Microglial Subtypes Research Articles

GM130-silencing may aggravate blood-brain barrier damage and affect microglia polarization by down-regulating PD-L1 expression after experimental intracerebral hemorrhage.

In addition to primary injury, secondary injuries related to BBB disruption and immune-inflammatory response also play an important role in intracerebral hemorrhage (ICH). And the Golgi apparatus play an important role in the state of ICH. ICH model and GM130-silencing ICH model were established in SD rats. The Garcia score was used to score the neurological defects of the rats. Blood-brain barrier (BBB) integrity were assessed by amount of extravasated Evans blue, and tight junction proteins. The expression of PD-L1 and GM130were detected through Western-blot and the subtype of microglia was showing with Immunofluorescence staining. Compared with the ICH group, GM130-silencing ICH rats got a worsened neurological deficit and enlarged volume of the hematoma. Evan's blue extravasation aggravated as well. The expression of GM130 in peri-hematoma tissue was further decreased, and the morphology and structure of the Golgi apparatus were further damaged. Meanwhile, the GM130 deficit resulted in decreased expression of PD-L1 and more polarization of microglia to the M1 subtype. We demonstrate that GM130 could influence the integrity of BBB and plays a role in neuroinflammation via regulation of PD-L1 after ICH. The manipulation of GM130 might be a promising therapeutical target in ICH.

Single-cell RNA Sequencing Identifies a Novel Subtype of Microglia with High Cd74 Expression that Facilitates White Matter Inflammation During Chronic Cerebral Hypoperfusion.

Vascular dementia (VaD) causes progressive cognitive decline in the elderly population, but there is short of available therapeutic measures. Microglia-mediated neuroinflammation is vigorously involved in the pathogenesis of VaD, but the traditional classification of microglial M1/M2 phenotypes remains restrictive and controversial. This study aims to investigate whether microglia transform into novel subtypes in VaD. Chronic cerebral hypoperfusion (CCH) rat model was constructed to mimic VaD. Microglia were isolated via magnetic-activated cell sorting and analyzed by single-cell RNA sequencing (scRNA-seq) and bioinformatics. The findings inferred from scRNA-seq and bioinformatics were further validated through in vivo experiments. In this study, microglia were divided into eight clusters. The proportion of MG5 cluster was significantly increased in the white matter of the CCH group compared with the Sham group and was named chronic ischemia-associated microglia (CIAM). Immunity- and inflammation-related genes, including RT1-Db1, RT1-Da, RT1-Ba, Cd74, Spp1, C3, and Cd68, were markedly upregulated in CIAM. Enrichment analysis illustrated that CIAM possessed the function of evoking neuroinflammation. Further studies unveiled that Cd74 is associated with the most abundant GO terms involved in inflammation as well as cell proliferation and differentiation. In addition, microglia-specific Cd74 knockdown mediated by adeno-associated virus decreased the abundance of CIAM in the white matter, thereby mitigating inflammatory cytokine levels, alleviating white matter lesions, and improving cognitive impairment for CCH rats. These findings indicate that Cd74 is the core molecule of CIAM to trigger neuroinflammation and induce microglial differentiation to CIAM, suggesting that Cd74 may be a potential therapeutic target for VaD.

Single cell transcriptomes and multiscale networks from persons with and without Alzheimer’s disease

The emergence of single nucleus RNA sequencing (snRNA-seq) offers to revolutionize the study of Alzheimer’s disease (AD). Integration with complementary multiomics data such as genetics, proteomics and clinical data provides powerful opportunities to link cell subpopulations and molecular networks with a broader disease-relevant context. We report snRNA-seq profiles from superior frontal gyrus samples from 101 well characterized subjects from the Banner Brain and Body Donation Program in combination with whole genome sequences. We report findings that link common AD risk variants with CR1 expression in oligodendrocytes as well as alterations in hematological parameters. We observed an AD-associated CD83(+) microglial subtype with unique molecular networks and which is associated with immunoglobulin IgG4 production in the transverse colon. Our major observations were replicated in two additional, independent snRNA-seq data sets. These findings illustrate the power of multi-tissue molecular profiling to contextualize snRNA-seq brain transcriptomics and reveal disease biology.

Spatial transcriptomics combined with single-nucleus RNA sequencing reveals glial cell heterogeneity in the human spinal cord.

Glial cells play crucial roles in regulating physiological and pathological functions, including sensation, the response to infection and acute injury, and chronic neurodegenerative disorders. Glial cells include astrocytes, microglia, and oligodendrocytes in the central nervous system, and satellite glial cells and Schwann cells in the peripheral nervous system. Despite the greater understanding of glial cell types and functional heterogeneity achieved through single-cell and single-nucleus RNA sequencing in animal models, few studies have investigated the transcriptomic profiles of glial cells in the human spinal cord. Here, we used high-throughput single-nucleus RNA sequencing and spatial transcriptomics to map the cellular and molecular heterogeneity of astrocytes, microglia, and oligodendrocytes in the human spinal cord. To explore the conservation and divergence across species, we compared these findings with those from mice. In the human spinal cord, astrocytes, microglia, and oligodendrocytes were each divided into six distinct transcriptomic subclusters. In the mouse spinal cord, astrocytes, microglia, and oligodendrocytes were divided into five, four, and five distinct transcriptomic subclusters, respectively.The comparative results revealed substantial heterogeneity in all glial cell types between humans and mice. Additionally, we detected sex differences in gene expression in human spinal cord glial cells. Specifically, in all astrocyte subtypes, the levels of NEAT1 and CHI3L1 were higher in males than in females, whereas the levels of CST3 were lower in males than in females. In all microglial subtypes, all differentially expressed genes were located on the sex chromosomes. In addition to sex-specific gene differences, the levels of MT-ND4, MT2A, MT-ATP6, MT-CO3, MT-ND2, MT-ND3, and MT-CO2 in all spinal cord oligodendrocyte subtypes were higher in females than in males. Collectively, the present dataset extensively characterizes glial cell heterogeneity and offers a valuable resource for exploring the cellular basis of spinal cord-related illnesses, including chronic pain, amyotrophic lateral sclerosis, and multiple sclerosis.

MiR-223 enhances lipophagy by suppressing CTSB in microglia following lysolecithin-induced demyelination in mice

BackgroundLipid droplet (LD)-laden microglia is a key pathological hallmark of multiple sclerosis. The recent discovery of this novel microglial subtype, lipid-droplet-accumulating microglia (LDAM), is notable for increased inflammatory factor secretion and diminished phagocytic capability. Lipophagy, the autophagy-mediated selective degradation of LDs, plays a critical role in this context. This study investigated the involvement of microRNAs (miRNAs) in lipophagy during demyelinating diseases, assessed their capacity to modulate LDAM subtypes, and elucidated the potential underlying mechanisms involved.MethodsC57BL/6 mice were used for in vivo experiments. Two weeks post demyelination induction at cervical level 4 (C4), histological assessments and confocal imaging were performed to examine LD accumulation in microglia within the lesion site. Autophagic changes were observed using transmission electron microscopy. miRNA and mRNA multi-omics analyses identified differentially expressed miRNAs and mRNAs under demyelinating conditions and the related autophagy target genes. The role of miR-223 in lipophagy under these conditions was specifically explored. In vitro studies, including miR-223 upregulation in BV2 cells via lentiviral infection, validated the bioinformatics findings. Immunofluorescence staining was used to measure LD accumulation, autophagy levels, target gene expression, and inflammatory mediator levels to elucidate the mechanisms of action of miR-223 in LDAM.ResultsOil Red O staining and confocal imaging revealed substantial LD accumulation in the demyelinated spinal cord. Transmission electron microscopy revealed increased numbers of autophagic vacuoles at the injury site. Multi-omics analysis revealed miR-223 as a crucial regulatory gene in lipophagy during demyelination. It was identified that cathepsin B (CTSB) targets miR-223 in autophagy to integrate miRNA, mRNA, and autophagy gene databases. In vitro, miR-223 upregulation suppressed CTSB expression in BV2 cells, augmented autophagy, alleviated LD accumulation, and decreased the expression of the inflammatory mediator IL-1β.ConclusionThese findings indicate that miR-223 plays a pivotal role in lipophagy under demyelinating conditions. By inhibiting CTSB, miR-223 promotes selective LD degradation, thereby reducing the lipid burden and inflammatory phenotype in LDAM. This study broadens the understanding of the molecular mechanisms of lipophagy and proposes lipophagy induction as a potential therapeutic approach to mitigate inflammatory responses in demyelinating diseases.

Inhibition of Notch1 signal promotes brain recovery by modulating glial activity after stroke

BackgroundNotch1 signaling inhibiton with N-[N-(3,5-difluorophenacetyl)-1-alanyl]-S-phenylglycine t-butylester] (DAPT) treatment could promote brain recovery and the intervention effect is different between striatum (STR) and cortex (CTX), which might be accounted for different changes of glial activities, but the in-depth mechanism is still unknown. The purpose of this study was to identify whether DAPT could modulate microglial subtype shifts and astroglial-endfeet aquaporin-4 (AQP4) mediated waste solute drainage. MethodsSprague-Dawley rats (n=10) were subjected to 90min of middle cerebral artery occlusion (MCAO) and were treated with DAPT (n=5) or act as control with no treatment (n=5). Two groups of rats underwent MRI scans at 24h and 4 week, and sacrificed at 4 week after stroke for immunofluorescence (IF). ResultsCompared with control rats, MRI data showed structural recovery in ipsilateral STR but not CTX. And IF showed decreased pro-inflammatory M1 microglia and increased anti-inflammatory M2 microglia in striatal lesion core and peri-lesions of STR, CTX. Meanwhile, IF showed decreased AQP4 polarity in ischemic brain tissue, however, AQP4 polarity in striatal peri-lesions of DAPT treated rats was higher than that in control rats but shows no difference in cortical peri-lesions between control and treated rats. ConclusionsThe present study indicated that DAPT could promote protective microglia subtype shift and striatal astrocyte mediated waste solute drainage, that the later might be the major contributor of waste solute metabolism and one of the accounts for discrepant recovery of STR and CTX.

Super-enhancer-driven LIF promotes the mesenchymal transition in glioblastoma by activating ITGB2 signaling feedback in microglia.

The mesenchymal (MES) subtype of glioblastoma (GBM) is believed to be influenced by both cancer cell-intrinsic alterations and extrinsic cellular interactions, yet the underlying mechanisms remain unexplored. Identification of microglial heterogeneity by bioinformatics analysis. Transwell migration, invasion assays, and tumor models were used to determine gene function and the role of small molecule inhibitors. RNA sequencing, chromatin immunoprecipitation, and dual-luciferase reporter assays were performed to explore the underlying regulatory mechanisms. We identified the inflammatory microglial subtype of tumor-associated microglia (TAM) and found that its specific gene integrin beta 2 (ITGB2) was highly expressed in TAM of MES GBM tissues. Mechanistically, the activation of ITGB2 in microglia promoted the interaction between the SH2 domain of STAT3 and the cytoplasmic domain of ITGB2, thereby stimulating the JAK1/STAT3/IL-6 signaling feedback to promote the MES transition of GBM cells. Additionally, microglia communicated with GBM cells through the interaction between the receptor ITGB2 on microglia and the ligand ICAM-1 on GBM cells, while an increased secretion of ICAM-1 was induced by the proinflammatory cytokine leukemia inhibitory factor (LIF). Further studies demonstrated that inhibition of cyclin-dependent kinase 7 substantially reduced the recruitment of SNW1 to the super-enhancer of LIF, resulting in transcriptional inhibition of LIF. We identified notoginsenoside R1 as a novel LIF inhibitor that exhibited synergistic effects in combination with temozolomide. Our research reveals that the epigenetic-mediated interaction of GBM cells with TAM drives the MES transition of GBM and provides a novel therapeutic avenue for patients with MES GBM.

New Insights into Microglia as Therapeutic Targets in Alzheimer’s Disease

Abstract: Alzheimer's disease (AD) is the most common neurodegenerative disease, accounting for 60–70% of dementia cases globally. Inflammation of the central nervous system (CNS) caused by microglia is a common characteristic of neurodegenerative illnesses such as Parkinson's disease and AD. Research has recently examined the relationship between neurodegenerative diseases and CNS microglia. Microglial cells comprise 10–15% of all CNS cells and are brain-resident myeloid cells mediating critical processes to support the CNS. Microglia have a variety of receptors that operate as molecular sensors, detecting exogenous and endogenous CNS injuries and triggering an immune response. Microglia serve as brain guardians by boosting phagocytic clearance and providing trophic support to enable tissue repair and maintain cerebral homeostasis, in addition to their traditional immune cell activity. At rest, microglia manage CNS homeostasis by phagocytic action, which removes pathogens and cell debris. Microglia cells that have been "resting" convert into active cells that create inflammatory mediators, protecting neurons and protecting against invading pathogens. Neuronal damage and neurodegenerative disorders are caused by excessive inflammation. Different microglial cells reply at different phases of the disease can lead to new therapy options and reduced inflammatory activity. This review focuses on the potential function of microglia, microglia subtypes, and M1/M2 phenotypic changes associated with neurodegenerative disorders. Microglial membrane receptors, the involvement of microglia in neuroinflammation, microglial targets in AD and the double role of microglia in AD pathogenesis are also discussed in this review.

Single-cell RNA sequencing reveals roles of unique retinal microglia types in early diabetic retinopathy

BackgroundThe pathophysiological mechanisms of diabetic retinopathy (DR), a blinding disease, are intricate. DR was thought to be a microvascular disease previously. However, growing studies have indicated that the retinal microglia-induced inflammation precedes microangiopathy. The binary concept of microglial M1/M2 polarization paradigms during inflammatory activation has been debated. In this study, we confirmed microglia had the most significant changes in early DR using single-cell RNA sequencing.MethodsA total of five retinal specimens were collected from donor SD rats. Changes in various cells of the retina at the early stage of DR were analyzed using single-cell sequencing technology.ResultsWe defined three new microglial subtypes at cellular level, including two M1 types (Egr2+ M1 and Egr2− M1) and one M2 type. We also revealed the anatomical location between these subtypes, the dynamic changes of polarization phenotypes, and the possible activation sequence and mutual activation regulatory mechanism of different cells. Furthermore, we constructed an inflammatory network involving microglia, blood-derived macrophages and other retinal nonneuronal cells. The targeted study of new disease-specific microglial subtypes can shorten the time for drug screening and clinical application, which provided insight for the early control and reversal of DR.ConclusionsWe found that microglia show the most obvious differential expression changes in early DR and reveal the changes in microglia in a high-glucose microenvironment at the single-cell level. Our comprehensive analysis will help achieve early reversal and control the occurrence and progression of DR.

Truncated GPNMB, a microglial transmembrane protein, serves as a scavenger receptor for oligomeric β-amyloid peptide1-42 in primary type 1 microglia.

Glycoprotein non-metastatic melanoma protein B (GPNMB) is up-regulated in one subtype of microglia (MG) surrounding senile plaque depositions of amyloid-beta (Aβ) peptides. However, whether the microglial GPNMB can recognize the fibrous Aβ peptides as ligands remains unknown. In this study, we report that the truncated form of GPNMB, the antigen for 9F5, serves as a scavenger receptor for oligomeric Aβ1-42 (o-Aβ1-42) in rat primary type 1 MG. 125I-labeled o-Aβ1-42 exhibited specific and saturable endosomal/lysosomal degradation in primary-cultured type 1 MG from GPNMB-expressing wild-type mice, whereas the degradation activity was markedly reduced in cells from Gpnmb-knockout mice. The Gpnmb-siRNA significantly inhibits the degradation of 125I-o-Aβ1-42 by murine microglial MG5 cells. Therefore, GPNMB contributes to mouse MG's o-Aβ1-42 clearance. In rat primary type 1 MG, the cell surface expression of truncated GPNMB was confirmed by a flow cytometric analysis using a previously established 9F5 antibody. 125I-labeled o-Aβ1-42 underwent endosomal/lysosomal degradation by rat primary type 1 MG in a dose-dependent fashion, while the 9F5 antibody inhibited the degradation. The binding of 125I-o-Aβ1-42 to the rat primary type 1 MG was inhibited by 42% by excess unlabeled o-Aβ1-42, and by 52% by the 9F5 antibody. Interestingly, the 125I-o-Aβ1-42 degradations by MG-like cells from human-induced pluripotent stem cells was inhibited by the 9F5 antibody, suggesting that truncated GPNMB also serve as a scavenger receptor for o-Aβ1-42 in human MG. Our study demonstrates that the truncated GPNMB (the antigen for 9F5) binds to oligomeric form of Aβ1-42 and functions as a scavenger receptor on MG, and 9F5 antibody can act as a blocking antibody for the truncated GPNMB.

Abstract 103: Acute Ischemia Induces Spatially and Transcriptionally Distinct Microglial Subclusters

Introduction: Damage in the ischemic core and penumbra after stroke affects patient prognosis. Microglia immediately respond to ischemic insult after stroke. However, the microglial heterogeneity and the underlying mechanisms remain unclear. In this study, we sought to uncover stroke-associated microglia and identify their heterogenous features. Methods: We performed single-cell RNA-sequencing and spatial transcriptomics on middle cerebral artery occlusion (MCAO) mice to determine stroke-associated microglial clusters. The expression of marker genes and the localization of different microglial subclusters were verified through RNAscope and immunofluorescence. GSVA was employed to reveal functional characteristics of microglial subclusters. IPA was used to explore upstream regulators of microglial clusters, which was confirmed by immunofluorescence, RT-qPCR, and targeted metabolomics. Finally, the infarct size and neurological deficits were evaluated in MCAO mice after pharmacological manipulation of specific microglial subcluster. Results: We discovered stroke-associated microglial subclusters in MCAO mice. We also identified novel marker genes of these microglial subclusters and defined these cells as ischemic core-associated (ICAM) and ischemic penumbra-associated (IPAM) microglia, according to their spatial distribution. ICAM, induced by damage-associated molecular patterns, are fueled by glycolysis, and exhibit increased pro-inflammatory cytokines and chemokines production. BACH1 is a key transcription factor driving ICAM generation. In contrast, glucocorticoids enriched in the penumbra triggers IPAM formation, which are powered by citrate cycle and oxidative phosphorylation and are characterized by moderate pro-inflammatory responses, inflammation-alleviating metabolic features, and myelinotrophic properties. Conclusions: ICAM induce excessive neuroinflammation, aggravating brain injury, whereas IPAM could be essential for the homeostasis and survival of cells in the penumbra. Our findings provide a biological basis for targeting specific microglial subtypes as a potential therapeutic strategy for ischemic stroke.

Discovery of disease‐associated, tau, and inflammatory microglial subpopulations and molecular drivers in Alzheimer’s disease using network‐based deep learning integration of human brain single‐cell RNA‐sequencing data

AbstractBackgroundMicroglia have been implicated in the pathophysiology of Alzheimer’s disease (AD) for over 100 years, and substantial progress has been made in characterizing microglial heterogeneities and biology, including disease‐associated microglia (or DAM, amyloid‐pathology related, neuro‐protective), tau microglia (tau‐pathology related), and inflammatory microglia (neuro‐toxic). However, the crucial next step knowing how to apply these findings to develop new treatments for AD, through uncovering the functional roles and molecular drivers of each microglial subtype, has not yet been accomplished.MethodIn this study, we conducted single‐cell RNA‐sequencing data integration analyses of ∼0.8 million cells/nuclei by leveraging frozen brain samples from AD subjects across different brain regions from The Alzheimer’s Cell Atlas (https://taca.lerner.ccf.org/) using a deep learning analytic approach. We annotated three microglial subtypes (including DAM, tau, and inflammatory microglia) using well‐established marker genes and then used a human protein‐protein interactome network‐based approach to identify potential molecular drivers of different microglial subtypes across the spectrum of AD. We also applied transition gene network analysis to characterize microglial subtype transition from non‐disease associated to disease‐associated stages at transcriptomic levels.ResultVia trajectory analysis we pinpointed the existence of three microglial subtypes across AD progression. We also identified that tau microglia were significantly associated with synaptic processes. Compared to DAM, upregulated genes within inflammatory microglia were more significantly enriched within key immune pathways (e.g., NF‐kappa B and toll‐like receptor signaling pathways). In addition, transition gene networks of inflammatory microglia and DAM were found to contain potential AD pathobiology regulators (e.g., SYK, LYN, IRF8, and CSF1R) and genetic risk genes (including INPP5D, PICALM, and MEF2A). We further conducted network‐based drug repurposing prediction by simultaneously activating DAM while inhibiting inflammatory microglia, which indicated that several predicted repurposable drugs (i.e., fluticasone and mometasone) are significantly associated with reduced risk of AD in large patient databases.ConclusionThis study identifies potential underlying mechanisms of microglial subtypes involved in human AD brains, leading to potential drug targets for future development of microglia‐targeted therapies for AD.

Integration of multiple single cell microglial datasets reveals radial differentiation into long‐lived substates associated with Alzheimer’s pathology

AbstractBackgroundUnsupervised classification of brain cell types can be readily achieved through analysis of gene expression patterns in multidimensional space. However, the small transcriptional differences between microglia subtypes require more principled, robust, and reproducible approaches to expose and distinguish biological differences from noise.MethodHere we leverage Fokker‐Planck diffusion maps [1,2] to examine the dynamics of microglia subtypes by finding a low dimensional representation of the underlying stochastic dynamical system responsible for generating these transcriptional states. Clustering directly on this projection allows us to reduce noise, assign time scales to these substates, and generate a hierarchy of microglial cell states. Once these subtypes have been defined, we apply pseudobulk differential expression analysis to identify marker genes, and use those genes to build a boosted tree model to classify cells states. We then apply Shapley additive explanations (SHAP) to identify which genes are most important for distinguishing between cell states.ResultWe observe a radial transition pattern emanating, with a subtype that appears homeostatic, inactivated, and poised for transition. This is consistent with the observation that microglia dynamics are sensitive to their microenvironment. Deconvolution of an independent bulk microglia dataset (n = 130) showed depletion in subtypes linked to protein misfolding and myelin phagocytosis, as well as enrichment in immunoreactive microglia in Alzheimer’s patients. SHAP reveals DUSP1, CD83, PLCG2, OLR1,CCL2, and JUN among the most important genes for differentiating into these AD‐associated clusters.ConclusionOur approach enables us to reproducibly identify microglial subtypes across sets of single cell‐resolution high dimensional molecular profile data, identifying likely drivers of subtype differentiation. With several of our driver genes already associated with Alzheimer’s via GWAS and other analyses, our results will enable us to elucidate the role of these genes in determining microglial subtypes.

Targeting Microglia/Macrophages Notch1 Protects Neurons from Pyroptosis in Ischemic Stroke.

The immune-inflammatory cascade and pyroptosis play an important role in the pathogenesis of cerebral ischemia-reperfusion injury (CIRI). The maintenance of immune homeostasis is inextricably linked to the Notch signaling pathway, but whether myeloid Notch1 affects microglia polarization as well as neuronal pyroptosis in CIRI is not fully understood. This study was designed to clarify the role of myeloid Notch1 in CIRI, providing new therapeutic strategies for ischemic stroke. Myeloid-specific Notch1 knockout (Notch1M-KO) mice and the floxed Notch1 (Notch1FL/FL) mice were subjected to middle cerebral artery occlusion (MCAO). After 3 days of CIRI, we evaluated the neurological deficit score and cerebral infarction volume. Immunofluorescence staining was used to detect the expression of Notch1 and microglial subtype markers. Cerebral infiltrating macrophages were detected by flow cytometry. RT-qPCR was used to detect pro-inflammatory cytokines. Western blot was used to detect the expression of pyroptosis related proteins. The Notch1-siRNA transfected BV2 cells were co-cultured with HT22 cells to investigate the potential mechanisms by which microglial Notch1 affects neuronal pyroptosis induced by anoxia/reoxygenation in vitro. We found that Notch1 was activated in cerebral microglia/macrophages after CIRI. Myeloid Notch1 deficiency decreased the cerebral infarct volume (24.17 ± 3.29 vs. 36.17 ± 2.27, p < 0.001), neurological function scores (2.33 ± 0.47 vs. 3.17 ± 0.37, p < 0.001) and the infiltration of peripheral monocytes/macrophages (3.26 ± 0.53 vs. 5.67 ± 0.57, p < 0.01). Strikingly, myeloid-specific Notch1 knockout alleviated pyroptosis. Compared with microglia M1, increased microglia M2 were detected in the ischemic penumbra. In parallel in vitro co-culture experiments, we found that Notch1 knockdown in microglial BV2 cells inhibited anoxia/reoxygenation-induced JAK2/STAT3 activation and pyroptosis in hippocampal neuron HT22 cells. Our findings elucidate the underlying mechanism of the myeloid Notch1 signaling pathway in regulating neuronal pyroptosis in CIRI, suggesting that targeting myeloid-specific Notch1 is an effective strategy for the treatment of ischemic stroke.

Effects of minocycline on dendrites, dendritic spines, and microglia in immature mouse brains after kainic acid-induced status epilepticus.

This study aimed to investigate whether minocycline could influence alterations of microglial subtypes, the morphology of dendrites and dendritic spines, the microstructures of synapses and synaptic proteins, or even cognition outcomes in immature male mice following status epilepticus (SE) induced by kainic acid. Golgi staining was performed to visualize the dendrites and dendritic spines of neurons of the hippocampus. The microstructures of synapses and synaptic proteins were observed using transmission electron microscopy and western blotting analysis, respectively. Microglial reactivation and their markers were evaluated using flow cytometry. The Morris water maze (MWM) test was used to analyze spatial learning and memory ability. Significant partial spines increase (predominate in thin spines) was observed in the dendrites of neurons after acute SE and partial loss (mainly in thin spines) was presented by days 14 and 28 post-SE. The postsynaptic ultrastructure was impaired on the 7th and 14th days after SE. The proportion of M1 microglia increased significantly only after acute SE Similarly, the proportion of M2 microglia increased in the acute stage with high expression levels of all surface markers. In contrast, a decrease in M2 microglia and their markers was noted by day 14 post-SE. Minocycline could reverse the changes in dendrites and synaptic proteins caused by SE, and increase the levels of synaptic proteins. Meanwhile, minocycline could inhibit the reactivation of M1 microglia and the expression of their markers, except for promoting CD200R. In addition, treatment with minocycline could regulate the expression of M2 microglia and their surface markers, as well as ameliorating the impaired spatial learning and memory on the 28th day after SE. Dendritic spines and microglia are dynamically changed after SE. Minocycline could ameliorate the impaired cognition in the kainic acid-induced mouse model by decreasing the damage to dendrites and altering microglial reactivation.

Microglia subtypes in acute, subacute, and chronic multiple sclerosis.

The study was designed to examine microglia morphology in early and late forms of multiple sclerosis (MS). Archival paraffin embedded tissue samples from 25 cases were examined immunohistochemically. Pío del Río Hortega reported that phagocytes in acute focal destructive CNS lesions develop from microglia with no early contribution from infiltrating monocytes. In this study, we were unable to identify the changes cited by del Río Hortega in support of his theory. Instead, myelin phagocytes in MS appear to originate chiefly from infiltrating monocytes. In 4 cases, walls composed of MHC class II antigen-positive "wall microglia" were observed at plaque margins separating demyelinated and bordering myelinated tissue. Wall microglia in 2 plaques were accompanied by AQP4-positive fiber-forming astrocytes. In chronic but not early disease MS cases, microglia were seen to interact with infiltrating monocytes to form microglial nodules of several types. Also, MHC II-positive "activated" microglia in bordering intact tissue were exceptionally prominent where there was little evidence of ongoing myelin loss. It is concluded that myelin phagocytes in MS derive entirely from infiltrating MRP14-positive monocytes and not from resident microglia and that Río Hortega's microglia play an anti-inflammatory role in MS and not the destructive role favored by the current literature.

The role of signaling crosstalk of microglia in hippocampus on progression of ageing and Alzheimer's disease

Based on single-cell sequencing of the hippocampi of 5× familiar Alzheimer's disease (5× FAD) and wild type mice at 2-, 12-, and 24-month of age, we found an increased percentage of microglia in aging and Alzheimer's disease (AD) mice. Blood brain barrier injury may also have contributed to this increase. Immune regulation by microglia plays a major role in the progression of aging and AD, according to the functions of 41 intersecting differentially expressed genes in microglia. Signaling crosstalk between C−C motif chemokine ligand (CCL) and major histocompatibility complex-1 bridges intercellular communication in the hippocampus during aging and AD. The amyloid precursor protein (APP) and colony stimulating factor (CSF) signals drive 5× FAD to deviate from aging track to AD occurrence among intercellular communication in hippocampus. Microglia are involved in the progression of aging and AD can be divided into 10 functional types. The strength of the interaction among microglial subtypes weakened with aging, and the CCL and CSF signaling pathways were the fundamental bridge of communication among microglial subtypes.

ARG1-expressing microglia show a distinct molecular signature and modulate postnatal development and function of the mouse brain

Molecular diversity of microglia, the resident immune cells in the CNS, is reported. Whether microglial subsets characterized by the expression of specific proteins constitute subtypes with distinct functions has not been fully elucidated. Here we describe a microglial subtype expressing the enzyme arginase-1 (ARG1; that is, ARG1+ microglia) that is found predominantly in the basal forebrain and ventral striatum during early postnatal mouse development. ARG1+ microglia are enriched in phagocytic inclusions and exhibit a distinct molecular signature, including upregulation of genes such as Apoe, Clec7a, Igf1, Lgals3 and Mgl2, compared to ARG1– microglia. Microglial-specific knockdown of Arg1 results in deficient cholinergic innervation and impaired dendritic spine maturation in the hippocampus where cholinergic neurons project, which in turn results in impaired long-term potentiation and cognitive behavioral deficiencies in female mice. Our results expand on microglia diversity and provide insights into microglia subtype-specific functions.

Abstract 144: Spatial transcriptomic landscape of the primary and recurrent glioblastoma tumor microenvironment

Abstract Differences in clonality, genomic and epigenetic alterations are known to occur between primary Glioblastoma (pGBM) and recurrent (rGBM), but this has been insufficient to yield a promising therapeutic approach. Recent studies show that the GBM tumor microenvironment (TME), composed of reactive glial cells, microglial subtypes, neuronal activity, myeloid cells, lymphocytes, etc., plays a crucial role in the invasiveness and evolution of the glioma cells. Nevertheless, how the spatial architecture and interplay among these cells facilitates GBM resurgence is largely unknown. To this point, we developed a pipeline for characterizing the TME changes using single nucleus and spatial transcriptomics (ST). We retrospectively collected matched, fresh-frozen, IDH-wildtype pGBM and rGBM samples (n=14, 7 patients) from the Mayo Clinic Neuroscience Biobank. Single nucleus RNA sequencing was performed using the 10x Chromium platform. Matched FFPE blocks from 8 samples were collected from the Mayo Clinic pathology department for 4 of the 7 patients. A region of interest was identified from the corresponding histopathology slide by a pathologist, avoiding areas of necrosis and hemorrhage. The region was collected from the block using a 5mm biopsy punch. The tissue was re-paraffinized, and a 5µm thick section was used to perform ST with the 10x Visium for FFPE platform. We performed transcriptomic and image texture-based clustering of capture dots along with differential gene expression analysis. Based on gene expression, we identified and characterized conserved regions across samples. We used single nucleus data to infer cell composition. Spatial correlation and autocorrelation statistics were calculated on features, such as gene expression, gene set scores, cell type prediction, and antibody capture. The features’ relative spatial changes were compared between pGBM and rGBM. Our analyses showed high heterogeneity within and across samples, but we found higher molecular and textural heterogeneity in rGBM samples compared to pGBM. We found higher androgen response activity (p &amp;lt; 0.001) presents diffusely (I: 0.09) throughout rGBM tissue but localized to mesenchymal-like regions in pGBM (I: 0.15). KRAS signaling signature was decreased in pGBM (p &amp;lt; 0.01) and increased in rGBM (p &amp;lt; 0.01). This increase was significantly overexpressed in cluster 0 for rGBM (p=0.02), which is an area characterized by neural progenitor signature. No significant difference in KRAS or EGFR magnitude or distribution of expression was present for either GBM type, indicating an independent mechanism for pGBM. We established a pipeline for assessing the spatio-temporal changes of the GBM TME. We found evidence indicating differences in the type of growth signaling, the cells bing targeted, and their distribution across the TME between pGBM and rGBM. Our work suggests how ST can help us better understand the intricate dynamics of GBM. Citation Format: Jean R. Clemenceau, Paola Suarez Meade, Mark E. Jentoft, Alfredo Quiñones-Hinojosa, Tae Hyun Hwang. Spatial transcriptomic landscape of the primary and recurrent glioblastoma tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 144.

Abstract 5871: Pan-cancer myeloid cell analysis at the single cell level reveals the influence of distinct organ sites in myeloid cell phenotypes and support targeting S100A4 to reverse immune suppression

Abstract With abundant pro-tumorigenic myeloid cells and few tumoricidal tumor-infiltrating lymphocytes (&amp;lt;5%), GBM is representative of “immune cold” tumors. As such, many different types of immunotherapies have failed to show significant benefits for most glioma patients. Hence, a better understanding of drivers of the immune suppressive microenvironment in GBM and other immune cold tumors is urgently needed to guide future immunotherapy development and application. We recently analyzed 201,986 human glioma and immune cells from 44 tissue fragments from 18 human glioma patients, and present a comprehensive and high-resolution cellular, molecular, and spatial heterogeneity atlas of human glioma. We report an extensive spatial and molecular heterogeneity of glioma and immune cells within the same patient. In addition, we discovered that cell:cell communication between glioma:myeloid cells is considerably more robust than glioma:T-cells, indicating that myeloid cells form a communication hub in vivo. To gain a deeper understanding of these important immune cells, we analyzed 83,479 glioma-infiltrating myeloid cells and identified 9 molecularly distinct myeloid subtypes: 3 microglia subtypes, 3 bone marrow-derived macrophage (BMDM) subtypes, MDSCs, neutrophils, and dendritic cells. Notably, we found that five of these myeloid cell subtype gene signatures are significant predictors of glioma patient survival, independent of glioma cell mutational profiles or gene expression patterns. Leveraging our dataset, we also identified a novel immunotherapy target that is highly expressed in immune-suppressive macrophages and T cells but not in anti-tumor leukocytes: S100A4. We provide both in vitro and in vivo evidence that S100a4 deletion in stromal cells is sufficient to reprogram the immune microenvironment and significantly extend the survival of two independent glioma models. To broaden the potential impact of targeting S100A4 as a selective modulator of immune suppressive leukocytes, we compared the molecular signatures of glioma-associated myeloid cells to those from 12 other cancer types and peripheral blood myeloid cells. We found that S100A4 expression pattern is highly consistent among all tumor types, where its expression is highest in the monocytes and MDSCs and low in most DCs and tissue-resident macrophages. Our preliminary analysis also shows that myeloid cells in gliomas are molecularly distinct from corresponding cell types in other cancers, strongly indicating the role brain microenvironment in influencing the infiltrating BMDM maturation and polarization. Citation Format: Nourhan Abdelfattah, Parveen Kumar, Caiyi Wang, Jia-Shiun Leu, David Baskin, William Flynn, Ruli Gao, Kumar Pichumani, Omkar Ijare, Stephanie Wood, Suzanne Powell, David Haviland, Brittany Parker Kerrigan, Frederick Lang, Sujit Prabhu, Kristin Huntoon, Wen Jiang, Betty Kim, Joshy George, Kyuson Yun. Pan-cancer myeloid cell analysis at the single cell level reveals the influence of distinct organ sites in myeloid cell phenotypes and support targeting S100A4 to reverse immune suppression. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5871.