Microglial Transcriptome Research Articles

Microglial and neuronal fates following inhibition of CSF-1R in synucleinopathy mouse model

Synucleinopathies are age-related neurological disorders characterized by the abnormal accumulation of α-synuclein (α-syn) in neuronal and non-neuronal cells. It has been proposed that microglial cells play an important role in synucleinopathy neuroinflammation, as well as homeostatically, such as in the clearance of α-syn aggregates in the brain. Here, we examined the effects of microglia on the pathogenesis of synucleinopathies by cell depletion in a mouse model of synucleinopathies. For this purpose, we treated non-transgenic (Non-tg) and α-synuclein transgenic (α-syn-tg) mice with pexidartinib (PLX3397), a tyrosine kinase inhibitor of colony-stimulating factor 1 receptor (CSF-1R). Neuropathological and immunoblot analysis confirmed that Iba-1 immunoreactive microglial cells were decreased by 95% following PLX3397 treatment in Non-tg and α-syn-tg mice. The level of total α-syn in the Triton X-insoluble fraction of brain homogenate was significantly decreased by microglial depletion in the α-syn-tg mice, while the level of Triton X-soluble human α-syn was not affected. Furthermore, the number of p-α-syn immunoreactive inclusions was reduced in α-syn-tg mice treated with PLX3397. Microglial depletion also ameliorated neuronal and synaptic degeneration in α-syn-tg mice, thereby resulted partially improving the motor behavioral deficit in α-syn-tg mice. Moreover, we demonstrated that microglia that survived post-PLX3397 treatment (PLX-resistant microglia) have lower expressions of CSF-1R, and microglial transcriptome analysis further elucidated that PLX-resistant microglia have unique morphology and transcriptomic signatures relative to vehicle-treated microglia of both genotypes; these include differences in definitive microglial functions such as their immune response, cell mobility, cell–cell communications, and regulation of neural homeostasis. Therefore, we suggest that microglia play a critical role in the pathogenesis of synucleinopathies, and that modulation of microglial status might be an effective therapeutic strategy for synucleinopathies.

Targeting dysfunctional endocannabinoid signaling in a mouse model of Gulf War illness

Gulf War Illness (GWI) is a chronic disorder characterized by a heterogeneous set of symptoms that include pain, fatigue, anxiety, and cognitive impairment. These are thought to stem from damage caused by exposure under unpredictable stress to toxic Gulf War (GW) chemicals, which include pesticides, nerve agents, and prophylactic drugs. We hypothesized that GWI pathogenesis might be rooted in long-lasting disruption of the endocannabinoid (ECB) system, a signaling complex that serves important protective functions in the brain. Using a mouse model of GWI, we found that tissue levels of the ECB messenger, anandamide, were significantly reduced in the brain of diseased mice, compared to healthy controls. In addition, transcription of the Faah gene, which encodes for fatty acid amide hydrolase (FAAH), the enzyme that deactivates anandamide, was significant elevated in prefrontal cortex of GWI mice and brain microglia. Behavioral deficits exhibited by these animals, including heightened anxiety-like and depression-like behaviors, and defective extinction of fearful memories, were corrected by administration of the FAAH inhibitor, URB597, which normalized brain anandamide levels. Furthermore, GWI mice displayed unexpected changes in the microglial transcriptome, implying persistent dampening of homeostatic surveillance genes and abnormal expression of pro-inflammatory genes upon immune stimulation. Together, these results suggest that exposure to GW chemicals produce a deficit in brain ECB signaling which is associated with persistent alterations in microglial function. Pharmacological normalization of anandamide-mediated ECB signaling may offer an effective therapeutic strategy for ameliorating GWI symptomology.

Effect of ultrasound-mediated blood-spinal cord barrier opening on survival and motor function in females in an amyotrophic lateral sclerosis mouse model

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by a progressive loss of motor neurons. The limited efficacy of recent therapies in clinical development may be linked to lack of drug penetration to the affected motor neurons due to the blood-brain barrier (BBB) and blood-spinal cord barrier (BSCB). In this work, the safety and efficacy of repeated short transient opening of the BSCB by low intensity pulsed ultrasound (US, sonication) was studied in females of an ALS mouse model (B6.Cg-Tg(SOD1∗G93A)1Gur/J). The BSCB was disrupted using a 1MHz ultrasound transducer coupled to the spinal cord, with and without injection of insulin-like growth factor 1 (IGF1), a neurotrophic factor that has previously shown efficacy in ALS models. Results in wild-type (WT) animals demonstrated that the BSCB can be safely disrupted and IGF1 concentrations significantly enhanced after a single session of transient BSCB disruption (176±32μg/g vs. 0.16±0.008μg/g, p<0.0001). Five repeated weekly US sessions performed in female ALS mice demonstrated a survival advantage in mice treated with IGF1 and US (US IGF1) compared to treatment with IGF1 alone (176 vs. 166 days, p=0.0038). Surprisingly, this survival advantage was also present in mice treated with US alone vs. untreated mice (178.5 vs. 166.5 days, p=0.0061). Muscle strength did not show difference among the groups. Analysis of glial cell immunoreactivity and microglial transcriptome showing reduced cell proliferation pathways, in addition to lymphocyte infiltration, suggested that the beneficial effect of US or US IGF1 could act through immune cell modulation. These results show the first step towards a possible beneficial impact of transient BSCB opening for ALS therapy and suggest implication of immune cells. Fondation pour la Recherche Médicale (FRM). Investissements d'avenirANR-10-IAIHU-06, Société Française de Neurochirurgie (SFNC), Fond d'étude et de Recherche du Corps Medical (FERCM), Aide à la Recherche des Maladies du Cerveau (ARMC), SLA Fondation Recherche (SLAFR), French Ministry for High Education and Research (MENR), Carthera, Laboratoire de Recherche en Technologies Chirurgicales Avancées (LRTCA).

Fokker-Planck diffusion maps of multiple single cell microglial transcriptomes reveals radial differentiation into substates associated with Alzheimer's pathology.

The identification of microglia subtypes is important for understanding the role of innate immunity in neurodegenerative diseases. Current methods of unsupervised cell type identification assume a small noise-to-signal ratio of transcriptome measurements that would produce well-separated cell clusters. However, identification of subtypes is obscured by gene expression noise, diminishing the distances in transcriptome space between distinct cell types and blurring boundaries. Here we use Fokker-Planck (FP) diffusion maps to model cellular differentiation as a stochastic process whereby cells settle into local minima, corresponding to cell subtypes, in a potential landscape constructed from transcriptome data using a nearest neighbor graph approach. By applying critical transition fields, we identify individual cells on the verge of transitioning between subtypes, revealing microglial cells in inactivated, homeostatic state before radially transitioning into various specialized subtypes. Specifically, we show that cells from Alzheimer's disease patients are enriched in a microglia subtype associated to antigen presentation and T-cell recruitment.

LRRK2 G2019S variant is associated with transcriptional changes in Parkinson's disease human myeloid cells under proinflammatory environment.

The G2019S mutation in the leucine-rich repeat kinase 2 (LRRK2) gene is a major risk factor for the development of Parkinson's disease (PD). LRRK2, although ubiquitously expressed, is highly abundant in cells of the innate immune system. Given the importance of central and peripheral immune cells in the development of PD, we sought to investigate the consequences of the G2019S mutation on microglial and monocyte transcriptome and function. We have generated large-scale transcriptomic profiles of isogenic human induced microglial cells (iMGLs) and patient derived monocytes carrying the G2019S mutation under baseline culture conditions and following exposure to the proinflammatory factors IFNγ and LPS. We demonstrate that the G2019S mutation exerts a profound impact on the transcriptomic profile of these myeloid cells, and describe corresponding functional differences in iMGLs. The G2019S mutation led to an upregulation in lipid metabolism and phagolysosomal pathway genes in untreated and LPS/IFNγ stimulated iMGLs, which was accompanied by an increased phagocytic capacity of myelin debris. We also identified dysregulation of cell cycle genes, with a downregulation of the E2F4 regulon. Transcriptomic characterization of human-derived monocytes carrying the G2019S mutation confirmed alteration in lipid metabolism associated genes. Altogether, these findings reveal the influence of G2019S on the dysregulation of the myeloid cell transcriptome under proinflammatory conditions.

The Microglial Transcriptome of Age-Associated Deep Subcortical White Matter Lesions Suggests a Neuroprotective Response to Blood-Brain Barrier Dysfunction.

Age-associated deep-subcortical white matter lesions (DSCLs) are an independent risk factor for dementia, displaying high levels of CD68+ microglia. This study aimed to characterize the transcriptomic profile of microglia in DSCLs and surrounding radiologically normal-appearing white matter (NAWM) compared to non-lesional control white matter. CD68+ microglia were isolated from white matter groups (n = 4 cases per group) from the Cognitive Function and Ageing Study neuropathology cohort using immuno-laser capture microdissection. Microarray gene expression profiling, but not RNA-sequencing, was found to be compatible with immuno-LCM-ed post-mortem material in the CFAS cohort and identified significantly differentially expressed genes (DEGs). Functional grouping and pathway analysis were assessed using the Database for Annotation Visualization and Integrated Discovery (DAVID) software, and immunohistochemistry was performed to validate gene expression changes at the protein level. Transcriptomic profiling of microglia in DSCLs compared to non-lesional control white matter identified 181 significant DEGs (93 upregulated and 88 downregulated). Functional clustering analysis in DAVID revealed dysregulation of haptoglobin-haemoglobin binding (Enrichment score 2.5, p = 0.017), confirmed using CD163 immunostaining, suggesting a neuroprotective microglial response to blood-brain barrier dysfunction in DSCLs. In NAWM versus control white matter, microglia exhibited 347 DEGs (209 upregulated, 138 downregulated), with significant dysregulation of protein de-ubiquitination (Enrichment score 5.14, p < 0.001), implying an inability to maintain protein homeostasis in NAWM that may contribute to lesion spread. These findings enhance understanding of microglial transcriptomic changes in ageing white matter pathology, highlighting a neuroprotective adaptation in DSCLs microglia and a potentially lesion-promoting phenotype in NAWM microglia.

Abstract 111: Membrane Lipid-Sensing Receptor MS4A Modulates Microglial Reparative Phenotype in Acute Germinal Matrix Hemorrhage-Intraventricular Hemorrhage (GMH-IVH)

Background: GMH-IVH refers to spontaneous bleeding in the periventricular germinal zone and ventricles of an immature brain. Preterm infants with GMH-IVH often have lifelong neurologic deficits. Microglia play crucial roles during development; however, what role they play in neonatal cerebrovascular injury such as GMH-IVH is less known. Here we established a mouse GMH-IVH model and unveiled an immature brain-specific microglial response. Methods: GMH-IVH and adult intracerebral hemorrhage (ICH)-IVH were induced by blood injection into the caudate-putamen and periventricular region of postnatal day 5 and 8-week-old mice. Astrogliosis, myelin loss, MS4A receptor expression, neuron morphology, and spine density were evaluated by immunofluorescence and Golgi’s staining. Transcriptome of microglia was profiled by NanoString. Cx3cr1 CreER ; iDRT mice and PLX3397 treatment were used to deplete microglia. Cognitive and affective functions were evaluated. In vivo siRNA delivery was used to explore the mechanism underlying acute microglia reparative activation after GMH-IVH. Result: Astrogliosis and myelin loss were sustained till at least 8 weeks after GMH-IVH. Reduced neuronal dendritic complexity in the dentate gyrus and medial prefrontal cortex suggested compromised neuronal development in GMH-IVH. Upon adulthood, GMH-IVH mice had impaired cognitive and affective functions . Microglia showed heterogeneous phenotypes on day 3 after GMH-IVH. Interestingly, acute depletion of microglia aggravated myelin loss and neurobehavioral deficits, implying a beneficial role of these cells in the early stage of GMH-IVH. Compared with day 28 after GMH-IVH and day 3 after ICH-IVH, microglia at day 3 GMH-IVH uniquely upregulated lipid-sensing receptor MS4A. Inhibition of MS4A worsened long-term GMH-IVH outcomes. Conclusion: MS4A-expressing microglia are beneficial for GMH-IVH and targeting their responses could be a therapeutic approach for treating GMH-IVH.

Abstract TP27: Transcriptional and Functional Characterization of Microglia in Vascular Dementia

Introduction: Proper function of the brain relies on sufficient blood supply to meet local energy demand. Age-related pathologies like cerebral small vessel disease (cSVD) compromise the ability of blood vessels to meet local energy demands and lead, over time, to the development of vascular dementia (VaD). Microglia are essential for maintaining homeostasis and have been shown to exacerbate inflammation and disease progression in neurodegenerative diseases. Activated microglia are present in the white matter lesions of human post-mortem VaD brains. However, their role in VaD progression is poorly understood. Hypothesis: Our main objective is to identify microglial pathways that contribute to VaD. We hypothesize microglial dysregulation interferes with brain homeostasis and wound healing, ultimately contributing to disease progression. Methods: Post-mortem samples of periventricular white matter from male and female elderly individuals exhibiting brain pathology consistent with VaD (n=4), along with age- and sex-matched controls without VaD (n=4) were acquired from the NIH NeuroBioBank. We performed single-nuclei RNA sequencing followed by differential gene expression analysis using Seurat and downstream analyses using Ingenuity Pathway Analysis. Results: Over 30,000 nuclei were analyzed. Transcriptomic results reveal profound differences in the microglial transcriptome in VaD, with 690 differentially regulated genes in microglia between cases and controls. Genes related to pathways such as complement production, phagocytosis, macrophage activation and overall protein production are downregulated in VaD while IL-10 production is upregulated (Figure 1B). Conclusions: Our data suggests microglia in VaD adopt a unique transcriptional signature, distinct from that observed in disease-associated microglia in other neurodegenerative conditions, which may impact their ability to maintain homeostasis in VaD.

Bruton’s tyrosine kinase-bearing B cells and microglia in neuromyelitis optica spectrum disorder

BackgroundNeuromyelitis optica spectrum disorder (NMOSD) is an inflammatory autoimmune disease of the central nervous system that involves B-cell receptor signaling as well as astrocyte–microglia interaction, which both contribute to evolution of NMOSD lesions.Main bodyThrough transcriptomic and flow cytometry analyses, we found that Bruton’s tyrosine kinase (BTK), a crucial protein of B-cell receptor was upregulated both in the blood and cerebrospinal fluid of NMOSD patients. Blockade of BTK with zanubrutinib, a highly specific BTK inhibitor, mitigated the activation and maturation of B cells and reduced production of causal aquaporin-4 (AQP4) autoantibodies. In a mouse model of NMO, we found that both BTK and pBTK expression were significantly increased in microglia. Transmission electron microscope scan demonstrated that BTK inhibitor ameliorated demyelination, edema, and axonal injury in NMO mice. In the same mice colocalization of GFAP and Iba-1 immunofluorescence indicated a noticeable increase of astrocytes–microglia interaction, which was alleviated by zanubrutinib. The smart-seq analysis demonstrated that treatment with BTK inhibitor instigated microglial transcriptome changes including downregulation of chemokine-related genes and genes involved in the top 5 biological processes related to cell adhesion and migration, which are likely responsible for the reduced crosstalk of microglia and astrocytes.ConclusionsOur results show that BTK activity is enhanced both in B cells and microglia and BTK inhibition contributes to the amelioration of NMOSD pathology. These data collectively reveal the mechanism of action of BTK inhibition and corroborate BTK as a viable therapeutic target.

Human iPSC‐microglia expressing TREM2 p.R47H risk variant for Alzheimer’s disease do not show a loss‐of‐function phenotype

AbstractBackgroundThe most common TREM2 variant associated with increased risk to develop Alzheimer’s disease (AD) is p.R47H. This variant has been classified as loss‐of‐function, mostly by comparing p.R47H with the common variant in TREM2 over‐expressing cell lines. Whether this holds true when TREM2 is expressed at physiological levels in a disease‐relevant cell type such as human microglia, has not been fully characterized.MethodWe established and thoroughly characterized human iPSC‐derived microglia endogenously expressing TREM2 to assess the effect of p.R47H on TREM2 cellular distribution, TREM2 signaling and microglial functioning. For this study microglia generated from a set of isogenic gene‐edited iPSC lines homozygous for TREM2 knockout (KO) and TREM2 p.R47H were evaluated and compared to microglia from the parental control iPSC line. In addition, microglia generated from iPSC lines obtained from several p.R47H carriers were compared to microglia from iPSC lines obtained from several healthy control non‐carriers. SNP genotyping confirmed the presence of R47H.ResultThe p.R47H and common variant TREM2 were present at similar levels at the cell surface of iPSC‐derived microglia, whereas no expression of TREM2 was detected in the TREM2 KO microglia. Microarray profiling indicated that the microglial transcriptome is very similar between the common variant and p.R47H yet distinct from TREM2 KO microglia. Biochemical evaluation of TREM2 signaling indicated no reduction in TREM2 downstream signaling activation in the p.R47H variant expressing microglia. Similarly, soluble TREM2 levels were increased in culture medium of p.R47H microglia. Finally, analysis of microglial functioning including phagocytosis of artificial and disease‐relevant substrates showed no difference between the p.R47H variant and the common variant, in contrast to TREM2 KO microglia.ConclusionWe conclude that in cultured human iPSC‐derived microglia, the p.R47H variant shows no evidence of a loss‐of‐function mechanism. Future experiments will determine whether this is also the case in microglia in their natural brain environment. Our long‐term goal is to better understand TREM2 biology to support the development of novel therapeutics for AD and other neurodegenerative diseases characterized by TREM2‐mediated neuroinflammation.

Advances in the study of the effects of gut microflora on microglia in Alzheimer’s disease

Alzheimer’s disease (AD) is a central nervous system (CNS) degenerative disorder, is caused by various factors including β-amyloid toxicity, hyperphosphorylation of tau protein, oxidative stress, and others. The dysfunction of microglia has been associated with the onset and advancement of different neurodevelopmental and neurodegenerative disorders, such as AD. The gut of mammals harbors a vast and complex population of microorganisms, commonly referred to as the microbiota. There’s a growing recognition that these gut microbes are intrinsically intertwined with mammalian physiology. Through the circulation of metabolites, they establish metabolic symbiosis, enhance immune function, and establish communication with different remote cells, including those in the brain. The gut microbiome plays a crucial part in influencing the development and performance of microglia, as indicated by recent preclinical studies. Dysbiosis of the intestinal flora leads to alterations in the microglia transcriptome that regulate the interconversion of microglia subtypes. This conversation explores recent research that clarifies how gut bacteria, their byproducts, and harmful elements affect the activation and characteristics of microglia. This understanding opens doors to innovative microbial-based therapeutic strategies for early identification and treatment goals in AD.

The γ-secretase substrate proteome and its role in cell signaling regulation

γ-Secretases mediate the regulated intramembrane proteolysis (RIP) of more than 150 integral membrane proteins. We developed an unbiased γ-secretase substrate identification (G-SECSI) method to study to what extent these proteins are processed in parallel. We demonstrate here parallel processing of at least 85 membrane proteins in human microglia in steady-state cell culture conditions. Pharmacological inhibition of γ-secretase caused substantial changes of human microglial transcriptomes, including the expression of genes related to the disease-associated microglia (DAM) response described in Alzheimer disease (AD). While the overall effects of γ-secretase deficiency on transcriptomic cell states remained limited in control conditions, exposure of mouse microglia to AD-inducing amyloid plaques strongly blocked their capacity to mount this putatively protective DAM cell state. We conclude that γ-secretase serves as a critical signaling hub integrating the effects of multiple extracellular stimuli into the overall transcriptome of the cell.

Identification of methylation-regulated genes modulating microglial phagocytosis in hyperhomocysteinemia-exacerbated Alzheimer’s disease

BackgroundHyperhomocysteinemia (HHcy) has been linked to development of Alzheimer’s disease (AD) neuropathologically characterized by the accumulation of amyloid β (Aβ). Microglia (MG) play a crucial role in uptake of Aβ fibrils, and its dysfunction worsens AD. However, the effect of HHcy on MG Aβ phagocytosis remains unstudied.MethodsWe isolated MG from the cerebrum of HHcy mice with genetic cystathionine-β-synthase deficiency (Cbs−/−) and performed bulk RNA-seq. We performed meta-analysis over transcriptomes of Cbs−/− mouse MG, human and mouse AD MG, MG Aβ phagocytosis model, human AD methylome, and GWAS AD genes.ResultsHHcy and hypomethylation conditions were identified in Cbs−/− mice. Through Cbs−/− MG transcriptome analysis, 353 MG DEGs were identified. Phagosome formation and integrin signaling pathways were found suppressed in Cbs−/− MG. By analyzing MG transcriptomes from 4 AD patient and 7 mouse AD datasets, 409 human and 777 mouse AD MG DEGs were identified, of which 37 were found common in both species. Through further combinatory analysis with transcriptome from MG Aβ phagocytosis model, we identified 130 functional-validated Aβ phagocytic AD MG DEGs (20 in human AD, 110 in mouse AD), which reflected a compensatory activation of Aβ phagocytosis. Interestingly, we identified 14 human Aβ phagocytic AD MG DEGs which represented impaired MG Aβ phagocytosis in human AD. Finally, through a cascade of meta-analysis of transcriptome of AD MG, functional phagocytosis, HHcy MG, and human AD brain methylome dataset, we identified 5 HHcy-suppressed phagocytic AD MG DEGs (Flt1, Calponin 3, Igf1, Cacna2d4, and Celsr) which were reported to regulate MG/MΦ migration and Aβ phagocytosis.ConclusionsWe established molecular signatures for a compensatory response of Aβ phagocytosis activation in human and mouse AD MG and impaired Aβ phagocytosis in human AD MG. Our discoveries suggested that hypomethylation may modulate HHcy-suppressed MG Aβ phagocytosis in AD.

B cell depletion modulates glial responses and enhances blood vessel integrity in a model of multiple sclerosis

Multiple sclerosis (MS) is characterized by a compromised blood-brain barrier (BBB) resulting in central nervous system (CNS) entry of peripheral lymphocytes, including T cells and B cells. While T cells have largely been considered the main contributors to neuroinflammation in MS, the success of B cell depletion therapies suggests an important role for B cells in MS pathology. Glial cells in the CNS are essential components in both disease progression and recovery, raising the possibility that they represent targets for B cell functions. Here, we examine astrocyte and microglia responses to B cell depleting treatments in an animal model of MS, experimental autoimmune encephalomyelitis (EAE). B cell depleted EAE animals had markedly reduced disease severity and myelin damage accompanied by reduced microglia and astrocyte reactivity 20 days after symptom onset. To identify potential initial mechanisms mediating functional changes following B cell depletion, astrocyte and microglia transcriptomes were analyzed 3 days following B cell depletion. In control EAE animals, transcriptomic analysis revealed astrocytic inflammatory pathways were activated and microglial influence on neuronal function were inhibited. Following B cell depletion, initial functional recovery was associated with an activation of astrocytic pathways linked with restoration of neurovascular integrity and of microglial pathways associated with neuronal function. These studies reveal an important role for B cell depletion in influencing glial function and CNS vasculature in an animal model of MS.

Human microglial state dynamics in Alzheimer’s disease progression

Altered microglial states affect neuroinflammation, neurodegeneration, and disease but remain poorly understood. Here, we report 194,000 single-nucleus microglial transcriptomes and epigenomes across 443 human subjects and diverse Alzheimer's disease (AD) pathological phenotypes. We annotate 12 microglial transcriptional states, including AD-dysregulated homeostatic, inflammatory, and lipid-processing states. We identify 1,542 AD-differentially-expressed genes, including both microglia-state-specific and disease-stage-specific alterations. By integrating epigenomic, transcriptomic, and motif information, we infer upstream regulators of microglial cell states, gene-regulatory networks, enhancer-gene links, and transcription-factor-driven microglial state transitions. We demonstrate that ectopic expression of our predicted homeostatic-state activators induces homeostatic features in human iPSC-derived microglia-like cells, while inhibiting activators of inflammation can block inflammatory progression. Lastly, we pinpoint the expression of AD-risk genes in microglial states and differential expression of AD-risk genes and their regulators during AD progression. Overall, we provide insights underlying microglial states, including state-specific and AD-stage-specific microglial alterations at unprecedented resolution.

Dietary Fiber and Microbiota Metabolite Receptors Enhance Cognition and Alleviate Disease in the 5xFAD Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is a neurodegenerative disorder with poorly understood etiology. AD has several similarities with other "Western lifestyle" inflammatory diseases, where the gut microbiome and immune pathways have been associated. Previously, we and others have noted the involvement of metabolite-sensing GPCRs and their ligands, short-chain fatty acids (SCFAs), in protection of numerous Western diseases in mouse models, such as Type I diabetes and hypertension. Depletion of GPR43, GPR41, or GPR109A accelerates disease, whereas high SCFA yielding diets protect in mouse models. Here, we extended the concept that metabolite-sensing receptors and SCFAs may be a more common protective mechanism against Western diseases by studying their role in AD pathogenesis in the 5xFAD mouse model. Both male and female mice were included. Depletion of GPR41 and GPR43 accelerated cognitive decline and impaired adult hippocampal neurogenesis in 5xFAD and WT mice. Lack of fiber/SCFAs accelerated a memory deficit, whereas diets supplemented with high acetate and butyrate (HAMSAB) delayed cognitive decline in 5xFAD mice. Fiber intake impacted on microglial morphology in WT mice and microglial clustering phenotype in 5xFAD mice. Lack of fiber impaired adult hippocampal neurogenesis in both W and AD mice. Finally, maternal dietary fiber intake significantly affects offspring's cognitive functions in 5xFAD mice and microglial transcriptome in both WT and 5xFAD mice, suggesting that SCFAs may exert their effect during pregnancy and lactation. Together, metabolite-sensing GPCRs and SCFAs are essential for protection against AD, and reveal a new strategy for disease prevention.Significance Statement Alzheimer's disease (AD) is one of the most common neurodegenerative diseases; currently, there is no cure for AD. In our study, short-chain fatty acids and metabolite receptors play an important role in cognitive function and pathology in AD mouse model as well as in WT mice. SCFAs also impact on microglia transcriptome, and immune cell recruitment. Out study indicates the potential of specialized diets (supplemented with high acetate and butyrate) releasing high amounts of SCFAs to protect against disease.

Microglial senescence contributes to female-biased neuroinflammation in the aging mouse hippocampus: implications for Alzheimer’s disease

BackgroundMicroglia, the brain’s principal immune cells, have been implicated in the pathogenesis of Alzheimer’s disease (AD), a condition shown to affect more females than males. Although sex differences in microglial function and transcriptomic programming have been described across development and in disease models of AD, no studies have comprehensively identified the sex divergences that emerge in the aging mouse hippocampus. Further, existing models of AD generally develop pathology (amyloid plaques and tau tangles) early in life and fail to recapitulate the aged brain environment associated with late-onset AD. Here, we examined and compared transcriptomic and translatomic sex effects in young and old murine hippocampal microglia.MethodsHippocampal tissue from C57BL6/N and microglial NuTRAP mice of both sexes were collected at young (5–6 month-old [mo]) and old (22–25 mo) ages. Cell sorting and affinity purification techniques were used to isolate the microglial transcriptome and translatome for RNA-sequencing and differential expression analyses. Flow cytometry, qPCR, and imaging approaches were used to confirm the transcriptomic and translatomic findings.ResultsThere were marginal sex differences identified in the young hippocampal microglia, with most differentially expressed genes (DEGs) restricted to the sex chromosomes. Both sex chromosomally and autosomally encoded sex differences emerged with aging. These sex DEGs identified at old age were primarily female-biased and enriched in senescent and disease-associated microglial signatures. Normalized gene expression values can be accessed through a searchable web interface (https://neuroepigenomics.omrf.org/). Pathway analyses identified upstream regulators induced to a greater extent in females than in males, including inflammatory mediators IFNG, TNF, and IL1B, as well as AD-risk genes TREM2 and APP.ConclusionsThese data suggest that female microglia adopt disease-associated and senescent phenotypes in the aging mouse hippocampus, even in the absence of disease pathology, to a greater extent than males. This sexually divergent microglial phenotype may explain the difference in susceptibility and disease progression in the case of AD pathology. Future studies will need to explore sex differences in microglial heterogeneity in response to AD pathology and determine how sex-specific regulators (i.e., sex chromosomal or hormonal) elicit these sex effects.

Single-nucleus RNA sequencing reveals cell type-specific transcriptome alterations of Down syndrome hippocampus using the Dp16 mouse model.

Down syndrome (DS), the most frequently occurring human chromosomal disorder, is caused by trisomy 21. The exact molecular effects of trisomy on certain cell populations in the brain remain poorly understood. The purpose of this study was to investigate the effects of trisomy on the transcriptomes of various types of neurons and nonneuronal cells in the hippocampus. A total of 8993 nuclei from the WT and 6445 nuclei from the Dp16 hippocampus were analyzed by single-nucleus RNA sequencing (snRNA-seq). Cell clustering was achieved by the Seurat program. Hippocampal cells were grouped into multiple neuronal and nonneuronal populations. Only a limited number of trisomic genes were upregulated (q < 0.001) over 1.25-fold in a specific type of hippocampal cell. Specifically, deregulation of genes associated with synaptic signaling and organization was observed in multiple cell populations, including excitatory neurons, oligodendrocytes, and microglia. This observation suggests the potential importance of synapse deficits in DS. Interestingly, GO annotation of the upregulated genes suggested potential activation of the immune system by hippocampal excitatory neurons. Fewer trisomic genes were altered in nonneuronal cells than in neurons. Notably, microglial transcriptome analysis revealed significantly (q < 0.001) increased expression of C1qb and C1qc, which suggested potential involvement of complement-mediated synapse loss mediated by microglia in DS. The trisomy-related hippocampal deficits should be driven by a small amount, not all, of the trisomic genes in a specific type of cell. Our work may help to narrow down both the molecular and cellular targets for future gene therapies in DS.

The effects of chronic high-dose morphine on microgliosis and the microglial transcriptome in rat spinal cord.

Background: Opioids are efficacious and safe analgesic drugs in short-term use for acute pain but chronic use can lead to tolerance and dependence. Opioid-induced microglial activation may contribute to the development of tolerance and this process may differ between males and females. A link is suggested between this microglial activation and inflammation, disturbances of circadian rhythms, and neurotoxic effects. We set out to further delineate the effects of chronic morphine on pain behaviour, microglial and neuronal staining, and the transcriptome of spinal microglia, to better understand the role of microglia in the consequences of long-term high-dose opioid administration. Experimental Approach: In two experiments, we administered increasing subcutaneous doses of morphine hydrochloride or saline to male and female rats. Thermal nociception was assessed with the tail flick and hot plate tests. In Experiment I, spinal cord (SC) samples were prepared for immunohistochemical staining for microglial and neuronal markers. In Experiment II, the transcriptome of microglia from the lumbar SC was analysed. Key Results: Female and male rats had similar antinociceptive responses to morphine and developed similar antinociceptive tolerance to thermal stimuli following chronic increasing high doses of s.c. morphine. The area of microglial IBA1-staining in SC decreased after 2weeks of morphine administration in both sexes. Following morphine treatment, the differentially expressed genes identified in the microglial transcriptome included ones related to the circadian rhythm, apoptosis, and immune system processes. Conclusions: Female and male rats showed similar pain behaviour following chronic high doses of morphine. This was associated with decreased staining of spinal microglia, suggesting either decreased activation or apoptosis. High-dose morphine administration also associated with several changes in gene expression in SC microglia, e.g., those related to the circadian rhythm (Per2, Per3, Dbp). These changes should be considered in the clinical consequences of long-term high-dose administration of opioids.

Innovations advancing our understanding of microglia in Alzheimer's disease: From in vitro to in vivo models.

Microglia have been implicated in Alzheimer's disease (AD) pathogenesis through the identification of risk factor genes that are specifically or predominantly expressed in this cell type. Additional evidence suggests that microglia undergo dramatic morphological and phenotypic state changes during AD progression, as observed in human post-mortem tissue and animal model research. Although valuable, these studies are often hampered by either representing one time point in human tissue (end point) or because of the lack of conservation between species of microglial transcriptomes, proteomes and cell states. Thus, the development and application of novel human model systems have been beneficial in the study of microglia in neurodegeneration. Recent innovations include the use of human pluripotent stem cell (hPSC)-derived microglia in 2D or 3D culture systems, the transdifferentiation of microglia from patient monocytes and the xenotransplantation of hPSC-derived microglia into mouse brains. This review summarizes the recent innovations that have advanced our understanding of microglia in AD, through the use of single-cell RNA sequencing, hPSC-derived microglia culture within brain organoids and xenotransplantation into mouse brain. Through outlining the strengths and limitations of these approaches, we provide recommendations that will aid future endeavours in advancing our understanding of the complex role of microglia in AD onset and progression.