed brain regions, it can spread and progressively affect adjacent and distant brain locales. This process may be controlled by cellular receptors of α-synuclein fibrils, one of which was proposed to be the LAG3 immune checkpoint molecule. Here, we analysed the expression pattern of LAG3 in human and mouse brains. Using a variety of methods and model systems, we found no evidence for LAG3 expression by neurons. While we confirmed that LAG3 interacts with α-synuclein fibrils, the specificity of this interaction appears limited. Moreover, overexpression of LAG3 in cultured human neural cells did not cause any worsening of α-synuclein pathology ex vivo. The overall survival of A53T α-synuclein transgenic mice was unaffected by LAG3 depletion, and the seeded induction of α-synuclein lesions in hippocampal slice cultures was unaffected by LAG3 knockout. These data suggest that the proposed role of LAG3 in the spreading of α-synucleinopathies is not universally valid

Correction: Pathogenic LRRK2 Mutations Do Not Alter Gene Expression in Cell Model Systems or Human Brain Tissue.

Point mutations in LRRK2 cause autosomal dominant Parkinson's disease. Despite extensive efforts to determine the mechanism of cell death in patients with LRRK2 mutations, the aetiology of LRRK2 PD is not well understood. To examine possible alterations in gene expression linked to the presence of LRRK2 mutations, we carried out a case versus control analysis of global gene expression in three systems: fibroblasts isolated from LRRK2 mutation carriers and healthy, non-mutation carrying controls; brain tissue from G2019S mutation carriers and controls; and HEK293 inducible LRRK2 wild type and mutant cell lines. No significant alteration in gene expression was found in these systems following correction for multiple testing. These data suggest that any alterations in basal gene expression in fibroblasts or cell lines containing mutations in LRRK2 are likely to be quantitatively small. This work suggests that LRRK2 is unlikely to play a direct role in modulation of gene expression, although it remains possible that this protein can influence mRNA expression under pathogenic cicumstances

Pathogenic LRRK2 Mutations Do Not Alter Gene Expression in Cell Model Systems or Human Brain Tissue

Point mutations in LRRK2 cause autosomal dominant Parkinson's disease. Despite extensive efforts to determine the mechanism of cell death in patients with LRRK2 mutations, the aetiology of LRRK2 PD is not well understood. To examine possible alterations in gene expression linked to the presence of LRRK2 mutations, we carried out a case versus control analysis of global gene expression in three systems: fibroblasts isolated from LRRK2 mutation carriers and healthy, non-mutation carrying controls; brain tissue from G2019S mutation carriers and controls; and HEK293 inducible LRRK2 wild type and mutant cell lines. No significant alteration in gene expression was found in these systems following correction for multiple testing. These data suggest that any alterations in basal gene expression in fibroblasts or cell lines containing mutations in LRRK2 are likely to be quantitatively small. This work suggests that LRRK2 is unlikely to play a direct role in modulation of gene expression, although it remains possible that this protein can influence mRNA expression under pathogenic cicumstances

Contribution of microglia to the pathogenic mechanisms behind LRRK2-associated Parkinson's disease

Microglia are immune cells of the brain playing critical roles during the inflammatory response. Among the genes mutated in familial Parkinson’s disease, those encoding LRRK2 and α-synuclein have been associated with neuroinflammatory processes. Previous work showed that LRRK2 is a positive regulator of inflammation, while aggregated α-synuclein released by dying neurons can activate microglia triggering the neuroinflammatory process. The main goal of this project was to gain novel insights into the contribution of microglial cells to the pathogenic mechanisms of LRRK2-associated Parkinson’s disease. To this aim, we initially performed RNA-sequencing (RNA-seq) of Lrrk2 wild-type (WT) and knockout (KO) primary microglia treated with α-synuclein pre-formed fibrils (PFFs) or lipopolysaccharide (LPS). We found that LPS and α-synuclein triggered in part overlapping but also different responses, while loss of Lrrk2 had a subtle effect in attenuating the pro-inflammatory response. Moreover, treatment with α-synuclein PFFs caused a significant induction of the antioxidant superoxide dismutase-2 (SOD2) enzyme in WT cells, with the effect attenuated in Lrrk2 KO microglia cells. Since loss of Lrrk2 in primary microglia revealed only small differences in gene expression both under resting or pro-inflammatory conditions, we next moved to an experimental condition that more closely resembles the physiological situation, e.g. acutely isolated microglia cells from LPS-injected adult mouse brains. Using single cell RNA sequencing (scRNA-seq), we confirmed a subset of the genes nominated in primary culture experiments, including IL-1, SOD2 and TXNIP as differentially expressed upon inflammatory stimulation with LPS. However, the overall effect on gene transcription due to loss of Lrrk2 remained subtle. To further explore the impact of LRRK2 on microglia function, we performed ex vivo phagocytic assays. Lrrk2 KO microglia displayed the highest phagocytic activity compared to microglia isolated from WT and pathogenic mutants G2019S and R1441C mice upon treatment with α-synuclein PFFs, suggesting that LRRK2 negatively regulates phagocytosis or delays lysosomal degradation. Additionally, we performed intrastriatal brain injections with lipopolysaccharide (LPS)-inflammatory agent or with PBS as a control, that demonstrated a significant morphological changes in microglial shape (increase in circularity and soma area) in LPS-stimulated animals compare to PBS-treated and a modest differences between LRRK2 genotypes. Additionally, the branch complexity was reduced in LPS-injected animals with stronger effect in Lrrk2-KO and R1441C genotypes and a less response in G2019S animals. Next, we evaluated LRRK2 mRNA and protein expression at the single cell level. We found that LRRK2 has a low and sparse pattern of expression in unstimulated resident microglia. However, upon stimulation with LPS, the proportion of microglia cells expressing LRRK2 increased, while LRRK2 levels remained unchanged per cell. This is, to date, the first attempt to analyze LRRK2 protein expression at the single cell level in brain cells. In summary, the present work provides new insights into the biology of LRRK2 in the brain resident microglia and shows that inflammatory stimulations with α-synuclein PFFs cause a significant induction of pro-inflammatory and anti-oxidant responses, which are attenuated in LRRK2-KO microglia