Primary Rat Cortical Astrocytes Research Articles

Tau depletion in human neurons mitigates Aβ-driven toxicity.

Alzheimer's disease (AD) is an age-related neurodegenerative condition and the most common type of dementia, characterised by pathological accumulation of extracellular plaques and intracellular neurofibrillary tangles that mainly consist of amyloid-β (Aβ) and hyperphosphorylated tau aggregates, respectively. Previous studies in mouse models with a targeted knock-out of the microtubule-associated protein tau (Mapt) gene demonstrated that Aβ-driven toxicity is tau-dependent. However, human cellular models with chronic tau lowering remain unexplored. In this study, we generated stable tau-depleted human induced pluripotent stem cell (iPSC) isogenic panels from two healthy individuals using CRISPR-Cas9 technology. We then differentiated these iPSCs into cortical neurons in vitro in co-culture with primary rat cortical astrocytes before conducting electrophysiological and imaging experiments for a wide range of disease-relevant phenotypes. Both AD brain derived and recombinant Aβ were used in this study to elicit toxic responses from the iPSC-derived cortical neurons. We showed that tau depletion in human iPSC-derived cortical neurons caused considerable reductions in neuronal activity without affecting synaptic density. We also observed neurite outgrowth impairments in two of the tau-depleted lines used. Finally, tau depletion protected neurons from adverse effectsby mitigating the impact of exogenous Aβ-induced hyperactivity, deficits in retrograde axonal transport of mitochondria, and neurodegeneration. Our study established stable human iPSC isogenic panels with chronic tau depletion from two healthy individuals. Cortical neurons derived from these iPSC lines showed that tau is essential in Aβ-driven hyperactivity, axonal transport deficits, and neurodegeneration, consistent with studies conducted in Mapt-/- mouse models. These findings highlight the protective effects of chronic tau lowering strategies in AD pathogenesis and reinforce the potential in clinical settings. The tau-depleted human iPSC models can now be applied at scale to investigate the involvement of tau in disease-relevant pathways and cell types.

Analysis of the Effect of Substrate Stiffness on the Efficacy of Fibroblast Growth Factor 2 and Bone Morphogenetic Protein 4 in Inducing Pro-Regenerative Astrocyte Phenotype: A Research Protocol

Introduction: Astrocytes are glial cells essential for neuronal development and repair and are thus a highly promising target for regenerative therapies for neurodegenerative disease. Neurodegeneration has been connected to the degradation of extracellular matrix (ECM) components. ECM's structural properties, such as stiffness, influence the phenotypic outcomes of growing cells. Notably, astrocytes are induced into an A2 (pro-regenerative) phenotype when grown on stiff substrates or exposed to specific signalling molecules, particularly fibroblast growth factor 2 (FGF-2) and bone morphogenetic protein 4 (BMP-4). Given that the ECM can bind and sequester growth factors, it is possible that matrix stiffness may modulate the efficacy of these molecules. Methods: In this in vitro study, rat primary cortical astrocytes will be cultured on soft and stiff substrates and exposed to varying amounts of FGF-2 and BMP-4. This study aims to determine the effect of substrate stiffness on the efficacy of FGF-2 and BMP-4 in promoting pro-regenerative phenotype. Cell proliferation and glial fibrillary acidic protein (GFAP) expression are key indicators of reactive astrocytes, including pro-regenerative astrocytes. 5-bromo-2-deoxyuridine staining will be used to analyze proliferation. GFAP expression will be determined using anti-GFAP antibody conjugated with Alexa Fluor 594. Further, pro-regenerative phenotypic genes Clcf1, Tgm2, and Ptgs2 will be detected via polymerase chain reaction to differentiate from pro-inflammatory astrocytes, a separate category of reactive astrocytes. Anticipated Results: We hypothesize there will be a greater positive correlation between the concentration of FGF-2 or BMP-4 and expression of markers of pro-regenerative phenotype under stiff substrate conditions, compared to softer substrate, thus indicating dependency or synergy between FGF-2 or BMP-4 and extracellular matrix-dependent pathways. Discussion: The correlation between FGF-2 or BMP-4 concentration and the prominence of A2 astrocyte phenotype indicators will be graphed and reported along with a comparison of these correlations between soft and stiff substrate groups. The authors will attempt to conclude whether substrate stiffness significantly effects the activities of FGF-2 or BMP-4. Conclusion: We hope the results of this proposed study will inform the development of neuroregenerative therapies involving astrocytes by indicating the necessity for greater focus on either the application of signalling molecules or modification of ECM.

Docosahexaenoic acid increased MeCP2 mediated mitochondrial respiratory complexes II and III enzyme activities in cortical astrocytes.

Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the methyl-CpG-binding protein 2 (MeCP2) in the neurons and glial cells of the central nervous system. Currently, therapeutics for RTT is aimed at restoring the loss-of-function by MeCP2 gene therapy, but that approach has multiple challenges. We have already reported impaired mitochondrial bioenergetics in MeCP2 deficient astrocytes. Docosahexaenoic acid (DHA), a polyunsaturated fatty acid, has been shown with health benefits, but its impact on mitochondrial functions in MeCP2 deficient astrocytes has never been paid much attention. The present study aimed to investigate the effects of DHA on mitochondrial respiratory chain regulation in MeCP2 knockdown astrocytes. We determined NADH dehydrogenase (ubiquinone) flavoprotein 2 (Ndufv2-complex-I), Ubiquinol cytochrome c reductase core protein (Uqcrc1-complex-III) genes expression, Ndufv2 protein expression, respiratory electron transport chain complex I, II, III, and IV enzyme activities, intracellular Ca+2 , reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) in DHA pre-incubated MeCP2 knock-down rat primary cortical astrocytes. Our study demonstrates that 100 µM DHA increases MeCP2 gene and protein expression. Increases brain-derived neurotrophic factor (BDNF) and Uqcrc1 gene expression, Ndufv2 protein expression, but has no effect on glial fibrillary acidic protein (GFAP) gene expression. DHA treatment also increases mitochondrial respiratory Complexes II and III activities and reduces intracellular calcium levels. Taken together, the effects of DHA seem independent of MeCP2 deficiency in astrocytes. Hence, further studies are warranted to understand the complicated mechanisms of DHA and for its therapeutic significance in MeCP2-mediated mitochondrial dysfunction and in RTT disease.

Senescence in Primary Rat Astrocytes Induces Loss of the Mitochondrial Membrane Potential and Alters Mitochondrial Dynamics in Cortical Neurons.

The decline in brain function during aging is one of the most critical health problems nowadays. Although senescent astrocytes have been found in old-age brains and neurodegenerative diseases, their impact on the function of other cerebral cell types is unknown. The aim of this study was to evaluate the effect of senescent astrocytes on the mitochondrial function of a neuron. In order to evaluate neuronal susceptibility to a long and constant senescence-associated secretory phenotype (SASP) exposure, we developed a model by using cellular cocultures in transwell plates. Rat primary cortical astrocytes were seeded in transwell inserts and induced to premature senescence with hydrogen peroxide [stress-induced premature senescence (SIPS)]. Independently, primary rat cortical neurons were seeded at the bottom of transwells. After neuronal 6 days in vitro (DIV), the inserts with SIPS-astrocytes were placed in the chamber and cocultured with neurons for 6 more days. The neuronal viability, the redox state [reduced glutathione/oxidized glutathione (GSH/GSSG)], the mitochondrial morphology, and the proteins and membrane potential were determined. Our results showed that the neuronal mitochondria functionality was altered after being cocultured with senescent astrocytes. In vivo, we found that old animals had diminished mitochondrial oxidative phosphorylation (OXPHOS) proteins, redox state, and senescence markers as compared to young rats, suggesting effects of the senescent astrocytes similar to the ones we observed in vitro. Overall, these results indicate that the microenvironment generated by senescent astrocytes can affect neuronal mitochondria and physiology.

Selective in vitro anticancer effect of blueberry extract (Vaccinium virgatum) against C6 rat glioma: exploring their redox status.

The aim of this study was to investigate the anticancer potential of blueberry extract (Vaccinium virgatum) against a C6 rat glioma lineage. Cultures of the C6 cells were exposed to blueberry extract at concentrations of 50 to 600µg/mL for 12, 24, 48, or 72h and then evaluated for cell viability, proliferation, migration, colony formation and oxidative stress. We also evaluated the effects of blueberry extract on primary rat cortical astrocytes. Our results show that treatment with blueberry extract did not alter the viability or proliferation of normal primary astrocytes but it did significantly reduce the viability in 21.54 % after 48h and proliferation in 8.59 % after 24h of C6 cells at 200µg/mL. We also observed a reduction in the size of the colonies of 29.99 % at 100µg/mL when compared to the control cells and cell migration was also reduced at 50µg/mL. After 72h, there was a reduction in the reactive oxygen species levels ranging from 46.26 to 34.73 %, in addition to a 380.2 % increase in total thiol content. Superoxide dismutase, catalase, and glutathione S-transferase activities were also enhanced when compared to the control. Taken together this data suggests that blueberry extract exerts some selective anticancer activity in C6 glioma cells.

Effects of ethanol-and choline-treated astrocytes on hippocampal neuron neurite outgrowth in vitro.

Exposure to ethanol in utero can result in Fetal Alcohol Spectrum Disorders, which may cause long-lasting cognitive and behavioral abnormalities. Preclinical studies indicate that choline ameliorates the behavioral effects of developmental alcohol exposure in rodents, and clinical studies on the effectiveness of choline, administered early in pregnancy, showed that the adverse effects of heavy prenatal alcohol exposure on postnatal growth, and cognition in human infants were mitigated. However, little is known on the mechanisms behind the effects of choline. We have previously reported that astrocyte pre-treatment with 75 mM ethanol, in vitro, reduces neurite outgrowth in hippocampal neurons co-cultured with the pre-treated astrocytes. Our in vitro system allows us to study the effects of chemicals on astrocyte functions, able to modulate neuronal development. The main objective was to test the hypothesis that choline can ameliorate the astrocyte-mediated effects of ethanol on neurite growth. In this study, we exposed primary rat cortical astrocytes to ethanol, choline, ethanol plus choline, or control conditions for 24 h. Culture media was then removed, replaced with fresh media containing no ethanol or choline treatments and primary rat hippocampal neurons were plated on top of the astrocyte monolayer and cultured for 16 h. Neurons were then stained for β-III Tubulin and neurite outgrowth was measured. Astrocyte exposure to ethanol (25, 50, and 75 mM) decreases neurite outgrowth in co-cultured hippocampal pyramidal neurons, while astrocyte treatment with choline had no effect. Astrocyte treatment with ethanol and choline in combination, however, prevented the effect of ethanol, leading to levels of neurite outgrowth similar the control condition. Choline prevents the inhibitory effect of ethanol-treated astrocytes on neurite outgrowth while not altering normal neuronal development. These results suggest a new, astrocyte-mediated mechanism by which choline ameliorates the effects of developmental alcohol exposure.

The Anemonia sulcata Toxin BDS-I Protects Astrocytes Exposed to Aβ1-42 Oligomers by Restoring [Ca2+]i Transients and ER Ca2+ Signaling.

Intracellular calcium concentration ([Ca2+]i) transients in astrocytes represent a highly plastic signaling pathway underlying the communication between neurons and glial cells. However, how this important phenomenon may be compromised in Alzheimer’s disease (AD) remains unexplored. Moreover, the involvement of several K+ channels, including KV3.4 underlying the fast-inactivating currents, has been demonstrated in several AD models. Here, the effect of KV3.4 modulation by the marine toxin blood depressing substance-I (BDS-I) extracted from Anemonia sulcata has been studied on [Ca2+]i transients in rat primary cortical astrocytes exposed to Aβ1–42 oligomers. We showed that: (1) primary cortical astrocytes expressing KV3.4 channels displayed [Ca2+]i transients depending on the occurrence of membrane potential spikes, (2) BDS-I restored, in a dose-dependent way, [Ca2+]i transients in astrocytes exposed to Aβ1–42 oligomers (5 µM/48 h) by inhibiting hyperfunctional KV3.4 channels, (3) BDS-I counteracted Ca2+ overload into the endoplasmic reticulum (ER) induced by Aβ1–42 oligomers, (4) BDS-I prevented the expression of the ER stress markers including active caspase 12 and GRP78/BiP in astrocytes treated with Aβ1–42 oligomers, and (5) BDS-I prevented Aβ1–42-induced reactive oxygen species (ROS) production and cell suffering measured as mitochondrial activity and lactate dehydrogenase (LDH) release. Collectively, we proposed that the marine toxin BDS-I, by inhibiting the hyperfunctional KV3.4 channels and restoring [Ca2+]i oscillation frequency, prevented Aβ1–42-induced ER stress and cell suffering in astrocytes.

Establishment and Preliminary Characterization of Three Astrocytic Cells Lines Obtained from Primary Rat Astrocytes by Sub-Cloning.

Gliomas are complex and heterogeneous tumors that originate from the glial cells of the brain. The malignant cells undergo deep modifications of their metabolism, and acquire the capacity to invade the brain parenchyma and to induce epigenetic modifications in the other brain cell types. In spite of the efforts made to define the pathology at the molecular level, and to set novel approaches to reach the infiltrating cells, gliomas are still fatal. In order to gain a better knowledge of the cellular events that accompany astrocyte transformation, we developed three increasingly transformed astrocyte cell lines, starting from primary rat cortical astrocytes, and analyzed them at the cytogenetic and epigenetic level. In parallel, we also studied the expression of the differentiation-related H1.0 linker histone variant to evaluate its possible modification in relation with transformation. We found that the most modified astrocytes (A-FC6) have epigenetic and chromosomal alterations typical of cancer, and that the other two clones (A-GS1 and A-VV5) have intermediate properties. Surprisingly, the differentiation-specific somatic histone H1.0 steadily increases from the normal astrocytes to the most transformed ones. As a whole, our results suggest that these three cell lines, together with the starting primary cells, constitute a potential model for studying glioma development.

Itaconate modulates tricarboxylic acid and redox metabolism to mitigate reperfusion injury

ObjectivesCerebral ischemia/reperfusion (IR) drives oxidative stress and injurious metabolic processes that lead to redox imbalance, inflammation, and tissue damage. However, the key mediators of reperfusion injury remain unclear, and therefore, there is considerable interest in therapeutically targeting metabolism and the cellular response to oxidative stress. MethodsThe objective of this study was to investigate the molecular, metabolic, and physiological impact of itaconate treatment to mitigate reperfusion injuries in in vitro and in vivo model systems. We conducted metabolic flux and bioenergetic studies in response to exogenous itaconate treatment in cultures of primary rat cortical neurons and astrocytes. In addition, we administered itaconate to mouse models of cerebral reperfusion injury with ischemia or traumatic brain injury followed by hemorrhagic shock resuscitation. We quantitatively characterized the metabolite levels, neurological behavior, markers of redox stress, leukocyte adhesion, arterial blood flow, and arteriolar diameter in the brains of the treated/untreated mice. ResultsWe demonstrate that the “immunometabolite” itaconate slowed tricarboxylic acid (TCA) cycle metabolism and buffered redox imbalance via succinate dehydrogenase (SDH) inhibition and induction of anti-oxidative stress response in primary cultures of astrocytes and neurons. The addition of itaconate to reperfusion fluids after mouse cerebral IR injury increased glutathione levels and reduced reactive oxygen/nitrogen species (ROS/RNS) to improve neurological function. Plasma organic acids increased post-reperfusion injury, while administration of itaconate normalized these metabolites. In mouse cranial window models, itaconate significantly improved hemodynamics while reducing leukocyte adhesion. Further, itaconate supplementation increased survival in mice experiencing traumatic brain injury (TBI) and hemorrhagic shock. ConclusionsWe hypothesize that itaconate transiently inhibits SDH to gradually “awaken” mitochondrial function upon reperfusion that minimizes ROS and tissue damage. Collectively, our data indicate that itaconate acts as a mitochondrial regulator that controls redox metabolism to improve physiological outcomes associated with IR injury.

Cellular Model of Endotoxin Tolerance in Astrocytes: Role of Interleukin 10 and Oxylipins.

A phenomenon of endotoxin tolerance where prior exposure of cells to minute amounts of lipopolysaccharide (LPS) causes them to become refractory to a subsequent high-amount endotoxin challenge is well described for innate immune cells such as monocytes/macrophages, but it is still obscure for brain cells. We exposed primary rat cortical astrocytes to a long-term low-grade concentration of LPS, followed by stimulation with a middle-grade concentration of LPS. Inflammatory markers, i.e., pro-inflammatory cytokine TNFα, inducible enzymes COX-2 and iNOS, anti-inflammatory cytokine interleukin 10 (IL-10) detected at the mRNA and protein levels reveal similarities between astrocytes and macrophages in the model, i.e., tolerance in pro-inflammatory markers and priming in IL-10. Long-term or short-term treatment with IL-10 does not change cell sensitivity for LPS, which makes doubtful its involvement in the mechanisms of cell tolerance development. Significant changes occur in the oxylipin profiles measured by UPLC-MS/MS analysis. The priming occurs in the following compounds: 11-HETE, PGD2, PGE2, cyclopentenone prostaglandins, and TXB2. Tolerance is observed for 12-HHT, PGF2α, and 6-keto-PGF1α. As far as we know, this is the first report on changes in oxylipin profiles in the endotoxin tolerance model. The data can greatly improve the understanding of oxylipins’ role in inflammatory and resolution processes in the brain and mechanisms of astrocyte involvement in neuroinflammation.

TRAUMATIC INJURY OF ASTROCYTES: AN IN VITRO STUDY

The objective of this work is to determine the injury criterion for primary rat cortical astrocytes through an in vitro traumatic injury model. The compressed air pressure was used to reproduce typical blast pressure profile, which could induce biaxial strain up to 100% in millisecond for cells cultured on flexible membrane utilizing a controlled cellular injury (CCI) device. The nominal pressure and time settings could be adjusted to accommodate a wide range of membrane strain and strain rate, which was estimated from finite element models. The relationship between the peak membrane displacement/strain and the nominal settings of the CCI device was then established. The model was calibrated using both high-speed imaging system and a theoretical model. The viability and morphology of the astrocytes were characterized and correlated with the strain level. Three different regimes were identified in the stretch-induced dose-response curves of the primary cortical astrocytes, with a sharp decline from live to dead in a narrow range of membrane strain (18%–35%). The level of actin organization of the astrocytes decreased as the membrane strain increased. This work could facilitate the understanding of cellar behaviors subjected to mild blast loadings and the potential tissue engineering therapeutics.

Biomimetic graphene for enhanced interaction with the external membrane of astrocytes.

Graphene and graphene substrates display huge potential as material interfaces for devices and biomedical tools targeting the modulation or recovery of brain functionality. However, to be considered reliable neural interfaces, graphene-derived substrates should properly interact with astrocytes, favoring their growth and avoiding adverse gliotic reactions. Indeed, astrocytes are the most abundant cells in the human brain and they have a crucial physiological role to maintain its homeostasis and modulate synaptic transmission. In this work, we describe a new strategy based on the chemical modification of graphene oxide (GO) with a synthetic phospholipid (PL) to improve interaction of GO with brain astroglial cells. The PL moieties were grafted on GO sheets through polymeric brushes obtained by atom-transfer radical-polymerization (ATRP) between acryloyl-modified PL and GO nanosheets modified with a bromide initiator. The adhesion of primary rat cortical astrocytes on GO-PL substrates increased by about three times with respect to that on glass substrates coated with standard adhesion agents (i.e. poly-d-lysine, PDL) as well as with respect to that on non-functionalized GO. Moreover, we show that astrocytes seeded on GO-PL did not display significant gliotic reactivity, indicating that the material interface did not cause a detrimental inflammatory reaction when interacting with astroglial cells. Our results indicate that the reported biomimetic approach could be applied to neural prosthesis to improve cell colonization and avoid glial scar formation in brain implants. Additionally, improved adhesion could be extremely relevant in devices targeting neural cell sensing/modulation of physiological activity.

Metformin limits apoptosis in primary rat cortical astrocytes subjected to oxygen and glucose deprivation.

Metformin, atype 2 anti-diabetic drug and an activator of AMP-activated protein kinase (AMPK), has been shown to reduce infarct size and pathological changes affecting astroglia in animal models of ischemic stroke. In this study, we evaluated how metformin affects cell viability, apoptosis and determined the role of AMPK, as well as JNK p46/p54 and p38 kinases, in the observed phenomena in the culture of primary rat cortical astrocytes subjected to 12 h of oxygen and glucose deprivation (OGD). Metformin improved cell viability, reduced the fraction of apoptotic nuclei, and inhibited the activation of the executive caspase-3. Decreased activation of JNK p54 and p38 was associated with increased Bcl-XL expression and decreased mitochondrial leakage of cytochrome c. However, only cell viability and partially the fraction of apoptotic nuclei varied concomitantly with changes in AMPK activity, suggesting that AMPK is critical for metformin-mediated effects and regulates programmed cell death in acaspase-independent manner. Experiments with the inhibitors of JNK and p38 supports the role of these kinases in the drug-related inhibition of mitochondrial and extrinsic pathway of apoptosis.

Lanthanum chloride reduces lactate production in primary culture rat cortical astrocytes and suppresses primary co-culture rat cortical astrocyte-neuron lactate transport.

Lanthanum (La) can impair learning memory and induce behavioral abnormalities in animals. However, the mechanism underlying these adverse effects of La is still elusive. It has been demonstrated that lactate derived from astrocytes is the major energy source for neurons during long-term memory (LTM) formation and the deficiency of lactate supply can result in LTM damage. However, little work has been done with respect to the impact of La on the lactate production in astrocytes and astrocyte-neuron lactate transport (ANLT). Herein, experiments were undertaken to explore if there was such an adverse effect of La. Primary culture rat cortical astrocytes and primary co-culture rat cortical astrocyte-neuron were treated with (0.125, 0.25 and 0.5mM) lanthanum chloride (LaCl3) for 24h. The results showed that LaCl3 treatment significantly downregulated the mRNA and protein expression of glucose transporter 1 (GLUT1), glycogen synthase (GS), glycogen phosphorylase (GP), lactate dehydrogenase A (LDHA), and monocarboxylate transporter 1, 2 and 4 (MCT 1 2 and 4); upregulated the mRNA and protein expression of lactate dehydrogenase B (LDHB); and decreased the glycogen level, total LDH and GP activity, GS/p-GS ratio and lactate contents. Moreover, rolipram (20, 40μM) or forskolin (20, 40μM) could increase the lactate content by upregulating GP expression and the GS/p-GS ratio, as well as antagonize the effects of La. These results suggested that La-induced learning-memory damage was probably related to its suppression of lactate production in astrocytes and ANLT. This study provides some novel clues for clarifying the mechanism underlying the neurotoxicity of La.

Sex differences in the mitochondrial bioenergetics of astrocytes but not microglia at a physiologically relevant brain oxygen tension

Biological sex is thought to influence mitochondrial bioenergetic function. Previous respiration measurements examining brain mitochondrial sex differences were made at atmospheric oxygen using isolated brain mitochondria. Oxygen is 160 mm Hg (21%) in the atmosphere, while the oxygen tension in the brain generally ranges from ∼5 to 45 mm Hg (∼1–6% O2). This study tested the hypothesis that sex and/or brain physiological oxygen tension influence the mitochondrial bioenergetic properties of primary rat cortical astrocytes and microglia. Oxygen consumption was measured with a Seahorse XF24 cell respirometer in an oxygen-controlled environmental chamber. Strikingly, male astrocytes had a higher maximal respiration than female astrocytes when cultured and assayed at 3% O2. Three percent O2 yielded a low physiological dissolved O2 level of ∼1.2% (9.1 mm Hg) at the cell monolayer during culture and 1.2–3.0% O2 during assays. No differences in bioenergetic parameters were observed between male and female astrocytes at 21% O2 (dissolved O2 of ∼19.7%, 150 mm Hg during culture) or between either of these cell populations and female astrocytes at 3% O2. In contrast to astrocytes, microglia showed no sex differences in mitochondrial bioenergetic parameters at either oxygen level, regardless of whether they were non-stimulated or activated to a proinflammatory state. There were also no O2- or sex-dependent differences in proinflammatory TNF-α or IL-1β cytokine secretion measured at 18 h activation. Overall, results reveal an intriguing sex variance in astrocytic maximal respiration that requires additional investigation. Findings also demonstrate that sex differences can be masked by conducting experiments at non-physiological O2.

Rhein and rhubarb similarly protect the blood-brain barrier after experimental traumatic brain injury via gp91phox subunit of NADPH oxidase/ROS/ERK/MMP-9 signaling pathway.

Oxidative stress chiefly contributes to the disruption of the BBB following traumatic brain injury (TBI). The Chinese herbal medicine rhubarb is a promising antioxidant in treating TBI. Here we performed in vivo and in vitro experiments to determine whether rhubarb and its absorbed bioactive compound protected the BBB after TBI by increasing ZO-1 expression through inhibition of gp91phox subunit of NADPH oxidase/ROS/ERK/MMP-9 pathway. Rats were subjected to the controlled cortical impact (CCI) model, and primary rat cortical astrocytes were exposed to scratch-wound model. The liquid chromatography with tandem mass spectrometry method showed that rhein was the compound absorbed in the brains of CCI rats after rhubarb administration. The wet-dry weights and Evans blue measurements revealed that rhubarb and rhein ameliorated BBB damage and brain edema in CCI rats. Western blots showed that rhubarb and rhein downregulated GFAP in vitro. RT-PCR, immunohistochemistry, Western blot and dichlorodihydrofluorescein diacetate analysis indicated that rhubarb prevented activation of gp91phox subunit of NADPH oxidase induced ROS production, subsequently inhibited ERK/MMP-9 pathway in vivo and in vitro. Interestingly, rhein and rhubarb similarly protected the BBB by inhibiting this signaling cascade. The results provide a novel herbal medicine to protect BBB following TBI via an antioxidative molecular mechanism.

Regulation and the Mechanism of Estrogen on Cav1.2 Gene in Rat-Cultured Cortical Astrocytes.

L-type calcium channel (LTCC) gene Cav1.2 is believed to play an important role in the alteration of Ca(2+) homeostasis in brain astrocytes. Increasing evidence shows that alteration of intracellular Ca(2+) concentration is related to the effect of 17β-estradiol (E2) in a variety of neurophysiological and neuropathological conditions. In this study, we measured immunoreactivity of Cav1.2 protein expression in rat primary cortical astrocytes by using Western blots. We demonstrated that E2 upregulated Cav1.2 expression in a dose- and time-dependent manner and the effect of E2 on Cav1.2 expression were blocked by an estrogen receptor (ER) antagonist, ICI-182,780. The ER subtype-selective ERα agonists propylpyrazole triole (PPT) and ERβ agonist diarylpropionitrile (DPN) both increase the expression of Cav1.2 in a dose-dependent manner. Also, the PPT most closely mimicked the upregulation of Cav1.2 protein expression by E2. Similar experiments of 10nM E2-treated ERα- or ERβ-knockdown astrocytes have also shown that the E2 regulation of Cav1.2 protein expression is mediated through an ERα-dependent pathway. Furthermore, we established that E2 did not change the level of Cav1.2 mRNA. The induction of E2-mediated Cav1.2 expression was inhibited by cycloheximide (CHX) but not by actinomycin D (Act-D), suggesting that E2 regulation of Cav1.2 expression occurred at a posttranscriptional level. We also found that E2 may increase Cav1.2 levels by decreasing its ubiquitination and degradation rate. These findings provide new information about the effect of E2 on Cav1.2 in astrocytes, particularly necessary for the treatment of neurological disease.

Enhanced expression of the calcium-sensing receptor in reactive astrocytes following ischemic injury in vivo and in vitro

We recently demonstrated that the G protein-coupled calcium-sensing receptor (CaSR) is associated with the pathogenesis of ischemic stroke and may be involved in vascular remodeling and astrogliosis. To further substantiate the involvement of CaSR in the astroglial reaction common to ischemic insults, we investigated the temporal and cell type-specific expression patterns of CaSR in the hippocampus after transient forebrain ischemia. CaSR was constitutively expressed in neurons of the pyramidal and granule cell layers, whereas increased CaSR immunoreactivity was observed in reactive astrocytes, but not in activated microglia or macrophages, in the CA1 region of the post-ischemic hippocampus. Astroglial induction of CaSR expression was evident on days 3–7 after reperfusion and appeared to increase progressively through day 28, at which time CaSR expression was prominent in astrocytes with a highly reactive hypertrophic phenotype and elevated levels of glial fibrillary acidic protein. This expression pattern was supported by results of immunoblot analyses. Furthermore, CaSR expression was upregulated in rat primary cortical astrocytes exposed to oxygen–glucose deprivation, which undergo reactive gliosis-like changes. Thus, our results demonstrate that selective and long-lasting astroglial induction of CaSR expression is a common characteristic of ischemic injury and suggest its involvement in the ischemia-induced astroglial reaction.

PFKFB3-mediated glycolysis is involved in reactive astrocyte proliferation after oxygen-glucose deprivation/reperfusion and is regulated by Cdh1

Reactive astrocyte proliferation is involved in many central degenerative diseases. The enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isoform 3 (PFKFB3), an allosteric activator of 6-phosphofructo-1-kinase (PFK1), controls glycolytic flux. Furthermore, APC/C-Cdh1 plays a crucial role in brain metabolism by regulating PFKFB3 expression. Previous studies have defined the roles of PFKFB3-mediated glycolysis in pathological angiogenesis, cell autophagy, and amyloid plaque deposition in proliferating cells. However, the role of PFKFB3 in reactive astrocyte proliferation after cerebral ischemia is unknown. In this study, we cultured rat primary cortical astrocytes and established an oxygen-glucose deprivation/reperfusion (OGD/R) model to mimic cerebral ischemia in vivo. Astrocyte proliferation was measured by western blotting for proliferating cell nuclear antigen (PCNA) and by EdU incorporation. We found that OGD/R up-regulated PFKFB3 and PFK1 expression, which was accompanied by reactive astrocyte proliferation. Knockdown of PFKFB3 by siRNA transfection significantly inhibited reactive astrocyte proliferation and lactate release, an indicator of glycolysis. We found that PFKFB3 and PFK1 expression were down-regulated and lactate release was decreased when OGD/R-induced astrocyte proliferation was inhibited by a Cdh1-expressing lentivirus. Thus, reactive astrocyte proliferation can be effectively suppressed by down-regulation of PFKFB3 through control of glycolytic flux, which is downstream of APC/C-Cdh1.

Abstract 1792: Radiation-induced downregulation of GLT-1 glutamate transporter mRNA expression is reversed by renin-angiotensin system inhibitors

Abstract Fractionated partial or whole brain irradiation (fWBI) is often required to treat metastatic brain cancer. Unfortunately, fWBI can lead to the development of a progressive, irreversible cognitive impairment in patients who survive longer than six months following treatment. Prior studies suggest that neuroinflammation and alterations in neuronal plasticity may play a role in this treatment-related side effect. In the current study we determined that radiation-induced inflammatory activation of primary rat cortical astrocytes results in downregulation of mRNA and protein for the GLT-1 glutamate transporter, which functions to regulate glutamate signaling at excitatory synapses. Furthermore, we show that delivery of clinically-relevant fWBI (40Gy, 5Gy/fraction, 2x/week) to young adult male rats results in 51% decrease in Glt-1 mRNA in the cortex at 48 hours. Two months after completion of fWBI, cortical GLT-1 mRNA is further decreased to 25% the levels observed in sham controls, while at six months following fWBI GLT-1 levels remain 43% decreased compared to controls. We have previously identified two classes of compounds, the renin-angiotensin system inhibitors and PPAR agonists, which ameliorate the fWBI-induced cognitive impairment in this rodent model, although the full mechanisms by which these compounds protect cognition remain unknown. We found that dietary administration of the angiotensin receptor blocker L-158-809, but not the ACE inhibitor Ramipril, prevents fWBI-induced loss of cortical GLT-1 mRNA at 48 hours, while both cognition-sparing compounds restored GLT-1 expression at 2 months post-fWBI. Similarly, administration of the PPARδ agonist GW0742 prevented the fWBI-induced decrease of GLT-1 mRNA observed 6 months post-fWBI. Together these data suggest that radiation-induced downregulation of GLT-1 is a potential mechanism contributing to the development of radiation-induced brain injury, including cognitive impairment, and that GLT-1 may represent a druggable target to prevent this treatment-related cognitive decline. (Supported by NIH CA112593) Citation Format: Mitra Kooshki, Christine Naczki, Michael E. Robbins, Linda J. Metheny-Barlow. Radiation-induced downregulation of GLT-1 glutamate transporter mRNA expression is reversed by renin-angiotensin system inhibitors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1792. doi:10.1158/1538-7445.AM2015-1792