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.

Novel insight into astrocyte-mediated gliotransmission modulates the synaptic plasticity in major depressive disorder.

Major depressive disorder (MDD) is a form of glial cell-based synaptic dysfunction disease in which glial cells interact closely with neuronal synapses and perform synaptic information processing. Glial cells, particularly astrocytes, are active components of the brain and are responsible for synaptic activity through the release gliotransmitters. A reduced density of astrocytes and astrocyte dysfunction have both been identified the brains of patients with MDD. Furthermore, gliotransmission, i.e., active information transfer mediated by gliotransmitters between astrocytes and neurons, is thought to be involved in the pathogenesis of MDD. However, the mechanism by which astrocyte-mediated gliotransmission contributes to depression remains unknown. This review therefore summarizes the alterations in astrocytes in MDD, including astrocyte marker, connexin 43 (Cx43) expression, Cx43 gap junctions, and Cx43 hemichannels, and describes the regulatory mechanisms of astrocytes involved in synaptic plasticity. Additionally, we investigate the mechanisms acting of the glutamatergic, gamma-aminobutyric acidergic, and purinergic systems that modulate synaptic function and the antidepressant mechanisms of the related receptor antagonists. Further, we summarize the roles of glutamate, gamma-aminobutyric acid, d-serine, and adenosine triphosphate in depression, providing a basis for the identification of diagnostic and therapeutic targets for MDD.

Autism-associated CHD8 controls reactive gliosis and neuroinflammation via remodeling chromatin in astrocytes.

Reactive changes of glial cells during neuroinflammation impact brain disorders and disease progression. Elucidating the mechanisms that control reactive gliosis may help us to understand brain pathophysiology and improve outcomes. Here, we report that adult ablation of autism spectrum disorder (ASD)-associated CHD8 in astrocytes attenuates reactive gliosis via remodeling chromatin accessibility, changing gene expression. Conditional Chd8 deletion in astrocytes, but not microglia, suppresses reactive gliosis by impeding astrocyte proliferation and morphological elaboration. Astrocyte Chd8 ablation alleviates lipopolysaccharide-induced neuroinflammation and septic-associated hypothermia in mice. Astrocytic CHD8 plays an important role in neuroinflammation by altering the chromatin landscape, regulating metabolic and lipid-associated pathways, and astrocyte-microglia crosstalk. Moreover, we show that reactive gliosis can be directly mitigated in vivo using an adeno-associated virus (AAV)-mediated Chd8 gene editing strategy. These findings uncover a role of ASD-associated CHD8 in the adult brain, which may warrant future exploration of targeting chromatin remodelers in reactive gliosis and neuroinflammation in injury and neurological diseases.

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.

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.

Targeting microglial GLP1R in epilepsy: A novel approach to modulate neuroinflammation and neuronal apoptosis.

BACKGROUND: Epilepsy is a prevalent disorder of the central nervous system. Approximately, one-third of patients show resistance to pharmacological interventions. The pathogenesis of epilepsy is complex, and neuronal apoptosis plays a critical role. Aberrantly reactive astrocytes, induced by cytokine release from activated microglia, may lead to neuronal apoptosis. This study investigated the role of glucagon-like peptide 1 receptor (GLP1R) in microglial activation in epilepsy and its impact on astrocyte-mediated neurotoxicity. METHODS: We used human hippocampal tissue from patients with temporal lobe epilepsy and a pilocarpine-induced epileptic mouse model to assess neurobiological changes in epilepsy. BV2 microglial cells and primary astrocytes were used to evaluate cytokine release and astrocyte activation in vitro. The involvement of GLP1R was explored using the GLP1R agonist, Exendin-4 (Ex-4). RESULTS: Our findings indicated that reduced GLP1R expression in hippocampal microglia in both epileptic mouse models and human patients, correlated with increased cytokine release and astrocyte activation. Ex-4 treatment restored microglial homeostasis, decreased cytokine secretion, and reduced astrocyte activation, particularly of the A1 phenotype. These changes were associated with a reduction in neuronal apoptosis. In addition, Ex-4 treatment significantly decreased the frequency and duration of seizures in epileptic mice. CONCLUSIONS: This study highlights the crucial role of microglial GLP1R in epilepsy pathophysiology. GLP1R downregulation contributes to microglial- and astrocyte-mediated neurotoxicity, exacerbating neuronal death and seizures. Activation of GLP1R with Ex-4 has emerged as a promising therapeutic strategy to reduce neuroinflammation, protect neuronal cells, and control seizures in epilepsy. This study provides a foundation for developing novel antiepileptic therapies targeting microglial GLP1R, with the potential to improve outcomes in patients with epilepsy.

A2 reactive astrocyte-derived exosomes alleviate cerebral ischemia-reperfusion injury by delivering miR-628.

Ischemia and hypoxia activate astrocytes into reactive types A1 and A2, which play roles in damage and protection, respectively. However, the function and mechanism of A1 and A2 astrocyte exosomes are unknown. After astrocyte exosomes were injected into the lateral ventricle, infarct volume, damage to the blood-brain barrier (BBB), apoptosis and the expression of microglia-related proteins were measured. The dual luciferase reporter assay was used to detect the target genes of miR-628, and overexpressing A2-Exos overexpressed and knocked down miR-628 were constructed. qRT-PCR, western blotting and immunofluorescence staining were subsequently performed. A2-Exos obviously reduced the infarct volume, damage to the BBB and apoptosis and promoted M2 microglial polarization. RT-PCR showed that miR-628 was highly expressed in A2-Exos. Dual luciferase reporter assays revealed that NLRP3, S1PR3 and IRF5 are target genes of miR-628. After miR-628 was overexpressed or knocked down, the protective effects of A2-Exos increased or decreased, respectively. A2-Exos reduced pyroptosis and BBB damage and promoted M2 microglial polarization through the inhibition of NLRP3, S1PR3 and IRF5 via the delivery of miR-628. This study explored the mechanism of action of A2-Exos and provided new therapeutic targets and concepts for treating cerebral ischemia.

Astrocytic pyruvate dehydrogenase kinase-lactic acid axis involvement in glia-neuron crosstalk contributes to morphine-induced hyperalgesia in mice.

The activation of spinal astrocytes accounts for opioid-induced hyperalgesia (OIH), but the underlying mechanisms remain elusive. The presence of astrocyte-neuron lactate shuttle (ANLS) makes astrocytes necessary for some neural function and communication. The aim of this study was to explore the role of ANLS in the occurrence and maintenance of OIH. After 7 days consecutive morphine injection, a mice OIH model was established and astrocytic pyruvate dehydrogenase kinase 4 (PDK4), phosphorylated pyruvate dehydrogenase (p-PDH) and accumulation of L-lactate was elevated in the spinal dorsal horn. Intrathecally administration of inhibitors of PDK, lactate dehydrogenase 5 and monocarboxylate transporters to decrease the supply of L-lactate on neurons was observed to attenuate hypersensitivity behaviors induced by repeated morphine administration and downregulate the expression of markers of central sensitization in the spinal dorsal horns. The astrocyte line and the neuronal line were co-cultured to investigate the mechanisms in vitro. In this study, we demonstrated that morphine-induced hyperalgesia was sustained by lactate overload consequent upon aberrant function of spinal ANLS. In this process, PDK-p-PDH-lactate axis serves a pivotal role, which might therefore be a new target to improve long-term opioid treatment strategy in clinical practice.

Acetate enables metabolic fitness and cognitive performance during sleep disruption.

Sleep is essential for overall health, and its disruption is linked to increased risks of metabolic, cognitive, and cardiovascular dysfunctions; however, the molecular mechanisms remain poorly understood. This study investigated how sleep disturbances contribute to metabolic imbalance and cognition impairment using a chronic sleep fragmentation (SF) mouse model. SF mice exhibited impaired cognition, glucose metabolism, and insulin sensitivity compared with controls. We identified increased acetate levels in hypothalamic astrocytes as a defensive response in SF mice. Through acetate infusion or astrocyte-specific Acss1 deletion to elevate acetate levels, we observed mitigated metabolic and cognitive impairments in SF mice. Mechanistically, acetate binds and activates pyruvate carboxylase, thereby restoring glycolysis and the tricarboxylic acid cycle. Among individuals most commonly affected by SF, patients with obstructive sleep apnea exhibited elevated acetate levels when coupled with type 2 diabetes. Our study uncovers the protective effect of acetate against sleep-induced metabolic and cognitive impairments.

Astrocyte Gi-GPCR signaling corrects compulsive-like grooming and anxiety-related behaviors in Sapap3 knockout mice.

Astrocytes are morphologically complex cells that serve essential roles. They are widely implicated in central nervous system (CNS) disorders, with changes in astrocyte morphology and gene expression accompanying disease. In the Sapap3 knockout (KO) mouse model of compulsive and anxiety-related behaviors related to obsessive-compulsive disorder (OCD), striatal astrocytes display reduced morphology and altered actin cytoskeleton and Gi-G-protein-coupled receptor (Gi-GPCR) signaling proteins. Here, we show that normalizing striatal astrocyte morphology, actin cytoskeleton, and essential homeostatic support functions by targeting the astrocyte Gi-GPCR pathway using chemogenetics corrected phenotypes in Sapap3 KO mice, including anxiety-related and compulsive behaviors. Our data portend an astrocytic pharmacological strategy for rescuing phenotypes in brain disorders that include compromised astrocyte morphology and tissue support.

Endothelin-1 increases Na+-K+-2Cl- cotransporter-1 expression in cultured astrocytes and in traumatic brain injury model: An involvement of HIF1α activation.

Na+-K+-2Cl- cotransporter-1 (NKCC1) is present in brain cells, including astrocytes. The expression of astrocytic NKCC1 increases in the acute phase of traumatic brain injury (TBI), which induces brain edema. Endothelin-1 (ET-1) is a factor that induces brain edema and regulates the expression of several pathology-related genes in astrocytes. In the present study, we investigated the effect of ET-1 on NKCC1 expression in astrocytes. ET-1 (100 nM)-treated cultured astrocytes showed increased NKCC1 mRNA and protein levels. The effect of ET-1 on NKCC1 expression in cultured astrocytes was reduced by BQ788 (1 µM), an ETB antagonist, but not by FR139317 (1 µM), an ETA antagonist. The involvement of ET-1 in NKCC1 expression in TBI was examined using a fluid percussion injury (FPI) mouse model that replicates the pathology of TBI with high reproducibility. Administration of BQ788 (15 nmol/day) decreased FPI-induced expressions of NKCC1 mRNA and protein, accompanied with a reduction of astrocytic activation. FPI-induced brain edema was attenuated by BQ788 and NKCC1 inhibitors (azosemide and bumetanide). ET-1-treated cultured astrocytes showed increased mRNA and protein expression of hypoxia-inducible factor-1α (HIF1α). Immunohistochemical observations of mouse cerebrum after FPI showed co-localization of HIF1α with GFAP-positive astrocytes. Increased HIF1α expression in the TBI model was reversed by BQ788. FM19G11 (an HIF inhibitor, 1 µM) and HIF1α siRNA suppressed ET-induced increase in NKCC1 expression in cultured astrocytes. These results indicate that ET-1 increases NKCC1 expression in astrocytes through the activation of HIF1α.

CREB-regulated transcription during glycogen synthesis in astrocytes.

Glycogen storage, conversion and utilization in astrocytes play an important role in brain energy metabolism. The conversion of glycogen to lactate through glycolysis occurs through the coordinated activities of various enzymes and inhibition of this process can impair different brain processes including formation of long-lasting memories. To replenish depleted glycogen stores, astrocytes undergo glycogen synthesis, a cellular process that has been shown to require transcription and translation during specific stimulation paradigms. However, the detail nuclear signaling mechanisms and transcriptional regulation during glycogen synthesis in astrocytes remains to be explored. In this report, we study the molecular mechanisms of vasoactive intestinal peptide (VIP)-induced glycogen synthesis in astrocytes. VIP is a potent neuropeptide that triggers glycogenolysis followed by glycogen synthesis in astrocytes. We show evidence that VIP-induced glycogen synthesis requires CREB-mediated transcription that is calcium dependent and requires conventional Protein Kinase C but not Protein Kinase A. In parallel to CREB activation, we demonstrate that VIP also triggers nuclear accumulation of the CREB coactivator CRTC2 in astrocytic nuclei. Transcriptome profiles of VIP-induced astrocytes identified robust CREB transcription, including a subset of genes linked to glucose and glycogen metabolism. Finally, we demonstrate that VIP-induced glycogen synthesis shares similar as well as distinct molecular signatures with glucose-induced glycogen synthesis, including the requirement of CREB-mediated transcription. Overall, our data demonstrates the importance of CREB-mediated transcription in astrocytes during stimulus-driven glycogenesis.

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.

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.

Induction of astrocyte reactivity promotes neurodegeneration in human pluripotent stem cell models.

Reactive astrocytes are known to exert detrimental effects upon neurons in several neurodegenerative diseases, yet our understanding of how astrocytes promote neurotoxicity remains incomplete, especially in human systems. In this study, we leveraged human pluripotent stem cell (hPSC) models to examine how reactivity alters astrocyte function and mediates neurodegeneration. hPSC-derived astrocytes were induced to a reactive phenotype, at which point they exhibited a hypertrophic profile and increased complement C3 expression. Functionally, reactive astrocytes displayed decreased intracellular calcium, elevated phagocytic capacity, and decreased contribution to the blood-brain barrier. Subsequently, co-culture of reactive astrocytes with a variety of neuronal cell types promoted morphological and functional alterations. Furthermore, when reactivity was induced in astrocytes from patient-specific hPSCs (glaucoma, Alzheimer's disease, and amyotrophic lateral sclerosis), the reactive state exacerbated astrocytic disease-associated phenotypes. These results demonstrate how reactive astrocytes modulate neurodegeneration, significantly contributing to our understanding of a role for reactive astrocytes in neurodegenerative diseases.

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.

Ghost cells: Wilder Penfield and the characterization of glia and glial pathology, 1924-1932.

Wilder Penfield is known for his contributions to the structure-function relationship of the brain and for the surgical treatment of focal epilepsy. Less well known are his contributions to the study of glial cells and his investigation of their role in human neuropathology. Penfield learned the gold and silver methods for staining neurons, glial cells, and their projections from Charles Sherrington and Pío del Río-Hortega. He and his colleague William Cone established a laboratory for the study of glial cells and human neuropathology using metallic stains, initially at the Presbyterian Hospital in New York City in 1925, and then at the Montreal Neurological Institute in 1928. Penfield, Cone, and their research fellows, building on the findings of Río-Hortega, confirmed the existence of oligodendrocytes and their relationship with myelin, and investigated the putative mesodermal origin of microglia. They discovered the reaction of oligodendrocytes to pathological stressors, and the phagocytic activity of microglia in human gliomas. In this article, we argue that Penfield's studies of astrocytes, oligodendrocytes, and microglia, and their responses to craniocerebral trauma, epilepsy, malignant brain tumors, and other pathologies of the central nervous system inaugurated a new era in clinical neurocytology and neuropathology.

Heterogeneous brain region-specific responses to astrocytic mitochondrial DNA damage in mice.

Astrocytes display context-specific diversity in their functions and respond to noxious stimuli between brain regions. Astrocytic mitochondria have emerged as key players in governing astrocytic functional heterogeneity, given their ability to dynamically adapt their morphology to regional demands on ATP generation and Ca2+ buffering functions. Although there is reciprocal regulation between mitochondrial dynamics and mitochondrial Ca2+ signaling in astrocytes, the extent of this regulation in astrocytes from different brain regions remains unexplored. Brain-wide, experimentally induced mitochondrial DNA (mtDNA) loss in astrocytes showed that mtDNA integrity is critical for astrocyte function, however, possible diverse responses to this noxious stimulus between brain areas were not reported in these experiments. To selectively damage mtDNA in astrocytes in a brain-region-specific manner, we developed a novel adeno-associated virus (AAV)-based tool, Mito-PstI expressing the restriction enzyme PstI, specifically in astrocytic mitochondria. Here, we applied Mito-PstI to two brain regions, the dorsolateral striatum and dentate gyrus, and we show that Mito-PstI induces astrocytic mtDNA loss in vivo, but with remarkable brain-region-dependent differences on mitochondrial dynamics, Ca2+ fluxes, and astrocytic and microglial reactivity. Thus, AAV-Mito-PstI is a novel tool to explore the relationship between astrocytic mitochondrial network dynamics and astrocytic mitochondrial Ca2+ signaling in a brain-region-selective manner.

Impacts of exposure to and subsequent discontinuation of clozapine on tripartite synaptic transmission.

BACKGROUND AND PURPOSE: Clozapine is an effective antipsychotic for treatment-resistant schizophrenia, but its discontinuation leads to discontinuation syndrome/catatonia complicated by benzodiazepine-resistance and rhabdomyolysis. EXPERIMENTAL APPROACH: This study determined time-dependent effects of exposure and subsequent discontinuation of clozapine on expression of connexin43, 5-HT receptors, intracellular L-ß-aminoisobutyrate (L-BAIBA) and 2nd-messengers and signalling of AMPK, PP2A and Akt in cultured astrocytes and rat frontal cortex. KEY RESULTS: Intracellular L-BAIBA levels increased during clozapine exposure but immediately recovered after discontinuation. Both exposure to clozapine and L-BAIBA increased connexin43 and signalling of AMPK/Akt time-dependently, but reduced PP2A signalling, 5-HT receptor expression and IP3 level. These changes recovered within 2 weeks after discontinuation, while 5-HT receptors and IP3 transiently increased during the recovery process. L-BAIBA activated AMPK signalling, leading to attenuated PP2A signalling. Astroglial D-serine release was increased by clozapine exposure but continued to increase within 1 week after discontinuation via activation of IP3 receptor function. CONCLUSION AND IMPLICATIONS: Clozapine discontinuation restored PP2A signalling due to decreased L-BAIBA, increased 5-HT receptor expression via probably enhanced 5-HT receptor recycling, but increased astroglial D-serine release persisted by transiently activated IP3 receptors via transiently increased IP3 level. Decreased L-BAIBA caused by clozapine discontinuation is, at least partially, involved in the transiently increased 5-HT receptor and astroglial D-serine release.