Astrocytic Response Research Articles

Ca2+ excitability of glia to neuromodulator octopamine in Drosophila living brain is greater than that of neurons.

Octopamine in the Drosophila brain has a neuromodulatory role similar to that of noradrenaline in mammals. After release from Tdc2 neurons, octopamine/tyramine may trigger intracellular Ca2+ signaling via adrenoceptor-like receptors on neural cells, modulating neurotransmission. Octopamine/tyramine receptors are expressed in neurons and glia, but how each of these cell types responds to octopamine remains elusive. This study aimed to characterize Ca2+ responses of neurons and astrocytes to neuromodulatory octopamine signals. We expressed Ca2+ indicator jGCaMP7b in specific cell type in adult Drosophila brains and performed intracellular Ca2+ imaging in the brain optic lobes upon bath application of octopamine by confocal microscopy. Octopamine-stimulated Ca2+ responses in neurons were different from those of glial cells. The amplitude of octopamine-mediated Ca2+ signals in neurons was 3.4-fold greater than in astrocytes. However, astrocytes were more sensitive to octopamine; the median effective concentration that triggered Ca2+ responses was nearly 6-fold lower in astrocytes than in neurons. In both cell types, Ca2+ transients are shaped by Gq and Gs protein-coupled octopamine/tyramine receptors. Our snRNA-seq database screening uncovered differential expression patterns of these receptors between brain cell types, which may explain the difference in Ca2+ signaling. In the brain optic lobes, astrocytes, not neurons, appear to be the sole responders to low concentration octopamine signals, and therefore likely drive synaptic plasticity and visual processing. Given the interconnectivity of the optic lobes with other brain regions, octopaminergic signals acting through the optic lobe astrocytes may also influence higher-order brain functions including learning and memory.

Astragaloside IV Relieves Central Sensitization by Regulating Astrocytic ROS/NF-κB Nuclear Translocation Signaling in Chronic Migraine Male Rats.

Chronic migraine (CM) is a disabling neurological disease. Astragaloside IV (AS-IV), a natural product derived from Astragalus membranaceus, shows great potential in treating chronic pain by relieving inflammation and oxidative stress. This study aimed to investigate the effects and mechanisms of action of AS-IV on CM. An inflammatory soup comprising histamine, bradykinin, serotonin, and prostaglandin E2 was used to establish a CM rat model, while lipopolysaccharide was applied to induce an inflammatory response in primary astrocytes. Pain threshold measurements were used to evaluate nociceptive hypersensitivity, while qPCR and Western blotting were applied to detect inflammatory indicators and synaptic protein expression, and Golgi-Cox staining was applied to observe dendritic spine density, while transmission electron microscopy was used to observe synaptic ultrastructure. Mitochondrial function and oxidative stress were assessed using JC-1 staining, Mitotracker staining, reactive oxygen species (ROS) quantification, and glutathione content. AS-IV pretreatment alleviated central sensitization and ameliorated astrocyte activation and neuroinflammation. AS-IV pretreatment alleviated mitochondrial dysfunction invitro, and reduced the nuclear translocation of NF-κB and the production of IL-1β, which were reversed by ROS scavengers invitro or mitochondrial respiratory chain disruptors invivo. Our study indicates that AS-IV can inhibit neuroinflammation by alleviating astrocyte mitochondrial dysfunction to mitigate central sensitization in CM, thereby providing an experimental basis for AS-IV and A. membranaceus in CM prevention and treatment.

Characterization of the Astrocyte Calcium Response to Norepinephrine in the Ventral Tegmental Area.

Astrocytes from different brain regions respond with Ca2+ elevations to the catecholamine norepinephrine (NE). However, whether this noradrenergic-mediated signaling is present in astrocytes from the ventral tegmental area (VTA), a dopaminergic circuit receiving noradrenergic inputs, has not yet been investigated. To fill in this gap, we applied a pharmacological approach along with two-photon microscopy and an AAV strategy to express a genetically encoded calcium indicator in VTA astrocytes. We found that VTA astrocytes from both female and male young adult mice showed a strong Ca2+ response to NE at both soma and processes. Our results revealed that Gq-coupled α1 adrenergic receptors, which elicit the production of IP3, are the main mediators of the astrocyte response. In mice lacking the IP3 receptor type-2 (IP3R2-/- mice), we found that the astrocyte response to NE, even if reduced, is still present. We also found that in IP3R2-/- astrocytes, the residual Ca2+ elevations elicited by NE depend on the release of Ca2+ from the endoplasmic reticulum, through IP3Rs different from IP3R2. In conclusion, our results reveal VTA astrocytes as novel targets of the noradrenergic signaling, opening to new interpretations of the cellular and molecular mechanisms that mediate the NE effects in the VTA.

Long-term impact of congenital Zika virus infection on the rat hippocampus: Neuroinflammatory, glial alterations and sex-specific effects.

Congenital Zika Syndrome (CZS) is a condition that arises when a neonate presents with abnormalities resulting from Zika virus infection during gestation. While microcephaly is a prominent feature of the syndrome, other forms of brain damage are also observed, often accompanied by significant neurological complications. It is therefore essential to investigate the long-term effects of CZS, with special attention to sex differences, particularly concerning hippocampal function, given its vulnerability to viral infections. The aim of this study was to evaluate the long-term impacts on cognitive and memory functions, as well as neuroinflammatory and glial alterations in the hippocampus, in offspring of both sexes exposed to a model of congenital Zika virus infection. Pregnant rats were subcutaneously inoculated with ZIKV-BR at a dose of 1 × 10^7 plaque-forming units (PFU mL^-1) of ZIKV isolated in Brazil (ZIKV-BR) on gestational day 18 (G18). From postnatal day 70, the animals underwent behavioral tests. On postnatal day 80, the animals were euthanized, and hippocampal samples were collected for biochemical and histological analyses. In the open field test, females displayed more exploratory behavior and less grooming, while no significant differences in locomotion were observed between the sexes. Additionally, ZIKV-exposed females showed a reduction in grooming behavior compared to ZIKV-exposed males. In the memory test, males in the ZIKV group exhibited greater memory impairment, spending more time to locate the correct quadrant, while females showed relatively better performance. Neuroinflammatory markers, such as TNF-α, were significantly elevated in the hippocampus of ZIKV-exposed animals, regardless of sex. However, microglial and astrocytic responses, indicated by higher IBA1 and GFAP density, were only observed in male ZIKV rats. In conclusion, our findings suggest that congenital ZIKV exposure leads to sex-specific behavioral and neuroinflammatory alterations. While both males and females exhibited some behavioral changes, males were more significantly impacted in memory performance. Additionally, increased neuroinflammatory markers and glial activation were observed in the hippocampus of ZIKV-exposed animals, with a pronounced response in males. These results highlight the long-term impact of ZIKV infection on neurodevelopment, emphasizing the importance of considering sex differences in studies of congenital ZIKV syndrome.

A spatial threshold for astrocyte calcium surge

Astrocytes are active cells involved in brain function through the bidirectional communication with neurons, in which astrocyte calcium plays a crucial role. Synaptically evoked calcium increases can be localized to independent subcellular domains or expand to the entire cell, i.e., calcium surge. Because a single astrocyte may contact ~100,000 synapses, the control of the intracellular calcium signal propagation may have relevant consequences on brain function. Yet, the properties governing the spatial dynamics of astrocyte calcium remains poorly defined. Imaging subcellular responses of cortical astrocytes to sensory stimulation in mice, we show that sensory-evoked astrocyte calcium responses originated and remained localized in domains of the astrocytic arborization, but eventually propagated to the entire cell if a spatial threshold of >23% of the arborization being activated was surpassed. Using Itpr2-/- mice, we found that type-2 IP3 receptors were necessary for the generation of astrocyte calcium surge. We finally show using in situ electrophysiological recordings that the spatial threshold of the astrocyte calcium signal consequently determined the gliotransmitter release. Present results reveal a fundamental property of astrocyte physiology, i.e., a spatial threshold for astrocyte calcium propagation, which depends on astrocyte intrinsic properties and governs astrocyte integration of local synaptic activity and subsequent neuromodulation.

Pharmacological Inhibition of Astrocytic Transglutaminase 2 Facilitates the Expression of a Neurosupportive Astrocyte Reactive Phenotype in Association with Increased Histone Acetylation.

Astrocytes play critical roles in supporting structural and metabolic homeostasis in the central nervous system (CNS). CNS injury leads to the development of a range of reactive phenotypes in astrocytes whose molecular determinants are poorly understood. Finding ways to modulate astrocytic injury responses and leverage a pro-recovery phenotype holds promise in treating CNS injury. Recently, it has been demonstrated that ablation of astrocytic transglutaminase 2 (TG2) shifts reactive astrocytes towards a phenotype that improves neuronal injury outcomes both in vitro and in vivo. Additionally, in an in vivo mouse model, pharmacological inhibition of TG2 with the irreversible inhibitor VA4 phenocopied the neurosupportive effects of TG2 deletion in astrocytes. In this study, we extended our comparisons of VA4 treatment and TG2 deletion to provide insights into the mechanisms by which TG2 attenuates neurosupportive astrocytic function after injury. Using a neuron-astrocyte co-culture model, we found that VA4 treatment improves the ability of astrocytes to support neurite outgrowth on an injury-relevant matrix, as we previously showed for astrocytic TG2 deletion. We hypothesize that TG2 mediates its influence on astrocytic phenotype through transcriptional regulation, and our previous RNA sequencing suggests that TG2 is primarily transcriptionally repressive in astrocytes, although it can facilitate both up- and downregulation of gene expression. Therefore, we asked whether VA4 inhibition could alter TG2's interaction with Zbtb7a, a transcription factor that we previously identified as a functionally relevant TG2 nuclear interactor. We found that VA4 significantly decreased the interaction of TG2 and Zbtb7a. Additionally, we assessed the effect of TG2 deletion and VA4 treatment on transcriptionally permissive histone acetylation and found significantly greater acetylation in both experimental groups. Consistent with these findings, our present proteomic analysis further supports the predominant transcriptionally repressive role of TG2 in astrocytes. Our proteomic data additionally unveiled pronounced changes in lipid and antioxidant metabolism in astrocytes with TG2 deletion or inhibition, which likely contribute to the enhanced neurosupportive function of these astrocytes.

Single-nucleus RNA sequencing of human periventricular white matter in vascular dementia.

Vascular dementia (VaD) refers to a variety of dementias driven by cerebrovascular disease and is the second leading cause of dementia globally. VaD may be caused by ischemic strokes, intracerebral hemorrhage, and/or cerebral small vessel disease, commonly identified as white matter hyperintensities on MRI. The mechanisms underlying these white matter lesions in the periventricular brain are poorly understood. In this study we perform an extensive transcriptomic analysis on human postmortem periventricular white matter lesions in patients with VaD with the goal of identifying molecular pathways in the disease. We find increased cellular stress responses in astrocytes, oligodendrocytes, and oligodendrocyte precursor cells as well as transcriptional and translational repression in microglia in our dataset. We show that several genes identified by GWAS as being associated with white matter disease are differentially expressed in cells in VaD. Finally, we compare our dataset to an independent snRNAseq dataset of PVWM in VaD and a scRNAseq dataset on human iPSC-derived microglia exposed to oxygen glucose deprivation (OGD). We identify the increase of the heat shock protein response as a conserved feature of VaD across celltypes and show that this increase is not linked to OGD exposure. Overall, our study is the first to show that increased heat shock protein responses are a common feature of lesioned PVWM in VaD and may represent a potential therapeutic target.

Extracellular vesicles from fifth-stage larval Angiostrongylus cantonensis upregulate cholesterol biosynthesis and suppress NLRP2-associated inflammatory responses in mouse astrocytes.

Angiostrongylus cantonensis is a zoonotic parasite that causes severe symptoms in humans, including eosinophilic meningitis and eosinophilic meningoencephalitis. Extracellular vesicles (EVs) derived from helminthes have been implicated in regulating host survival and immune response. However, the roles of A. cantonensis EVs in modulating parasite pathogenesis and host immune response remain poorly understood. Herein, we characterized EVs derived from A. cantonensis fifth-stage larvae (L5) and adult worms. Ultrastructural features showed that EVs from adult worms are smaller in size compared with those from L5. Proteomic analysis identified stage-specific proteins packaged in L5 and adult worm EVs. To investigate the crosstalk between L5 EVs and host cells, RNA sequencing analysis was conducted to identify the differentially expressed genes (DEGs) and enriched biological pathways in mouse astrocytes treated with L5 EVs. GO and KEGG enrichment analysis demonstrated that the pathways related to "cholesterol biosynthesis" are significantly upregulated in L5 EV-treated astrocytes. Based on the transcriptomic data, we observed a downregulated trend of NOD-like receptors (NLRs) protein 2 (NLRP2), a key regulator of brain inflammation, in mouse astrocytes treated with L5 EVs. To validate this result, we utilized ATP to induce the expression of NLRP2 inflammasome-related genes and proteins, as well as the secretion of downstream cytokines. Notably, ATP-induced overexpression of NLRP2 inflammasome-related molecules was significantly reduced in mouse astrocytes upon L5 EV treatment. Collectively, our data suggest that A. cantonensis L5 EVs enhance cholesterol synthesis and potentially modulate immune response by reducing NLRP2 inflammasome-related signaling in non-permissive host cells.IMPORTANCEAngiostrongylus cantonensis is a significant causative agent of eosinophilic meningitis and eosinophilic meningoencephalitis in humans. Helminth-derived extracellular vesicles (EVs) are known to play a crucial role in parasite pathogenesis and host immunomodulation. However, the protein compositions of A. cantonensis EVs and their roles in parasite pathogenesis and host immune response remain unclear. Our results demonstrate for the first time the distinct protein compositions in A. cantonensis L5 and adult worm EVs. The highly abundant proteins in L5 EVs that have immunomodulatory or pathogenic potential in the host deserve further investigation. Additionally, the uptake of L5 EVs by mouse astrocytes significantly upregulates cholesterol synthesis and suppresses ATP-induced NLRP2 inflammasome-related signaling. This study highlights the immunomodulatory roles of L5 EVs in non-permissive hosts, suggesting their potential as therapeutic targets and vaccine candidates against A. cantonensis.

Monosodium Glutamate Treatment Elevates the Immunoreactivity of GFAP and S100β in Caudate Nucleus of the Striatum in Rats.

Fifteen rats were divided into a control group receiving saline and MSG2 and MSG4 groups receiving 2 g/kg b.w. MSG and 4 g/kg b.w. MSG, respectively, for 3 days. An immunohistochemical reaction was conducted on frontal sections containing the caudate nucleus with use of antibodies against GFAP and S100β. Analyses indicated elevated density of astrocytes immunoreactive for the studied proteins in the caudate nucleus in animals receiving MSG. The studied glial cells also demonstrated increased immunostaining intensity for both GFAP and S100β immunoreactive cells especially in the MSG4 group. The number of GFAP-positive processes in astrocytes was similar in all studied groups. The studies demonstrate a potential response of astrocytes to the effect of MSG administration in the caudate nucleus. It was shown that GFAP- and S100β-positive astrocytes in the caudate nucleus may act differently, suggesting distinct roles of these proteins against glutamate excitotoxicity.

Acute astrocytic reaction is associated with 3-month functional outcome after stroke treated with endovascular therapy.

More than 50% of large vessel occlusion (LVO) acute ischemic stroke (AIS) patients treated with endovascular therapy (EVT) remain severely disabled at 3 months. We hypothesized that acute astrocytic inflammatory response may play a pivotal role in post-AIS brain changes associated with poor functional outcome. We proposed to evaluate the level of YKL-40, a glycoprotein mainly released by reactive astrocytes. A monocentric prospective cohort study was conducted on consecutive LVO AIS patients treated with EVT. Three blood samples (before, within 1 and 24-hour post-EVT) were collected to measure plasma YKL-40 concentrations. Functional outcome was assessed according to the modified Rankin Scale (mRS) score at 3 months. Between 2016 and 2020, 120 patients were included. The plasma concentration of YKL-40 before EVT was statistically and independently associated with 3-month worse functional outcome (adjusted cOR, 1.59; 95% CI [1.05-2.44], p = 0.027) but not the two following samples 1-hour and 24-hour post-EVT. Accordingly, we found that excellent functional outcome was associated with a lower level of YKL-40 before and within 1 h after EVT (p = 0.005 and p = 0.003, respectively) but not when measured 24 h after EVT (p = 0.2). This study suggests that the astrocytic reaction to acute brain hypoxia, especially before recanalization, is associated with worse functional outcome. Such early biomarker of the astrocytic response in AIS may optimize individualized care in the future. http://www.clinicaltrials.gov. Unique identifier: NCT02900833.

Human VCP mutant ALS/FTD microglia display immune and lysosomal phenotypes independently of GPNMB

BackgroundMicroglia play crucial roles in maintaining neuronal homeostasis but have been implicated in contributing to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). However, the role of microglia in ALS/FTD remains incompletely understood.MethodsHere, we generated highly enriched cultures of VCP mutant microglia derived from human induced pluripotent stem cells (hiPSCs) to investigate their cell autonomous and non-cell autonomous roles in ALS pathogenesis. We used RNA-sequencing, proteomics and functional assays to study hiPSC derived VCP mutant microglia and their effects on hiPSC derived motor neurons and astrocytes.ResultsTranscriptomic, proteomic and functional analyses revealed immune and lysosomal dysfunction in VCP mutant microglia. Stimulating healthy microglia with the inflammatory inducer lipopolysaccharide (LPS) showed partial overlap with VCP mutant microglia in their reactive transformation. LPS-stimulated VCP mutant microglia displayed differential activation of inflammatory pathways compared with LPS-stimulated healthy microglia. Conserved gene expression changes were identified between VCP mutant microglia, SOD1 mutant mice microglia, and postmortem ALS spinal cord microglial signatures, including increased expression of the transmembrane glycoprotein GPNMB. While knockdown of GPNMB affected inflammatory and phagocytosis processes in microglia, this was not sufficient to ameliorate cell autonomous phenotypes in VCP mutant microglia. Secreted factors from VCP mutant microglia were sufficient to activate the JAK-STAT pathway in hiPSC derived motor neurons and astrocytes.ConclusionsVCP mutant microglia undergo cell autonomous reactive transformation involving immune and lysosomal dysfunction that partially recapitulate key phenotypes of microglia from other ALS models and post mortem tissue. These phenotypes occur independently of GPNMB. Additionally, VCP mutant microglia elicit non cell autonomous responses in motor neurons and astrocytes involving the JAK-STAT pathway.

Anti-inflammatory effects of quercetin in lipopolysaccharide-activated cortical astrocytes

Dysregulated neuroinflammation is a hallmark of multiple neurodegenerative diseases, characterized by increased pro-inflammatory cytokines, and reduced anti-inflammatory cytokines, and is mediated by resident astrocytes. Previous studies have demonstrated the neuroprotective effects of quercetin hydrate, a plant flavonoid, however, limited studies exist showing the targets of quercetin in astrocytes. Here, we demonstrate the anti-inflammatory effects of quercetin hydrate at the gene expression level and the involvement of signalling pathway molecules. The rat primary cortical astrocytes were preincubated with different concentrations of quercetin (25–200 μM) followed by lipopolysaccharide (LPS) stimulation. Semi-quantitative PCR analysis showed the therapeutic potential of quercetin by decreasing the gene expression of pro-inflammatory cytokines (IL-1β, IL-6), and Cyclooxygenase-2, and increasing Hemeoxygenase-1 in LPS-stimulated astrocytes. By Immunoblotting analysis, we document the modulatory effects of quercetin on phospho p38 and phospho ERK1/2 in LPS-stimulated astrocytes. The study indicates the anti-inflammatory effects of quercetin against inflammatory responses in astrocytes.

Comparison of Drug Delivery Systems with Different Types of Nanoparticles in Terms of Cellular Uptake and Responses in Human Endothelial Cells, Pericytes, and Astrocytes.

Background/Objectives: The key components of the blood-brain barrier (BBB) are endothelial cells, pericytes, astrocytes, and the capillary basement membrane. The BBB serves as the main barrier for drug delivery to the brain and is the most restrictive endothelial barrier in the body. Nearly all large therapeutic molecules and over 90% of small-molecule drugs cannot cross the BBB. To overcome this challenge, nanotechnology, particularly drug delivery systems such as nanoparticles (NPs), have gained significant attention. Methods: Poly(lactide-co-glycolide) (PLGA) and albumin-based NPs (bovine/human), with or without transferrin (Tf) ligands (BSA, HSA, BSA-Tf, HSA-Tf), and nanolipid carriers (NLC) were synthesized. The interactions of these NPs with human brain microvascular endothelial cells (hBMECs), human brain vascular pericytes (hBVPs), and human astrocytes (hASTROs) were analyzed. Results: At doses of 15.62 µg/mL, 31.25 µg/mL, and 62.5 µg/mL, none of the NPs caused toxic effects on hBMECs, hBVPs, or hASTROs after 3 h of incubation. All NPs were internalized by the cells, but BSA-Tf and HSA-Tf showed significantly higher uptake in hBMECs in a dose-dependent manner. Ultrastructural analysis revealed notable differences between NP formulation and cell type. Conclusions: Our findings underscore the potential of ligand-targeted NPs to selectively interact with BBB endothelial cells. Ultrastructural analysis reveals distinct cellular processing pathways for various NP formulations across BBB-associated cell types, with autophagy emerging as a crucial mechanism for NP handling in pericytes and astrocytes. Changes in NP chemical properties upon biological exposure present significant challenges for nanomedicine design, emphasizing the need for further investigation into NP interactions at the cellular and subcellular levels.

Astrocyte Dysfunction Reflected in Ischemia-Induced Astrocyte-Derived Extracellular Vesicles: A Pilot Study on Acute Ischemic Stroke Patients.

Extracellular vesicles (EVs) secreted by astrocytes (ADEVs) mediate numerous biological processes, providing insights into damage, repair, and protection following ischemic stroke (IS). This pilot study aimed to broaden the current knowledge on the astrocyte response to ischemia by dynamically assessing the aquaporin-4 (AQP4) and glial cell line-derived neurotrophic factor (GDNF) as cargo proteins of these vesicles in eighteen acute IS patients and nine controls. EV proteins were detected by Western blotting and followed 24 h (D1), 7 days (D7), and one month (M1) after symptoms onset. The post-ischemic ADEV AQP4 and GDNF levels were higher at D1 compared to the control group (p = 0.006 and p = 0.023). Significant differences were observed in ADEV AQP4 during the three evaluated time points (n = 12, p = 0.013) and between D1 and D7 (z = 2.858, p = 0.012), but not in EV GDNF. There was a positive relationship between the severity of stroke at D1 according to the National Institutes of Health Stroke Scale, and ADEV AQP4 at D1 (r = 0.50, p = 0.031), as well as ADEV GDNF at D1 and D7 (r = 0.49, p = 0.035 and r = 0.53, p = 0.021, respectively). The release of EVs with distinct protein profiles can be an attractive platform for the development of biomarkers in IS.

The Mechanisms of Neuroprotection by Topical Rho Kinase Inhibition in Experimental Mouse Glaucoma and Optic Neuropathy.

The purpose of this study was to delineate the neuroprotective mechanisms of topical 2% ripasudil (Rip), a Rho kinase (ROCK) inhibitor. In 340 mice, scheduled 2% Rip or balanced salt solution (BSS) saline drops were intermittently, unilaterally delivered. Intracameral microbead glaucoma (GL) injection increased intraocular pressure (IOP) from 1day to 6 weeks (6W), whereas other mice underwent optic nerve (ON) crush. Retinal ganglion cell (RGC) loss was assessed using retinal wholemount anti-RNA Binding Protein with Multiple Splicing (RBPMS) labeling and ON axon counts. Axonal transport was quantified with β-amyloid precursor protein (APP) immunolocalization. Micro-Western (Wes) analysis quantified protein expression. Immunofluorescent expression of ROCK pathway molecules, quantitative astrocyte structural changes, and ON biomechanical strains (explanted eyes) were evaluated. ROCK activity assays were conducted in separate ON regions. At 6W GL, mean RGC axon loss was 6.6 ± 13.3% in Rip and 36.3 ± 30.9% in BSS (P = 0.04, n = 10/group). RGC soma loss after crush was lower with Rip (68.6 ± 8.2%) than BSS (80.5 ± 5.7%, P = 0.006, n = 10/group). After 6W GL, RGC soma loss was lower with Rip (34 ± 5.0%) than BSS (51 ± 8.1%, P = 0.03, n = 10/group). Axonal transport of APP within the unmyelinated ON (UON) was unaffected by Rip. Maximum principal mechanical strains increased similarly in Rip and BSS-treated mice. Retinal ROCK 1 and 2 activity was reduced by Rip in GL eyes. The pROCK2/ROCK2 protein ratio rose in the retina of BSS GL eyes, but not in Rip GL eyes. Topical Rip reduced RGC loss in GL and ON crush, with suppression of ROCK signaling in the retina and ON. The neuroprotection mechanisms appear to involve effects on both RGC and astrocyte responses to IOP elevation.

R-Spondin 1 Suppresses Inflammatory Cytokine Production in Human Cortical Astrocytes

Background/Objectives: Wnt signaling pathways are essential in various biological processes, including embryonic development and tissue homeostasis, and are implicated in many diseases. The R-Spondin (RSpo) family, particularly RSpo1, plays a significant role in modulating Wnt signaling. This study aims to explore how RSpo1 binding to astrocytic LGR6 receptors influences central nervous system (CNS) homeostasis, particularly in the context of inflammation. Methods: Human-induced pluripotent stem cell-derived astrocytes were treated with RSpo1 to assess its impact on inflammatory cytokine release. A proteomic analysis was conducted using a Human Cytokine Array Kit to measure differential protein expression. Pathway enrichment analysis was performed to identify affected signaling pathways. Results: RSpo1 treatment led to a suppression of inflammatory cytokines such as IL-10, IFN-γ, and IL-23 in astrocytes, while TNF-α and CXCL12 levels were increased. Pathway analysis revealed significant alterations in key signaling pathways, including cytokine–cytokine receptor interaction, chemokine signaling, and TNF signaling pathways, suggesting RSpo1’s role in modulating immune responses within the CNS. Conclusions: RSpo1 significantly influences inflammatory responses in astrocytes by modulating cytokine release and altering key signaling pathways. These findings enhance our understanding of the interaction between cell-specific Wnt signaling and CNS inflammation, suggesting potential therapeutic applications of RSpo1 in neuroinflammatory and neurodegenerative diseases.

Pharmacological inhibition of astrocytic transglutaminase 2 facilitates the expression of a neurosupportive astrocyte reactive phenotype in association with increased histone acetylation.

Astrocytes play critical roles in supporting structural and metabolic homeostasis in the central nervous system (CNS). CNS injury leads to the development of a range of reactive phenotypes in astrocytes whose molecular determinants are poorly understood. Finding ways to modulate astrocytic injury responses and leverage a pro-recovery phenotype holds promise in treating CNS injury. Recently, it has been demonstrated that ablation of astrocytic transglutaminase 2 (TG2) modulates the phenotype of reactive astrocytes in a way that improves neuronal injury outcomes both in vitro and in vivo . In an in vivo mouse model, pharmacological inhibition of TG2 with the irreversible inhibitor VA4 phenocopies the neurosupportive effects of TG2 deletion in astrocytes. In this study, we provide insights into the mechanisms by which TG2 deletion or inhibition result in a more neurosupportive astrocytic phenotype. Using a neuron-astrocyte co-culture model, we show that VA4 treatment improves the ability of astrocytes to support neurite outgrowth on an injury-relevant matrix. To better understand how pharmacologically altering TG2 affects its ability to regulate reactive astrocyte phenotypes, we assessed how VA4 inhibition impacts TG2's interaction with Zbtb7a, a transcription factor we have previously identified as a functionally relevant TG2 nuclear interactor. The results of these studies demonstrate that VA4 significantly decreases the interaction of TG2 and Zbtb7a. TG2's interactions with Zbtb7a, as well as a wide range of other transcription factors and chromatin regulatory proteins, suggest that TG2 may act as an epigenetic regulator to modulate gene expression. To begin to understand if TG2-mediated epigenetic modification may impact astrocytic phenotypes in our models, we interrogated the effect of TG2 deletion and VA4 treatment on histone acetylation and found significantly greater acetylation in both experimental groups. Consistent with these findings, previous RNA-sequencing and our present proteomic analysis also supported a predominant transcriptionally suppressive role of TG2 in astrocytes. Our proteomic data additionally unveiled pronounced changes in lipid and antioxidant metabolism in astrocytes with TG2 deletion or inhibition, which likely contribute to the enhanced neurosupportive function of these astrocytes.

Astrocyte transcriptomic changes along the spatiotemporal progression of Alzheimer’s disease

Astrocytes are crucial to brain homeostasis, yet their changes along the spatiotemporal progression of Alzheimer’s disease (AD) neuropathology remain unexplored. Here we performed single-nucleus RNA sequencing of 628,943 astrocytes from five brain regions representing the stereotypical progression of AD pathology across 32 donors spanning the entire normal aging to severe AD continuum. We mapped out several unique astrocyte subclusters that exhibited varying responses to neuropathology across the AD-vulnerable neural network (spatial axis) or AD pathology stage (temporal axis). The proportion of homeostatic, intermediate and reactive astrocytes changed only along the spatial axis, whereas two other subclusters changed along the temporal axis. One of these, a trophic factor-rich subcluster, declined along pathology stages, whereas the other increased in the late stage but returned to baseline levels in the end stage, suggesting an exhausted response with chronic exposure to neuropathology. Our study underscores the complex dynamics of astrocytic responses in AD.

Mitochondrial complex I inhibition enhances astrocyte responsiveness to pro-inflammatory stimuli

Inhibition of the mitochondrial oxidative phosphorylation (OXPHOS) system can lead to metabolic disorders and neurodegenerative diseases. In primary mitochondrial disorders, reactive astrocytes often accompany neuronal degeneration and may contribute to neurotoxic inflammatory cascades that elicit brain lesions. The influence of mitochondria to astrocyte reactivity as well as the underlying molecular mechanisms remain elusive. Here we report that mitochondrial Complex I dysfunction promotes neural progenitor cell differentiation into astrocytes that are more responsive to neuroinflammatory stimuli. We show that the SWItch/Sucrose Non-Fermentable (SWI/SNF/BAF) chromatin remodeling complex takes part in the epigenetic regulation of astrocyte responsiveness, since its pharmacological inhibition abrogates the expression of inflammatory genes. Furthermore, we demonstrate that Complex I deficient human iPSC-derived astrocytes negatively influence neuronal physiology upon cytokine stimulation. Together, our data describe the SWI/SNF/BAF complex as a sensor of altered mitochondrial OXPHOS and a downstream epigenetic regulator of astrocyte-mediated neuroinflammation.

Tau, synapse loss and gliosis progress in an Alzheimer's mouse model after amyloid-β immunotherapy.

While preclinical studies assessing drugs for Alzheimer's disease (AD) are conducted in animal models that usually display only one neuropathological feature of AD, patients present with a complex combination of comorbidities and neuropathologies. Importantly, it is well-established that amyloid (Aβ) plaque and tau tangle accumulation interact in a phase-dependent manner, making it difficult to predict how targeting one might influence the other, as well as downstream degeneration. We developed a transgenic mouse model, APP/PS1xTau22, with progressive cortical Aβ deposition and hippocampal tau neurofibrillary inclusions to investigate how both neuropathologies act jointly to bring about neural degeneration, synapse loss, and glial phenotypes. We then assessed whether applying murine chimeric Aducanumab, an anti-amyloid immunotherapy, could impact the synergistic relationship between amyloid and tau. Drug treatment resulted in a ∼70% reduction in Aβ deposition in hippocampal and cortical areas and produced a robust peri-plaque microglial and astrocytic response. Removing amyloid from the brain did not reverse or slow tau pathology or alter synapse loss. Our findings suggest that, once the interaction between amyloid and tau is set in motion, reducing plaque burden by Aβ immunotherapy may stimulate glial responses, but is insufficient to curb degenerative phenotypes in this model.