Microglial Cell Death Research Articles

Cellular Therapy in Experimental Autoimmune Encephalomyelitis as an Adjuvant Treatment to Translate for Multiple Sclerosis.

This study aims to evaluate and compare cellular therapy with human Wharton's jelly (WJ) mesenchymal stem cells (MSCs) and neural precursors (NPs) in experimental autoimmune encephalomyelitis (EAE), a preclinical model of Multiple Sclerosis. MSCs were isolated from WJ by an explant technique, differentiated to NPs, and characterized by cytometry and immunocytochemistry analysis after ethical approval. Forty-eight rats were EAE-induced by myelin basic protein and Freund's complete adjuvant. Forty-eight hours later, the animals received intraperitoneal injections of 250 ng/dose of Bordetella pertussis toxin. Fourteen days later, the animals were divided into the following groups: a. non-induced, induced: b. Sham, c. WJ-MSCs, d. NPs, and e. WJ-MSCs plus NPs. 1 × 105. Moreover, the cells were placed in a 10 µL solution and injected via a stereotaxic intracerebral ventricular injection. After ten days, the histopathological analysis for H&E, Luxol, interleukins, and CD4/CD8 was carried out. Statistical analyses demonstrated a higher frequency of clinical manifestation in the Sham group (15.66%) than in the other groups; less demyelination was seen in the treated groups than the Sham group (WJ-MSCs, p = 0.016; NPs, p = 0.010; WJ-MSCs + NPs, p = 0.000), and a lower cellular death rate was seen in the treated groups compared with the Sham group. A CD4/CD8 ratio of <1 showed no association with microglial activation (p = 0.366), astrocytes (p = 0.247), and cell death (p = 0.577) in WJ-MSCs. WJ-MSCs and NPs were immunomodulatory and neuroprotective in cellular therapy, which would be translated as an adjunct in demyelinating diseases.

Targeting MAPK14 in microglial cells: neuroimmune implications of Panax ginseng in post-stroke inflammation.

This study investigates the molecular mechanisms through which Panax ginseng and Panax notoginseng saponin (PNS) mitigate neuroinflammatory damage and promote neural repair postischemic stroke, utilizing bioinformatics, and experimental approaches. Cerebral infarction significantly contributes to disability worldwide, with chronic neuroinflammation worsening cognitive impairments and leading to neurodegenerative diseases. Addressing neuroimmune interactions is crucial for slowing disease progression and enhancing patient recovery, highlighting the need for advanced research in neuroimmune regulatory mechanisms and therapeutic strategies. To elucidate the effects of the traditional Chinese medicine components Panax ginseng and PNS on neuroinflammatory damage following ischemic stroke, focusing on the molecular pathways involved in mitigating inflammation and facilitating neural repair. The study employs single-cell sequencing and transcriptomic analysis to investigate gene expression changes associated with cerebral infarction. Gene set enrichment analysis and weighted gene co-expression network analysis are used to identify key molecular markers and core genes. Furthermore, pharmacological profiling, including functional assays, assesses the impact of Ginsenoside-Rc, a PNS derivative, on microglial cell viability, cytokine production, and reactive oxygen species (ROS) levels. Our analysis revealed that MAPK14 is a critical mediator in the neuroinflammatory response to ischemic stroke. Ginsenoside-Rc potentially targets and modulates MAPK14 activity to suppress inflammation. Experimental validation showed that Ginsenoside-Rc treatment, combined with MAPK14 silencing, significantly alters MAPK14 expression and mitigates neuroinflammatory damage, evidenced by reduced microglial cell death, inflammatory factor secretion, and ROS production. Ginsenoside-Rc's modulation of MAPK14 offers a promising therapeutic strategy for reducing neuroinflammation and potentially improving cognitive recovery post-ischemic stroke. This supports the therapeutic application of the traditional Chinese medicine Sanqi in ischemic stroke care, providing a theoretical and experimental foundation for its use. Future work will focus on extending these findings through clinical trials to evaluate the efficacy and safety of Ginsenoside-Rc in human subjects, aiming to translate these promising preclinical results into practical therapeutic interventions for ischemic stroke recovery.

NLRX1 Inhibits LPS-Induced Microglial Death via Inducing p62-Dependent HO-1 Expression, Inhibiting MLKL and Activating PARP-1.

The activation of microglia and the production of cytokines are key factors contributing to progressive neurodegeneration. Despite the well-recognized neuronal programmed cell death regulated by microglial activation, the death of microglia themselves is less investigated. Nucleotide-binding oligomerization domain, leucine-rich repeat-containing X1 (NLRX1) functions as a scaffolding protein and is involved in various central nervous system diseases. In this study, we used the SM826 microglial cells to understand the role of NLRX1 in lipopolysaccharide (LPS)-induced cell death. We found LPS-induced cell death is blocked by necrostatin-1 and zVAD. Meanwhile, LPS can activate poly (ADP-ribose) polymerase-1 (PARP-1) to reduce DNA damage and induce heme oxygenase (HO)-1 expression to counteract cell death. NLRX1 silencing and PARP-1 inhibition by olaparib enhance LPS-induced SM826 microglial cell death in an additive manner. Less PARylation and higher DNA damage are observed in NLRX1-silencing cells. Moreover, LPS-induced HO-1 gene and protein expression through the p62-Keap1-Nrf2 axis are attenuated by NLRX1 silencing. In addition, the Nrf2-mediated positive feedback regulation of p62 is accordingly reduced by NLRX1 silencing. Of note, NLRX1 silencing does not affect LPS-induced cellular reactive oxygen species (ROS) production but increases mixed lineage kinase domain-like pseudokinase (MLKL) activation and cell necroptosis. In addition, NLRX1 silencing blocks bafilomycin A1-induced PARP-1 activation. Taken together, for the first time, we demonstrate the role of NLRX1 in protecting microglia from LPS-induced cell death. The underlying protective mechanisms of NLRX1 include upregulating LPS-induced HO-1 expression via Nrf2-dependent p62 expression and downstream Keap1-Nrf2 axis, mediating PARP-1 activation for DNA repair via ROS- and autophagy-independent pathway, and reducing MLKL activation.

Indole-3-carbinol (I3C) reduces apoptosis and improves neurological function after cerebral ischemia–reperfusion injury by modulating microglia inflammation

Indole-3-carbinol(I3C) is a tumor chemopreventive substance that can be extracted from cruciferous vegetables. Indole-3-carbinol (I3C) has been shown to have antioxidant and anti-inflammatory effects. In this study, we investigated the cerebral protective effects of I3C in an in vivo rats model of middle cerebral artery occlusion (MCAO). 8–10 Week-Old male SD rat received I3C (150 mg/kg, once daily) for 3 days and underwent 3 h of middle cerebral artery occlusion (MCAO) followed by reperfusion. The results showed that I3C pretreatment (150 mg/kg, once daily) prevented CIRI-induced cerebral infarction in rats. I3C pretreatment also decreased the mRNA expression levels of several apoptotic proteins, including Bax, caspase-3 and caspase-9, by increasing the mRNA expression levels of the anti-apoptotic protein Bcl-2. Inhibited apoptosis in the brain cells of MCAO rats. In addition, we found that I3C pretreatment reduced neuronal loss, promoted neurological recovery after ischemia–reperfusion injury and increased seven-day survival in MCAO rats. I3C pretreatment also significantly reduced the expression of inducible nitric oxide synthase (INOS), interleukin-1β (IL-1β) and interleukin-6 (IL-6) mRNA in ischemic brain tissue; Increased expression of interleukin-4 (IL-4) and interleukin-10 (IL-10) mRNA. At the same time, I3C pretreatment significantly decreased the expression of the M1 microglial marker IBA1 after cerebral ischemia–reperfusion injury and increased the expression of these results in the M2 microglial marker CD206. I3C pretreatment also significantly decreased apoptosis and death of HAPI microglial cells after hypoxia induction, decreased interleukin-1β (IL-1β) and interleukin-6 (IL-6) mRNA The expression of interleukin-4 (IL-4) and interleukin-10 (IL-10) mRNAs was increased. These results suggest that I3C protects the brain from CIRI by regulating the anti-inflammatory and anti-apoptotic effects of microglia.

Extracellular vesicles of Weizmannia coagulans lilac-01 reduced cell death of primary microglia and increased mitochondrial content in dermal fibroblasts in vitro.

We investigated the properties of extracellular vesicles from the probiotic Weizmannia coagulans lilac-01 (Lilac-01EVs). The phospholipids in the Lilac-01EV membrane were phosphatidylglycerol and mitochondria-specific cardiolipin. We found that applying Lilac-01EVs to primary rat microglia in vitro resulted in a reduction in primary microglial cell death (P<.05). Lilac-01EVs, which contain cardiolipin and phosphatidylglycerol, may have the potential to inhibit cell death in primary microglia. The addition of Lilac-01EVs to senescent human dermal fibroblasts suggested that Lilac-01 EVs increase the mitochondrial content without affecting their membrane potential in these cells.

MTOR signaling regulates microglia metabolism and survival via 4E‐BP‐controlled mRNA translation

AbstractBackgroundRare loss‐of‐function variants in TREM2 (Triggering Receptor Expressed in Myeloid cells 2) are associated with an increased risk of AD. There are ongoing efforts to therapeutically boost microglial neuroprotective functions by targeting TREM2. However, the intracellular mechanisms underlying these functions in AD are not fully understood. TREM2 signaling maintains metabolic fitness through activation of the mechanistic target of rapamycin (mTOR) allowing microglia to sustain their metabolism during amyloidosis (Ulland et al., 2017). Detrimental decreases in microglial mTOR signalling during amyloidosis independent of TREM2 loss‐of‐function has also been reported (Baik et al., 2019).The mTOR pathway acts upstream of the eukaryotic mRNA translation by suppressing the translation repressor, eukaryotic translation initiation factor 4E (eIF4E)‐binding proteins (4E‐BPs). mTOR inhibits the 4E‐BPs via phosphorylation, thereby releasing eIF4E and allowing mRNA translation to initiate (Hay and Sonenberg, 2004). Work from our group demonstrated that mTOR boosts mitochondrial metabolism through 4E‐BP‐controlled mRNA translation (Morita et al., 2013). Therefore, we aimed to understand the dysregulated mechanisms at the intersection of mTOR‐controlled mRNA translation and metabolism in microglia response to Amyloid‐β (Aß).MethodWe used immunoblotting to investigate the effect on 4E‐BP phosphorylation following chronic exposure to Aß aggregates and TREM2 signaling inhibition in a microglia cell line. We manipulated the mTOR pathway by knocking out the downstream effectors, 4E‐BPs, to promote the translation of 4E‐BP‐controlled mRNAs in microglia in vitro and in vivo. We crossed the microglia‐specific 4E‐BPs knock out mouse with a RiboTag mouse to pull‐down ribosome‐bound mRNAs, providing a genome‐wide pool of actively translating mRNAs (the translatome) in the absence of 4E‐BPs in microglia.ResultWe showed that 4E‐BP‐controlled mRNA translation is inhibited upon chronic exposure to Aß or TREM2 signaling inhibition. The deletion of 4E‐BPs in microglia in‐vitro leads to a decrease in expression of pro‐inflammatory mediators and microglia cell death, and an increase in reliance on oxidative phosphorylation upon exposure to Aß. We also observed that 4E‐BP deletion changes the microglia translatome and decreases expression of inflammatory mediators in vivo after an intracranial Aß infusion.ConclusionWe conclude that mTOR signaling exerts key effects on microglia function and metabolism through 4E‐BP‐controlled mRNA translation.

Myeloid Differentiation Factor 2 Inhibition Protects Against Doxorubicin‐induced Microglial Activation and Senescence

AbstractBackgroundDoxorubicin, an anti‐tumor drug, generates neuroinflammation that leads to microglial senescence and chemo‐brain (1, 2). Toll‐like receptor 4 (TLR4) signaling pathway is mainly expressed in microglia and it plays a key role in developing doxorubicin‐induced neuroinflammation, microglial activation and chemo‐brain (3). Myeloid differentiation factor 2 (MD‐2) is known as an essential protein in activation of TLR4 signaling pathway (1). However, the effect of blocking TLR4 signaling via targeting MD‐2 on doxorubicin‐induced microglial activation and senescence has never been investigated.MethodThe human microglial clone 3 cell line (HMC3) was treated with three different doses of MD‐2 inhibitor (L6H21: 5μM, 10μM, 20μM) for 30 minutes, then with doxorubicin hydrochloride (DOX) (50nM) for 24 hours. After 24 hours treatment with L6H21 and DOX, the microglial cells were assessed for the TLR4 signaling, microglial activation, cell death and cellular senescence pathways.ResultDOX treatment activated TLR4 signaling pathway as well as microglia, as evidenced by significantly increased protein expressions of TLR4, MD‐2, tumor necrosis factor alpha and ionized calcium‐binding adapter molecule 1. In addition, DOX treatment induced microglial cell death and senescence, as shown by significantly increased protein expressions of Bcl‐2 associated X‐protein to B‐cell lymphoma 2 ratio and p16Ink4a. Treatment with L6H21 (5μM) attenuated not only TLR4 signaling along with microglial activation, but also apoptosis and cellular senescence in DOX‐treated microglia. L6H21 (10μM and 20μM) protected against TLR4 signaling along with microglial activation, apoptosis as well as cellular senescence in DOX‐treated microglia.ConclusionThese findings imply that MD‐2 inhibition could be the potential therapeutic strategy for attenuating DOX‐induced microglial activation and senescence.

Nrf2 activator Diethyl Maleate attenuates ROS mediated NLRP3 inflammasome activation in murine microglia.

Microglia are the tissue-resident immune cells of the central nervous system. As a part of the innate immune response, NLR Family Pyrin Domain Containing Protein 3 (NLRP3) inflammasome activation leads to cleavage of caspase-1 and triggers secretion of proinflammatory cytokines and may also result in pyroptotic cell death. Inflammasome activation plays a crucial role in inflammatory conditions; aberrant activation of inflammasome contributes to the pathogenesis of neurodegenerative diseases. Diethyl Maleate (DEM) is a promising antiinflammatory chemical to alleviate inflammasome activation. In this study, NLRP3 inflammasome was activated in N9 murine microglia via 1µg/ml LPS (Lipopolysaccharide) for 4h and 5 mM ATP (Adenosine 5'-triphosphate) for 1h, respectively. We demonstrated that 1h pretreatment of DEM attenuated NLRP3 inflammasome activation in microglial cells. Besides, mitochondrial ROS decreased upon DEM pretreatment in inflammasome-induced cells. Likewise, it ameliorated pyroptotic cell death in microglia. DEM is a potent activator of Nrf2 transcription factor, the key regulator of the antioxidant response pathway. Nrf2 has been a significant target to decrease aberrant inflammasome activation through the antioxidant compounds, including DEM. Here, we have shown that DEM increased Nrf2 translocation to the nucleus, resulting in Nrf2 target gene expression in microglia. In conclusion, DEM is a promising protective agent against NLRP3 inflammasome activation.

Inflammasome Activation Mediates Apoptotic and Pyroptotic Death in Astrocytes Under Ischemic Conditions.

Inflammation is a hallmark mechanism of ischemic stroke-induced brain injury. Recent studies have shown that an intracellular multimeric protein complex known as an inflammasome is a key factor for inducing an inflammatory response, and apoptotic and pyroptotic cell death in ischemic stroke. Inflammasome assembly leads to the activation of pro-inflammatory caspases, and the maturation and secretion of pro-inflammatory cytokines IL-1β and IL-18. While the role of inflammasomes in ischemic stroke-induced neuronal death, and microglial activation and cell death have been established, little is known about the role of inflammasomes in astrocytes under ischemic conditions. In this study, we investigated the expression and activation of inflammasome components in protoplasmic and fibrous astrocytes under ischemic conditions. We found that both protoplasmic and fibrous astrocytes expressed a differential increase in inflammasome protein components, and that their activation promoted maturation of IL-1β and IL-18, and secretion of IL-1β, as well as initiating apoptotic and pyroptotic cell death. Pharmacological inhibition of caspase-1 decreased expression of cleaved caspase-1 and production of mature IL-1β, and protected against inflammasome-mediated apoptotic and pyroptotic cell death. Overall, this study provides novel insights into the role of inflammasome signaling in astrocytes under ischemic conditions.

Dose-dependent modulation of microglia activation in rats after penetrating traumatic brain injury (pTBI) by transplanted human neural stem cells.

Traumatic brain injury (TBI) often results in long-lasting patterns of neurological deficits including motor, sensory, and cognitive abnormalities. Cranial gunshot survivors are among the most disabled TBI patients and face a lifetime of disability with no approved strategies to protect or repair the brain after injury. Recent studies using a model of penetrating TBI (pTBI) have reported that human neural stem cells (hNSCs) transplantation can lead to dose and location-dependent neuroprotection. Evidence for regional patterns of microglial activation has also been reported after pTBI with evidence for microglial cell death by pyroptosis. Because of the importance of injury-induced microglial activation in the pathogenesis of TBI, we tested the hypothesis that dose-dependent hNSC mediated neuroprotection after pTBI was associated with reduced microglial activation in pericontusional cortical areas. To test this hypothesis, quantitative microglial/macrophage Iba1 immunohistochemistry and Sholl analysis was conducted to investigate the arborization patterns using four experimental groups including, (i) Sham operated (no injury) + low dose (0.16 million cells/rat), (ii) pTBI + vehicle (no cells), (iii) pTBI + low dose hNSCs (0.16 million/rat), and (iv) pTBI + high dose hNSCs (1.6 million cells/rat). At 3 months post-transplantation (transplants at one week after pTBI), the total number of intersections was significantly reduced in vehicle treated pTBI animals versus sham operated controls indicating increased microglia/macrophage activation. In contrast, hNSC transplantation led to a dose-dependent increase in the number of intersections compared to pTBI vehicle indicating less microglia/macrophage activation. The peak of Sholl intersections at 1 μm from the center of the microglia/macrophages ranged from ~6,500-14,000 intersections for sham operated, ~250-500 intersections for pTBI vehicle, ~550-1,000 intersections for pTBI low dose, and ~2,500-7,500 intersections for pTBI high dose. Plotting data along the rostrocaudal axis also showed that pericontusional cortical areas protected by hNSC transplantation had increased intersections compared to nontreated pTBI animals. These studies using a non-biased Sholl analysis demonstrated a dose-dependent reduction in inflammatory cell activation that may be associated with a neuroprotective effect driven by the cellular transplant in perilesional regions after pTBI.

RNA binding protein RPS3 mediates microglial polarization by activating NLRP3 inflammasome via SIRT1 in ischemic stroke

BackgroundIschemic stroke is the obstruction of cerebral blood flow with a high morbidity. Microglial polarization is a contributing factor for ischemic stroke-induced injury. Here, we focused on function and mechanism of RNA binding protein RPS3 in microglial polarization after ischemic stroke. MethodsTransient middle cerebral artery occlusion (tMCAO) was conducted in SD rats. Infarct area was detected by TTC staining and neurological score was assessed. Fluorescence staining tested neuronal apoptosis and microglial differentiation. Oxygen and glucose deprivation/reoxygenation (OGD/R) was applied for treating microglia. Levels of RPS3, SIRT1, M1 and M2 polarization markers (CD86, iNOS, CD206, Arg-1) were determined by RT-qPCR. Western blot detected RPS3, SIRT1, NLRP3, ASC and Cleaved-caspase-1 expression. RIP assay validated that RPS3 interacted with SIRT1. CCK-8 measured cell viability. Flow cytometry and ELISA assessed M1 and M2 polarization markers. LDH release was detected using colorimetric CytoTox 96 Cytotoxicity kit. ResultsRPS3 depletion improved neurological dysfunction and reduced infarction area in rats after tMCAO. Knockdown of RPS3 resulted in increased SIRT1 expression and decreased NLRP3 inflammasome activation, and induced microglia M2 polarization after ischemia-reperfusion (I/R). Besides, RPS3 directly targeted SIRT1 and reduced its expression in microglia. RPS3 silencing suppressed OGD/R-triggered neuronal and microglial cell death through SIRT1. Moreover, RPS3 activated NLRP3 inflammasome and regulated microglial polarization via SIRT1. ConclusionRPS3 regulates microglial polarization and neuronal injury through SIRT1/NLRP3 pathway, suggesting a novel target for ischemic stroke treatment.

Clinical parameters related to the glial activation and neuronal alterations present in the retinas of COVID‐19 patients

AbstractPurpose: Ocular and eye fundus alterations have been described in COVID‐19 patients. In this study, the aim was to establish several stages of response to damage in the retinal cells from human donors with COVID‐19 and analyse their relationship with clinical parameters.Methods: Retinal sections and flat‐mount retinas from human donors with COVID‐19 (n = 16) and control (n = 12) were analysed with immunohistochemistry. The location of angiotensin‐converting enzyme 2 (ACE2) and the morphology of microglial cells, Müller cells, astrocytes, and photoreceptors were evaluated by confocal microscopy. Microglial analysis and cell death were measured. Clinical data were correlated with different retinal parameters.Results: Müller cells, outer segment of cones and retinal pigment epithelium presented the ACE2 protein. Larger staining of ACE2 and cellular retinaldehyde–binding protein (CRALBP) was found in the cell bodies of Müller cells in COVID‐19 group. Disorganization of honeycomb‐like pattern formed by Müller cells and disruption of external limiting membrane were observed in the 81.3% of COVID‐19 patients. The 53.3% of COVID‐19 patients showed reactive gliosis as well as astrocytes protruding and epiretinal membranes. Cone degeneration was aggravated in the COVID‐19 group. Activated or ameboid‐shape microglia was found in the 93.8% of COVID‐19 patients. Most retinas showed a reduction of the area occupied by microglia and presented microglial nodules around vessels. Retinal degeneration in COVID‐19 group correlated with the duration of the disease (ro: 0.655; p &lt; 0.01) and age (ro: −0.5; p &lt; 0.05). Moreover, microglia activation was correlated with blood oxygen levels (ro: −0.554; p &lt; 0.05).Conclusions: Different stages of glial activation and neuronal alterations are present in COVID‐19 retinas and are significantly correlated with the duration of the disease, age and respiratory failure.

Necrotic-like BV-2 microglial cell death due to methylmercury exposure.

Methylmercury (MeHg) is a dangerous environmental contaminant with strong bioaccumulation in the food chain and neurotoxic properties. In the nervous system, MeHg may cause neurodevelopment impairment and potentially interfere with immune response, compromising proper control of neuroinflammation and aggravating neurodegeneration. Human populations are exposed to environmental contamination with MeHg, especially in areas with strong mining or industrial activity, raising public health concerns. Taking this into consideration, this work aims to clarify pathways leading to acute toxic effects caused by MeHg exposure in microglial cells. BV-2 mouse microglial cells were incubated with MeHg at different concentrations (0.01, 0.1, 1 and 10µM) for 1h prior to continuous Lipopolysaccharide (LPS, 0.5μg/ml) exposure for 6 or 24h. After cell exposure, reactive oxygen species (ROS), IL-6 and TNF-α cytokines production, inducible nitric oxide synthase (iNOS) expression, nitric oxide (NO) release, metabolic activity, propidium iodide (PI) uptake, caspase-3 and -9 activities and phagocytic activity were assessed. MeHg 10µM decreased ROS formation, the production and secretion of pro-inflammatory cytokines IL-6, TNF-α, iNOS immunoreactivity, the release of NO in BV-2 cells. Furthermore, MeHg 10µM decreased the metabolic activity of BV-2 and increased the number of PI-positive cells (necrotic-like cell death) when compared to the respective control group. Besides, MeHg did not interfere with caspase activity or the phagocytic profile of cells. The short-term effects of a high concentration of MeHg on BV-2 microglial cells lead to impaired production of several pro-inflammatory mediators, as well as a higher microglial cell death via necrosis, compromising their neuroinflammatory response. Clarifying the mechanisms underlying MeHg-induced neurotoxicity and neurodegeneration in brain cells is relevant to better understand acute and long-term chronic neuroinflammatory responses following MeHg exposure.

Single-nucleus chromatin accessibility and RNA sequencing reveal impaired brain development in prenatally e-cigarette exposed neonatal rats

SummaryAlthough emerging evidence reveals that vaping alters the function of the central nervous system, the effects of maternal vaping on offspring brain development remain elusive. Using a well-established in utero exposure model, we performed single-nucleus ATAC-seq (snATAC-seq) and RNA sequencing (snRNA-seq) on prenatally e-cigarette-exposed rat brains. We found that maternal vaping distorted neuronal lineage differentiation in the neonatal brain by promoting excitatory neurons and inhibiting lateral ganglionic eminence-derived inhibitory neuronal differentiation. Moreover, maternal vaping disrupted calcium homeostasis, induced microglia cell death, and elevated susceptibility to cerebral ischemic injury in the developing brain of offspring. Our results suggest that the aberrant calcium signaling, diminished microglial population, and impaired microglia-neuron interaction may all contribute to the underlying mechanisms by which prenatal e-cigarette exposure impairs neonatal rat brain development. Our findings raise the concern that maternal vaping may cause adverse long-term brain damage to the offspring.

Gut bacterial isoamylamine promotes age-related cognitive dysfunction by promoting microglial cell death

The intestinal microbiome releases a plethora of small molecules. Here, we show that the Ruminococcaceae metabolite isoamylamine (IAA) is enriched in aged mice and elderly people, whereas Ruminococcaceae phages, belonging to the Myoviridae family, are reduced. Young mice orally administered IAA show cognitive decline, whereas Myoviridae phage administration reduces IAA levels. Mechanistically, IAA promotes apoptosis of microglial cells by recruiting the transcriptional regulator p53 to the S100A8 promoter region. Specifically, IAA recognizes and binds the S100A8 promoter region to facilitate the unwinding of its self-complementary hairpin structure, thereby subsequently enabling p53 to access the S100A8 promoter and enhance S100A8 expression. Thus, our findings provide evidence that small molecules released from the gut microbiome can directly bind genomic DNA and act as transcriptional coregulators by recruiting transcription factors. These findings further unveil a molecular mechanism that connects gut metabolism to gene expression in the brain with implications for disease development.

The antioxidant enzyme Peroxiredoxin-1 controls stroke-associated microglia against acute ischemic stroke

Ischemic stroke is the leading cause of immortal disability and death worldwide. For treatment in the acute phase, it is necessary to control excessive reactive oxygen species (ROS) damage during ischemia/reperfusion (I/R). Microglia are well known to be closely associated with excessive ROS response in the early stage of I/R. However, the precise roles of microglia associated with mitigating ROS damage, and molecular markers of heterogenetic microglia in the I/R damaged brain has not been clarified. Here, we identified a new type of microglia associated with stroke in the I/R injured brain. Single-cell RNA sequencing (scRNA-seq) was used to assess transcriptional changes of microglia and immune cells in the contralateral (CL) and ipsilateral (IL) hemispheres after transient middle cerebral artery occlusion (tMCAO) surgery to mimic ischemic stroke. We classified a unique type of microglia with enhanced antioxidant function and markers similar to those of disease-associated microglia (DAM), designated them as stroke-associated microglia (SAM). The representative antioxidant enzyme, Peroxiredoxin-1 (Prdx1), was predominantly expressed in SAM and mediated ROS defense genes, including Txn1, Srx1, Mt1, and Mt2. In the Prdx1−/− I/R damaged brain, we observed significantly increased infarction, as assessed by TTC staining, and FACS analysis detected severe microglial cell death. Importantly, scRNA transcriptomics data showed that the SAM population was specifically decreased in Prdx1−/− mice and that these mice exhibited decreased ROS damage resistance. Inflammatory responses which were detected by ELISA and qPCR, were also increased in Prdx1−/− IL hemispheres. Finally, Prdx1-dependent antioxidative SAM were found to be essential for increasing the transcription levels of stroke-protective molecules, such as osteopontin and ferritin. A novel microglia type (SAM) is specifically activated in response to stroke I/R injury, and that Prdx1 expression is required for the activation and enhanced antioxidant function of SAM.

Neuroinvasion and Neurotropism by SARS-CoV-2 Variants in the K18-hACE2 Mouse.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) not only affects the respiratory tract but also causes neurological symptoms such as loss of smell and taste, headache, fatigue or severe cerebrovascular complications. Using transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2), we investigated the spatiotemporal distribution and pathomorphological features in the CNS following intranasal infection with SARS-CoV-2 variants, as well as after prior influenza A virus infection. Apart from Omicron, we found all variants to frequently spread to and within the CNS. Infection was restricted to neurons and appeared to spread from the olfactory bulb mainly in basally oriented regions in the brain and into the spinal cord, independent of ACE2 expression and without evidence of neuronal cell death, axonal damage or demyelination. However, microglial activation, microgliosis and a mild macrophage and T cell dominated inflammatory response was consistently observed, accompanied by apoptotic death of endothelial, microglial and immune cells, without their apparent infection. Microgliosis and immune cell apoptosis indicate a potential role of microglia for pathogenesis and viral effect in COVID-19 and the possible impairment of neurological functions, especially in long COVID. These data may also be informative for the selection of therapeutic candidates and broadly support the investigation of agents with adequate penetration into relevant regions of the CNS.

Anti‐Epileptic Drug and Teratogen Valproic Acid Induces Microglial Cell Death in a Valproic Acid Murine Model of Autism

Autism Spectrum Disorder (ASD) is a neurodevelopmental condition that affects approximately 1 in 44 children in North America, and its association with neuronal connectivity is an area of intense research. Valproic acid (VPA) is a multi‐target drug widely used to treat epilepsy. It is classified as a teratogen and as a histone deacetylase inhibitor (HDACi) and fetal exposure to VPA increases the risk of ASD. The VPA model has been well characterized for behavioral and neuronal deficits, including hyperconnectivity. The cause of hyperconnectivity is poorly understood however, it is speculated that lack of pruning may be partly responsible for this anomaly. Microglia, the principal immune cells of CNS regulate dendrite and synapse formation during early brain development. The effect of VPA on microglia during early development has not been well characterized and may provide potential hints regarding the etiology of this disorder. Preliminary studies from our lab indicate that VPA reduces microglial number during peak periods of postnatal synaptogenesis in the mouse brain primary cortex. Nevertheless, the cause of this reduction is not known. Therefore, in this study, we determined the mechanism for reduced microglial numbers in the VPA model of autism.Pregnant BALBc mice were administered 600 mg/kg of VPA by subcutaneous injection on day 13.5 of gestation. We harvested the mice brains at embryonic (E) day 14, E17, and postnatal day 1 (PD1). Brain tissue was double immunolabeled for microglia (IBA1) and cell death marker (TUNEL). We found a gradual increase in TUNEL staining beginning E14 to PD1 compared to age‐matched controls. The mechanism for reduced microglial number may be due to cell death. Thus, our findings possibly have pathophysiological/pathogenic implications in autism and may, ultimately, lead to the discovery of new therapeutic target(s) for autism.

Circulating extracellular vesicles activate the pyroptosis pathway in the brain following ventilation-induced lung injury

BackgroundMechanical ventilation of preterm newborns causes lung injury and is associated with poor neurodevelopmental outcomes. However, the mechanistic links between ventilation-induced lung injury (VILI) and brain injury is not well defined. Since circulating extracellular vesicles (EVs) are known to link distant organs by transferring their cargos, we hypothesized that EVs mediate inflammatory brain injury associated with VILI.MethodsNeonatal rats were mechanically ventilated with low (10 mL/kg) or high (25 mL/kg) tidal volume for 1 h on post-natal day 7 followed by recovery for 2 weeks. Exosomes were isolated from the plasma of these rats and adoptively transferred into normal newborn rats. We assessed the effect of mechanical ventilation or exosome transfer on brain inflammation and activation of the pyroptosis pathway by western blot and histology.ResultsInjurious mechanical ventilation induced similar markers of inflammation and pyroptosis, such as increased IL-1β and activated caspase-1/gasdermin D (GSDMD) in both lung and brain, in addition to inducing microglial activation and cell death in the brain. Isolated EVs were enriched for the exosomal markers CD9 and CD81, suggesting enrichment for exosomes. EVs isolated from neonatal rats with VILI had increased caspase-1 but not GSDMD. Adoptive transfer of these EVs led to neuroinflammation with microglial activation and activation of caspase-1 and GSDMD in the brain similar to that observed in neonatal rats that were mechanically ventilated.ConclusionsThese findings suggest that circulating EVs can contribute to the brain injury and poor neurodevelopmental outcomes in preterm infants with VILI through activation of GSDMD.

NeuroD1 induces microglial apoptosis and cannot induce microglia-to-neuron cross-lineage reprogramming

The regenerative capacity of neurons is limited in the central nervous system (CNS), with irreversible neuronal loss upon insult. In contrast, microglia exhibit extraordinary capacity for repopulation. Matsuda etal. (2019) recently reported NeuroD1-induced microglia-to-neuron conversion, aiming to provide an "unlimited" source to regenerate neurons. However, the extent to which NeuroD1 can exert cross-lineage reprogramming of microglia (myeloid lineage) to neurons (neuroectodermal lineage) is unclear. In this study, we unexpectedly found that NeuroD1 cannot convert microglia to neurons in mice. Instead, NeuroD1 expression induces microglial cell death. Moreover, lineage tracing reveals non-specific leakage of similar lentiviruses as previously used for microglia-to-neuron conversion, which confounds the microglia-to-neuron observation. In summary, we demonstrated that NeuroD1 cannot induce microglia-to-neuron cross-lineage reprogramming. We here propose rigid principles for verifying glia-to-neuron conversion. This Matters Arising paper is in response to Matsuda etal. (2019), published in Neuron.