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PARK7/DJ-1 deficiency impairs microglial activation in response to LPS-induced inflammation

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE256105 | GEO | 2024/08/09

REPOSITORIES: GEO

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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.

Project description:Microglia, the resident immune cells of the central nervous system (CNS), have two distinct phenotypes in the developing brain: amoeboid form, known to be amoeboid microglial cells (AMC) and ramified form, known to be ramified microglial cells (RMC) alongside several intermediate forms. The AMC are characterized by being proliferative, phagocytic and migratory whereas the RMC are quiescent and exhibit a slow turnover rate. The AMC transform into RMC with advancing age, and this transformation is indicative of the gradual shift in the microglial functions. Both AMC and RMC respond to CNS inflammation, and they become hypertrophic when they are activated by trauma, infection or neurodegenerative stimuli. The molecular mechanisms and functional significance of morphological transformation of microglia during normal development and in disease conditions is not clear. It is hypothesized that AMC and RMC are functionally regulated by a specific set of genes encoding various signaling molecules and transcription factors. To address this, we carried out cDNA microarray analysis using lectin-labeled AMC and RMC isolated from frozen tissue sections of the corpus callosum of 5-day and 4-week old rat brain respectively, by laser capture microdissection (LCM). The global gene expression profiles of both microglial phenotypes were compared and the differentially expressed genes in AMC and RMC were clustered based on their functional annotations. This genome wide comparative analysis helps in identifying genes that are specific to AMC and RMC. The novel and specific molecules identified in both microglial phenotypes can be targeted for therapeutic purposes in developing and adult brain diseases. We used microarrays to identify the genes specific to amoeboid and ramified microglia. RNA was isolated from the laser-captured amoeboid and ramified microglia from the corpus callosum of 5-day and 4-week old rat brain. The RNA was hybridised onto Affymetrix Rat 230 2.0 array.

Project description:Microglia colonize the brain parenchyma at early stages of development and accumulate in specific regions where they actively participate in cell death, angiogenesis, neurogenesis and synapse elimination. A recurring feature of embryonic microglial distribution is their association with developing axon tracts which, together with in vitro data, supports the idea of a physiological role for microglia in neurite development. Yet the demonstration of this role of microglia is still lacking. Here, we have studied the consequences of microglial dysfunction on the formation of the corpus callosum, the largest connective structure in the mammalian brain, which shows consistent microglial accumulation during development. We studied two models of microglial dysfunction: the loss-of-function of DAP12, a key microglial-specific signaling molecule, and a model of maternal inflammation by peritoneal injection of LPS at E15.5. We performed transcriptional profiling of maternally inflamed and Dap12-mutant microglia at E17.5. We found that both treatments principally down-regulated genes involved in nervous system development and function, particularly in neurite formation. We then analyzed the functional consequences of these microglial dysfunctions on the formation of the corpus callosum. We also took advantage of the Pu.1-/- mouse line, which is devoid of microglia. We now show that all three models of altered microglial activity resulted in the same defasciculation phenotype. Our study demonstrates that microglia are actively involved in the fasciculation of corpus callosum axons. To investigate possible roles for microglial during brain development, we challenged microglial function by two complementary approaches. First, we induced maternal inflammation by peritoneal injection of LPS into pregnant dams. Next, we analyzed the consequences of a loss of function of DAP12, a signaling molecule specifically expressed in microglia that is crucial for several aspects of microglia biology (references in Wakselman et al., 2008). We compared the gene expression profiles of microglia from control, maternally-inflamed by LPS (MI), and Dap12-mutated embryos. We isolated RNA from FACS sorted maternally inflamed (by LPS) and Dap12-mutant microglia at E17.5 pooled per pregnant dam; as a control we included PBS treated and untreated (UT) microglia. We compared gene expression between maternally inflamed microlgia (PBSvsLPS) and DAP12-mutant microglia (UTvsDAP12KO).

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PARK7/DJ-1 deficiency impairs microglial activation in response to LPS-induced inflammation

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