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Single cell RNA sequencing reveals limited effects of Lrrk2 genotype in a mouse model of acute CNS inflammation

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE265833 | GEO | 2024/04/29

REPOSITORIES: GEO

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Project description:Acute cognitive impairment (i.e., delirium) is common in elderly emergency department patients and frequently results from infections that are unrelated to the central nervous system. Since activation of the peripheral innate immune system induces brain microglia to produce inflammatory cytokines that are responsible for behavioral deficits, we investigated if aging exacerbated neuroinflammation and sickness behavior after peripheral injection of lipopolysaccharide (LPS). Microarray analysis revealed a transcriptional profile indicating the presence of primed or activated microglia and increased inflammation in the aged brain. Furthermore, aged mice had a unique gene expression profile in the brain after an intraperitoneal injection of LPS, and the LPS-induced elevation in the brain inflammatory cytokines and oxidative stress was both exaggerated and prolonged compared with adults. Aged mice were anorectic longer and lost more weight than adults after peripheral LPS administration. Moreover, reductions in both locomotor and social behavior remained 24 h later in aged mice, when adults had fully recovered, and the exaggerated neuroinflammatory response in aged mice was not reliably paralleled by increased circulating cytokines in the periphery. Taken together these data establish that activation of the peripheral innate immune system leads to exacerbated neuroinflammation in the aged as compared with adult mice. This dysregulated link between the peripheral and central innate immune system is likely to be involved in the severe behavioral deficits that frequently occur in older adults with systemic infections. Experiment Overall Design: In this study, adult and aged mice were injected intraperitoneal with sterile saline or Escherichia coli LPS (0.33 mg/kg, ~10 µg/mouse; serotype 0127:B8, Sigma). This dosage of LPS was used because it induces a mild transient sickness behavior in young adults. Mice were killed 4 h after saline or LPS injection by CO2 asphyxiation. Blood samples were collected and brains were removed, separated in half at the longitudinal fissure, frozen in liquid nitrogen, and stored (-80°C) until assaying. Total RNA was later isolated from some brain samples for microarray analysis (n=3).

Project description:Background: Specific microglia responses are thought to contribute to the development and progression of neurodegenerative diseases, including Parkinson’s disease (PD). However, the phenotypic acquisition of microglial cells and their role during the underlying neuroinflammatory processes remain largely elusive. Here, according to the multiple-hit hypothesis, which stipulates that PD etiology is determined by a combination of genetics and various environmental risk factors, we investigate microglial transcriptional programs and morphological adaptations under PARK7/DJ-1 deficiency, a genetic cause of PD, during lipopolysaccharide (LPS)-induced inflammation. Methods: Using a combination of single-cell RNA-sequencing, bulk RNA-sequencing, multicolor flow cytometry and immunofluorescence analyses, we comprehensively compared microglial cell phenotypic characteristics in PARK7/DJ-1 knock-out (KO) with wildtype littermate mice following 6- or 24-hour intraperitoneal injection with LPS. For translational perspectives, we conducted corresponding analyses in human PARK7/DJ-1 mutant induced pluripotent stem cell (iPSC)-derived microglia and murine bone marrow-derived macrophages (BMDMs). Results: By excluding the contribution of other immune brain resident and peripheral cells, we show that microglia acutely isolated from PARK7/DJ-1 KO mice display a distinct phenotype, specially related to type II interferon and DNA damage response signaling, when compared with wildtype microglia, in response to LPS. We also detected discrete signatures in human PARK7/DJ-1 mutant iPSC-derived microglia and BMDMs from PARK7/DJ-1 KO mice. These specific transcriptional signatures were reflected at the morphological level, with microglia in LPS-treated PARK7/DJ-1 KO mice showing a less amoeboid cell shape compared to wildtype mice, both at 6 and 24 hours after acute inflammation, as also observed in BMDMs. Conclusions: Taken together, our results show that, under inflammatory conditions, PARK7/DJ-1 deficiency skews microglia towards a distinct phenotype characterized by downregulation of genes involved in type II interferon signaling and a less prominent amoeboid morphology compared to wildtype microglia. These findings suggest that the underlying oxidative stress associated with the lack of PARK7/DJ-1 affects microglia neuroinflammatory responses, which may play a causative role in PD onset and progression.

Project description:Background: Specific microglia responses are thought to contribute to the development and progression of neurodegenerative diseases, including Parkinson’s disease (PD). However, the phenotypic acquisition of microglial cells and their role during the underlying neuroinflammatory processes remain largely elusive. Here, according to the multiple-hit hypothesis, which stipulates that PD etiology is determined by a combination of genetics and various environmental risk factors, we investigate microglial transcriptional programs and morphological adaptations under PARK7/DJ-1 deficiency, a genetic cause of PD, during lipopolysaccharide (LPS)-induced inflammation. Methods: Using a combination of single-cell RNA-sequencing, bulk RNA-sequencing, multicolor flow cytometry and immunofluorescence analyses, we comprehensively compared microglial cell phenotypic characteristics in PARK7/DJ-1 knock-out (KO) with wildtype littermate mice following 6- or 24-hour intraperitoneal injection with LPS. In a translational approach, we conducted corresponding analyses in human PARK7/DJ-1 mutant induced pluripotent stem cell (iPSC)-derived microglia and murine bone marrow-derived macrophages (BMDMs). Results: By excluding the contribution of other immune brain resident and peripheral cells, we show that microglia acutely isolated from PARK7/DJ-1 KO mice display a distinct phenotype, specially related to type II interferon and DNA damage response signaling, when compared with wildtype microglia, in response to LPS. We also detected discrete signatures in human PARK7/DJ-1 mutant iPSC-derived microglia and BMDMs from PARK7/DJ-1 KO mice. These specific transcriptional signatures were reflected at the morphological level, with microglia in LPS-treated PARK7/DJ-1 KO mice showing a less amoeboid cell shape compared to wildtype mice, both at 6 and 24 hours after acute inflammation, as also observed in BMDMs. Conclusions: Taken together, our results show that, under inflammatory conditions, PARK7/DJ-1 deficiency skews microglia towards a distinct phenotype characterized by downregulation of genes involved in type II interferon signaling and a less prominent amoeboid morphology compared to wildtype microglia. These findings suggest that the underlying oxidative stress associated with the lack of PARK7/DJ-1 affects microglia neuroinflammatory responses, which may play a causative role in PD onset and progression.

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Single cell RNA sequencing reveals limited effects of Lrrk2 genotype in a mouse model of acute CNS inflammation

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