Astrocytic P2X7 receptor in retrosplenial cortex drives electroacupuncture analgesia.

P2X7 receptor (P2X7R) has been found to contribute to the peripheral mechanism of acupuncture analgesia (AA). However, whether it plays an important role in central mechanism remains unknown. In this study, we aimed to reveal the role of astrocytic P2X7R in retrosplenial cortex (RSC) in AA and provide new evidence for underlying the central mechanism of AA. We applied the chemogenetic receptors hM3Dq to stimulate or hM4Di to inhibit astrocytes ligand clozapine-N-oxide (CNO) following injection of adeno-associated virus (AAV) into the bilateral RSC, or pharmacologically intervened in the activity of the purinergic receptor P2X7R. Current data indicated that chemogenetic inhibition of astrocytes or injection of P2X7R agonist Bz-ATP in the bilateral RSC significantly reverses the analgesic effect of electroacupuncture (EA) in formalin tests while the bilateral injection of the P2X7R antagonist A438079 alleviated formalin-induced nociceptive behavior. Additionally, chemogenetic suppression of astrocytic P2X7R by injection of AAV in the bilateral RSC decreased hind paw flinches induced by formalin in the mice. These findings indicate the participation of both astrocytes and P2X7R in the RSC in EA analgesic. Moreover, P2X7R on astrocytes in the RSC appears to play a critical role in the ability of EA to attenuate formalin-induced pain responses in mice.

Mitochondrial dysfunction and increased reactive oxygen species production in MECP2 mutant astrocytes and their impact on neurons.

Studies on MECP2 function and its implications in Rett Syndrome (RTT) have traditionally centered on neurons. Here, using human embryonic stem cell (hESC) lines, we modeled MECP2 loss-of-function to explore its effects on astrocyte (AST) development and dysfunction in the brain. Ultrastructural analysis of RTT hESC-derived cerebral organoids revealed significantly smaller mitochondria compared to controls (CTRs), particularly pronounced in glia versus neurons. Employing a multiomics approach, we observed increased gene expression and accessibility of a subset of nuclear-encoded mitochondrial genes upon mutation of MECP2 in ASTs compared to neurons. Analysis of hESC-derived ASTs showed reduced mitochondrial respiration and altered key proteins in the tricarboxylic acid cycle and electron transport chain in RTT versus CTRs. Additionally, RTT ASTs exhibited increased cytosolic amino acids under basal conditions, which were depleted upon increased energy demands. Notably, mitochondria isolated from RTT ASTs exhibited increased reactive oxygen species and influenced neuronal activity when transferred to cortical neurons. These findings underscore MECP2 mutation's differential impact on mitochondrial and metabolic pathways in ASTs versus neurons, suggesting that dysfunctional AST mitochondria may contribute to RTT pathophysiology by affecting neuronal health.

The Blood-Brain Barrier in Both Humans and Rats: A Perspective From 3D Imaging.

Background: The blood-brain barrier (BBB) is part of the neurovascular unit (NVU) which plays a key role in maintaining homeostasis. However, its 3D structure is hardly known. The present study is aimed at imaging the BBB using tissue clearing and 3D imaging techniques in both human brain tissue and rat brain tissue. Methods: Both human and rat brain tissue were cleared using the CUBIC technique and imaged with either a confocal or two-photon microscope. Image stacks were reconstructed using Imaris. Results: Double staining with various antibodies targeting endothelial cells, basal membrane, pericytes of blood vessels, microglial cells, and the spatial relationship between astrocytes and blood vessels showed that endothelial cells do not evenly express CD31 and Glut1 transporter in the human brain. Astrocytes covered only a small portion of the vessels as shown by the overlap between GFAP-positive astrocytes and Collagen IV/CD31-positive endothelial cells as well as between GFAP-positive astrocytes and CD146-positive pericytes, leaving a big gap between their end feet. A similar structure was observed in the rat brain. Conclusions: The present study demonstrated the 3D structure of both the human and rat BBB, which is discrepant from the 2D one. Tissue clearing and 3D imaging are promising techniques to answer more questions about the real structure of biological specimens.

NR1H4 ameliorates Parkinson's disease via inhibiting astrocyte activation and neuroinflammation in a CEBPß/NF-κB dependent manner.

Parkinson's Disease (PD) is a degenerative disease driven by neuroinflammation. Nuclear receptor subfamily 1 group H member 4 (NR1H4), a nuclear receptor involved in metabolic and inflammatory regulation, is found to be widely expressed in central nervous system. Previous studies suggested the protective role of NR1H4 in various diseases related to inflammation, whether NR1H4 participates in PD progression remains unknown. To investigate the role of NR1H4 in neuroinflammation regulation, especially astrocyte activation during PD, siRNA and adenovirus were used to manipulate Nr1h4 expression. RNA-sequencing (RNA-seq), quantitative real-time PCR, enzyme-linked immunosorbent assay, Chromatin immunoprecipitation and western blotting were performed to further study the underlying mechanisms. We identified that NR1H4 was down-regulated during PD progression. In vitro experiments suggested that Nr1h4 knockdown led to inflammatory response, reactive oxygen species generation and astrocytes activation whereasNr1h4 overexpressionhad the opposite effects. The results of RNA-seq on astrocytes revealed that NR1H4 manipulated neuroinflammation in a CEBPß/NF-κB dependent manner. Additionally, pharmacological activation of NR1H4 via Obeticholic acid ameliorated neuroinflammation and promoted neuronal survival. Our study first proved the neuroprotective effects of NR1H4against PD via inhibiting astrocyte activation and neuroinflammation in a CEBPß/NF-κB dependent manner.

Glial modulation of synapse development and plasticity: oligodendrocyte precursor cells as a new player in the synaptic quintet.

Synaptic communication is an important process in the central nervous system that allows for the rapid and spatially specified transfer of signals. Neurons receive various synaptic inputs and generate action potentials required for information transfer, and these inputs can be excitatory or inhibitory, which collectively determines the output. Non-neuronal cells (glial cells) have been identified as crucial participants in influencing neuronal activity and synaptic transmission, with astrocytes forming tripartite synapses and microglia pruning synapses. While it has been known that oligodendrocyte precursor cells (OPCs) receive neuronal inputs, whether they also influence neuronal activity and synaptic transmission has remained unknown for two decades. Recent findings indicate that OPCs, too, modulate neuronal synapses. In this review, we discuss the roles of different glial cell types at synapses, including the recently discovered involvement of OPCs in synaptic transmission and synapse refinement, and discuss overlapping roles played by multiple glial cell types.

Scaled Complexity of Mammalian Astrocytes: Insights From Mouse and Macaque.

Astrocytes intricately weave within the neuropil, giving rise to characteristic bushy morphologies. Pioneering studies suggested that primate astrocytes are more complex due to increased branch numbers and territory size compared to rodent counterparts. However, there has been no comprehensive comparison of astrocyte morphology across species. We employed several techniques to investigate astrocyte morphology and directly compared them between mice and rhesus macaques in cortical and subcortical regions. We assessed astrocyte density, territory size, branching structure, fine morphological complexity, and interactions with neuronal synapses using a combination of techniques, including immunohistochemistry, adeno-associated virus-mediated transduction of astrocytes, diOlistics, confocal imaging, and electron microscopy. We found significant morphological similarities between primate and rodent astrocytes, suggesting that astrocyte structure has scaled with evolution. Our findings show that primate astrocytes are larger and more numerous than those in rodents but contest the view that primate astrocytes are morphologically far more complex.

Membrane properties and coupling of macroglia in the optic nerve.

We established a longitudinal acute slice preparation of transgenic mouse optic nerve to characterize membrane properties and coupling of glial cells by patch-clamp and dye-filling, complemented by immunohistochemistry. Unlike in cortex or hippocampus, the majority of EGFP + cells in optic nerve of the hGFAP-EGFP transgenic mouse, a tool to identify astrocytes, were characterized by time and voltage dependent K+-currents including A-type K+-currents, properties previously described for NG2 glia. Indeed, the majority of transgene expressing cells in optic nerve were immunopositive for NG2 proteoglycan, whereas only a minority show GFAP immunoreactivity. Similar physiological properties were seen in YFP + cells from NG2-YFP transgenic mice, indicating that in optic nerve the transgene of hGFAP-EGFP animals is expressed by NG2 glia instead of astrocytes. Using Cx43kiECFP transgenic mice as another astrocyte-indicator revealed that astrocytes had passive membrane currents. Dye-filling showed that hGFAP-EGFP+ cells in optic nerve were coupled to none or few neighboring cells while hGFAP-EGFP+ cells in the cortex form large networks. Similarly, dye-filling of NG2-YFP+ and Cx43-CFP+ cells in optic nerve revealed small networks. Our work shows that identification of astrocytes in optic nerve requires distinct approaches, that the cells express membrane current patterns distinct from cortex and that they form small networks.

Targeting EGLN2/PHD1 protects motor neurons and normalizes the astrocytic interferon response.

Neuroinflammation and dysregulated energy metabolism are linked to motor neuron degeneration in amyotrophic lateral sclerosis (ALS). The egl-9 family hypoxia-inducible factor (EGLN) enzymes, also known as prolyl hydroxylase domain (PHD) enzymes, are metabolic sensors regulating cellular inflammation and metabolism. Using an oligonucleotide-based and a genetic approach, we showed that the downregulation of Egln2 protected motor neurons and mitigated the ALS phenotype in two zebrafish models and a mouse model of ALS. Single-nucleus RNA sequencing of the murine spinal cord revealed that the loss of EGLN2 induced an astrocyte-specific downregulation of interferon-stimulated genes, mediated via the stimulator of interferon genes (STING) protein. In addition, we found that the genetic deletion of EGLN2 restored this interferon response in patient induced pluripotent stem cell (iPSC)-derived astrocytes, confirming the link between EGLN2 and astrocytic interferon signaling. In conclusion, we identified EGLN2 as a motor neuron protective target normalizing the astrocytic interferon-dependent inflammatory axis in vivo, as well as in patient-derived cells.

Microglia contact cerebral vasculature through gaps between astrocyte endfeet.

The close spatial relationship between microglia and cerebral blood vessels implicates microglia in vascular development, homeostasis and disease. In this study we used the publicly available Cortical MM^3 electron microscopy dataset to systematically investigate microglial interactions with the vasculature. Our analysis revealed that approximately 20% of microglia formed direct contacts with blood vessels through gaps between adjacent astrocyte endfeet. We termed these contact points "plugs". Plug-forming microglia exhibited closer proximity to blood vessels than non-plug forming microglia and formed multiple plugs, predominantly near the soma, ranging in surface area from ∼0.01 µm2 to ∼15 µm2. Plugs were enriched at the venule end of the vascular tree and displayed a preference for contacting endothelial cells over pericytes at a ratio of 3:1. In summary, we provide novel insights into the ultrastructural relationship between microglia and the vasculature, laying a foundation for understanding how these contacts contribute to the functional cross-talk between microglia and cells of the vasculature in health and disease.

REV-ERBα Mitigates Astrocyte Activation and Protects Dopaminergic Neurons from Damage.

Parkinson's disease (PD) is characterized by astrocyte activation and disruptions in circadian rhythm. Within the astrocyte population, two distinct reactive states exist: A1 and A2. A1 astrocytes are associated with neurotoxicity and inflammation, while A2 astrocytes exhibit neuroprotective functions. Our investigation focused on the role of REV-ERBα, a member of the nuclear receptor superfamily and a key regulator of the circadian clock, in astrocyte activation. We observed that REV-ERBα expression in A1 astrocytes was reduced to one-third of its normal level. Notably, activation of REV-ERBα prompted a transformation of astrocytes from A1 to A2. Mechanistically, REV-ERBα inhibition was linked to the classical NF-κB pathway, while it concurrently suppressed the STAT3 pathway. Furthermore, astrocytes with low REV-ERBα expression were associated with dopaminergic neurons apoptosis. Intriguingly, the opposite effect was observed when using a REV-ERBα agonist, which mitigated astrocyte activation and reduced dopaminergic neuron damage by 50%. In summary, our study elucidates the pivotal role of REV-ERBα in modulating astrocyte function and its potential implications in PD pathogenesis.

Generation of Astrocyte-specific BEST1 Conditional Knockout Mouse with Reduced Tonic GABA Inhibition in the Brain.

Bestrophin-1 (BEST1) is a Ca2+-activated anion channel known for its role in astrocytes. Best1 is permeable to gliotransmitters, including GABA, to contribute to tonic GABA inhibition and modulate synaptic transmission in neighboring neurons. Despite the crucial functions of astrocytic BEST1, there is an absence of genetically engineered cell-type specific conditional mouse models addressing these roles. In this study, we developed an astrocyte-specific BEST1 conditional knock-out (BEST1 aKO) mouse line. Using the embryonic stem cell (ES cell) targeting method, we developed Best1 floxed mice (C57BL/6JCya-Best1em1flox/Cya), which have exon 3, 4, 5, and 6 of Best1 flanked by two loxP sites. By crossing with hGFAP-CreERT2 mice, we generated Best1 floxed/hGFAP-CreERT2 mice, which allowed for the tamoxifen-inducible deletion of Best1 under the human GFAP promoter. We characterized its features across various brain regions, including the striatum, hippocampal dentate gyrus (HpDG), and Parafascicular thalamic nucleus (Pf). Compared to the Cre-negative control, we observed significantly reduced BEST1 protein expression in immunohistochemistry (IHC) and tonic GABA inhibition in patch clamp recordings. The reduction in tonic GABA inhibition was 66.7% in the striatum, 46.4% in the HpDG, and 49.6% in the Pf. Our findings demonstrate that the BEST1 channel in astrocytes significantly contributes to tonic inhibition in the local brain areas. These mice will be valuable for future studies not only on tonic GABA release but also on tonic release of gliotransmitters mediated by astrocytic BEST1.

Upregulated expression of ubiquitin ligase TRIM21 promotes PKM2 nuclear translocation and astrocyte activation in experimental autoimmune encephalomyelitis.

Reactive astrocytes play critical roles in the occurrence of various neurological diseases such as multiple sclerosis. Activation of astrocytes is often accompanied by a glycolysis-dominant metabolic switch. However, the role and molecular mechanism of metabolic reprogramming in activation of astrocytes have not been clarified. Here, we found that PKM2, a rate-limiting enzyme of glycolysis, displayed nuclear translocation in astrocytes of EAE (experimental autoimmune encephalomyelitis) mice, an animal model of multiple sclerosis. Prevention of PKM2 nuclear import by DASA-58 significantly reduced the activation of mice primary astrocytes, which was observed by decreased proliferation, glycolysis and secretion of inflammatory cytokines. Most importantly, we identified the ubiquitination-mediated regulation of PKM2 nuclear import by ubiquitin ligase TRIM21. TRIM21 interacted with PKM2, promoted its nuclear translocation and stimulated its nuclear activity to phosphorylate STAT3, NF-κB and interact with c-myc. Further single-cell RNA sequencing and immunofluorescence staining demonstrated that TRIM21 expression was upregulated in astrocytes of EAE. TRIM21 overexpressing in mice primary astrocytes enhanced PKM2-dependent glycolysis and proliferation, which could be reversed by DASA-58. Moreover, intracerebroventricular injection of a lentiviral vector to knockdown TRIM21 in astrocytes or intraperitoneal injection of TEPP-46, which inhibit the nuclear translocation of PKM2, effectively decreased disease severity, CNS inflammation and demyelination in EAE. Collectively, our study provides novel insights into the pathological function of nuclear glycolytic enzyme PKM2 and ubiquitination-mediated regulatory mechanism that are involved in astrocyte activation. Targeting this axis may be a potential therapeutic strategy for the treatment of astrocyte-involved neurological disease.

Insulin and leptin acutely modulate the energy metabolism of primary hypothalamic and cortical astrocytes.

Astrocytes constitute a heterogeneous cell population within the brain, contributing crucially to brain homeostasis and playing an important role in overall brain function. Their function and metabolism are not only regulated by local signals, for example, from nearby neurons, but also by long-range signals such as hormones. Thus, two prominent hormones primarily known for regulating the energy balance of the whole organism, insulin, and leptin, have been reported to also impact astrocytes within the brain. In this study, we investigated the acute regulation of astrocytic metabolism by these hormones in cultured astrocytes prepared from the mouse cortex and hypothalamus, a pivotal region in the context of nutritional regulation. Utilizing genetically encoded, fluorescent nanosensors, the cytosolic concentrations of glucose, lactate, and ATP, along with glycolytic rate and the NADH/NAD+ redox state were measured. Under basal conditions, differences between the two populations of astrocytes were observed for glucose and lactate concentrations as well as the glycolytic rate. Additionally, astrocytic metabolism responded to insulin and leptin in both brain regions, with some unique characteristics for each cell population. Finally, both hormones influenced how cells responded to elevated extracellular levels of potassium ions, a common indicator of neuronal activity. In summary, our study provides evidence that insulin and leptin acutely regulate astrocytic metabolism within minutes. Additionally, while astrocytes from the hypothalamus and cortex share similarities in their metabolism, they also exhibit distinct properties, further underscoring the growing recognition of astrocyte heterogeneity.

Inhibition of PHLDA3 expression in human superoxide dismutase 1-mutant amyotrophic lateral sclerosis astrocytes protects against neurotoxicity.

Pleckstrin homology-like domain family A-member 3 (PHLDA3) has recently been identified as a player in adaptive and maladaptive cellular stress pathways. The outcome of pleckstrin homology-like domain family A-member 3 signalling was shown to vary across different cell types and states. It emerges that its expression and protein level are highly increased in amyotrophic lateral sclerosis (ALS) patient-derived astrocytes. Whether it orchestrates a supportive or detrimental function remains unexplored in the context of neurodegenerative pathologies. To directly address the role of pleckstrin homology-like domain family A-member 3 in healthy and ALS astrocytes, we used overexpression and knockdown strategies. We generated cultures of primary mouse astrocytes and also human astrocytes from control and ALS patient-derived induced pluripotent stem cells harbouring the superoxide dismutase 1 mutation. Then, we assessed astrocyte viability and the impact of their secretome on oxidative stress responses in human stem cell-derived cortical and spinal neuronal cultures. Here, we show that PHLDA3 overexpression or knockdown in control astrocytes does not significantly affect astrocyte viability or reactive oxygen species production. However, PHLDA3 knockdown in ALS astrocytes diminishes reactive oxygen species concentrations in their supernatants, indicating that pleckstrin homology-like domain family A-member 3 can facilitate stress responses in cells with altered homeostasis. In support, supernatants of PHLDA3-silenced ALS and even control spinal astrocytes with a lower pleckstrin homology-like domain family A-member 3 protein content could prevent sodium arsenite-induced stress granule formation in spinal neurons. Our findings provide evidence that reducing pleckstrin homology-like domain family A-member 3 levels may transform astrocytes into a more neurosupportive state relevant to targeting non-cell autonomous ALS pathology.

Astrocyte-derived exosomal miR-378a-5p mitigates cerebral ischemic neuroinflammation by modulating NLRP3-mediated pyroptosis.

Objective: This study aimed to investigate the regulatory role of astrocyte-derived exosomes and their microRNAs (miRNAs) in modulating neuronal pyroptosis during cerebral ischemia. Methods: Astrocyte-derived exosomes were studied for treating cerebral ischemia in both in vitro and in vivo models. The effects of astrocyte-derived exosomes on neuroinflammation were investigated by analyzing exosome uptake, nerve damage, and pyroptosis protein expression. High throughput sequencing was used to identify astrocyte-derived exosomal miRNAs linked to pyroptosis, followed by validation via qRT‒PCR. The relationship between these miRNAs and NLRP3 was studied using a dual luciferase reporter assay. This study used miR-378a-5p overexpression and knockdown to manipulate OGD injury in nerve cells. The impact of astrocyte-derived exosomal miR-378a-5p on the regulation of cerebral ischemic neuroinflammation was assessed through analysis of nerve injury and pyroptosis protein expression. Results: Our findings demonstrated that astrocyte-derived exosomes were internalized by neurons both in vitro and in vivo. Additionally, Astrocyte-derived exosomes displayed a neuroprotective effect against OGD-induced neuronal injury and brain injury in the ischemic cortical region of middle cerebral artery occlusion (MCAO) rats while also reducing pyroptosis. Further investigations revealed the involvement of astrocyte-derived exosomal miR-378a-5p in regulating pyroptosis by inhibiting NLRP3. The overexpression of miR-378a-5p mitigated neuronal damage, whereas the knockdown of miR-378a-5p increased NLRP3 expression and exacerbated pyroptosis, thus reversing this neuroprotective effect. Conclusion: Astrocyte-derived exosomal miR-378a-5p has a neuroprotective effect on cerebral ischemia by suppressing neuroinflammation associated with NLRP3-mediated pyroptosis.Further research is required to comprehensively elucidate the signaling pathways by which astrocyte-derived exosomal miR-378a-5p modulates neuronal pyroptosis.

Diospyros lotus leaf extract and its main component myricitrin inhibit itch­related IL­6 and IL­31 by suppressing microglial inflammation and microglial­mediated astrocyte activation.

Diospyros lotus has been traditionally used in Asia for medicinal purposes, exhibiting a broad spectrum of pharmacological effects including antioxidant, neuroprotective and anti­inflammatory properties. While the anti­itch effect of D. lotus leaves has been reported, studies on the detailed mechanism of action in microglia and astrocytes, which are members of the central nervous system, have yet to be revealed. The present study aimed to investigate effects of D. lotus leaf extract (DLE) and its main component myricitrin (MC) on itch­related cytokines and signaling pathways in lipopolysaccharide (LPS)­stimulated microglia. The effect of DLE and MC on activation of astrocyte stimulated by microglia was also examined. Cytokine production was evaluated through reverse transcription PCR and western blot analysis. Signaling pathway was analyzed by performing western blotting and immunofluorescence staining. The effect of microglia on astrocytes activation was evaluated via western blotting for receptors, signaling molecules and itch mediators and confirmed through gene silencing using short interfering RNA. DLE and MC suppressed the production of itch­related cytokine IL­6 and IL­31 in LPS­stimulated microglia. These inhibitory effects were mediated through the blockade of NF­κB, MAPK and JAK/STAT pathways. In astrocytes, stimulation by microglia promoted the expression of itch­related molecules such as oncostatin M receptor, interleukin 31 receptor a, inositol 1,4,5­trisphosphate receptor 1, lipocalin­2 (LCN2), STAT3 and glial fibrillary acidic protein. However, DLE and MC significantly inhibited these receptors. Additionally, astrocytes stimulated by microglia with IL­6, IL­31, or both genes silenced did not show activation of LCN2 or STAT3. The findings of the present study demonstrated that DLE and MC could suppress pruritic activity in astrocytes induced by microglia­derived IL­6 and IL­31. This suggested the potential of DLE and MC as functional materials capable of alleviating pruritus.

Translatome analysis in acute ischemic stroke: Astrocytes and microglia exhibit differences in poststroke alternative splicing of expressed transcripts.

Astrocytes and microglia undergo dynamic and complex morphological and functional changes following ischemic stroke, which are instrumental in both inflammatory responses and neural repair. While gene expression alterations poststroke have been extensively studied, investigations into posttranscriptional regulatory mechanisms, specifically alternative splicing (AS), remain limited. Utilizing previously reported Ribo-Tag-seq data, this study analyzed AS alterations in poststroke astrocytes and microglia from young adult male and female mice. Our findings reveal that in astrocytes, compared to the sham group, 109 differential alternative splicing (DAS) events were observed at 4 h poststroke, which increased to 320 at day 3. In microglia, these numbers were 316 and 266, respectively. Interestingly, the disparity between DAS genes and differentially expressed genes is substantial, with fewer than 10 genes shared at both poststroke time points in astrocytes and microglia. Gene ontology enrichment analysis revealed the involvement of these DAS genes in diverse functions, encompassing immune response (Adam8, Ccr1), metabolism (Acsl6, Pcyt2, Myo5a), and developmental cell growth (App), among others. Selective DAS events were further validated by semiquantitative RT-PCR. Overall, this study comprehensively describes the AS alterations in astrocytes and microglia during the hyperacute and acute phases of ischemic stroke and underscores the significance of certain hub DAS events in neuroinflammatory processes.

Spatial-temporal representation of the astroglial markers in the developing human cortex.

Specific spatiotemporal patterns of the normal glial differentiation during human brain development have not been thoroughly studied. Immunomorphological studies on postmortem material have remained a basic method for human neurodevelopmental studies so far. The main problem for the immunohistochemical research of astrogliogenesis is that now there are no universal astrocyte markers, that characterize the whole mature astrocyte population or precursors at each stage of development. To define the general course of astrogliogenesis in the developing human cortex, 25 fetal autopsy samples at the stages from eight postconceptional weeks to birth were collected for the immunomorphological analysis. Spatiotemporal immunoreactivity patterns with the panel of markers (ALDH1L1, GFAP, S100, SOX9, and Olig-2), related to glial differentiation were described and compared. The early S100 + cell population of ventral origin was described as well. This S100 + cell distribution deviated from the SOX9-immunoreactivity pattern and was similar to the Olig-2 one. In the given material the dorsal gliogenic wave was characterized by ALDH1L1-, GFAP-, and S100-immunoreactivity manifestation in the dorsal proliferative niche at the end of the early fetal period. The time point of dorsal astrogliogenesis was agreed upon not later than the 17 GW stage. ALDH1L1 + , GFAP + , S100 + , and SOX9 + cell expansion patterns from the ventricular and subventricular zones to the intermediate zone, subplate, and cortical plate were described at the end of early fetal, middle, and late fetal periods. The ALDH1L1-, GFAP-, and S100-immunoreactivity patterns were shown to be not completely identical.

Dissecting reactive astrocyte responses: lineage tracing and morphology-based clustering.

Brain damage triggers diverse cellular and molecular events, with astrocytes playing a crucial role in activating local neuroprotective and reparative signaling within damaged neuronal circuits. Here, we investigated reactive astrocytes using a multidimensional approach to categorize their responses into different subtypes based on morphology. This approach utilized the StarTrack lineage tracer, single-cell imaging reconstruction and multivariate data analysis. Our findings identified three profiles of reactive astrocyte responses, categorized by their effects on cell size- and shape- related morphological parameters: "moderate", "strong," and "very strong". We also examined the heterogeneity of astrocyte reactivity, focusing on spatial and clonal distribution. Our research revealed a notable enrichment of protoplasmic and fibrous astrocytes within the "strong" and "very strong" response subtypes. Overall, our study contributes to a better understanding of astrocyte heterogeneity in response to an injury. By characterizing the diverse reactive responses among astrocyte subpopulations, we provide insights that could guide future research aimed at identifying novel therapeutic targets to mitigate brain damage and promote neural repair.

Proteomic evidence of depression-associated astrocytic dysfunction in the human male olfactory bulb.

The olfactory bulb (OB), a major structure of the limbic system, has been understudied in human investigations of psychopathologies such as depression. To explore more directly the molecular features of the OB in depression, a global comparative proteome analysis was carried out with human post-mortem OB samples from 11 males having suffered from depression and 12 healthy controls. We identified 188 differentially abundant proteins (with adjusted p < 0.05) between depressed cases and controls. Gene ontology and gene enrichment analyses suggested that these proteins are involved in biological processes including the complement and coagulation cascades. Cell type enrichment analysis displayed a significant reduction in several canonical astrocytic proteins in OBs from depressed patients. Furthermore, using RNA-fluorescence in-situ hybridization, we observed a decrease in the percentage of ALDH1L1+ cells expressing canonical astrocytic markers including ALDOC, NFIA, GJA1 (connexin 43) and SLC1A3 (EAAT1). These results are consistent with previous reports of downregulated astrocytic marker expression in other brain regions in depressed patients. We also conducted a comparative phosphoproteomic analysis of OB samples and found a dysregulation of proteins involved in neuronal and astrocytic functions. To determine whether OB astrocytic abnormalities is specific to humans, we also performed proteomics on the OB of socially defeated male mice, a commonly used model of depression. Cell-type specific analysis revealed that in socially defeated animals, the most striking OB protein alterations were associated with oligodendrocyte-lineage cells rather than with astrocytes, highlighting an important species difference. Overall, this study further highlights cerebral astrocytic abnormalities as a consistent feature of depression in humans.