Primary Cultures Of Mouse Astrocytes Research Articles

Triclosan affects steroidogenesis in mouse primary astrocytes in vitro with engagement of Sirtuin 1 and 3

Triclosan (TCS) is a widely used antimicrobial, antifungal, and antiviral agent. To date, it has been reported that TCS can enter the human body and disrupt hormonal homeostasis. Therefore, the aim of our paper was to evaluate the impact of TCS on astrocytes, i.e. a crucial population of cells responsible for steroid hormone production. Our data showed that, in mouse primary astrocyte cultures, TCS can act as an endocrine disrupting chemical through destabilization of the production or secretion of progesterone (P4), testosterone (T), and estradiol (E2). TCS affects the mRNA expression of enzymes involved in neurosteroidogenesis, such as Cyp17a1, 17β-Hsd, and Cyp19a1. Our data showed that a partial PPARγ agonist (honokiol) prevented changes in Cyp17a1 mRNA expression caused by TCS. Similarly, honokiol inhibited TCS-stimulated P4 release. However, rosiglitazone (classic PPARγ agonist) or GW9662 (PPARγ antagonist) had a much stronger effect. Therefore, we believe that the changes observed in the P4, T, and E2 levels are a result of dysregulation of the activity of the aforementioned enzymes, whose expression can be affected by TCS through a Pparγ-dependent pathway. TCS was found to decrease the aryl hydrocarbon receptor (AhR) and Sirtuin 3 protein levels, which may be the result of the activation of the these proteins. Since our study showed dysregulation of the production or secretion of neurosteroids in astrocytes, it can be concluded that TCS reaching the brain may contribute to the development of neurodegenerative diseases in which an abnormal amount of neurosteroids is observed.

Cytotoxicity of phosphoramidate, bis-amidate and cycloSal prodrug metabolites against tumour and normal cells.

Phosphonate and phosphate prodrugs are integral to enhancing drug permeability, but the potential toxicity of their metabolites requires careful consideration. This study evaluates the impact of widely used phosphoramidate, bis-amidate, and cycloSal phosph(on)ate prodrug metabolites on BxPC3 pancreatic cancer cells, GL261-Luc glioblastoma cells, and primary cultured mouse astrocytes. 1-Naphthol and 2-naphthol demonstrated the greatest toxicity. Notably, 2-naphthol exhibited an ED50 of 21 μM on BxPC3 cells, surpassing 1-naphthol with an ED50 of 82 μM. Real-time xCELLigence experiments revealed notable activity for both metabolites at a low concentration of 16 μM. On primary cultured mouse astrocyte cells, all prodrugs exhibited reduced viability at 128 to 256 μM after only 4 hours of exposure. A cell-type-dependent sensitivity to phosph(on)ate prodrug metabolites was evident, with normal cells showing greater susceptibility than corresponding tumour cells. The results suggest it is essential to consider the potential cytotoxicity of phosph(on)ate prodrugs in the drug design and evaluation process.

Spike S1 peptide associated neuroinflammatory and cytokine induction is associated with modified behavior in in vivo models

AbstractBackgroundCoronavirus disease (COVID‐19), a severe acute respiratory syndrome coronavirus 2 (SARS CoV‐2) is a damaging illness leading to severe health crisis and economic crisis worldwide. Numerous brain complication has been reported in COVID‐19 patients and surprisingly has persisted after COVID‐19 infection recovery in patients. The underlying mechanism are not clear. So far, there is no proper therapeutic strategy against COVID‐19 and its complications on the brain.MethodInitially we validate the effect of recombinant Spike S1 in A5339 cells (Paidi, Jana et al. 2021, Paidi, Jana et al. 2021, Paidi, Jana et al. 2021) and as well as mouse primary astrocyte cultures, in terms of cytokine induction and neuroinflammatory markers. In the current study, mice were administered recombinant spike S1 intranasally for 14 days. Initially we evaluated the physiological and behavioral alteration for 12 days. SARS‐CoV‐2 binds to angiotensin‐converting enzyme 2 (ACE2) for entering host cells, we so investigate the lung histology and its cytokine induction. Further, we evaluate the cytokine and neuroinflammatory levels in the brain, and its relevance to behavioral modifications in the mouse.ResultIn the present study, Spike S1 treatment prominently elevate the cytokine levels and inflammatory markers in invitro models. Intranasal intoxication of C57/BL6 mice with recombinant SARS‐CoV‐2 spike S1 led to fever, increase in IL‐6 in the lungs and brain. We found that the impairment in spatial learning memory functions and motor disabilities, mimic some of the important symptoms of COVID‐19. Our studies show Spike S1 induced cytokine levels in lungs and as well as in the brain.ConclusionOur results suggest a simple, but reliable means of developing a Spike S1 mediated in vivo model which exhibits severe lung pathology and reinforce the validity of this animal model to study COVID‐19 pathogenesis and potential therapies. Keywords Spike S1, COVID‐19; IL‐6; Cognitive functions; Motor disabilities, Cytokine induction;

Ethanol-induced transcriptional and translational changes in Aldh1l1-Egfp/Rpl10a cortical astrocyte cultures.

The role astrocytes play in brain development and function has garnered greater attention as the diversity of roles they are involved in has become apparent. We have previously shown that ethanol-exposed astrocytes alter neuronal neurite outgrowth in an in vitro co-culture system and that ethanol alters the astrocyte-produced extracellular matrix (ECM) in vitro, with similar alterations in vivo. In this study, we utilized the translating ribosome affinity purification (TRAP) procedure in Aldh1l1-EGFP/Rpl10a transgenic mouse primary cortical astrocyte cultures to transcriptionally and translationally profile the astrocyte response to ethanol. We found a large number of differences between the total RNA pool and the translating RNA pool, indicating that the transcriptional state of astrocytes may not always reflect the translational state of astrocytes. In addition, there was a considerable overlap between ethanol-dysregulated genes in the total RNA pool and the translating RNA pool. Comparisons to published datasets indicate the in vitro model used here is most similar to PD1 or PD7 in vivo cortical astrocytes, and the ethanol-regulated genes showed a significant overlap with models of chronic ethanol exposure in astrocytes, a model of third-trimester ethanol exposure in the hippocampus and cerebellum, and an acute model of ethanol exposure in the hippocampus. These findings will further our understanding of the effects of ethanol on astrocyte gene expression and protein translation and how these changes may alter brain development and support the use of in vitro astrocyte cultures as models of neonatal astrocytes.

Expression analysis and targets prediction of microRNAs in OGD/R treated astrocyte-derived exosomes by smallRNA sequencing

Astrocytes activate and crosstalk with neurons influencing inflammatory responses following ischemic stroke. The distribution, abundance, and activity of microRNAs in astrocytes-derived exosomes after ischemic stroke remains largely unknown. In this study, exosomes were extracted from primary cultured mouse astrocytes via ultracentrifugation, and exposed to oxygen glucose deprivation/re‑oxygenation injury to mimic experimental ischemic stroke. SmallRNAs from astrocyte-derived exosomes were sequenced, and differentially expressed microRNAs were randomly selected and verified by stem-loop real time quantitative polymerase chain reaction. We found that 176 microRNAs, including 148 known and 28 novel microRNAs, were differentially expressed in astrocyte-derived exosomes following oxygen glucose deprivation/re‑oxygenation injury. In gene ontology enrichment, Kyoto encyclopedia of genes and genomes pathway analyses, and microRNA target gene prediction analyses, these alteration in microRNAs were associated to a broad spectrum of physiological functions including signaling transduction, neuroprotection and stress responses. Our findings warrant further investigating of these differentially expressed microRNAs in human diseases particularly ischemic stroke.

Heparan Sulfated Glypican-4 Is Released from Astrocytes by Proteolytic Shedding and GPI-Anchor Cleavage Mechanisms.

Astrocytes provide neurons with diffusible factors that promote synapse formation and maturation. In particular, glypican-4/GPC4 released from astrocytes promotes the maturation of excitatory synapses. Unlike other secreted factors, GPC4 contains the C-terminal GPI-anchorage signal. However, the mechanism by which membrane-tethered GPC4 is released from astrocytes is unknown. Using mouse primary astrocyte cultures and a quantitative luciferase-based release assay, we show that GPC4 is expressed on the astrocyte surface via a GPI-anchorage. Soluble GPC4 is robustly released from the astrocytes largely by proteolytic shedding and, to a lesser extent, by GPI-anchor cleavage, but not by vesicular release. Pharmacological, overexpression, and loss of function screens showed that ADAM9 in part mediates the release of GPC4 from astrocytes. The released GPC4 contains the heparan sulfate side chain, suggesting that these release mechanisms provide the active form that promotes synapse maturation and function. Overall, our studies identified the release mechanisms and the major releasing enzyme of GPC4 in astrocytes and will provide insights into understanding how astrocytes regulate synapse formation and maturation.

Palmitoylation of acetylated tubulin and association with ceramide-rich platforms is critical for ciliogenesis

AbstractMicrotubules are polymers composed of αβ-tubulin subunits that provide structure to cells and play a crucial role in in the development and function of neuronal processes and cilia, microtubule-driven extensions of the plasma membrane that have sensory (primary cilia) or motor (motile cilia) functions. To stabilize microtubules in neuronal processes and cilia, α tubulin is modified by the posttranslational addition of an acetyl group, or acetylation. We discovered that acetylated tubulin in microtubules interacts with the membrane sphingolipid, ceramide. However, the molecular mechanism and function of this interaction are not understood. Here, we show that in human induced pluripotent stem cell–derived neurons, ceramide stabilizes microtubules, which indicates a similar function in cilia. Using proximity ligation assays, we detected complex formation of ceramide with acetylated tubulin in Chlamydomonas reinhardtii flagella and cilia of human embryonic kidney (HEK293T) cells, primary cultured mouse astrocytes, and ependymal cells. Using incorporation of palmitic azide and click chemistry–mediated addition of fluorophores, we show that a portion of acetylated tubulin is S-palmitoylated. S-palmitoylated acetylated tubulin is colocalized with ceramide-rich platforms in the ciliary membrane, and it is coimmunoprecipitated with Arl13b, a GTPase that mediates transport of proteins into cilia. Inhibition of S-palmitoylation with 2-bromo palmitic acid or inhibition of ceramide biosynthesis with fumonisin B1 reduces formation of the Arl13b-acetylated tubulin complex and its transport into cilia, concurrent with impairment of ciliogenesis. Together, these data show, for the first time, that ceramide-rich platforms mediate membrane anchoring and interaction of S-palmitoylated proteins that are critical for cilium formation, stabilization, and function.

Astrocytic pyruvate dehydrogenase kinase-2 is involved in hypothalamic inflammation in mouse models of diabetes

Hypothalamic inflammation plays an important role in disrupting feeding behavior and energy homeostasis as well as in the pathogenesis of obesity and diabetes. Here, we show that pyruvate dehydrogenase kinase (PDK)-2 plays a role in hypothalamic inflammation and its sequelae in mouse models of diabetes. Cell type-specific genetic ablation and pharmacological inhibition of PDK2 in hypothalamic astrocytes suggest that hypothalamic astrocytes are involved in the diabetic phenotype. We also show that the PDK2-lactic acid axis plays a regulatory role in the observed metabolic imbalance and hypothalamic inflammation in mouse primary astrocyte and organotypic cultures, through the AMPK signaling pathway and neuropeptidergic circuitry governing feeding behavior. Our findings reveal that PDK2 ablation or inhibition in mouse astrocytes attenuates diabetes-induced hypothalamic inflammation and subsequent alterations in feeding behavior.

Elaphuri Davidiani Cornu Improves Depressive-Like Behavior in Mice and Increases Neurotrophic Factor Expression in Mouse Primary Astrocytes via cAMP and ERK-Dependent Pathways.

Elaphuri Davidiani Cornu (EDC) is the natural shedding horn of Elaphurus davidiauus Millne-Edwards that was used by people in ancient China for maintaining physical and mental health. We evaluated the antidepressant effect of EDC using depression-like animal models and explored possible mechanisms in mouse primary astrocyte cultures. We found that aqueous extracts of EDC significantly improved depression-like behavior in a mouse model of depression. The extracts enhanced expression of nerve growth factor and brain-derived neurotrophic factor neurotrophic factors in mouse prefrontal cortex and hippocampus tissues. In the mouse primary astrocyte cultures, the EDC aqueous extracts significantly increased the neurotrophic factor expression both at the transcriptional and protein levels. EDC extracts might exhibit these functions by regulating matrix metalloprotein-9 of the nerve growth factor and brain-derived neurotrophic factor metabolic pathways and might enhance expression of neurotrophic factors via the cAMP- and ERK-dependent pathways. We confirmed this possibility by showing the effects of related inhibitors, providing scientific evidence that supports the utility of EDC in the development of drugs to treat major depressive disorders.

Amyloid-β regulates gap junction protein connexin 43 trafficking in cultured primary astrocytes

Altered expression and function of astroglial gap junction protein connexin 43 (Cx43) has increasingly been associated to neurotoxicity in Alzheimer disease (AD). Although earlier studies have examined the effect of increased β-amyloid (Aβ) on Cx43 expression and function leading to neuronal damage, underlying mechanisms by which Aβ modulates Cx43 in astrocytes remain elusive. Here, using mouse primary astrocyte cultures, we have examined the cellular processes by which Aβ can alter Cx43 gap junctions. We show that Aβ25-35 impairs functional gap junction coupling yet increases hemichannel activity. Interestingly, Aβ25-35 increased the intracellular pool of Cx43 with a parallel decrease in gap junction assembly at the surface. Intracellular Cx43 was found to be partly retained in the endoplasmic reticulum-associated cell compartments. However, forward trafficking of the newly synthesized Cx43 that already reached the Golgi was not affected in Aβ25-35-exposed astrocytes. Supporting this, treatment with 4-phenylbutyrate, a well-known chemical chaperone that improves trafficking of several transmembrane proteins, restored Aβ-induced impaired gap junction coupling between astrocytes. We further show that interruption of Cx43 endocytosis in Aβ25-35-exposed astrocytes resulted in their retention at the cell surface in the form of functional gap junctions indicating that Aβ25-35 causes rapid internalization of Cx43 gap junctions. Additionally, in silico molecular docking suggests that Aβ can bind favorably to Cx43. Our study thus provides novel insights into the cellular mechanisms by which Aβ modulates Cx43 function in astrocytes, the basic understanding of which is vital for the development of alternative therapeutic strategy targeting connexin channels in AD.

Serum amyloid A inhibits astrocyte migration via activating p38 MAPK

BackgroundThe accumulation of astrocytes around senile plaques is one of the pathological characteristics in Alzheimer’s disease (AD). Serum amyloid A (SAA), known as a major acute-phase protein, colocalizes with senile plaques in AD patients. Here, we demonstrate the role of SAA in astrocyte migration.MethodsThe effects of SAA on astrocyte activation and accumulation around amyloid β (Aβ) deposits were detected in APP/PS1 transgenic mice mated with Saa3−/− mice. SAA expression, astrocyte activation, and colocalization with Aβ deposits were evaluated in mice using immunofluorescence staining and/or Western blotting. The migration of primary cultures of mouse astrocytes and human glioma U251 cells was examined using Boyden chamber assay and scratch-would assay. The actin and microtubule networks, protrusion formation, and Golgi apparatus location in astrocytes were determined using scratch-would assay and immunofluorescence staining.ResultsSaa3 expression was significantly induced in aged APP/PS1 transgenic mouse brain. Saa3 deficiency exacerbated astrocyte activation and increased the number of astrocytes around Aβ deposits in APP/PS1 mice. In vitro studies demonstrated that SAA inhibited the migration of primary cultures of astrocytes and U251 cells. Mechanistic studies showed that SAA inhibited astrocyte polarization and protrusion formation via disrupting actin and microtubule reorganization and Golgi reorientation. Inhibition of the p38 MAPK pathway abolished the suppression of SAA on astrocyte migration and polarization.ConclusionsThese results suggest that increased SAA in the brain of APP/PS1 mice inhibits the migration of astrocytes to amyloid plaques by activating the p38 MAPK pathway.

The metabolic response to inflammation in astrocytes is regulated by nuclear factor-kappa B signaling.

Inflammation and metabolism are intrinsically linked with inflammatory stimuli inducing metabolic changes in cells and, in turn, metabolic capacity determining cellular inflammatory responses. Although well characterized in peripheral immune cells there is comparatively less known about these "immunometabolic" responses in astrocytes. In this study, we tested the hypothesis that the astrocytic inflammatory response driven by nuclear factor-kappa B (NF-κB) signaling is dependent on glycolytic metabolism. Using mouse primary cortical astrocyte cultures, we assessed changes in cellular metabolism after exposure to lipopolysaccharide (LPS), with cytokine ELISAs and immunoblotting being used to measure inflammatory responses. Results indicate temporally distinct metabolic adaptations to pro-inflammatory stimulation in astrocytes: 3 hr LPS treatment increased glycolysis but did not alter mitochondrial metabolism, while following 24 hr of LPS treatment we observed increased oxidative phosphorylation, and decreased glycolytic capacity and glucose uptake, partly due to reduced glucose transporter 1 expression. Inhibition of NF-κB signaling with the IKK-beta inhibitor TPCA-1 prevented the LPS induced changes to glycolysis and oxidative phosphorylation. Furthermore, TPCA-1 treatment altered both glycolysis and oxidative phosphorylation independently from inflammatory stimulation, indicating a role for NF-κB signaling in regulation of basal metabolism in astrocytes. Inhibition of glycolysis with 2-deoxyglucose significantly attenuated LPS-induced cytokine release and NF-κB phosphorylation, indicating that intact glycolysis is required for the full inflammatory response to LPS. Together our data indicate that astrocytes display immunometabolic responses to acute LPS stimulation which may represent a potential therapeutic target for neuroinflammatory disorders.

Molecular mechanisms of cell death induced in glioblastoma by experimental and antineoplastic drugs: New and old drugs induce apoptosis in glioblastoma.

Glioblastoma multiforme (GBM) is one of the most aggressive astrocytic tumors; it is resistant to most chemotherapeutic agents currently available and is associated with a poor patient survival. Thus, the development of new anticancer compounds is urgently required. Herein, we studied the molecular mechanisms of cell death induced by the experimental drugs resveratrol and MG132 or the antineoplastic drugs cisplatin and etoposide on a human GBM cell line (D54) and on primary cultured mouse astrocytes (PCMAs). Caspases, Bcl-2, inhibitors of apoptosis proteins (IAP) family members, and p53 were identified as potential molecular targets for these drugs. All drugs had a cytotoxic effect on D54 cells and PCMAs, with a similar inhibitory concentration (IC50) after 24 h. However, MG132 and cisplatin were more effective to induce apoptosis and autophagy than resveratrol and etoposide. Cell death by apoptosis involved the activation of caspases-3/7, -8, and -9, increased lysosomal permeability, LC3 lipidation, poly-(ADP-ribose) polymerase (PARP)-1 fragmentation, and a differential expression of genes related with apoptosis and autophagy like Mcl-1, Survivin, Noxa, LC3, and Beclin. In addition, apoptosis activation was partially dependent on p53 activation. Since experimental and antineoplastic drugs yielded similar results, further work is required to justify their use in clinical protocols.

Stem Cell-Derived Exosomes Protect Astrocyte Cultures From in vitro Ischemia and Decrease Injury as Post-stroke Intravenous Therapy.

In the present study, we assessed efficacy of exosomes harvested from human and mouse stem cell cultures in protection of mouse primary astrocyte and neuronal cell cultures following in vitro ischemia, and against ischemic stroke in vivo. Cell media was collected from primary mouse neural stem cell (NSC) cultures or from human induced pluripotent stem cell-derived cardiomyocyte (iCM) cultures. Exosomes were extracted and purified by polyethylene glycol complexing and centrifugation, and exosome size and concentration were determined with a NanoSiteTM particle analyzer. Exosomes were applied to primary mouse cortical astrocyte or neuronal cultures prior to, and/or during, combined oxygen-glucose deprivation (OGD) injury. Cell death was assessed via lactate dehydrogenase (LHD) and propidium iodide staining 24 h after injury. NSC-derived exosomes afforded marked protection to astrocytes following OGD. A more modest (but significant) level of protection was observed with human iCM-derived exosomes applied to astrocytes, and with NSC-derived exosomes applied to primary neuronal cultures. In subsequent experiments, NSC-derived exosomes were injected intravenously into adult male mice 2 h after transient (1 h) middle cerebral artery occlusion (MCAO). Gross motor function was assessed 1 day after reperfusion and infarct volume was assessed 4 days after reperfusion. Mice treated post-stroke with intravenous NSC-derived exosomes exhibited significantly reduced infarct volumes. Together, these results suggest that exosomes isolated from mouse NSCs provide neuroprotection against experimental stroke possibly via preservation of astrocyte function. Intravenous NSC-derived exosome treatment may therefore provide a novel clinical adjuvant for stroke in the immediate post-injury period.

Bestrophin-3 Expression in a Subpopulation of Astrocytes in the Neonatal Brain After Hypoxic-Ischemic Injury

Bestrophin-3, a potential candidate for a calcium-activated chloride channel, recently was suggested to have cell-protective functions. We studied the expression and alternative splicing of bestrophin-3 in neonatal mouse brain and after hypoxic-ischemic (HI) injury and in human neonatal brain samples. HI brain injury was induced in 9-day old mice by unilateral permanent common carotid artery occlusion in combination with exposure to 10% oxygen for 50 min. Endoplasmic reticulum stress was induced by thapsigargin treatment in primary culture of mouse brain astrocytes. We also investigated expression of bestrophin-3 protein in a sample of human neonatal brain tissue. Bestrophin-3 protein expression was detected with immunohistochemical methods and western blot; mRNA expression and splicing were analyzed by RT-PCR. HI induced a brain tissue infarct and a pronounced increase in the endoplasmic reticulum-associated marker CHOP. Three days after HI a population of astrocytes co-expressed bestrophin-3 and nestin in a penumbra-like area of the injured hemisphere. However, total levels of Bestrophin-3 protein in mouse cortex were reduced after injury. Mouse astrocytes in primary culture also expressed bestrophin-3 protein, the amount of which was reduced by endoplasmic reticulum stress. Bestrophin-3 protein was detected in astrocytes in the hippocampal region of the human neonatal brain which had patchy white matter gliosis and neuronal loss in the Sommer’s sector of the Ammon’s horn (CA1). Analysis of bestrophin-3 mRNA in mouse brain with and without injury showed the presence of two truncated spliced variants, but no full-length mRNA. Total amount of bestrophin-3 mRNA increased after HI, but showed only minor injury-related change. However, the splice variants of bestrophin-3 mRNA were differentially regulated after HI depending on the presence of tissue injury. Our results show that bestrophin-3 is expressed in neonatal mouse brain after injury and in the human neonatal brain with pathology. In mouse brain bestrophin-3 protein is upregulated in a specific astrocyte population after injury and is co-expressed with nestin. Splice variants of bestrophin-3 mRNA respond differently to HI, which might indicate their different roles in tissue injury.

Dopamine D2 receptor restricts astrocytic NLRP3 inflammasome activation via enhancing the interaction of β-arrestin2 and NLRP3.

Astrocytes are involved in the neuroinflammation of neurodegenerative diseases, such as Parkinson's disease (PD). Among the numerous inflammatory cytokines, interleukin-1β (IL-1β) produced by astrocytic Nod-like receptor protein (NLRP) inflammasome is crucial in the pathogenesis of PD. β-arrestin2-mediated dopamine D2 receptor (Drd2) signal transduction has been regarded as a potential anti-inflammatory target. Our previous study revealed that astrocytic Drd2 suppresses neuroinflammation in the central nervous system. However, the role of Drd2 in astrocytic NLRP3 inflammasome activation and subsequent IL-1β production remains unclear. In the present study, we used 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced PD mouse model to investigate whether Drd2 could suppress astrocytic NLRP3 inflammasome activation. We showed that Drd2 agonist inhibited NLRP3 inflammasome activation, evidenced by decreased caspase-1 expression and reduced IL-1β release in the midbrain of wild type mice. The anti-inflammasome effect of Drd2 was abolished in β-arrestin2 knockout and β-arrestin2 small interfering RNA-injected mice, suggesting a critical role of β-arrestin2 in Drd2-regulated NLRP3 inflammasome activation. We also found that Drd2 agonists suppressed the upregulation of caspase-1 and IL-1β expression in primary cultured mouse astrocytes in response to the activation of NLRP3 inflammasome induced by lipopolysaccharide plus adenosine triphosphate. Furthermore, we demonstrated that β-arrestin2 mediated the inhibitory effect of Drd2 on NLRP3 inflammasome activation via interacting with NLRP3 and interfering the inflammasome assembly. Collectively, our study illustrates that astrocytic Drd2 inhibits NLRP3 inflammasome activation through a β-arrestin2-dependent mechanism, and provides a new strategy for treatment of PD.

Oxidative stress, caspase-3 activation and cleavage of ROCK-1 play an essential role in MeHg-induced cell death in primary astroglial cells

Methylmercury is a toxic environmental contaminant that elicits significant toxicity in humans. The central nervous system is the primary target of toxicity, and is particularly vulnerable during development. Rho-associated protein kinase 1 (ROCK-1) is a major downstream effector of the small GTPase RhoA and a direct substrate of caspase-3. The activation of ROCK-1 is necessary for membrane blebbing during apoptosis. In this work, we examined whether MeHg could affect the RhoA/ROCK-1 signaling pathway in primary cultures of mouse astrocytes. Exposure of cells with 10 μM MeHg decreased cellular viability after 24 h of incubation. This reduction in viability was preceded by a significant increase in intracellular and mitochondrial reactive oxygen species levels, as well as a reduced NAD+/NADH ratio. MeHg also induced an increase in mitochondrial-dependent caspase-9 and caspase-3, while the levels of RhoA protein expression were reduced or unchanged. We further found that MeHg induced ROCK-1 cleavage/activation and promoted LIMK1 and MYPT1 phosphorylation, both of which are the best characterized ROCK-1 downstream targets. Inhibiting ROCK-1 and caspases activation attenuated the MeHg-induced cell death. Collectively, these findings are the first to show that astrocytes exposed to MeHg showed increased cleavage/activation of ROCK-1, which was independent of the small GTPase RhoA.

Inhibition of Inwardly Rectifying Potassium (Kir) 4.1 Channels Facilitates Brain-Derived Neurotrophic Factor (BDNF) Expression in Astrocytes.

Inwardly rectifying potassium (Kir) 4.1 channels in astrocytes regulate neuronal excitability by mediating spatial potassium buffering. Although dysfunction of astrocytic Kir4.1 channels is implicated in the development of epileptic seizures, the functional mechanisms of Kir4.1 channels in modulating epileptogenesis remain unknown. We herein evaluated the effects of Kir4.1 inhibition (blockade and knockdown) on expression of brain-derived neurotrophic factor (BDNF), a key modulator of epileptogenesis, in the primary cultures of mouse astrocytes. For blockade of Kir4.1 channels, we tested several antidepressant agents which reportedly bound to and blocked Kir4.1 channels in a subunit-specific manner. Treatment of astrocytes with fluoxetine enhanced BDNF mRNA expression in a concentration-dependent manner and increased the BDNF protein level. Other antidepressants (e.g., sertraline and imipramine) also increased the expression of BDNF mRNA with relative potencies similar to those for inhibition of Kir4.1 channels. In addition, suppression of Kir4.1 expression by the transfection of small interfering RNA (siRNA) targeting Kir4.1 significantly increased the mRNA and protein levels of BDNF. The BDNF induction by Kir4.1 siRNA transfection was suppressed by the MEK1/2 inhibitor U0126, but not by the p38 MAPK inhibitor SB202190 or the JNK inhibitor SP600125. The present results demonstrated that inhibition of Kir4.1 channels facilitates BDNF expression in astrocytes primarily by activating the Ras/Raf/MEK/ERK pathway, which may be linked to the development of epilepsy and other neuropsychiatric disorders.

Maxi-anion channels play a key role in glutamate-induced ATP release from mouse astrocytes in primary culture.

Astrocytes are an abundant source of ATP, which might be released from the cytosol into extracellular spaces under various conditions and even affect cell fate under some circumstances. In the present study, we aimed to evaluate the pathway(s) contributing toward glutamate-induced ATP release from mouse astrocytes. Firstly, our study of cultured astrocytes showed marked ATP release in response to stimuli of glutamate at different concentrations (0.1-1 mM), with an interesting bimodal distribution in time course. Inhibitors or blockers of potential pathways for ATP release such as exocytotic vesicular release, gap junction hemichannels, P2X7 receptors, and volume-sensitive outwardly rectifying chloride channels had no significant effects on the observed ATP release. In contrast, glutamate-induced ATP release from astrocytes was significantly inhibited by gadolinium (50 µM), an inhibitor of a maxi-anion channel; meanwhile, the application of gadolinium can allay glutamate-induced cell injury significantly. Thus, we propose that the maxi-anion channel might play an important role in glutamate-induced ATP release from mouse astrocytes and inhibition of maxi-anion channel activities to reduce ATP release can produce protective effects in the case of glutamate stimuli.

HDLs and LDLs in the Brain: Characterizing the Lipid Secretome of Astrocytes

Comprising up to a hundred‐fold more lipid than a typical cell, a neuron has extensive processes like dendrites and axons. The magnitude of the neuronal membrane surface area suggests the importance of the regulation of the lipids in the membrane to maintain normal neuronal function. While particles like high density lipoproteins (HDL), low density lipoproteins (LDL), and very low density lipoproteins (VLDL) normally circulate throughout the body for lipid regulation, these particles cannot pass the blood brain barrier. The brain must synthesize lipids locally in order to fulfill the biosynthetic/repair needs of the neurons. Lipid synthesis and transportation in the brain are facilitated by astrocytes, the helper glial cells of the nervous system. Astrocytes package and secrete lipids into “HDL/LDL‐like” particles, comprising a lipid core and a shell of proteins, like apolipoproteins, on the exterior. These particles function to regulate the lipid membrane of adjacent neurons, but they also vary in functionality depending on the particles' ratio of lipids to proteins (or how “LDL‐like” or “HDL‐like” the particles are). For example, apolipoprotein E (apoE) is a protein that is synthesized by astrocytes and is essential for cholesterol transportation to neurons. In Alzheimer's Disease (AD) specifically, the apoE4 polymorphism is implicated in higher AD frequency than other apoE polymorphisms because apoE4 is a weaker carrier of cholesterol than the other apoE variants. The apoE variant functionality suggests the imperative role of cholesterol homeostasis to maintain normal function in neurons. ApoE has also been shown to be the main constituent of the HDL‐like particles in the brain. Our initial studies of the lipid secretome of primary cultured mouse astrocytes revealed that the younger astrocytes secreted nearly ten‐fold less cholesterol than the older astrocytes while protein secretions between the different ages were not significantly different. These results suggest the “HDL‐like” particles are secreted more by the younger astrocytes while the “LDL‐like” particles are secreted by older astrocytes, revealing how aging plays a role in lipid homeostasis. We are continuing to isolate the lipid secretome of our primary astrocytes by using the lipid‐binding resin, Cleanascite. In order to understand the composition of these lipid particles, we are using thin layer chromatography to observe the presence of different lipids qualitatively and quantitatively.Support or Funding InformationMach Research Fellowship and Cowles Professorship