Cell Death In Astrocytes Research Articles

Astrocytes initiate autophagic flux and maintain cell viability after internalizing non-active native extracellular α-synuclein

Astrocytes are tasked with regulating the synaptic environment. Early stages of various neurodegenerative diseases are characterized by synapse loss, and astrocytic atrophy and dysfunction has been proposed as a possible cause. α-Synuclein (αS) is a highly expressed neuronal protein located in the synapse that can be released in the extracellular space. Evidence points to astrocytes as being responsible for uptake and degradation of extracellular αS. Therefore, misfolded active fibrillized αS resulting in protein inclusions and aggregates could be due to astrocytic dysfunction. Despite these pathological hallmarks and lines of evidence, the autophagic function of astrocytes in response to monomeric non-active αS to model healthy conditions has not been investigated. Human primary cortical astrocytes were treated with 100 nM of extracellular monomeric non-active αS alone, and in combination with N-terminal binding monomeric γ-synuclein (γS) as a control. Western blot analysis and super resolution imaging of HiLyte-488 labeled αS confirmed successful internalization of αS at 12, 24 and 48 h after treatment, while αS dimers were only observed at 48 h. Western blot analysis also confirmed αS's ability to induce autophagic flux by 48 h. Annexin V/PI flow cytometry results revealed increased early apoptosis at 24 h, but which resolved itself by 48 h, indicating no cell death in cortical astrocytes at all time points, suggesting astrocytes can manage the protein degradation demand of monomeric αS in healthy physiological conditions. Likewise, astrocytes reduced secretion of apolipoprotein (ApoE), a protein involved in pro-inflammatory pathways, synapse regulation, and autophagy by 12 h. Similarly, total c-JUN protein levels, a transcription factor involved in pro-inflammatory pathways increased by 12 h in the nuclear fraction. Therefore, astrocytes are able to respond and degrade αS in healthy physiological conditions, and astrocyte dysfunction could precede detrimental αS accumulation.

NOX4-mediated astrocyte ferroptosis in Alzheimer’s disease

This study investigates NADPH oxidase 4 (NOX4) involvement in iron-mediated astrocyte cell death in Alzheimer’s Disease (AD) using single-cell sequencing data and transcriptomes. We analyzed AD single-cell RNA sequencing data, identified astrocyte marker genes, and explored biological processes in astrocytes. We integrated AD-related chip data with ferroptosis-related genes, highlighting NOX4. We validated NOX4’s role in ferroptosis and AD in vitro and in vivo. Astrocyte marker genes were enriched in AD, emphasizing their role. NOX4 emerged as a crucial player in astrocytic ferroptosis in AD. Silencing NOX4 mitigated ferroptosis, improved cognition, reduced Aβ and p-Tau levels, and alleviated mitochondrial abnormalities. NOX4 promotes astrocytic ferroptosis, underscoring its significance in AD progression.

Cell loss in retinal ischemia is associated with increased necroptosis

Retinal ischemia plays acentral pathophysiological role in numerous eye diseases, such as glaucoma. In addition to apoptosis, autophagy, necroptosis and ferroptosis are among the cell death mechanisms of ischemia; however, their role is not clearly understood and controversially discussed. The aim of this study is to gain an improved understanding of the role of alternative cell death mechanisms such as autophagy and necroptosis after retinal ischemia. Based on this, future autophagy-based or necroptosis-based therapeutic approaches could be developed. Retinal ischemia reperfusion was induced in one eye of 6 to 8‑week-old rats by temporarily increasing the intraocular pressure to 140 mm Hg (60 min), followed by reperfusion. The untreated contralateral eye served as acontrol. Retinas after ischemia and control retinas were examined 7days after ischemia immunohistochemically with markers for retinal ganglion cells (RGC), astrocytes (GFAP) as well as an autophagy (LAMP1) and anecroptosis marker (RIPK3) (n = 6/group). Ischemia reperfusion resulted in both significant RGC loss (p ≤ 0.001) and asignificant increase of astrocyte area (p = 0.026) after 7days. Interestingly, the number of autophagic LAMP1 positive cells was unchanged 7days after ischemia (p = 0.272), whereas the number of necroptotic RIPK3 positive cells was significantly increased (p ≤ 0.001). Necroptotic processes appear to be activated 7days after ischemia reperfusion, contributing to retinal cell death and activation of astrocytes. Late autophagic processes are not activated 7days after ischemia. Necroptosis-associated parameters could therefore be targeted as an early therapeutic approach after ischemia in the future.

Prolactin is an Endogenous Antioxidant Factor in Astrocytes That Limits Oxidative Stress-Induced Astrocytic Cell Death via the STAT3/NRF2 Signaling Pathway

Oxidative stress-induced death of neurons and astrocytes contributes to the pathogenesis of numerous neurodegenerative diseases. While significant progress has been made in identifying neuroprotective molecules against neuronal oxidative damage, little is known about their counterparts for astrocytes. Prolactin (PRL), a hormone known to stimulate astroglial proliferation, viability, and cytokine expression, exhibits antioxidant effects in neurons. However, its role in protecting astrocytes from oxidative stress remains unexplored. Here, we investigated the effect of PRL against hydrogen peroxide (H2O2)-induced oxidative insult in primary cortical astrocyte cultures. Incubation of astrocytes with PRL led to increased enzymatic activity of superoxide dismutase (SOD) and glutathione peroxidase (GPX), resulting in higher total antioxidant capacity. Concomitantly, PRL prevented H2O2-induced cell death, reactive oxygen species accumulation, and protein and lipid oxidation. The protective effect of PRL upon H2O2-induced cell death can be explained by the activation of both signal transducer and activator of transcription 3 (STAT3) and NFE2 like bZIP transcription factor 2 (NRF2) transduction cascades. We demonstrated that PRL induced nuclear translocation and transcriptional upregulation of Nrf2, concurrently with the transcriptional upregulation of the NRF2-dependent genes heme oxygenase 1, Sod1, Sod2, and Gpx1. Pharmacological blockade of STAT3 suppressed PRL-induced transcriptional upregulation of Nrf2, Sod1 and Gpx1 mRNA, and SOD and GPX activities. Furthermore, genetic ablation of the PRL receptor increased astroglial susceptibility to H2O2-induced cell death and superoxide accumulation, while diminishing their intrinsic antioxidant capacity. Overall, these findings unveil PRL as a potent antioxidant hormone that protects astrocytes from oxidative insult, which may contribute to brain neuroprotection.

Matrilin-3 supports neuroprotection in ischemic stroke by suppressing astrocyte-mediated neuroinflammation

In the brain, the role of matrilin-3, an extracellular matrix component in cartilage, is unknown. Here, we identify that matrilin-3 decreased in reactive astrocytes but was unchanged in neurons after ischemic stroke in animals. Importantly, it declined in serum of patients with acute ischemic stroke. Genetic or pharmacological inhibition or supplementation of matrilin-3 aggravates or reduces brain injury, astrocytic cell death, and glial scar, respectively, but has no direct effect on neuronal cell death. RNA sequencing demonstrates that Matn3-/- mice display an increased inflammatory response profile in the ischemic brain, including the nuclear factor κB (NF-κB) signaling pathway. Both endogenous and exogenous matrilin-3 reduce inflammatory mediators. Mechanistically, extracellular matrilin-3 enters astrocytes via caveolin-1-mediated endocytosis. Cytoplasmic matrilin-3 translocates into the nucleus by binding to NF-κB p65, suppressing inflammatory cytokine transcription. Extracellular matrilin-3 binds to BMP-2, blocking the BMP-2/Smads pathway. Thus, matrilin-3 is required for astrocytes to exert neuroprotection, at least partially, by suppressing astrocyte-mediated neuroinflammation.

The Effects of the S1P Receptor Agonist Fingolimod (FTY720) on Central and Peripheral Myelin in Twitcher Mice.

Krabbe's disease (KD) is caused by mutations in the lysosomal enzyme galactocerebrosidase and is associated with psychosine toxicity. The sphingosine 1-phosphate receptor (S1PR) agonist fingolimod (FTY720) attenuates psychosine-induced cell death of human astrocytes, demyelination in cerebellar slices, as well as demyelination in the central nervous system of twitcher mice. Psychosine also accumulates in the peripheral nervous system in twitcher mice; however, effects of fingolimod on this peripheral myelin have not been examined. The aim of this study was to investigate the effects of fingolimod administration on peripheral and central markers of myelination. Here, we report that fingolimod administration (1 mg/kg/day) from postnatal day 5 (PND) onwards did not alter peripheral demyelination in the sciatic nerve of twitcher mice, despite significantly reducing myelin debris, glial reactivity, and neuronal damage in the cerebellum. We also find fingolimod administration improves twitching and mobility scores in twitcher mice. Importantly, we find that fingolimod significantly increases the lifespan of twitcher mice by approximately 5 days. These findings suggest differential effects of fingolimod on peripheral and central neuropathy in twitcher mice, which may explain its modest efficacy on behavior and lifespan.

RAGE induces physiological activation of NADPH oxidase in neurons and astrocytes and neuroprotection.

The transmembrane receptor for advanced glycation end products (RAGE) is a signaling receptor for many damage- and pathogen-associated molecules. Activation of RAGE is associated with inflammation and an increase in reactive oxygen species (ROS) production. Although several sources of ROS have been previously suggested, how RAGE induces ROS production is still unclear, considering the multiple targets of pathogen-associated molecules. Here, using acute brain slices and primary co-culture of cortical neurons and astrocytes, we investigated the effects of a range of synthetic peptides corresponding to the fragments of the RAGE V-domain on redoxsignaling. We found that the synthetic fragment (60-76) of the RAGE V-domain induces activation of ROS production in astrocytes and neurons from the primary co-culture and acute brain slices. This effect occurred through activation of RAGE and could be blocked by a RAGE inhibitor. Activation of RAGE by the synthetic fragment stimulates ROS production in NADPH oxidase (NOX). This RAGE-induced NOX activation produced only minor decreases in glutathione levels and increased the rate of lipid peroxidation, although it also reduced basal and β-amyloid induced cell death in neurons and astrocytes. Thus, specific activation of RAGE induces redox signaling through NOX, which can be a part of a cell protective mechanism.

The role of microRNA-325-3p as a critical player in cell death in NSCs and astrocytes.

Neural stem cells (NSCs) are defined by their ability to self-renew and generate various cell types within the nervous system. Understanding the underlying mechanism by which NSCs proliferate and differentiate is crucial for the efficient modulation of in vivo neurogenesis. MicroRNAs are small non-coding RNAs controlling gene expression concerned in post-transcriptional control by blocking messenger RNA (mRNA) translation or degrading mRNA. MicroRNAs play a role as modulators by matching target mRNAs. Recent studies have discussed the biological mechanism of microRNA regulation in neurogenesis. To investigate the role of microRNAs in NSCs and NSC-derived glial cells, we screened out NSC-specific microRNAs by using miRNome-wide screening. Then, we induced downregulation by the sponge against the specific microRNA to evaluate the functional role of the microRNA in proliferation, differentiation, and apoptosis in NSCs and NSC-derived astrocytes. We found that microRNA-325-3p is highly expressed in NSCs and astrocytes. Furthermore, we showed that microRNA-325-3p is a regulator of apoptosis by targeting brain-specific angiogenesis inhibitor (BAI1), which is a receptor for apoptotic cells and expressed in the brain and cultured astrocytes. Downregulation of microRNA-325-3p using an inducible sponge system induced cell death by regulating BAI1 in NSCs and NSC-derived astrocytes. Overall, our findings can provide an insight into the potential roles of NSC-specific microRNAs in brain neurogenesis and suggest the possible usage of the microRNAs as biomarkers of neurodegenerative disease.

Elucidating the Therapeutic Utility of Olaparib in Sulfatide-Induced Human Astrocyte Toxicity and Neuroinflammation

Metachromatic leukodystrophy (MLD) is a severe demyelinating, autosomal recessive genetic leukodystrophy, with no curative treatment. The disease is underpinned by mutations in the arylsulfatase A gene (ARSA), resulting in deficient activity of this lysosomal enzyme, and consequential accumulation of galactosylceramide-3-O-sulfate (sulfatide) in the brain. Most of the effects in the brain have been attributed to the accumulation of sulfatides in oligodendrocytes and their cell damage. In contrast, less is known regarding sulfatide toxicity in astrocytes. Poly (ADP-ribose) polymerase (PARP) inhibitors are anti-cancer therapeutics that have proven efficacy in preclinical models of many neurodegenerative and inflammatory diseases, but have never been tested for MLD. Here, we examined the toxic effect of sulfatides on human astrocytes and restoration of this cell damage by the marketed PARP-1 inhibitor, Olaparib. Cultured human astrocytes were treated with increasing concentrations of sulfatides (5–100 μM) with or without Olaparib (100 nM). Cell viability assays were used to ascertain whether sulfatide-induced toxicity was rescued by Olaparib. Immunofluorescence, calcium (Ca2+) imaging, ROS, and mitochondrial damage assays were also used to explore the effects of sulfatides and Olaparib. ELISAs were performed and chemotaxis of peripheral blood immune cells was measured to examine the effects of Olaparib on sulfatide-induced inflammation in human astrocytes. Here, we established a concentration-dependent (EC50∼20 μM at 24 h) model of sulfatide-induced astrocyte toxicity. Our data demonstrate that sulfatide-induced astrocyte toxicity involves (i) PARP-1 activation, (ii) pro-inflammatory cytokine release, and (iii) enhanced chemoattraction of peripheral blood immune cells. Moreover, these sulfatide-induced effects were attenuated by Olaparib (IC50∼100 nM). In addition, sulfatide caused impairments of ROS production, mitochondrial stress, and Ca2+ signaling in human astrocytes, that were indicative of metabolic alterations and that were also alleviated by Olaparib (100 nM) treatment. Our data support the hypothesis that sulfatides can drive astrocyte cell death and demonstrate that Olaparib can dampen many facets of sulfatide-induced toxicity, including, mitochondrial stress, inflammatory responses, and communication between human astrocytes and peripheral blood immune cells. These data are suggestive of potential therapeutic utility of PARP inhibitors in the sphere of rare demyelinating diseases, and in particular MLD.Graphical Graphical abstract. Proposed mechanism of action of Olaparib in sulfatide-treated astrocytes. Human astrocytes treated for 24 h with sulfatides increase PARP-1 expression and die. PARP-1 overexpression is modulated by Ca2+ release from the endoplasmic reticulum, thus enhancing intracellular Ca2+ concentration. PARP-1 inhibition with Olaparib reduces Ca2+ influx and cell death. Olaparib also decreases IL-6, IL-8, IL-17, and CX3CL1 release from sulfatide-stimulated astrocytes, suggesting that PARP-1 plays a role in dampening neuroinflammation in MLD. This is confirmed by the reduction of immune cell migration such as lymphocytes, NK cells, and T cells towards sulfatide-treated astrocytes. Moreover, mitochondrial stress and ROS production induced by sulfatides are rescued by PARP-1 inhibition. Future studies will focus on the signaling cascades triggered by PARP-1-mediated currents in reactive astrocytes and Olaparib as a potential therapeutic target for MLD.

The antidepressant-like and glioprotective effects of the Y2 receptor antagonist SF-11 in the astroglial degeneration model of depression in rats: Involvement of glutamatergic inhibition

In this study, we explored the potential antidepressant-like properties of the brain-penetrant Y2 receptor (Y2R) antagonist SF-11 [N-(4-ethoxyphenyl)− 4-(hydroxydiphenylmethyl)− 1-piperidinecarbothioamide] in the astroglial degeneration model of depression with an emphasis on checking the possible mechanisms implicated in this antidepressant-like effect. The model of depression relies on the loss of astrocytes in the medial prefrontal cortex (mPFC) in Sprague-Dawley rats after administering the gliotoxin L-alpha-aminoadipic acid (L-AAA). SF-11 was administered intraperitoneally (i.p.) once (10 mg/kg) or for three consecutive days (10 mg/kg/day), and the effects of L-AAA and SF-11 injected alone or in combination were investigated using the forced swim test (FST), sucrose intake test (SIT), Western blotting, immunohistochemical staining, and microdialysis. SF-11 produced an antidepressant-like effect after single or three-day administration in rats subjected to astrocyte impairment, as demonstrated by the FST and SIT, respectively. Immunoblotting and immunohistochemical analyses showed that SF-11 reversed the L-AAA-induced astrocyte cell death in the mPFC, suggesting it is glioprotective. Microdialysis studies showed that SF-11 decreased extracellular glutamate (Glu) levels compared to basal value when administered alone and compared to the basal value and control group in LAAA-treated rats. The results from immunoblotting analysis indicated the involvement of Y2Rs in the astrocyte ablation model of depression and the antidepressant-like effect of SF-11. In addition, we observed the participation of the caspase-3 apoptotic pathway in the mechanism of gliotoxin action induced by L-AAA. These findings demonstrate that SF-11, a Y2R antagonist, elicited a rapid antidepressant-like response, possibly linked to its ability to inhibit glutamatergic neurotransmission.

Copper induces neuron-sparing, ferredoxin 1-independent astrocyte toxicity mediated by oxidative stress.

Copper is an essential enzyme cofactor in oxidative metabolism, anti-oxidant defenses, and neurotransmitter synthesis. However, intracellular copper, when improperly buffered, can also lead to cell death. Given the growing interest in the use of copper in the presence of the ionophore elesclomol (CuES) for the treatment of gliomas, we investigated the effect of this compound on the surround parenchyma-namely neurons and astrocytes in vitro. Here, we show that astrocytes were highly sensitive to CuES toxicity while neurons were surprisingly resistant, a vulnerability profile that is opposite of what has been described for zinc and other toxins. Bolstering these findings, a human astrocytic cell line was similarly sensitive to CuES. Modifications of cellular metabolic pathways implicated in cuproptosis, a form of copper-regulated cell death, such as inhibition of mitochondrial respiration or knock-down of ferredoxin 1 (FDX1), did not block CuES toxicity to astrocytes. CuES toxicity was also unaffected by inhibitors of apoptosis, necrosis or ferroptosis. However, we did detect the presence of lipid peroxidation products in CuES-treated astrocytes, indicating that oxidative stress is a mediator of CuES-induced glial toxicity. Indeed, treatment with anti-oxidants mitigated CuES-induced cell death in astrocytes indicating that oxidative stress is a mediator of CuES-induced glial toxicity. Lastly, prior induction of metallothioneins 1 and 2 in astrocytes with zinc plus pyrithione was strikingly protective against CuES toxicity. As neurons express high levels of metallothioneins basally, these results may partially account for their resistance to CuES toxicity. These results demonstrate a unique toxic response to copper in glial cells which contrasts with the cell selectivity profile of zinc, another biologically relevant metal.

Effect of maternal serum magnesium and calcium levels on umbilical glial fibrillary acidic protein levels in preterm labor

Magnesium can prevent astrocyte cell death and Glial Fibrillary Acidic Protein (GFAP) secretion as inflammatory marker in preterm delivery. This study was performed to analyze differences in umbilical cord GFAP levels in preterm labor given magnesium sulfate (MgSO4) as treatment group and control group and analyze the correlation between magnesium and calcium levels with umbilical GFAP levels. This quasi-experimental study was performed on 68 patients at Dr. Hasan Sadikin General Hospital from February-June 2021 consisting of 34 patients in each group. Maternal-umbilical cord magnesium levels, calcium levels, and GFAP levels were examined using ELISA test. The result was statistically measured by IBM SPSS 24.0. We found that there was a significant difference between maternal and umbilical magnesium levels and GFAP umbilical cord blood levels between the treatment and the control group (P < 0.05) in which GFAP level was higher in the control group. The multivariate analysis showed no significant relevance between mother magnesium and calcium level to umbilical cord GFAP level in the MgSO4 group. As conclusions, umbilical cord blood GFAP levels in preterm labor given MgSO4 were lower than in preterm deliveries who were not given MgSO4. There was no correlation between magnesium, calcium, and GFAP levels in the treatment group.

NOX4 as a critical effector mediating neuroinflammatory cytokines, myeloperoxidase and osteopontin, specifically in astrocytes in the hippocampus in Parkinson's disease

Oxidative stress and mitochondrial dysfunction have been believed to play an important role in the pathogenesis of aging and neurodegenerative diseases, including Parkinson's disease (PD). The excess of reactive oxygen species (ROS) increases with age and causes a redox imbalance, which contributes to the neurotoxicity of PD. Accumulating evidence suggests that NADPH oxidase (NOX)-derived ROS, especially NOX4, belong to the NOX family and is one of the major isoforms expressed in the central nervous system (CNS), associated with the progression of PD. We have previously shown that NOX4 activation regulates ferroptosis via astrocytic mitochondrial dysfunction. We have previously shown that activation of NOX4 regulates ferroptosis through mitochondrial dysfunction in astrocytes. However, it remains unclear why an increase in NOX4 in neurodegenerative diseases leads to astrocyte cell death by certain mediators. Therefore, this study was designed to evaluate how NOX4 in the hippocampus is involved in PD by comparing an MPTP-induced PD mouse model compared to human PD patients. We could detect that the hippocampus was dominantly associated with elevated levels of NOX4 and α-synuclein during PD and the neuroinflammatory cytokines, myeloperoxidase (MPO) and osteopontin (OPN), were upregulated particularly in astrocytes. Intriguingly, NOX4 suggested a direct intercorrelation with MPO and OPN in the hippocampus. Upregulation of MPO and OPN induces mitochondrial dysfunction by suppressing five protein complexes in the mitochondrial electron transport system (ETC) and increases the level of 4-HNE leading to ferroptosis in human astrocytes. Overall, our findings indicate that the elevation of NOX4 cooperated with the MPO and OPN inflammatory cytokines through mitochondrial aberration in hippocampal astrocytes during PD.

Carbon Monoxide Stimulates Both Mitophagy And Mitochondrial Biogenesis to Mediate Protection Against Oxidative Stress in Astrocytes.

Astrocytes are key glial cells for the metabolic and functional support of the brain. Mitochondrial quality control (MQC), in particular the balance between mitophagy and mitochondrial biogenesis, is a major event for the maintenance of cellular homeostasis. Carbon monoxide (CO) is an endogenous gasotransmitter that inhibits cell death and inflammation by targeting mitochondria. It is well established that CO promotes cytoprotection by increasing mitochondrial population and metabolism (oxidative phosphorylation). Thus, it is hypothesized that CO-induced cytoprotection may also be mediated by the balance between mitophagy and mitochondrial biogenesis. Herein, the carbon monoxide releasing molecule-A1 (CORM-A1) was used in primary cultures of astrocytes to assess CO role on mitochondrial turnover. PINK1/Parkin-dependent mitophagy was stimulated by CORM-A1 following 1h of treatment. While at 24h after treatment, CORM-A1 increased mitochondrial population, which may indicate mitochondrial biogenesis. In fact, mitochondrial biogenesis was confirmed by the enhancement of PGC-1α expression that upregulates several mitochondrial transcription factors. Furthermore, inhibition of mitophagy by knocking down PINK1 expression reverted CO-induced mitochondrial biogenesis, indicating that mitochondrial turnover is dependent on modulation of mitophagy. Finally, CORM-A1 prevented astrocytic cell death induced by oxidative stress in a mitophagy-dependent manner. In fact, whenever PINK1 was knocked down, CORM-A1-induced cytoprotection was lost. In summary, CORM-A1 stimulates mitochondrial turnover, which in turn prevents astrocytic cell death. CO cytoprotection depends on increasing mitochondrial population and on eliminating dysfunctional mitochondria.

HIV-induced bystander cell death in astrocytes requires cell-to-cell viral transmission.

Human immunodeficiency virus (HIV) neuroinvasion occurs early after infection through the trafficking of virus-infected immune cells into the central nervous system (CNS) and viral dissemination into the brain. There, it can infect resident brain cells including astrocytes, the most abundant cell type that is crucial to brain homeostasis. In this report, we examined the HIV-related mechanism able to induce bystander cell death in astrocytes mediated by cell-to-cell contact with productively infected (PI) ones. We first demonstrate that HIV-induced bystander cell death involves mitochondrial dysfunction that promotes exacerbated reactive oxygen species production. Such a phenomenon is a contagious cell death that requires contact with HIV-PI astrocytes that trigger caspase-dependent (apoptosis and pyroptosis) and caspase-independent cell death pathways. The HIV accessory proteins Nef, Vpu, and Vpr counteract astrocyte death among PI cells but, in contrast, participate to promote contagious bystander cell death by inducing mitochondrial reactive oxygen species production. Our findings indicate that astrocytes PI by HIV became capable to counteract infection-derived death signals, surviving, and spreading the bystander cell death into neighboring uninfected cells by a cell-to-cell contact-dependent mechanism. Considering that astrocytes have been proposed as a long-term HIV reservoir in the CNS, ascertaining the mechanism of survival and contagious bystander death will afford clear targets in the current goal to achieve a functional cure.

Cholesterol-induced robust Ca oscillation in astrocytes required for survival and lipid droplet formation in high-cholesterol condition

SummaryCholesterol, one of the major cell membrane components, stabilizes membrane fluidity and regulates signal transduction. Beside its canonical roles, cholesterol has been reported to directly activate signaling pathways such as hedgehog (Hh). We recently found that astrocytes, one of the glial cells, respond to Hh pathway stimulation by Ca signaling. These notions led us to test if extracellularly applied cholesterol triggers Ca signaling in astrocytes. Here, we found that cholesterol application induces robust Ca oscillation only in astrocytes with different properties from the Hh-induced Ca response. The Ca oscillation has a long delay which corresponds to the onset of cholesterol accumulation in the plasma membrane. Blockade of the Ca oscillation resulted in enhancement of astrocytic cell death and disturbance of lipid droplet formation, implying a possibility that the cholesterol-induced Ca oscillation plays important roles in astrocytic survival and cholesterol handling under pathological conditions of cholesterol load such as demyelination.

Z-DNA binding protein 1 mediates necroptotic and apoptotic cell death pathways in murine astrocytes following herpes simplex virus-1 infection

BackgroundThe mechanisms by which glia respond to viral central nervous system (CNS) pathogens are now becoming apparent with the demonstration that microglia and astrocytes express an array of pattern recognition receptors that include intracellular RNA and DNA sensors. We have previously demonstrated that glia express Z-DNA binding protein 1 (ZBP1) and showed that this cytosolic nucleic acid sensor contributes to the inflammatory/neurotoxic responses of these cells to herpes simplex virus-1 (HSV-1). However, the relative contribution made by ZBP1- to HSV-1-mediated cell death in glia has not been determined.MethodsWe have investigated the relative contribution made by ZBP1- to HSV-1-mediated cell death in primary astrocytes derived from mice genetically deficient in this sensor. We have used capture ELISAs and immunoblot analysis to assess inflammatory cytokine production and ZBP1 and phosphorylated mixed lineage kinase domain-like protein (MLKL) expression levels, respectively, following HSV-1 challenge. Furthermore, we have used a commercially available cell viability assay to determine the proportion and rate of cell death in cells following infection with laboratory and neuroinvasive clinical strains of HSV-1, and pharmacological inhibitors of necroptotic and apoptotic pathway components to assess the relative role of each.ResultsWe show that the loss of ZBP1 in astrocytes results in an increase in the number of viral particles released following HSV-1 infection. Importantly, we have confirmed that HSV-1 induces necroptosis in astrocytes and have established the ability of ZBP1 to mediate this cell death pathway. Interestingly, while ZBP1 is best known for its role in necroptotic signaling, our findings indicate that this sensor can also contribute to virally induced apoptosis in these glia.ConclusionsOur findings indicate that ZBP1 serves as a restriction factor for HSV-1 infection and is associated with the induction of both necroptotic and apoptotic cell death pathways in primary murine astrocytes. While it remains to be seen whether ZBP1-mediated activation of cell death in astrocytes contributes significantly to host protection or, rather, exacerbates HSV-1 encephalitis pathology, the identification of such a role in resident CNS cells may represent a novel target for therapeutic intervention to reduce HSV encephalitis-associated morbidity and mortality.

TNF-like weak inducer of apoptosis / nuclear factor κB axis feedback loop promotes spinal cord injury by inducing astrocyte activation

ABSTRACT Non-canonical signaling pathways have been proved to act as potent sites of astrocytes osmotic expanding or proliferation, which promotes the regeneration of axons in areas with non-neural spinal cord injury (SCI). However, the relevant signal pathway that induces autophagic cell death in astrocytes and its function relative to the TNF-like weak inducer of apoptosis/nuclear factor κB (TWEAK/NF-κB) axis remains elusive. The SCI model was established by vertically striking the spinal cord according to Allen’s model. Astrocytes and neuronal cells were prepared from spinal cells extracted from spinal cord tissues of SCI or normal C57BL/6 newborn mice. After co-culturing astrocytes and neurons, cell viability and autophagy were determined by CCK-8, transmission electron microscopy (TEM), and western blot. The expression of TWEAK, NF-κB and inflammatory cytokines was confirmed by qRT-PCR, western blot, Immunofluorescence and ELISA assay. Chromatin immunoprecipitation (CHIP) was used to evaluate the interaction between TWEAK and NF-κB. Our results demonstrated that knockdown of TWEAK and NF-κB inhibited secretion of high levels of TNF-α/IL-1β, partially counteracted by adding Rap. TWEAK/NF-κB was the positive correlation feedback loop regulating the proliferation and autophagy of astrocytes involved in SCI. Moreover, restraining the excess growth of astrocytes was beneficial to the growth of neurons. Collectively, our findings illustrated that the TWEAK/NF-κB pathway might act as a positive modulator of SCI by inducing astrocyte activation, shedding new insights for SCI treatment.

Astrocyte plasticity in mice ensures continued endfoot coverage of cerebral blood vessels following injury and declines with age

Astrocytes extend endfeet that enwrap the vasculature, and disruptions to this association which may occur in disease coincide with breaches in blood-brain barrier (BBB) integrity. Here we investigate if focal ablation of astrocytes is sufficient to disrupt the BBB in mice. Targeted two-photon chemical apoptotic ablation of astrocytes induced a plasticity response whereby surrounding astrocytes extended processes to cover vascular vacancies. In young animals, replacement processes occur in advance of endfoot retraction, but this is delayed in aged animals. Stimulation of replacement astrocytes results in constriction of pre-capillary arterioles, suggesting that replacement astrocytes are functional. Pharmacological inhibition of pSTAT3, as well as astrocyte specific deletion of pSTAT3, reduces astrocyte replacement post-ablation, without perturbations to BBB integrity. Similar endfoot replacement occurs following astrocyte cell death due to reperfusion in a stroke model. Together, these studies uncover the ability of astrocytes to maintain cerebrovascular coverage via substitution from nearby cells.

Valproic Acid Inhibits Glial Scar Formation after Ischemic Stroke

Introduction: Cerebral ischemia induces reactive proliferation of astrocytes (astrogliosis) and glial scar formation. As a physical and biochemical barrier, the glial scar not only hinders spontaneous axonal regeneration and neuronal repair but also deteriorates the neuroinflammation in the recovery phase of ischemic stroke. Objectives: Previous studies have shown the neuroprotective effects of the valproic acid (2-n-propylpentanoic acid, VPA) against ischemic stroke, but its effects on the ischemia-induced formation of astrogliosis and glial scar are still unknown. As targeting astrogliosis has become a therapeutic strategy for ischemic stroke, this study was designed to determine whether VPA can inhibit the ischemic stroke-induced glial scar formation and to explore its molecular mechanisms. Methods: Glial scar formation was induced by an ischemia-reperfusion (I/R) model in vivo and an oxygen and glucose deprivation (OGD)-reoxygenation (OGD/Re) model in vitro. Animals were treated with an intraperitoneal injection of VPA (250 mg/kg/day) for 28 days, and the ischemic stroke-related behaviors were assessed. Results: Four weeks of VPA treatment could markedly reduce the brain atrophy volume and improve the behavioral deficits in rats’ I/R injury model. The results showed that VPA administrated upon reperfusion or 1 day post-reperfusion could also decrease the expression of the glial scar makers such as glial fibrillary acidic protein, neurocan, and phosphacan in the peri-infarct region after I/R. Consistent with the in vivo data, VPA treatment showed a protective effect against OGD/Re-induced astrocytic cell death in the in vitro model and also decreased the expression of GFAP, neurocan, and phosphacan. Further studies revealed that VPA significantly upregulated the expression of acetylated histone 3, acetylated histone 4, and heat-shock protein 70.1B in the OGD/Re-induced glial scar formation model. Conclusion: VPA produces neuroprotective effects and inhibits the glial scar formation during the recovery period of ischemic stroke via inhibition of histone deacetylase and induction of Hsp70.1B.