Nrf2 pathway activation promotes the expression of genes related to glutathione metabolism in alcohol-exposed astrocytes.

Introduction: Oxidative and antioxidant pathways play essential roles in the development of alcohol-induced brain injury. The Nrf2 pathway is an endogenous antioxidant response pathway, but there has been little research on the role of Nrf2 in alcohol-related diseases. Thus, we examined the effects of alcohol and an Nrf2 agonist (TBHQ) on astrocyte function, mRNA expression, and metabolite content to further explore the protective mechanisms of Nrf2 agonists in astrocytes following alcohol exposure. Methods: CTX TNA2 astrocytes were cultured with alcohol and TBHQ and then subjected to transcriptome sequencing, LC-MS/MS analysis, quantitative reverse transcription polymerase chain reaction (qRT-PCR), and malondialdehyde (MDA) and superoxide dismutase (SOD) activity assays. Results: Alcohol exposure significantly increased malondialdehyde (MDA) levels while decreasing superoxide dismutase (SOD) levels in astrocytes. Treatment with TBHQ effectively reversed these effects, demonstrating its protective role against oxidative stress induced by alcohol. Transcriptome sequencing and qRT-PCR analysis revealed that TBHQ specifically upregulates genes involved in glutathione metabolism, including a notable increase in the expression of the glutathione S-transferase A5 (GSTA5) gene, which was suppressed by alcohol exposure. Additionally, metabolomic analysis showed that TBHQ regulates key components of ether lipid metabolism in alcohol-exposed astrocytes, with significant reductions in the levels of lysophosphatidylcholine (18:0) (LysoPC (18:0)) and 2-acetyl-1-alkyl-sn-glycero-3-phosphocholine, both of which are critical markers in the ether lipid metabolic pathway. Discussion: The findings underscore the role of TBHQ as an Nrf2 agonist in mitigating alcohol-induced oxidative damage in astrocytes by modulating glutathione metabolism and ether lipid metabolism. The regulation of GSTA5 gene expression emerges as a key mechanism through which Nrf2 agonists confer neuroprotection against oxidative stress and lipid oxidation. These insights pave the way for potential therapeutic strategies targeting the Nrf2 pathway to protect astrocytes from alcohol-induced damage.

Cell adhesion molecule CD44 is dispensable for reactive astrocyte activation during prion disease.

Prion diseases are fatal, infectious, neurodegenerative disorders resulting from accumulation of misfolded cellular prion protein in the brain. Early pathological changes during CNS prion disease also include reactive astrocyte activation with increased CD44 expression, microgliosis, as well as loss of dendritic spines and synapses. CD44 is a multifunctional cell surface adhesion and signalling molecule which is considered to play roles in astrocyte morphology and the maintenance of dendritic spine integrity and synaptic plasticity. However, the role of CD44 in prion disease was unknown. Here we used mice deficient in CD44 to determine the role of CD44 during prion disease. We show that CD44-deficient mice displayed no difference in their response to CNS prion infection when compared to wild type mice. Furthermore, the reactive astrocyte activation and microgliosis that accompanies CNS prion infection was unimpaired in the absence of CD44. Together, our data show that although CD44 expression is upregulated in reactive astrocytes during CNS prion disease, it is dispensable for astrocyte and microglial activation and the development of prion neuropathogenesis.

Empagliflozin alleviates neuroinflammation by inhibiting astrocyte activation in the brain and regulating gut microbiota of high-fat diet mice.

A high-fat diet can modify the composition of gut microbiota, resulting in dysbiosis. Changes in gut microbiota composition can lead to increased permeability of the gut barrier, allowing bacterial products like lipopolysaccharides (LPS) to enter circulation. This process can initiate systemic inflammation and contribute to neuroinflammation. Empagliflozin (EF), an SGLT2 inhibitor-type hypoglycemic drug, has been reported to treat neuroinflammation. However, there is a lack of evidence showing that EF regulates the gut microbiota axis to control neuroinflammation in HFD models. In this study, we explored whether EF could improve neuroinflammation caused by an HFD via regulation of the gut microbiota and the mechanism underlying this phenomenon. Our data revealed that EF alleviates pathological brain injury, reduces the reactive proliferation of astrocytes, and increases the expression of synaptophysin. In addition, the levels of inflammatory factors in hippocampal tissue were significantly decreased after EF intervention. Subsequently, the results of 16S rRNA gene sequencing showed that EF could change the microbial community structure of mice, indicating that the abundance of Lactococcus, Ligilactobacillus and other microbial populations decreased dramatically. Therefore, EF alleviates neuroinflammation by inhibiting gut microbiota-mediated astrocyte activation in the brains of high-fat diet-fed mice. Our study focused on the gut-brain axis, and broader research on neuroinflammation can provide a more holistic understanding of the mechanisms driving neurodegenerative diseases and inform the development of effective strategies to mitigate their impact on brain health. The results provide strong evidence supporting the larger clinical application of EF.

Digestion of whey peptide induces antioxidant and anti-inflammatory bioactivity on glial cells: Sequences identification and structural activity analysis.

Whey derived peptides have shown potential activity improving brain function in pathological condition. However, there is little information about their mechanism of action on glial cells, which have important immune functions in brain. Astrocytes and microglia are essential in inflammatory and oxidative defense that take place in neurodegenerative disease. In this work we evaluate antioxidant and anti-inflammatory potential bioactivity of whey peptide in glial cells. Peptides were formed during simulated gastrointestinal digestion (Infogest protocol), and low molecular weight (<5kDA) peptides (WPHf) attenuated reactive oxygen species (ROS) production induced by hydrogen peroxide stimulus in both cells in dose-dependent manner. WPHf induced an increase in the antioxidant glutathione (GSH) content and prevented GSH reduction induced by lipopolysaccharides (LPS) stimulus in astrocytes cells in a cell specific form. An increase in cytokine mRNA expression (TNFα and IL6) and nitric oxide secretion induced by LPS was attenuated by WPHf pre-treatment in both cells. The inflammatory pathway was dependent on NFκB activation. Bioactive peptide ranking analysis showed positive correlation with hydrophobicity and negative correlation with high molecular weights. The sequence identification revealed 19 peptides cross-referred with bioactive database. Whey peptides were rich in leucine, valine and tyrosine in the C-terminal region and lysine in the N-terminal region. The anti-inflammatory and antioxidant potential of whey peptides were assessed in glia cells and its mechanisms of action were related, such as modulation of antioxidant enzymes and anti-inflammatory pathways. Features of the peptide structure, such as molecular size, hydrophobicity and types of amino acids present in the terminal region are associated to bioactivity.

Astrocyte-Specific Inhibition of the Primary Cilium Suppresses C3 Expression in Reactive Astrocyte.

C3-positive reactive astrocytes play a neurotoxic role in various neurodegenerative diseases. However, the mechanisms controlling C3-positive reactive astrocyte induction are largely unknown. We found that the length of the primary cilium, a cellular organelle that receives extracellular signals was increased in C3-positive reactive astrocytes, and the loss or shortening of primary cilium decreased the count of C3-positive reactive astrocytes. Pharmacological experiments suggested that Ca2+ signalling may synergistically promote C3 expression in reactive astrocytes. Conditional knockout (cKO) mice that specifically inhibit primary cilium formation in astrocytes upon drug stimulation exhibited a reduction in the proportions of C3-positive reactive astrocytes and apoptotic cells in the brain even after the injection of lipopolysaccharide (LPS). Additionally, the novel object recognition (NOR) score observed in the cKO mice was higher than that observed in the neuroinflammation model mice. These results suggest that the primary cilium in astrocytes positively regulates C3 expression. We propose that regulating astrocyte-specific primary cilium signalling may be a novel strategy for the suppression of neuroinflammation.

cFLIP in the molecular regulation of astroglia-driven neuroinflammation in experimental glaucoma.

BACKGROUND: Recent experimental studies of neuroinflammation in glaucoma pointed to cFLIP as a molecular switch for cell fate decisions, mainly regulating cell type-specific caspase-8 functions in cell death and inflammation. This study aimed to determine the importance of cFLIP for regulating astroglia-driven neuroinflammation in experimental glaucoma by analyzing the outcomes of astroglia-targeted transgenic deletion of cFLIP or cFLIPL. METHODS: Glaucoma was modeled by anterior chamber microbead injections to induce ocular hypertension in mouse lines with or without conditional deletion of cFLIP or cFLIPL in astroglia. Morphological analysis of astroglia responses assessed quantitative parameters in retinal whole mounts immunolabeled for GFAP and inflammatory molecules or assayed for TUNEL. The molecular analysis included 36-plexed immunoassays of the retina and optic nerve cytokines and chemokines, NanoString-based profiling of inflammation-related gene expression, and Western blot analysis of selected proteins in freshly isolated samples of astroglia. RESULTS: Immunoassays and immunolabeling of retina and optic nerve tissues presented reduced production of various proinflammatory cytokines, including TNFα, in GFAP/cFLIP and GFAP/cFLIPL relative to controls at 12 weeks of ocular hypertension with no detectable alteration in TUNEL. Besides presenting a similar trend of the proinflammatory versus anti-inflammatory molecules displayed by immunoassays, NanoString-based molecular profiling detected downregulated NF-κB/RelA and upregulated RelB expression of astroglia in ocular hypertensive samples of GFAP/cFLIP compared to ocular hypertensive controls. Analysis of protein expression also revealed decreased phospho-RelA and increased phospho-RelB in parallel with an increase in caspase-8 cleavage products. CONCLUSIONS: A prominent response limiting neuroinflammation in ocular hypertensive eyes with cFLIP-deletion in astroglia values the role of cFLIP in the molecular regulation of glia-driven neuroinflammation during glaucomatous neurodegeneration. The molecular responses accompanying the lessening of neurodegenerative inflammation also seem to maintain astroglia survival despite increased caspase-8 cleavage with cFLIP deletion. A transcriptional autoregulatory response, dampening RelA but boosting RelB for selective expression of NF-κB target genes, might reinforce cell survival in cFLIP-deleted astroglia.

Myelin oligodendrocyte glycoprotein reactive Th17 cells drive Janus Kinase 1 dependent transcriptional reprogramming in astrocytes and alter cell surface cytokine receptor profiles during experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is an autoimmune demyelinating disease affecting the central nervous system (CNS). T helper (Th) 17 cells are involved in the pathogenesis of MS and its animal model of experimental autoimmune encephalomyelitis (EAE) by infiltrating the CNS and producing effector molecules that engage resident glial cells. Among these glial cells, astrocytes have a central role in coordinating inflammatory processes by responding to cytokines and chemokines released by Th17 cells. In this study, we examined the impact of pathogenic Th17 cells on astrocytes in vitro and in vivo. We identified that Th17 cells reprogram astrocytes by driving transcriptomic changes partly through a Janus Kinase (JAK)1-dependent mechanism, which included increased chemokines, interferon-inducible genes, and cytokine receptors. In vivo, we observed a region-specific heterogeneity in the expression of cell surface cytokine receptors on astrocytes, including those for IFN-γ, IL-1, TNF-α, IL-17, TGFß, and IL-10. Additionally, these receptors were dynamically regulated during EAE induced by adoptive transfer of myelin-reactive Th17 cells. This study overall provides evidence of Th17 cell reprogramming of astrocytes, which may drive changes in the astrocytic responsiveness to cytokines during autoimmune neuroinflammation.

ß-arrestin2 is indispensable for the antidepressant effects of fluoxetine via inhibiting astrocytic pyroptosis in chronic mild stress mouse model for depression.

ß-arrestin2 is a versatile protein for signaling transduction in brain physiology and pathology. Herein, we investigated the involvement of ß-arrestin2 in pharmacological effects of fluoxetine for depression. A chronic mild stress (CMS) model was established using wild-type (WT) and ß-arrestin2-/- mice. Behavioral results demonstrated that CMS mice showed increased immobility time in the tail suspension test and forced swimming test, elevated concentrations of pro-inflammatory factors in peripheral blood, increased expression of pyroptosis-related proteins, and increased co-labeling of glial fibrillary acidic protein and Caspase1 p10 in the hippocampus compared to the CON group. Treatment with fluoxetine (FLX) ameliorated these conditions. However, compared with the ß-arrestin2-/- CMS group, these results of the ß-arrestin2-/- CMS + FLX group showed no significant changes. These results suggested that the above effects of FLX could be eliminated by knocking out ß-arrestin2. Mass spectrometry implying that FLX promoted the binding of ß-arrestin2 to the NLRP2 inflammasome of depressed mice. Subsequently, the results of the cellular experiments suggested that the 5HT2B receptor antagonist may attenuate L-kynurenine + ATP-induced cell pyroptosis by attenuating NLRP2 binding to ß-arrestin2. We further found that the lack of ß-arrestin2 eliminated the anti-pyroptosis effect of fluoxetine. In conclusion, ß-arrestin2 is an essential protein for fluoxetine to alleviate pyroptosis in the hippocampal astrocytes of CMS mice. Mechanistically, we found that the 5-HT2BR-ß-arrestin2-NLRP2 axis is vital for maintaining the antidepressant effects of fluoxetine.

Inhibition of astroglial hemichannels prevents synaptic transmission decline during spreading depression.

BACKGROUND: Spreading depression (SD) is an intriguing phenomenon characterized by massive slow brain depolarizations that affect neurons and glial cells. This phenomenon is repetitive and produces a metabolic overload that increases secondary damage. However, the mechanisms associated with the initiation and propagation of SD are unknown. Multiple lines of evidence indicate that persistent and uncontrolled opening of hemichannels could participate in the pathogenesis and progression of several neurological disorders including acute brain injuries. Here, we explored the contribution of astroglial hemichannels composed of connexin-43 (Cx43) or pannexin-1 (Panx1) to SD evoked by high-K+ stimulation in brain slices. RESULTS: Focal high-K+ stimulation rapidly evoked a wave of SD linked to increased activity of the Cx43 and Panx1 hemichannels in the brain cortex, as measured by light transmittance and dye uptake analysis, respectively. The activation of these channels occurs mainly in astrocytes but also in neurons. More importantly, the inhibition of both the Cx43 and Panx1 hemichannels completely prevented high K+-induced SD in the brain cortex. Electrophysiological recordings also revealed that Cx43 and Panx1 hemichannels critically contribute to the SD-induced decrease in synaptic transmission in the brain cortex and hippocampus. CONCLUSIONS: Targeting Cx43 and Panx1 hemichannels could serve as a new therapeutic strategy to prevent the initiation and propagation of SD in several acute brain injuries.

TFEB/LAMP2 contributes to PM0.2-induced autophagy-lysosome dysfunction and alpha-synuclein dysregulation in astrocytes.

Atmospheric particulate matter (PM) exacerbates the risk factor for Alzheimer's and Parkinson's diseases (PD) by promoting the alpha-synuclein (α-syn) pathology in the brain. However, the molecular mechanisms of astrocytes involvement in α-syn pathology underlying the process remain unclear. This study investigated PM with particle size <200 nm (PM0.2) exposure-induced α-syn pathology in ICR mice and primary astrocytes, then assessed the effects of mammalian target of rapamycin inhibitor (PP242) in vitro studies. We observed the α-syn pathology in the brains of exposed mice. Meanwhile, PM0.2-exposed mice also exhibited the activation of glial cell and the inhibition of autophagy. In vitro study, PM0.2 (3, 10 and 30 µg/mL) induced inflammatory response and the disorders of α-syn degradation in primary astrocytes, and lysosomal-associated membrane protein 2 (LAMP2)-mediated autophagy underlies α-syn pathology. The abnormal function of autophagy-lysosome was specifically manifested as the expression of microtubule-associated protein light chain 3 (LC3II), cathepsin B (CTSB) and lysosomal abundance increased first and then decreased, which might both be a compensatory mechanism to toxic α-syn accumulation induced by PM0.2. Moreover, with the transcription factor EB (TFEB) subcellular localization and the increase in LC3II, LAMP2, CTSB, and cathepsin D proteins were identified, leading to the restoration of the degradation of α-syn after the intervention of PP242. Our results identified that PM0.2 exposure could promote the α-syn pathological dysregulation in astrocytes, providing mechanistic insights into how PM0.2 increases the risk of developing PD and highlighting TFEB/LAMP2 as a promising therapeutic target for antagonizing PM0.2 toxicity.

Spatially resolved multiomics on the neuronal effects induced by spaceflight in mice.

Impairment of the central nervous system (CNS) poses a significant health risk for astronauts during long-duration space missions. In this study, we employed an innovative approach by integrating single-cell multiomics (transcriptomics and chromatin accessibility) with spatial transcriptomics to elucidate the impact of spaceflight on the mouse brain in female mice. Our comparative analysis between ground control and spaceflight-exposed animals revealed significant alterations in essential brain processes including neurogenesis, synaptogenesis and synaptic transmission, particularly affecting the cortex, hippocampus, striatum and neuroendocrine structures. Additionally, we observed astrocyte activation and signs of immune dysfunction. At the pathway level, some spaceflight-induced changes in the brain exhibit similarities with neurodegenerative disorders, marked by oxidative stress and protein misfolding. Our integrated spatial multiomics approach serves as a stepping stone towards understanding spaceflight-induced CNS impairments at the level of individual brain regions and cell types, and provides a basis for comparison in future spaceflight studies. For broader scientific impact, all datasets from this study are available through an interactive data portal, as well as the National Aeronautics and Space Administration (NASA) Open Science Data Repository (OSDR).

A comprehensive exploration of astrocytes in migraine: a bibliometric and visual analysis.

BACKGROUND: Migraine, as a prevalent neurologic disorder, involves intricate and yet incompletely elucidated pathophysiological mechanisms. A plethora of research findings underscores the pivotal role played by astrocytes in the progression of migraines. In order to elucidate the current advances and directions in research pertaining to astrocytes in migraines, we conducted bibliometric analysis of relevant literature and visualized the results. Subsequently, we expound upon these findings to contribute to the evolving understanding of the role of astrocytes in migraine pathophysiology. METHODS: On November 21, 2023, we conducted a search on Web of Science (WOS), restricting the document type to articles or reviews and language to English. Following a meticulous selection process involving three researchers, we identified the literature to be included in our analysis. Subsequently, we employed Microsoft Office Excel programs, R, VOSviewer, Scimago Graphica, and CiteSpace software to conduct visualization analysis of basic information and trends regarding journals, countries/regions, and influential authors, institutions, keywords, and papers. RESULTS: As of November 21, 2023, relevant literature has been published in 71 journals across 27 countries/regions. This corpus comprises contributions from 576 authors affiliated with 220 institutions, encompassing 865 keywords and referencing 6065 scholarly articles. CEPHALALGIA stands out as the most influential journal in this field, while authors PIETROBON D and DALKARA T have significant impact. The United States is highly influential, with CNR and UNIV PADUA emerging as highly influential institutions. The predominant category is Neurosciences. CONCLUSIONS: Future investigators may continue to focus on migraines with aura, familial hemiplegic migraine (FHM), and the crucial calcitonin gene-related peptide (CGRP) system. Employing advanced observational techniques, such as imaging, researchers should pay attention to cellular and tissue structures, such as microglia and the trigeminal ganglion, as well as mechanisms involving inflammation and central sensitization. Moreover, animal models are paramount in obtaining high-quality evidence.

The Role of Glial Cells in Neurobiology and Prion Neuropathology.

Prion diseases are rare and neurodegenerative diseases that are characterized by the misfolding and infectious spread of the prion protein in the brain, causing progressive and irreversible neuronal loss and associated clinical and behavioral manifestations in humans and animals, ultimately leading to death. The brain has a complex network of neurons and glial cells whose crosstalk is critical for function and homeostasis. Although it is established that prion infection of neurons is necessary for clinical disease to occur, debate remains in the field as to the role played by glial cells, namely astrocytes and microglia, and whether these cells are beneficial to the host or further accelerate disease. Here, we review the current literature assessing the complex morphologies of astrocytes and microglia, and the crosstalk between these two cell types, in the prion-infected brain.

Comparison between sub-chronic and chronic sleep deprivation-induced behavioral and neuroimmunological abnormalities in mice: Focusing on glial cell phenotype polarization.

BACKGROUND: Sleep disorders, depression, and Alzheimer's disease (AD) are extensively reported as comorbidity. Although neuroinflammation triggered by microglial phenotype M1 activation, leading to neurotransmitter dysfunction and Aß aggregation, is considered as the leading cause of depression and AD, whether and how sub-chronic or chronic sleep deprivation (SD) contribute to the onset and development of these diseases remains unclear. METHODS: Memory and depression-like behaviors were evaluated in both SDs, and then circadian markers, glial cell phenotype polarization, cytokines, depression-related neurotransmitters, and AD-related gene/protein expressions were measured by qRT-PCR, enzyme-linked immunosorbent assay, high-performance liquid chromatography, and western-blotting respectively. RESULTS: Both SDs induced give-up behavior and anhedonia and increased circadian marker period circadian regulator 2 (PER2) expression, which were much worse in chronic than in the sub-chronic SD group, while brain and muscle ARNT-like protein-1 only decreased in the chronic-SD. Furthermore, increased microglial M1 and astrocyte A1 expression and proinflammatory cytokines, interleukin (IL)-1ß, IL-6, and tumor necrosis factor-α was observed in both SDs, which were more significant in chronic SD. Similarly, decreased norepinephrine and 5-hydroxytryptamine/5-hydroxyindoleacetic acid ratio were more significant, which corresponds to the worse depression-like behavior in chronic than sub-chronic-SD. With regard to AD, increased amyloid precursor protein (APP) and soluble (s)-APPß and decreased sAPPα in both SDs were more significant in the chronic. However, sAPPα/sAPPß ratio was only decreased in chronic SD. CONCLUSION: These findings suggest that both SDs induce depression-like changes by increasing PER2, leading to neuroinflammation and neurotransmitter dysfunction. However, only chronic SD induced memory impairment likely due to severer circadian disruption, higher neuroinflammation, and dysregulation of APP metabolism.

Bone morphogenetic protein 4 derived from the cerebrospinal fluid in patients with postherpetic neuralgia induces allodynia via the crosstalk between microglia and astrocyte.

INTRODUCTION: During postherpetic neuralgia (PHN), the cerebral spinal fluid (CSF) possesses the capability to trigger glial activation and inflammation, yet the specific changes in its composition remain unclear. Recent findings from our research indicate elevations of central bone morphogenetic protein 4 (BMP4) during neuropathic pain (NP), serving as an independent modulator of glial cells. Herein, the aim of the present study is to test the CSF-BMP4 expressions and its role in the glial modulation in the process of PHN. METHODS: CSF samples were collected from both PHN patients and non-painful individuals (Control) to assess BMP4 and its antagonist Noggin levels. Besides, intrathecal administration of both CSF types was conducted in normal rats to evaluate the impact on pain behavior, glial activity, and inflammation.; Additionally, both Noggin and STAT3 antagonist-Stattic were employed to treat the PHN-CSF or exogenous BMP4 challenged cultured astrocytes to explore downstream signals. Finally, microglial depletion was performed prior to the PHN-CSF intervention so as to elucidate the microglia-astrocyte crosstalk. RESULTS: BMP4 levels were significantly higher in PHN-CSF compared to Control-CSF (P < 0.001), with a positive correlation with pain duration (P < 0.05, r = 0.502). Comparing with the Control-CSF producing moderate paw withdrawal threshold (PWT) decline and microglial activation, PHN-CSF further exacerbated allodynia and triggered both microglial and astrocytic activation (P < 0.05). Moreover, PHN-CSF rather than Control-CSF evoked microglial proliferation and pro-inflammatory transformation, reinforced iron storage, and activated astrocytes possibly through both SMAD159 and STAT3 signaling, which were all mitigated by the Noggin application (P < 0.05). Next, both Noggin and Stattic effectively attenuated BMP4-induced GFAP and IL-6 upregulation, as well as SMAD159 and STAT3 phosphorylation in the cultured astrocytes (P < 0.05). Finally, microglial depletion diminished PHN-CSF induced astrogliosis, inflammation and endogenous BMP4 expression (P < 0.05). CONCLUSION: Our study highlights the role of CSF-BMP4 elevation in glial activation and allodynia during PHN, suggesting a potential therapeutic avenue for future exploration.

Astrocytic Slc4a4 regulates blood-brain barrier integrity in healthy and stroke brains via a CCL2-CCR2 pathway and NO dysregulation.

Astrocytes play vital roles in blood-brain barrier (BBB) maintenance, yet how they support BBB integrity under normal or pathological conditions remains poorly defined. Recent evidence suggests that ion homeostasis is a cellular mechanism important for BBB integrity. In the current study, we investigated the function of an astrocyte-specific pH regulator, Slc4a4, in BBB maintenance and repair. We show that astrocytic Slc4a4 is required for normal astrocyte morphological complexity and BBB function. Multi-omics analyses identified increased astrocytic secretion of CCL2 coupled with dysregulated arginine-NO metabolism after Slc4a4 deletion. Using a model of ischemic stroke, we found that loss of Slc4a4 exacerbates BBB disruption, which was rescued by pharmacological or genetic inhibition of the CCL2-CCR2 pathway in vivo. Together, our study identifies the astrocytic Slc4a4-CCL2 and endothelial CCR2 axis as a mechanism controlling BBB integrity and repair, while providing insights for a therapeutic approach against BBB-related CNS disorders.

Modulation of Paracellular Permeability in SARS-CoV-2 Blood-to-Brain Transcytosis.

SARS-CoV-2 primarily infects the lungs via the ACE2 receptor but also other organs including the kidneys, the gastrointestinal tract, the heart, and the skin. SARS-CoV-2 also infects the brain, but the hematogenous route of viral entry to the brain is still not fully characterized. Understanding how SARS-CoV-2 traverses the blood-brain barrier (BBB) as well as how it affects the molecular functions of the BBB are unclear. In this study, we investigated the roles of the receptors ACE2 and DPP4 in the SARS-CoV-2 infection of the discrete cellular components of a transwell BBB model comprising HUVECs, astrocytes, and pericytes. Our results demonstrate that direct infection on the BBB model does not modulate paracellular permeability. Also, our results show that SARS-CoV-2 utilizes clathrin and caveolin-mediated endocytosis to traverse the BBB, resulting in the direct infection of the brain side of the BBB model with a minimal endothelial infection. In conclusion, the BBB is susceptible to SARS-CoV-2 infection in multiple ways, including the direct infection of endothelium, astrocytes, and pericytes involving ACE2 and/or DPP4 and the blood-to-brain transcytosis, which is an event that does not require the presence of host receptors.

Molecular signatures of astrocytes and microglia maladaptive responses to acute stress are rescued by a single administration of ketamine in a rodent model of PTSD.

Stress affects the brain and alters its neuroarchitecture and function; these changes can be severe and lead to psychiatric disorders. Recent evidence suggests that astrocytes and microglia play an essential role in the stress response by contributing to the maintenance of cerebral homeostasis. These cells respond rapidly to all stimuli that reach the brain, including stressors. Here, we used a recently validated rodent model of post-traumatic stress disorder in which rats can be categorized as resilient or vulnerable after acute inescapable footshock stress. We then investigated the functional, molecular, and morphological determinants of stress resilience and vulnerability in the prefrontal cortex, focusing on glial and neuronal cells. In addition, we examined the effects of a single subanesthetic dose of ketamine, a fast-acting antidepressant recently approved for the treatment of resistant depression and proposed for other stress-related psychiatric disorders. The present results suggest a prompt glial cell response and activation of the NF-κB pathway after acute stress, leading to an increase in specific cytokines such as IL-18 and TNF-α. This response persists in vulnerable individuals and is accompanied by a significant change in the levels of critical glial proteins such as S100B, CD11b, and CX43, brain trophic factors such as BDNF and FGF2, and proteins related to dendritic arborization and synaptic architecture such as MAP2 and PSD95. Administration of ketamine 24 h after the acute stress event rescued many of the changes observed in vulnerable rats, possibly contributing to support brain homeostasis. Overall, our results suggest that pivotal events, including reactive astrogliosis, changes in brain trophic factors, and neuronal damage are critical determinants of vulnerability to acute traumatic stress and confirm the therapeutic effect of acute ketamine against the development of stress-related psychiatric disorders.

High-Caloric Diets in Adolescence Impair Specific GABAergic Subpopulations, Neurogenesis, and Alter Astrocyte Morphology.

We compared the effects of two different high-caloric diets administered to 4-week-old rats for 12 weeks: a diet rich in sugar (30% sucrose) and a cafeteria diet rich in sugar and high-fat foods. We focused on the hippocampus, particularly on the gamma-aminobutyric acid (GABA)ergic system, including the Ca2+-binding proteins parvalbumin (PV), calretinin (CR), calbindin (CB), and the neuropeptides somatostatin (SST) and neuropeptide Y (NPY). We also analyzed the density of cholinergic varicosities, brain-derived neurotrophic factor (BDNF), reelin (RELN), and cyclin-dependent kinase-5 (CDK-5) mRNA levels, and glial fibrillary acidic protein (GFAP) expression. The cafeteria diet reduced PV-positive neurons in the granular layer, hilus, and CA1, as well as NPY-positive neurons in the hilus, without altering other GABAergic populations or overall GABA levels. The high-sugar diet induced a decrease in the number of PV-positive cells in CA3 and an increase in CB-positive cells in the hilus and CA1. No alterations were observed in the cholinergic varicosities. The cafeteria diet also reduced the relative mRNA expression of RELN without significant changes in BDNF and CDK5 levels. The cafeteria diet increased the number but reduced the length of the astrocyte processes. These data highlight the significance of determining the mechanisms mediating the observed effects of these diets and imply that the cognitive impairments previously found might be related to both the neuroinflammation process and the reduction in PV, NPY, and RELN expression in the hippocampal formation.

Interplay between Energy Supply and Glutamate Toxicity in the Primary Cortical Culture.

Limited substrate availability because of the blood-brain barrier (BBB) has made the brain develop specific molecular mechanisms to survive, using lactate synthesized by astrocytes as a source of energy in neurons. To understand if lactate improves cellular viability and susceptibility to glutamate toxicity, primary cortical cells were incubated in glucose- or lactate-containing media and toxic concentrations of glutamate for 24 h. Cell death was determined by immunostaining and lactate dehydrogenase (LDH) release. Mitochondrial membrane potential and nitric oxide (NO) levels were measured using Tetramethylrhodamine, methyl ester (TMRM) and 4-Amino-5-Methylamino-2',7'-Difluorofluorescein Diacetate (DAF-FM) live staining, respectively. LDH activity was quantified in single cells in the presence of lactate (LDH substrate) and oxamate (LDH inhibitor). Nuclei of cells were stained with DAPI and neurons with MAP2. Based on the distance between neurons and glial cells, they were classified as linked (<10 µm) and non-linked (>10 µm) neurons. Lactate increased cell death rate and the mean value of endogenous NO levels compared to glucose incubations. Mitochondrial membrane potential was lower in the cells cultured with lactate, but this effect was reversed when glutamate was added to the lactate medium. LDH activity was higher in linked neurons compared to non-linked neurons, supporting the hypothesis of the existence of the lactate shuttle between astrocytes and at least a portion of neurons. In conclusion, glucose or lactate can equally preserve primary cortical neurons, but those neurons having a low level of LDH activity and incubated with lactate cannot cover high energetic demand solely with lactate and become more susceptible to glutamate toxicity.