Microglial Gene Expression Research Articles

Resolving neuroinflammation in AD

AbstractBackgroundHuman genetics point towards a key role of microglia and microglia expressed genes like TREM2 in neuroinflammation and AD pathology, yet our understanding of microglia spatial and temporal dynamics, therapeutic directionality and translational biomarkers remains limited.Method and ResultsMuna Therapeutics applies structural biology, computational chemistry and human IPSC derived cell assays to develop small molecule agonists for microglial drug targets including TREM2. Introduction of human mutations allows specific drug – target interactions to be probed and has revealed novel small molecule binding pockets, enabling optimized drug design. To identify novel drug targets involved in neuroinflammation, we have built an all‐in‐human spatial transcriptomics and bioinformatics‐based discovery and validation platform. We have assessed >50 human brain samples for pathology‐induced changes in gene expression in microglia and other cells from samples with AD pathology from patients with and without cognitive impairment. This approach has revealed patterns of differentially expressed genes and pathways in brains linked to pathological hallmarks such as Ab plaques and tau tangles, as well as to resilience to the effects of these pathologies. Candidate targets and mechanisms are tested for their impact on neuroinflammation, including directionality, in humanized model systems that also enable translational biomarker discovery and validation.ConclusionAt Muna Tx, state‐of‐the‐art target discovery and validation as well as structural biology and computational chemistry platforms enable microglia targeted drug discovery and can accelerate therapeutic advancement to clinical development.

The effect of microglial DMT1 inhibition on neuroinflammation in male and female mice

AbstractBackgroundNeuroinflammation and microglial activation are critical components of Alzheimer’s Disease neuropathology as observed in patients and animal models. Microglia have multiple functions in addition to their inflammatory phenotype that can contribute to disease. Specifically, iron accumulates in AD‐associated brain regions, and we and others have shown enhanced iron load in activated microglia surrounding amyloid‐beta (Aβ) plaques. Furthermore, we and others have shown a distinct upregulation in the microglial iron importer, divalent metal transporter 1 (DMT1) in response to Aβ. Our aim is to further elucidate the role of microglial iron‐handling in age and AD‐associated inflammation.MethodWe measured DMT1 gene expression to assess changes in iron‐handling in response to age and inflammation. Microglia isolated from aged mice were treated with an inflammatory Aβ stimulus before measuring DMT1 expression and immunohistochemical markers of iron load. To determine the role of microglial DMT1 during inflammatory pathology in vivo, we targeted DMT1 expression utilizing a microglial‐specific tamoxifen‐inducible genetic mouse model in males and females. Transgenic mice were given intraperitoneal lipopolysaccharide (LPS) as a widely‐used model of neuroinflammation. We isolated microglia 24hr after LPS administration and measured DMT1 expression via qRT‐PCR for knockdown confirmation. Sickness behavior and markers of inflammatory state were also assessed.ResultMicroglia from 2‐year‐old aged mice display an augmented increase in Aβ‐induced DMT1 expression compared to young controls. Our in vivo studies also suggest a sex‐dependent increase in microglial DMT1 expression following systemic LPS. In our genetic mouse model, we obtained a 79% and 89% knockdown of microglial DMT1 gene expression in males and females, respectively. Male knockdown animals exhibited a trending decrease in Il‐1β expression in response to LPS, indicating an intimate link between pro‐inflammatory signaling and microglial iron‐handling.ConclusionThese studies link changes in microglial iron‐handling and the neuroinflammatory response. Additional in vivo work is further being analyzed to comprehensively determine the effects of DMT1 inhibition on the neuroinflammatory response. Considering the prominence of iron‐loaded microglia and chronic neuroinflammation in late‐stage AD, early intervention aimed at a specific aspect of microglial iron‐handling could be a target to decrease inflammation and AD‐related pathology.

Characterizing microglial gene expression in a model of secondary progressive multiple sclerosis.

Multiple sclerosis (MS) is the most common inflammatory, demyelinating and neurodegenerative disease of the central nervous system in young adults. Chronic-relapsing experimental autoimmune encephalomyelitis (crEAE) in Biozzi ABH mice is an experimental model of MS. This crEAE model is characterized by an acute phase with severe neurological disability, followed by remission of disease, relapse of neurological disease and remission that eventually results in a chronic progressive phase that mimics the secondary progressive phase (SPEAE) of MS. In both MS and SPEAE, the role of microglia is poorly defined. We used a crEAE model to characterize microglia in the different phases of crEAE phases using morphometric and RNA sequencing analyses. At the initial, acute inflammation phase, microglia acquired a pro-inflammatory phenotype. At the remission phase, expression of standard immune activation genes was decreased while expression of genes associated with lipid metabolism and tissue remodeling were increased. Chronic phase microglia partially regain inflammatory gene sets and increase expression of genes associated with proliferation. Together, the data presented here indicate that microglia obtain different features at different stages of crEAE and a particularly mixed phenotype in the chronic stage. Understanding the properties of microglia that are present at the chronic phase of EAE will help to understand the role of microglia in secondary progressive MS, to better aid the development of therapies for this phase of the disease.

EPCO-20. EXPLORATION OF DNA METHYLATION PATTERNS IN GLIOBLASTOMA REPROGRAMMED MICROGLIAL CELLS

Abstract Glioblastoma (GBM) is the most frequent and malignant adult-type diffuse glioma (WHO grade IV) with a median patient survival of less than 15 months despite multimodal treatment. The poor prognosis of GBM, among others, is associated with a tumor-supporting microenvironment characterized by a rather anti-inflammatory state, where the tumor cells have been shown to hijack microglial gene expression profiles. These so-called tumor-associated microglia cells (TAMs) repress normal immune functions, such as phagocytosis and pro-inflammatory cytokine secretion through transcriptional reprogramming, further underlying morphological changes. It is currently starting to emerge that the exceptional cellular plasticity required to orchestrate such microglial reactivity could originate from pre-imposed epigenetic modifications. We thus hypothesize that TAM reprogramming in GBM is controlled by epigenetic mechanisms, and therefore aim to understand if modifications in DNA methylation patterns can lead to pro-tumor transcriptomic and phenotypical changes.Reactive microglia are classically described as either ‘pro-inflammatory’ M1 or ‘anti-inflammatory’ M2 phenotypes, inducable by Lipopolysaccharide (LPS) and Interferon-gamma (IFNɣ) or Interleukin-4 (IL-4), respectively. In a GBM context, microglial cells have been mostly classified as M2, but exhibit heterogeneous features among the M1 to M2 activation spectrum. In this study, we exposed human and murine microglial cell lines to LPS, IFNɣ, IL-4, and GBM-conditioned medium (GBM-CM), followed by morphological assessment, transcript expression and secretome characterization. The onset of activation was followed in time series every 12h to interrogate epigenetic and transcriptomic changes together with protein expression and secretion. After exposure to GBM-CM, microglial cells exhibited a bipolar and more elongated phenotype. We observed significant variations in cytokine production together with a decrease in phagocytosis in LPS-treated microglia, whereas the secretome in GBM-CM was only slightly altered. We furthermore observed differentially methylated regions (DMRs) using the novel Infinium® Mouse Methylation BeadChip, suggesting a role of DNA methylation in microglial reprogramming.

Single cell profiling of CD45+ spinal cord cells reveals microglial and B cell heterogeneity and crosstalk following spinal cord injury

BackgroundImmune cells play crucial roles after spinal cord injury (SCI). However, incomplete knowledge of immune contributions to injury and repair hinders development of SCI therapies. We leveraged single-cell observations to describe key populations of immune cells present in the spinal cord and changes in their transcriptional profiles from uninjured to subacute and chronic stages of SCI.MethodsDeep-read single-cell sequencing was performed on CD45+ cells from spinal cords of uninjured and injured Swiss-webster mice. After T9 thoracic contusion, cells were collected 3-, 7-, and 60-day post-injury (dpi). Subpopulations of CD45+ immune cells were identified informatically, and their transcriptional responses characterized with time. We compared gene expression in spinal cord microglia and B cell subpopulations with those in published models of disease and injury. Microglia were compared with Disease Associated Microglia (DAM) and Injury Responsive Microglia (IRM). B cells were compared to developmental lineage states and to an Amyotrophic Lateral Sclerosis (ALS) model.ResultsIn uninjured and 7 dpi spinal cord, most CD45+ cells isolated were microglia while chronically B cells predominated. B cells accumulating in the spinal cord following injury included immature B to mature stages and were predominantly found in the injury zone. We defined diverse subtypes of microglia and B cells with altered gene expression with time after SCI. Spinal cord microglia gene expression indicates differences from brain microglia at rest and in inflammatory states. Expression analysis of signaling ligand–receptor partners identified microglia–B cell interactions at acute and chronic stages that may be involved in B cell recruitment, retention, and formation of ectopic lymphoid follicles.ConclusionsImmune cell responses to SCI have region-specific aspects and evolve with time. Developmentally diverse populations of B cells accumulate in the spinal cord following injury. Microglia at subacute stages express B cell recruitment factors, while chronically, they express factors predicted to reduce B cell inflammatory state. In the injured spinal cord, B cells create ectopic lymphoid structures, and express secreted factors potentially acting on microglia. Our study predicts previously unidentified crosstalk between microglia and B cells post-injury at acute and chronic stages, revealing new potential targets of inflammatory responses for SCI repair warranting future functional analyses.

TPM1 mediates inflammation downstream of TREM2 via the PKA/CREB signaling pathway

BackgroundMicroglia, the innate immune cells in the central nervous system, play an essential role in brain homeostasis, neuroinflammation and brain infections. Dysregulated microglia, on the other hand, are associated with neurodegenerative diseases, yet the mechanisms underlying pro-inflammatory gene expression in microglia are incompletely understood.MethodsWe investigated the role of the actin-associated protein tropomyosin 1 (TPM1) in regulating pro-inflammatory phenotype of microglia in the retina by using a combination of cell culture, immunocytochemistry, Western blot, qPCR, TUNEL, RNA sequencing and electroretinogram analysis. TREM2−/− mice were used to investigate whether TPM1 regulated pro-inflammatory responses downstream of TREM2. To conditionally deplete microglia, we backcrossed CX3CR1CreER mice with Rosa26iDTR mice to generate CX3CR1CreER:Rosa26iDTR mice.ResultsWe revealed a vital role for TPM1 in regulating pro-inflammatory phenotype of microglia. We found that TPM1 drove LPS-induced inflammation and neuronal death in the retina via the PKA/CREB pathway. TPM1 knockdown ameliorated LPS-induced inflammation in WT retinas yet exaggerated the inflammation in TREM2−/− retinas. RNA sequencing revealed that genes associated with M1 microglia and A1 astrocytes were significantly downregulated in LPS-treated WT retinas but upregulated in LPS-treated TREM2−/− retinas after TPM1 knockdown. Mechanistically, we demonstrated that CREB activated by TPM1 knockdown mediated anti-inflammatory genes in LPS-treated WT retinas but pro-inflammatory genes in LPS-treated TREM2−/− retinas, suggesting a novel role for TREM2 as a brake on TPM1-mediated inflammation. Furthermore, we identified that TPM1 regulated inflammation downstream of TREM2 and in a microglia-dependent manner.ConclusionsWe demonstrate that TPM1 mediates inflammation downstream of TREM2 via the PKA/CREB signaling pathway. Our findings suggest that TPM1 could be a potential target for therapeutic intervention in brain diseases.

Work hard, play hard: how sexually differentiated microglia work to shape social play and reproductive behavior.

Microglia are brain-resident immune cells that play a critical role in synaptic pruning and circuit fine-tuning during development. In the adult brain, microglia actively survey their local environment and mobilize inflammatory responses to signs of damage or infection. Sex differences in microglial gene expression and function across the lifespan have been identified, which play a key role in shaping brain function and behavior. The levels of sex hormones such as androgens, estrogens, and progesterone vary in an age-dependent and sex-dependent manner. Microglia respond both directly and indirectly to changes in hormone levels, altering transcriptional gene expression, morphology, and function. Of particular interest is the microglial function in brain regions that are highly sexually differentiated in development such as the amygdala as well as the pre-optic and ventromedial hypothalamic regions. With a focus on hormone-sensitive developmental windows, this review compares male and female microglia in the embryonic, developing, and adult brain with a particular interest in the influence of sex hormones on microglial wiring of social, reproductive, and disordered behavior circuits in the brain.

Aging exacerbates the brain inflammatory micro-environment contributing to α-synuclein pathology and functional deficits in a mouse model of DLB/PD

BackgroundAlthough ɑ-synuclein (ɑ-syn) spreading in age-related neurodegenerative diseases such as Parkinson’s disease (PD) and Dementia with Lewy bodies (DLB) has been extensively investigated, the role of aging in the manifestation of disease remains unclear.MethodsWe explored the role of aging and inflammation in the pathogenesis of synucleinopathies in a mouse model of DLB/PD initiated by intrastriatal injection of ɑ-syn preformed fibrils (pff).ResultsWe found that aged mice showed more extensive accumulation of ɑ-syn in selected brain regions and behavioral deficits that were associated with greater infiltration of T cells and microgliosis. Microglial inflammatory gene expression induced by ɑ-syn-pff injection in young mice had hallmarks of aged microglia, indicating that enhanced age-associated pathologies may result from inflammatory synergy between aging and the effects of ɑ-syn aggregation. Based on the transcriptomics analysis projected from Ingenuity Pathway Analysis, we found a network that included colony stimulating factor 2 (CSF2), LPS related genes, TNFɑ and poly rl:rC-RNA as common regulators.ConclusionsWe propose that aging related inflammation (eg: CSF2) influences outcomes of pathological spreading of ɑ-syn and suggest that targeting neuro-immune responses might be important in developing treatments for DLB/PD.

C5aR1 antagonism alters microglial polarization and mitigates disease progression in a mouse model of Alzheimer’s disease

Multiple studies have recognized the involvement of the complement cascade during Alzheimer’s disease pathogenesis. However, the specific role of C5a-C5aR1 signaling in the progression of this neurodegenerative disease is still not clear. Furthermore, its potential as a therapeutic target to treat AD still remains to be elucidated. Canonically, generation of the anaphylatoxin C5a as the result of complement activation and interaction with its receptor C5aR1 triggers a potent inflammatory response. Previously, genetic ablation of C5aR1 in a mouse model of Alzheimer’s disease exerted a protective effect by preventing cognitive deficits. Here, using PMX205, a potent, specific C5aR1 antagonist, in the Tg2576 mouse model of Alzheimer’s disease we show a striking reduction in dystrophic neurites in parallel with the reduced amyloid load, rescue of the excessive pre-synaptic loss associated with AD cognitive impairment and the polarization of microglial gene expression towards a DAM-like phenotype that are consistent with the neuroprotective effects seen. These data support the beneficial effect of a pharmacological inhibition of C5aR1 as a promising therapeutic approach to treat Alzheimer’s disease. Supportive of the safety of this treatment is the recent FDA-approval of another other C5a receptor 1 antagonist, Avacopan, as a treatment for autoimmune inflammatory diseases.

Circadian modulation of microglial physiological processes and immune responses.

Microglia is considered the central nervous system (CNS) resident macrophages that establish an innate immune response against pathogens and toxins. However, the recent studies have shown that microglial gene and protein expression follows a circadian pattern; several immune activation markers and clock genes are expressed rhythmically without the need for an immune stimulus. Furthermore, microglia responds to an immune challenge with different magnitudes depending on the time of the day. This review examines the circadian control of microglia function and the possible physiological implications. For example, we discuss that synaptic prune is performed in the cortex at a certain moment of the day. We also consider the implications of daily microglial function for maintaining biological rhythms like general activity, body temperature, and food intake. We conclude that the developmental stage, brain region, and pathological state are not the only factors to consider for the evaluation of microglial functions; instead, emerging evidence indicates that circadian time as an essential aspect for a better understanding of the role of microglia in CNS physiology.

Maternal Allergic Asthma Induces Prenatal Neuroinflammation.

Autism spectrum disorder (ASD) is a class of neurodevelopmental disorders characterized by impaired social interactions and communication skills and repetitive or stereotyped behaviors. Rates of ASD diagnosis continue to rise, with current estimates at 1 in 44 children in the US (Maenner 2021). Epidemiological studies have suggested a link between maternal allergic asthma and an increased likelihood of having a child diagnosed with ASD. However, a lack of robust laboratory models prevents mechanistic research from being carried out. We developed a novel mouse model of maternal asthma-allergy (MAA) and previously reported that offspring from these mothers exhibit behavioral deficits compared to controls. In addition, it was shown that epigenetic regulation of gene expression in microglia was altered in these offspring, including several autism candidate genes. To further elucidate if there is neuroinflammation in the fetus following MAA, we investigated how allergic asthma impacts the maternal environment and inflammatory markers in the placenta and fetal brain during gestation. Female C57Bl/6 mice were primed with ovalbumin (OVA) prior to allergic asthma induction during pregnancy by administering aerosolized ovalbumin or PBS control to pregnant dams at gestational days (GD)9.5, 12.5, and 17.5. Four hours after the final induction, placenta and fetal brains were collected and measured for changes in cytokines using a Luminex bead-based multiplex assay. Placental MAA tissue showed a decrease in interleukin (IL)-17 in male and female offspring. There was a sex-dependent decrease in female monocyte chemoattractant protein 1 (MCP-1). In male placentas, IL-4, C–X–C motif chemokine 10 (CXCL10)—also known as interferon γ-induced protein 10 kDa (IP-10)—and chemokine (C-C motif) ligand 5 (RANTES) were decreased. In fetal brains, elevated inflammatory cytokines were found in MAA offspring when compared to controls. Specifically, interferon-gamma (IFN-γ), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 1α (IL-1α), IL-6, and tumor necrosis factor α (TNFα) were elevated in both males and females. In contrast, a decrease in the cytokine IL-9 was also observed. There were slight sex differences after OVA exposures. Male fetal brains showed elevated levels of macrophage inflammatory protein-2 (MIP-2), whereas female brains showed increased keratinocytes-derived chemokine (KC). In addition, IL-1𝛽 and IP-10 in male fetal brains were decreased. Together, these data indicate that repeated exposure to allergic asthma during pregnancy alters cytokine expression in the fetal environment in a sex-dependent way, resulting in homeostatic and neuroinflammatory alterations in the fetal brain.

Glucose and fructose directly stimulate brain-derived neurotrophic factor gene expression in microglia.

Brain-derived neurotrophic factor (BDNF) is expressed in both hypothalamic neurons and microglia, and plays a critical role in the regulation of metabolism. Although hypothalamic expression of BDNF is regulated by metabolic signals such as nutrients and hormones, it remains unknown whether these signals differentially regulate BDNF expression in different cell types. The present study aimed to determine whether glucose and fructose regulate BDNF expression in microglia via the specific glucose transporter. To determine the effect of glucose and fructose on Bdnf mRNA and protein expression, murine microglial cell line SIM-A9 cells were exposed to the maintenance concentration of glucose (17.5 mmol/l), high glucose (25 mmol/l), or fructose (7.5 mmol/l) for 40 min to 24 h. To determine whether the blockade of glucose transporter 5 (GLUT5) negates the effect of glucose on Bdnf mRNA expression, cells were exposed to 25 mmol/l glucose in the presence or absence of the GLUT5 inhibitor for 4 h. Levels of Bdnf mRNA and protein were measured by real-time PCR and ELISA, respectively. High glucose caused a significant increase in both pan-Bdnf and long-form Bdnf (L-Bdnf) mRNA as well as protein levels when compared with the maintenance of concentration of glucose in a time-dependent manner. Fructose treatment also increased L-Bdnf mRNA expression. Pharmacological blockade of GLUT5 did not affect glucose-induced Bdnf mRNA expression. These findings suggest that glucose and fructose directly stimulate Bdnf mRNA expression in microglia and these responses may mediate the metabolic actions of glucose and fructose.

Repopulated microglia induce expression of Cxcl13 with differential changes in Tau phosphorylation but do not impact amyloid pathology

BackgroundAdult microglia rely on self-renewal through division to repopulate and sustain their numbers. However, with aging, microglia display morphological and transcriptional changes that reflect a heightened state of neuroinflammation. This state threatens aging neurons and other cells and can influence the progression of Alzheimer’s disease (AD). In this study, we sought to determine whether renewing microglia through a forced partial depletion/repopulation method could attenuate AD pathology in the 3xTg and APP/PS1 mouse models.MethodsWe pharmacologically depleted the microglia of two cohorts of 21- to 22-month-old 3xTg mice and one cohort of 14-month-old APP/PS1 mice using PLX5622 formulated in chow for 2 weeks. Following depletion, we returned the mice to standard chow diet for 1 month to allow microglial repopulation. We assessed the effect of depletion and repopulation on AD pathology, microglial gene expression, and surface levels of homeostatic markers on microglia using immunohistochemistry, single-cell RNAseq and flow cytometry.ResultsAlthough we did not identify a significant impact of microglial repopulation on amyloid pathology in either of the AD models, we observed differential changes in phosphorylated-Tau epitopes after repopulation in the 3xTg mice. We provide evidence that repopulated microglia in the hippocampal formation exhibited changes in the levels of homeostatic microglial markers. Lastly, we identified novel subpopulations of microglia by performing single-cell RNAseq analysis on CD45int/+ cells from hippocampi of control and repopulated 3xTg mice. In particular, one subpopulation induced after repopulation is characterized by heightened expression of Cxcl13.ConclusionOverall, we found that depleting and repopulating microglia causes overexpression of microglial Cxcl13 with disparate effects on Tau and amyloid pathologies.

Glucose Stimulates Glial Cell Line-Derived Neurotrophic Factor Gene Expression in Microglia through a GLUT5-Independent Mechanism.

Feeding-regulating neurotrophic factors are expressed in both neurons and glial cells. However, nutritional regulation of anorexigenic glial cell line-derived neurotrophic factor (GDNF) and orexigenic mesencephalic astrocyte-derived neurotrophic factor (MANF) expression in specific cell types remains poorly understood. Hypothalamic glucose sensing plays a critical role in the regulation of food intake. It has been theorized that local glucose concentration modulates microglial activity partially via glucose transporter 5 (GLUT5). We hypothesized that an increased local glucose concentration stimulates GDNF expression while inhibiting MANF expression in the hypothalamus and microglia via GLUT5. The present study investigated the effect of glucose on Gdnf and Manf mRNA expression in the mouse hypothalamus and murine microglial cell line SIM-A9. Intracerebroventricular glucose treatment significantly increased Gdnf mRNA levels in the hypothalamus without altering Manf mRNA levels. Exposure to high glucose caused a significant increase in Gdnf mRNA expression and a time-dependent change in Manf mRNA expression in SIM-A9 cells. GLUT5 inhibitor treatment did not block glucose-induced Gdnf mRNA expression in these cells. These findings suggest that microglia are responsive to changes in the local glucose concentration and increased local glucose availability stimulates the expression of microglial GNDF through a GLUT5-independent mechanism, contributing to glucose-induced feeding suppression.

The transcription factor CCAAT/enhancer-binding protein β in spinal microglia contributes to pre-operative stress-induced prolongation of postsurgical pain.

Prolongation of postsurgical pain caused by pre-operative stress is a clinically significant problem, although the mechanisms are not fully understood. Stress can promote the pro-inflammatory activation of microglia, and the transcription factor CCAAT/enhancer-binding protein (C/EBP) β regulates pro-inflammatory gene expression in microglia. Therefore, we speculated that C/EBPβ in spinal microglia may have critical roles in the development of chronic postsurgical pain. Accordingly, in this study, we used a single prolonged stress (SPS) procedure and plantar incisions to evaluate the roles of C/EBPβ in postsurgical pain. Our experiments showed that SPS exposure prolonged mechanical allodynia, increased the expression of C/EBPβ and pro-inflammatory cytokines, and potentiated the activation of spinal microglia. Subsequently, microinjection of C/EBPβ siRNA attenuated the duration of SPS-prolonged postoperative mechanical allodynia and inhibited microglial activation in the spinal cord. Conversely, mimicking this increase in C/EBPβ promoted microglial activation via pretreatment with a pre-injection of AAV5-C/EBPβ, leading to prolongation of postsurgical pain. Overall, these results suggested that spinal microglia may play key roles in prolongation of postsurgical pain induced by pre-operative stress and that C/EBPβ may be a potential target for disease treatment.

Small extracellular vesicles induced gene expression changes in microglia

BackgroundSmall extracellular vesicles (sEVs) are 30‐150 nm membranous particles released by a variety of cells and serve as a mediator in intercellular communication between adjacent and distal cells. sEVs carry biomolecular cargo including miRNAs, mRNAs, proteins, and lipids, which are selectively sorted and packaged and mirror the physiological state of the donor cells. Disease states can alter sEV composition affecting the message carried and thereby, its functional impact. Microglia, as the tissue‐resident macrophages and primary innate immune cells of the central nervous system, play an important role in neuropathic pain. Here, we investigated alterations in the composition of serum sEVs from a mouse model of neuropathic pain and assessed the functional consequences of sEV uptake by primary cortical microglia.MethodssEVs were purified from the serum of a spared nerve injury (SNI) mouse model of neuropathic pain and sham‐operated mice four weeks after surgery. sEV uptake by primary cortical microglia was confirmed by incubating microglia with 1 µg of PKH26‐labeled sEVs for 24 h and visualized using confocal microscopy. Transcriptome and cytokine alterations induced by sEV uptake in microglia (1 µg for 24 h) were determined using RNA‐seq and cytokine array.ResultsWe identified a differential expression of 22 miRNAs in serum sEVs derived from SNI and sham control mice. Confocal microscopy confirmed the presence of sEVs in microglia following 24 h sEV incubation. We observed a significant difference in gene expression resulting from the uptake of distinct serum sEVs – alterations were more prominent in microglia treated with sham sEVs. Gene Ontology analysis indicated gene expression changes induced by the uptake of sEVs from both SNI and sham surgery were both implicated in immune responses, but genes upregulated by sham sEVs were also enriched in inflammatory responses. Secretions of pro‐inflammatory cytokines and chemokines including CXCL10, CXCL2, and TNF‐α were higher in microglia incubated with sEVs from mice that had sham surgery compared to PBS control or SNI sEVs.ConclusionsOur data demonstrate alterations in sEV composition from both SNI and sham surgeries, and the molecular changes captured in donor sEVs could impact gene expression in the recipient cells. The differences observed in microglial phenotypes and gene expression suggest sEVs from sham surgery are directed at wound healing and thus differ from nerve injury‐induced sEVs.

Neuronal apoptosis drives remodeling states of microglia and shifts in survival pathway dependence.

Microglia serve critical remodeling roles that shape the developing nervous system, responding to the changing neural environment with phagocytosis or soluble factor secretion. Recent single-cell sequencing (scRNAseq) studies have revealed the context-dependent diversity in microglial properties and gene expression, but the cues promoting this diversity are not well defined. Here, we ask how interactions with apoptotic neurons shape microglial state, including lysosomal and lipid metabolism gene expression and dependence on Colony-stimulating factor 1 receptor (CSF1R) for survival. Using early postnatal mouse retina, a CNS region undergoing significant developmental remodeling, we performed scRNAseq on microglia from mice that are wild-type, lack neuronal apoptosis (Bax KO), or are treated with CSF1R inhibitor (PLX3397). We find that interactions with apoptotic neurons drive multiple microglial remodeling states, subsets of which are resistant to CSF1R inhibition. We find that TAM receptor Mer and complement receptor 3 are required for clearance of apoptotic neurons, but that Mer does not drive expression of remodeling genes. We show TAM receptor Axl is negligible for phagocytosis or remodeling gene expression but is consequential for microglial survival in the absence of CSF1R signaling. Thus, interactions with apoptotic neurons shift microglia toward distinct remodeling states and through Axl, alter microglial dependence on survival pathway, CSF1R.

Anti-Inflammatory and Neuroprotective Mechanisms of GTS-21, an α7 Nicotinic Acetylcholine Receptor Agonist, in Neuroinflammation and Parkinson’s Disease Mouse Models

Neuroinflammation is crucial in the progression of neurodegenerative diseases. Thus, controlling neuroinflammation has been proposed as an important therapeutic strategy for neurodegenerative disease. In the present study, we examined the anti-inflammatory and neuroprotective effects of GTS-21, a selective α7 nicotinic acetylcholine receptor (α7 nAChR) agonist, in neuroinflammation and Parkinson’s disease (PD) mouse models. GTS-21 inhibited the expression of inducible nitric oxide synthase (iNOS) and proinflammatory cytokines in lipopolysaccharide (LPS)-stimulated BV2 microglial cells and primary microglia. Further research revealed that GTS-21 has anti-inflammatory properties by inhibiting PI3K/Akt, NF-κB, and upregulating AMPK, Nrf2, CREB, and PPARγ signals. The effects of GTS-21 on these pro-/anti-inflammatory signaling molecules were reversed by treatment with an α7 nAChR antagonist, suggesting that the anti-inflammatory effects of GTS-21 are mediated through α7 nAChR activation. The anti-inflammatory and neuroprotective properties of GTS-21 were then confirmed in LPS-induced systemic inflammation and MPTP-induced PD model mice. In LPS-injected mouse brains, GTS-21 reduced microglial activation and production of proinflammatory markers. Furthermore, in the brains of MPTP-injected mice, GTS-21 restored locomotor activity and dopaminergic neuronal cell death while inhibiting microglial activation and pro-inflammatory gene expression. These findings suggest that GTS-21 has therapeutic potential in neuroinflammatory and neurodegenerative diseases such as PD.

Modulatory effect of IL-1 inhibition following lipopolysaccharide-induced neuroinflammation in neonatal microglia and astrocytes.

Inflammation-induced white matter injury (WMI) in preterm infants is characterized by microglia activation, astrogliosis, oxidative stress and neurodevelopmental impairments. Microglia and astrocytes activation can be described under a broad spectrum of activation profile with extremes described as pro-inflammatory/neurotoxic (M1 microglia or A1 astrocyte) or anti-inflammatory/neuroprotective (M2 microglia or A2 astrocyte) in response to stimuli including lipopolysaccharide (LPS) and interleukin 1 (IL-1). As IL-1 signalling pathway has been posited as a major driver of inflammation-induced perinatal WMI, our aim was to evaluate the contribution of IL-1 modulation in LPS-induced microglia and astrocyte activation. Primary neonatal cell co-cultures of astrocytes and microglia were treated with LPS (100 ng/ml) for 8h or 24 h. Two distinct IL-1 receptor antagonists, Rytvela or Kineret (1μg/ml), were added simultaneously with LPS to respectively modulate or block IL-1 receptor. Medium was collected to measure levels of IL-1β. Expression of markers related to pro- and anti-inflammatory microglia and astrocyte activation profiles and antioxidant genes were assessed. At 8h, LPS exposure induced pro- (M1/A1) and anti-inflammatory (M2/A2) marker expression and inhibited antioxidant gene expression in microglia and astrocytes. By 24 h, continuous LPS exposure increased pro-inflammatory and neurotoxic microglial and astrocytic markers (M1/A1), as well as antioxidant genes. Administration of IL-1 antagonists Rytvela and Kineret with continuous LPS exposure had no significant effect on modulation of specific microglia and astrocyte activation pathways. We show that LPS effects on in vitro neonatal microglia and astrocytes co-cultures are time dependent eliciting a number of pro- and anti-inflammatory responses during the acute phase of inflammation (8h), which shift towards pro-inflammatory and neurotoxic factors by 24 h. Although LPS-induced inflammation led to abundant IL-1 expression, IL-1 inhibition had no significant impact on in vitro modulation of microglia and astrocyte activation pathways towards M2 and A2 activation profile.

Dietary Fiber as a Counterbalance to Age-Related Microglial Cell Dysfunction.

With increasing age, microglia shift toward a pro-inflammatory phenotype that may predispose individuals to neurodegenerative disease. Because fiber fermentation in the colon produces bioactive short-chain fatty acids (SCFAs; e.g., acetate, butyrate, and propionate) that signal through the gut-brain axis, increasing dietary fiber may prevent or reverse age-related dysregulation of microglia. Adult (3–4 months old) and aged (23–24 months old) male and female mice were given ad libitum access to a modified AIN-93M diet with 1% cellulose or the same diet with 2.5 or 5.0% inulin for 8 weeks. Several adult and aged male mice fed 0 or 5% inulin were randomly selected for whole brain single-cell RNA sequencing (scRNA-seq) and differential gene expression analysis to classify brain microglia according to gene expression profile; and identify additional genetic markers of aging as possible targets for dietary interventions. Microglia were isolated from remaining mice and expression of selected aging-, inflammatory-, and sensome-related genes was assessed by Fluidigm as was the ex vivo secretion of tumor necrosis factor-alpha (TNF-α). SCFAs were measured in samples collected from the cecum. Microglia from adult and aged mice segregated into distinct phenotypes according to their gene expression profile. In aged mice, a considerably greater proportion of the population of microglia was identified being “activated” and a considerably smaller proportion was identified being “quiescent.” These findings using whole brain scRNA-seq were largely corroborated using highly purified microglia and Fluidigm analysis to assess a selected panel of genes. Aged mice compared to adults had lower levels of SCFA’s in cecum. Dietary inulin increased SCFAs in cecum and mostly restored microglial cell gene expression and TNF-α secretion to that seen in adults. Sex differences were observed with females having lower levels of SCFAs in cecum and increased neuroinflammation. Overall, these data support the use of fiber supplementation as a strategy to counterbalance the age-related microglial dysregulation.