The Alzheimer’s disease risk factor INPP5D restricts neuroprotective microglial responses in amyloid beta-mediated pathology [snRNA-seq]
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ABSTRACT: INTRODUCTION: Gain-of-function mutations in INPP5D, which encodes for the SH2-domain-containing inositol phosphatase SHIP-1, have recently been linked to an increased risk of developing late-onset Alzheimer’s disease (LOAD). Yet, little is currently known in regards to how SHIP-1 affects neurobiology or neurodegenerative disease pathogenesis. METHODS: To bridge this knowledge gap, we generated 5xFAD Inpp5dfl/flCx3cr1Ert2Cre mice to investigate the function of microglial SHIP-1 signaling in response to amyloid beta (Aβ)-mediated pathology. RESULTS: In our studies, we found that SHIP-1 deletion in microglia leads to substantially enhanced recruitment of microglia to Aβ plaques, altered microglial gene expression, and marked improvements in neuronal health. Further, SHIP-1 loss enhanced microglial plaque containment and Aβ engulfment when compared to microglia from Cre-negative 5xFAD Inpp5dfl/fl littermate controls. DISCUSSION: These results define SHIP-1 as a pivotal regulator of microglial responses during Aβ-driven neurological disease.
Project description:We crossed Tyrobp-deficient and Inpp5d-deficient mice with App(NL-G-F/NL-G-F) Alzheimer model mice (hereafter, NLGF) and generate Tyrobp-/-;Inpp5d+/-;NLGF mice to assess the role of Inpp5d in Tyrobp-/- mice. We isolated microglia from NLGF, Tyrobp-/-;NLGF, and Tyrobp-/-;Inpp5d+/-;NLGF mice and analyzed microglial transcriptome using RNA-sequencing. Tyrobp-/- microglia showed substantially different transcriptome compared with NLGF microglia. Previously described "disease asscociated microglia" genes were enriched in the differentially expressed genes in Tyrobp-/- microglia. On the other hand, Tyrobp-/-;Inpp5d+/-;NLGF microglia showed similar transcriptome to Tyrobp-/- microglia except several genes (e.g., Wdfy1, Mamdc2, Klrb1f). These data suggest Inpp5d modulates microglial functions without affecting disease associated microglial gene expression in Tyrobp-/-;NLGF micrglia.
Project description:Microglia are innate immune cells of the brain that perform phagocytic and inflammatory functions in disease conditions. Transcriptomic studies of acutely-isolated microglia have provided novel insights into their molecular and functional diversity in homeostatic and neurodegenerative disease states. State-of-the-art mass spectrometric methods can comprehensively characterize proteomic alterations in microglia in neurodegenerative disorders, potentially providing novel functionally-relevant molecular insights that are not provided by transcriptomics. However, proteomic profiling of adult primary microglia in neurodegenerative disease conditions has not been performed. We performed quantitative proteomic analyses of purified CD11b+ acutely-isolated microglia adult mice in normal, acute neuroinflammatory (LPS-treatment) and chronic neurodegenerative states (5xFAD model of Alzheimer’s disease [AD]) using tandem mass tag mass spectrometry. Differential expression analyses were performed to characterize specific microglial proteomic changes in 5xFAD mice and identify overlap with LPS-induced pro-inflammatory changes. Our results were also contrasted with existing proteomic data from wild-type mouse microglia and from existing microglial transcriptomic data from wild-type and 5xFAD mice. Neuropathological validation studies of select proteins were performed in human AD and 5xFAD brains. Of 4,133 proteins identified, 187 microglial proteins were differentially expressed in the 5xFAD mouse model of AD pathology, including proteins with previously known (Apoe, Clu and Htra1) as well as previously unreported relevance to AD biology (Cotl1 and Hexb). Proteins upregulated in 5xFAD microglia shared significant overlap with pro-inflammatory changes observed in LPS-treated mice. Several proteins increased in human AD brain were also upregulated by 5xFAD microglia (Aβ peptide, Apoe, Htra1, Cotl1 and Clu). Cotl1 was identified as a novel microglia-specific marker with increased expression and strong association with AD neuropathology. Apoe protein was also detected within plaque-associated microglia in which Apoe and Aβ were highly co-localized suggesting a role for Apoe in phagocytic clearance of Aβ. We report the first comprehensive comparative proteomic study of adult mouse microglia derived from acute neuroinflammatory and AD models, representing a valuable resource to the neuroscience research community. We highlight shared and unique microglial proteomic changes in acute neuroinflammatory, aging and AD mouse models in addition to identifying novel roles for microglial proteins in human neurodegeneration.
Project description:It was recently revealed that gut microbiota promote amyloid-beta (Aβ) burden in mouse models of Alzheimer’s disease (AD). However, the underlying mechanisms when using either germ-free (GF) housing conditions or treatments with antibiotics (ABX) remained unknown. In this study, we show that GF and ABX-treated 5x familial AD (5xFAD) mice developed attenuated hippocampal Aβ pathology and associated neuronal loss, and thereby delayed disease-related memory deficits. While Ab production remained unaffected in both GF and ABX-treated 5xFAD mice, we noticed in GF 5xFAD mice enhanced microglial Aβ uptake at early stages of the disease compared to ABX-treated 5xFAD mice. Furthermore, RNA-sequencing of hippocampal microglia from SPF, GF and ABX-treated 5xFAD mice revealed distinct microbiota-dependent gene expression profiles associated with phagocytosis and altered microglial activation states. Taken together, we observed that constitutive or induced microbiota modulation in 5xFAD mice differentially controls microglial Aβ clearance mechanisms preventing neurodegeneration and cognitive deficits.
Project description:Microglia and neuroinflammation are implicated in the development and progression of Alzheimer’s disease (AD). To better understand microglia-mediated processes in AD, we studied the function of INPP5D/SHIP1, a gene linked to AD through GWAS. Immunostaining and single nucleus RNA sequencing confirmed that INPP5D expression in the adult human brain is enriched in microglia. Examination of prefrontal cortex across a large cohort revealed reduced full-length soluble INPP5D protein levels in AD patient brains compared to cognitively normal controls. However, elevated INPP5D immunostaining in microglia in the AD brain was observed, which was driven by elevations in plaque-associated microglia suggesting that these two methods quantify different pools of INPP5D. Examination of INPP5D overexpression and bi-allelic loss-of-function in induced pluripotent stem cell derived microglia (iMGs) revealed that INPP5D LOF most closely resemble microglia in the AD brain with respect to immune signaling alterations, supporting the hypothesis that INPP5D function is reduced in AD microglia. The functional consequences of reduced INPP5D activity were evaluated in human iMGs, using both pharmacological inhibition of the phosphatase activity of INPP5D and genetic reduction in copy number. Unbiased transcriptional and proteomic profiling of these iMGs suggested an upregulation of innate immune signaling pathways, lower levels of scavenger receptors, reduced lysosomal proteins and altered inflammasome signaling with INPP5D reduction. INPP5D inhibition induced the secretion of IL-1ß and IL-18, further implicating inflammasome activation. Inflammasome activation was confirmed through visualization of inflammasome formation through ASC immunostaining in INPP5D-inhibited iMGs, increased cleaved caspase-1 and through rescue of elevated IL-1ß and IL-18 with caspase-1 and NLRP3 inhibitors. In accord, lower INPP5D is associated with higher IL-18 levels and an elevation in microglia with ASC specks in the AD brain. This work implicates INPP5D as a regulator of inflammasome signaling in human microglia.
Project description:Microglia and neuroinflammation play an important role in the development and progression of Alzheimer’s disease (AD). Inositol polyphosphate-5-phosphatase D (INPP5D) is a myeloid-expressed gene genetically-associated with AD. Through unbiased analyses of RNA and protein profiles in INPP5D-disrupted iPSC-derived human microglia, we find that reduction in INPP5D activity is associated with molecular profiles consistent with disrupted autophagy and inflammasome activation. These findings are validated through targeted pharmacological experiments which demonstrate that reduced INPP5D activity induces the formation of the NLRP3 inflammasome, cleavage of CASP1, and secretion of IL-1 and IL-18. Further, in-depth analyses of human brain tissue across hundreds of individuals using a multi-analytic approach provides evidence that a reduction in function of INPP5D in microglia results in inflammasome activation in AD. These findings provide insights into the molecular mechanisms underlying microglia-mediated processes in AD and highlight the inflammasome as a potential therapeutic target for modulating INPP5D-mediated vulnerability to AD.
Project description:Triggering receptor expressed on myeloid cells 2 (TREM2) sustains microglia response to brain injury stimuli including apoptotic cells, myelin damage, and amyloid β (Aβ). Alzheimer’s Disease (AD) risk is associated with the TREM2R47H variant, which impairs ligand binding and consequently microglia responses to Aβ pathology. Here we tested whether TREM2 engagement by an agonistic mAb, hT2AB, designated as a surrogate ligand facilitates microglia responses in 5XFAD transgenic mice that accumulate Aβ and express either the common TREM2 variant (TREM2CV) or TREM2R47H. scRNA-seq of microglia from TREM2CV-5XFAD mice treated once with control IgG exposed trajectories representative of activated microglial populations including disease-associated microglia (DAM), interferon-responsive (IFN-R), cycling (Cyc-M), and MHC-II expressing (MHC-II) microglia. All of these were underrepresented in TREM2R47H-5XFAD mice, suggesting that TREM2 ligand engagement is required for microglia transitions. Moreover, Cyc-M and IFN-R microglia were better represented in female than male TREM2CV-5XFAD mice, likely reflecting a greater Aβ load in female 5XFAD mice. A single systemic injection of hT2AB replenished the Cyc-M, IFN-R, and MHC-II pools in TREM2R47H-5XFAD mice. In TREM2CV-5XFAD mice, however, hT2AB brought the representation of male Cyc-M and IFN-R microglia closer to that of females, in which microglia transitions in response to Aβ had already reached their peak. Repeated treatment with a murinized version mT2AB over a 10 days period increased brain content of chemokines in TREM2R47H-5XFAD mice, consistent with microglia expansion and shifts in gene expression patterns. Thus, the impact of hT2AB on microglia responses is shaped by the extent of TREM2 endogenous ligand engagement and basal microglia activation.
Project description:We define a pathogenic subset of microglia that is distinguished by expression of the CD11c protein and by production of osteopontin (OPN). OPN production by this CD11c+ microglial subset correlates positively with disease pathology and severity in the 5XFAD mouse model and in AD patients. Genetic ablation of OPN in 5XFAD mice leads to reduced development of pro-inflammatory CD11c+ microglia, increased amyloid beta (Aβ) phagocytosis and improved cognitive function.
Project description:INTRODUCTION: INPP5D is genetically associated with Alzheimer’s disease risk. Loss of Inpp5d alters amyloid pathology in models of amyloidosis. Inpp5d is predominantly expressed in microglia but its function in brain is poorly understood. METHODS: We performed single-cell RNA-sequencing to study the effect of Inpp5d loss on wild-type mouse brain transcriptomes. RESULTS: Loss of Inpp5d has sex-specific effects on the brain transcriptome. Affected genes are enriched for multiple neurodegeneration terms. Network analyses reveal a gene co-expression module centered around Inpp5d in female mice. Inpp5d loss alters PTN, PSAP, and VEGFA signaling probability between cell types. DISCUSSION: Our data suggest Inpp5d’s normal function is entangled with mechanisms involved in neurodegeneration. We report the effect of Inpp5d loss without pathology and show that this has dramatic effects on gene expression. Our study provides a critical reference for researchers of neurodegeneration, allowing separation of disease-specific changes mediated by Inpp5d in disease from baseline effects of Inpp5d loss.
Project description:The gut microbiota and innate immune system play critical roles in Alzheimer’s disease (AD). Bacteroides is elevated in AD patients and correlates with cerebrospinal fluid levels of Aβ and tau. We found that increased amyloid-β (Aβ) plaques in Bacteroides fragilis treated APP/PS1-21 mice were associated with altered cortical expression Aβ processing genes. B. fragilis suppressed peripheral CD4+ T cell production of GM-CSF and IL-4 and transcriptional changes in microglia related to GM-CSF and IL-4 signaling, phagocytosis, and protein degradation. Furthermore, B. fragilis impaired the microglial uptake of intracranially injected Aβ42, whereas Erysipelotrichaceae strains increased uptake. Depleting murine Bacteroidetes with metronidazole decreased amyloid load in aged 5xFAD mice, increased CD4+ T cell GM-CSF production, and activated microglial pathways related to cytokine signaling, phagocytosis and lysosomal degradation. These data suggest that the gut microbiome may contribute to AD pathogenesis by suppressing peripheral cytokines and microglia phagocytic function, leading to impaired immune-mediated Aβ clearance.
Project description:The gut microbiota and innate immune system play critical roles in Alzheimer’s disease (AD). Bacteroides is elevated in AD patients and correlates with cerebrospinal fluid levels of Aβ and tau. We found that increased amyloid-β (Aβ) plaques in Bacteroides fragilis treated APP/PS1-21 mice were associated with altered cortical expression Aβ processing genes. B. fragilis suppressed peripheral CD4+ T cell production of GM-CSF and IL-4 and transcriptional changes in microglia related to GM-CSF and IL-4 signaling, phagocytosis, and protein degradation. Furthermore, B. fragilis impaired the microglial uptake of intracranially injected Aβ42, whereas Erysipelotrichaceae strains increased uptake. Depleting murine Bacteroidetes with metronidazole decreased amyloid load in aged 5xFAD mice, increased CD4+ T cell GM-CSF production, and activated microglial pathways related to cytokine signaling, phagocytosis and lysosomal degradation. These data suggest that the gut microbiome may contribute to AD pathogenesis by suppressing peripheral cytokines and microglia phagocytic function, leading to impaired immune-mediated Aβ clearance.