•MC-LF and MC-LW, but not MC-LR, are toxic to astrocytes.•MC-LW and MC-LF induce apoptosis of rat cortical astrocytes.•MC-LW and MC-LF intoxication induce GFAP, actin and tubulin network ...degradation.•Rat cortical astrocytes express Oatp1a4, Oatp1c1 and Oatp1a5, but not Oatp1b2.
Microcystins (MCs) comprise a group of cyanobacterial toxins with hepatotoxic, nephrotoxic and, possibly, neurotoxic activity in mammals. In order to understand the development of their neurotoxicity we investigated the toxic effects of MC variants, MC-LR, MC-LW and MC-LF, in astrocytes that play a central role in maintaining brain homeostasis. 24h exposure of cultured rat cortical astrocytes to MCs revealed dose-dependent toxicity of MC-LF and MC-LW, but not of MC-LR, observed by significant reduction in cell number, declined viability monitored by MTT test and an increased percentage of apoptotic cells, confirmed by Annexin-V labelling. The cultured astrocytes expressed organic anion-transporting polypeptides (Oatp) Oatp1a4, Oatp1c1 and Oatp1a5, but not Oatp1b2. Intracellular localisation of MC-LF and MC-LW, proven by anti-Adda primary antibody, demonstrated transport of tested MCs into cultured astrocytes. Acute MC-LW and MC-LF intoxication induced cytoskeletal disruption as seen by the degradation of glial fibrillary acid protein (GFAP), actin and the tubulin network.
In this in vitro study, MC-LF and MC-LW, but not MC-LR, are shown to cause the dysfunction of astrocytic homeostatic capabilities, already at low concentrations, suggesting that astrocyte atrophy, with loss of function, could be expected in the brain response to the toxic insult.
The influence of cannabidiol (CBD) on brain development is inadequately understood. Since CBD is considered a non-intoxicating drug, it has attracted great interest concerning its potential medical ...applicability, including in pregnant women and children. Here, we elucidated the response of perinatal rat cortical neurons and astrocytes to CBD at submicromolar (0.1, 0.5, 1, 5 µM) concentrations attainable in humans. The effect of CBD was concentration- and time-dependent and cell-specific. In neurons, 0.1 µM CBD induced an early and transient change in mitochondrial membrane potential (ΔΨm), ATP depletion, and caspase-8 activation, followed by rapid ATP recovery and progressive activation of caspase-9 and caspase-3/7, resulting in early apoptotic cell death with reduction and shortening of dendrites, cell shrinkage, and chromatin condensation. The decrease in neuronal viability, ATP depletion, and caspase activation due to CBD exposure was prevented by transient receptor potential vanilloid 1 (TRPV1) antagonist. In astrocytes, 0.5 µM CBD caused an immediate short-term dysregulation of ΔΨm, followed by ATP depletion with transient activation of caspase-8 and progressive activation of caspase-9 and caspase-3/7, leading to early apoptosis and subsequent necroptosis. In astrocytes, both TRPV1 and cannabinoid receptor 1 (CB
) antagonists protected viability and prevented apoptosis. Given that CBD is a non-intoxicating drug, our results clearly show that this is not the case during critical periods of brain development when it can significantly interfere with the endogenous cannabinoid system.
Astrocytes as an active part of the tripartite synapse can respond to the synaptically released neurotransmitters. Because brain-derived neurotrophic factor (BDNF) is produced by astrocytes, in ...addition to neurons, we focused our present study on the regulatory effects of monoamines noradrenaline (NA), serotonin (5-HT), and dopamine (DA) on the synthesis of BDNF protein in rat neonatal astrocytes from specific brain regions (cortex, cerebellum). All tested neurotransmitters are able to potently and transiently increase BDNF cellular contents; their maximal effects are dose and time dependent and differ between the two brain regions. In cultured cortical astrocytes, NA (1
μM; 6
h) elevates BDNF levels by a 4-fold, 5-HT (1
μM; 4
h) by a 2.3-fold, and DA (150
μM; 4
h) by a 2.2-fold. In cerebellar astrocytes, NA (1
μM; 4
h) increases BDNF content by a 4.7-fold, 5-HT (1
μM; 4
h) by a 1.7-fold, and DA (150
μM; 4
h) by a 1.4-fold. The initial increase in the BDNF levels return to basal levels when incubation with monoamines is extended beyond 12
h (for 5-HT) or 24
h (for NA and DA). Our results confirm the involvement of monoaminergic systems in the regulation of BDNF production in astrocytes and suggest the existence of a positive reciprocal interaction between monoaminergic neuronal activity and astrocytic neurotrophic support in neuron–astrocyte crosstalk, which has a dynamic role in mediating neuronal plasticity and trophic functions in the brain.
Brain-derived neurotrophic factor (BDNF) synthesis in astrocytes induced by noradrenaline (NA) is a receptor-mediated process utilizing two parallel adrenergic pathways: β1/β2-adrenergic/cAMP and the ...novel α1-adrenergic/PKC pathway.
BDNF is produced by astrocytes, in addition to neurons, and the noradrenergic system plays a role in controlling BDNF synthesis. Since astrocytes express various subtypes of α- and β-adrenergic receptors that have the potential to be activated by synaptically released NA, we focused our present study on the mediatory role of adrenergic receptors in the noradrenergic up-regulation of BDNF synthesis in cultured neonatal rat cortical astrocytes.
NA (1μM) elevates BDNF levels by four-fold after 6h of incubation. Its stimulation was partly inhibited by either the β1-adrenergic antagonist atenolol, the β2-adrenergic antagonist ICI 118,551, or by the α1-adrenergic antagonist prazosin, while the α2-adrenergic antagonist yohimbine showed no effect. BDNF levels in astrocytes were increased by the specific β1-adrenergic agonist dobutamine and the β2-adrenergic agonist salbutamol, as well as by adenylate cyclase activation (by forskolin) and PKA activation (by dBcAMP). However, none of the tested agonists or mediators of the intracellular β-adrenergic pathways were able to reach the level of NA's stimulatory effect. BDNF cellular levels were also elevated by the α1-adrenergic agonist methoxamine, but not by the α2-adrenergic agonist clonidine. The increase in intracellular Ca2+ by ionophore A23187 showed no effect, whereas PKC activation by phorbol 12-myristate 13-acetate (TPA) potently stimulated BDNF levels in the cells. The methoxamine-stimulated BDNF synthesis was inhibited by desensitizing pretreatment with TPA, indicating that the α1-stimulation was mediated via PKC activation. In conclusion, the synthesis of astrocytic BDNF stimulated by noradrenergic neuronal activity is an adaptable process using multiple types (α1 and β1/β2) of adrenergic receptor activation.
Izhodišča: Zastrupitev z ogljikovim monoksidom (CO) močno okrne funkcijo astrocitov in nevronov. Pozne nevropsihološke posledice zastrupitve lahko preprečimo z zdravljenjem s hiperbaričnim kisikom ...(HBO). V raziskavi smo preučevali učinek CO in HBO na zgodnje procese celične smrti v nevronski in mešani kulturi ter ugotavljali, ali se raven glutationa v astrocitih po izpostavljenosti CO in HBO spremeni in ali bi lahko le-ta bil možna nova tarča za zdravljenje zastrupitve s CO.
Metode: Primarne astrocitne, nevronske in mešane celične kulture možganske skorje podgane smo izpostavili 3.000 ppm CO v zraku, nato pa jih v obdobju 24-urne normoksije v različnih časovnih presledkih za 1 uro izpostavili 100-odstotnemu kisiku pri tlaku 3 barov. V celicah mešane in nevronske kulture smo merili aktivnost laktat dehidrogenaze (LDH) in kaspaz 3/7, v astrocitih pa raven glutationa.
Rezultati: CO je povzročil zvišanje aktivnosti LDH in kaspaz 3/7 v nevronskih kulturah, v mešanih pa le zvišanje aktivnosti kaspaz 3/7. Po izpostavitvi CO in HBO se je zvišala aktivnost LDH v nevronskih kulturah in znižala aktivnost kaspaz 3/7 v mešanih kulturah. CO je v astrocitih povzročil znižanje celokupnega glutationa (GSHt), zvišanje glutation disulfida (GSSG) in znižanje GSH/GSSG, po izpostavitvi CO in HBO pa se je zvišala GSHt, znižala GSSG in zvišala GSH/GSSG.
Zaključek: Razlike v citotoksičnem delovanju CO in zaščitni vlogi HBO v nevronski, mešani in astrocitni kulturi kažejo, da so nevroni, ki rastejo brez astrocitov, v primerjavi z mešano kulturo dovzetnejši za škodljive učinke CO ter nakazujejo, da astrociti ob oksidativnem stresu poskušajo ščititi nevrone, ki so pri sintezi glutationa odvisni od njih.
Metrifonate is an inhibitor of acetylcholinesterase (AChE). Several studies confirmed its positive effects on cognitive impairment in Alzheimer's disease but it was due to adverse events withdrawn ...from clinical trials. Based on the importance of astrocytes in physiological and pathological brain activities we investigated the impact of metrifonate and, for comparison, acetylcholine on intrinsic neurotrophic activity in these cells.
Metabolic activity, intracellular adenosine 5'-triphosphate (ATP) levels and lactate dehydrogenase (LDH) release was measured to examine the impact of metrifonate on viability and integrity of cultured rat cortical astrocytes. The influence of metrifonate, acetylcholine and selective cholinergic ligands on nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) synthesis and secretion was determined by specific two-site enzyme immunoassays.
Exposure of cultured astrocytes to metrifonate displayed no toxic effects on cell viability. Metrifonate and acetylcholine potently and transiently elevated NGF and BDNF, but not NT-3, protein levels and secretion with different intensity and time frame of their maximal response. Stimulatory effect on NGF was mimicked by selective nicotinic receptor agonist nicotine and completely blocked by nicotinic antagonist mecamylamine. The impact on BDNF synthesis was mimicked by muscarinic receptor agonist pilocarpine and abolished by selective muscarinic antagonist scopolamine.
Metrifonate up-regulates astrocytic NGF and BDNF synthesis in the same manner as acetylcholine, their effect depends on different cholinergic pathways. These results suggest a trophic role of metrifonate, based on a well-known neurotrophic activity of NGF and BDNF in vivo.
Recently we reported that cultured rat cortical astrocytes express histamine H3 receptor that is functionally coupled to Gi/o proteins and participates to the stimulatory effect of histamine. Due to ...the lack of data on the distribution of histamine H3 receptors on glial cells we further investigated their presence in cultured astrocytes from different brain regions.
Real-time PCR was performed to examine the expression of native histamine H3 receptor in cultured rat astrocytes from cortex, cerebellum, hippocampus and striatum. Double-antigen immunofluorescence staining and 3HN-α-methylhistamine (3HNαMH) binding studies were utilized to specifically identify and characterize receptor binding sites in astrocytes.
Histamine H3 receptor mRNA was detected in rat astrocytes from all the regions under investigation with the highest levels in striatal astrocytes followed by hippocampal astrocytes and approximately equal levels in cerebellar and cortical astrocytes. Double-antigen immunofluorescence confirmed the presence of histamine H3 receptors on the membrane of all examined astroglial populations. 3HNαMH bound with high affinity and specificity to an apparently single class of saturable sites on cortical astrocytic membranes (KD=4.55±0.46nM; Bmax=5.63±0.21fmol/mg protein) and competition assays with selective agonists and antagonists were consistent with labeling of histamine H3 receptor (range of pKi values 7.50–8.87).
Our study confirmed the ability of cultured astrocytes from different rat brain regions to express histamine H3 receptors. The observed diverse distribution of the receptors within various astrocytic populations possibly mirrors their heterogeneity in the brain and indicates their active involvement in histamine-mediated effects.
Astrocytes actively control neuronal activity and synaptic transmission and by producing various neurotrophic factors represent an important local cellular source of trophic support in the normal and ...diseased brain. Our present study showed the ability of astrocytes to synthesize neurotrophin-3 (NT-3) and the active involvement of the monoamine neurotransmitters noradrenaline, adrenaline, dopamine, and serotonin, as well as basic intracellular second messenger systems, in the regulation of NT-3 production in neonatal rat cortical astrocytes. Using a new NT-3 specific enzyme-immunoassay, we showed that neonatal rat cortical and, for comparison, cerebellar astrocytes in primary culture can synthesize NT-3; the basal cellular content of NT-3 protein was 23.2+/-0.4 pg NT-3/mg cell protein and 23.6+/-0.9 pg NT-3/mg cell protein, respectively. The examined neurotransmitters, with the exception of serotonin, were able to potently and transiently increase NT-3 mRNA and NT-3 protein content; their maximal effects were dose- and time-dependent. Noradrenaline (1 microM), adrenaline (1 microM), and dopamine (100 microM) showed a maximal increase in NT-3 cellular content after 6h treatment causing a 1.9-, 1.8- and 2.7-fold elevation, respectively. Prior to the observed increase in NT-3 protein levels, the examined catecholamines increased NT-3 mRNA levels with maximal effects observed after 1h (noradrenaline) and 2h (adrenaline and dopamine) of incubation causing 2.4-, 2.6- and 3-fold elevation, respectively. Screening different activators of basic intracellular second messenger systems for their influence on NT-3 synthesis revealed that forskolin (20 microM), dibutyryl cAMP (dBcAMP) (100 microM), as well as calcimycin (1 microM) (Ca(2+) ionophore A23187) and phorbol 12-myristate 13-acetate (TPA) (100 nM), markedly increased the cellular level of NT-3 protein. Neurotransmitter-induced NT-3 levels were susceptible (to varying degrees) to inhibition by H-89 (protein kinase A inhibitor) or staurosporin (protein kinase C inhibitor), which led us to conclude that downstream signaling responsible for the stimulation of NT-3 synthesis by monoamines in astrocytes consists of multiple, complex intracellular mechanisms involving the cAMP/protein kinase A pathway, activation of protein kinase C, as well as mobilization of Ca(2+) ions. Our results indicate for the first time that monoaminergic neurotransmitters play an important role in the regulation of NT-3 synthesis in cultured rat astrocytes.
Background Astrocytes maintain central nerve system homeostasis and are relatively resistant to cell death. Dysfunction of cell death mechanisms may underlie glioblastoma genesis and resistance to ...cancer therapy; therefore more detailed understanding of astrocytic death modalities is needed in order to design effective therapy. The purpose of this study was to determine the effect of VAS2870, a pan-NADPH oxidase inhibitor, on staurosporine-induced cell death in astrocytes. Materials and methods Cultured rat astrocytes were treated with staurosporine as activator of cell death. Cell viability, production of reactive oxygen species (ROS), and mitochondrial potential were examined using flow cytometric analysis, while chemiluminescence analysis was performed to assess caspase 3/7 activity and cellular ATP. Results We show here for the first time, that VAS2870 is able to prevent staurosporine-induced cell death. Staurosporine exerts its toxic effect through increased generation of ROS, while VAS2870 reduces the level of ROS. Further, VAS2870 partially restores mitochondrial inner membrane potential and level of ATP in staurosporine treated cells. Conclusions Staurosporine induces cell death in cultured rat astrocytes through oxidative stress. Generation of ROS, mitochondrial membrane potential and energy level are sensitive to VAS2870, which suggests NADPH oxidases as an important effector of cell death. Consequently, NADPH oxidases activation pathway could be an important target to modulate astrocytic death.
Recently we reported that cultured rat cortical astrocytes express histamine H
receptor that is functionally coupled to G
proteins and participates to the stimulatory effect of histamine. Due to the ...lack of data on the distribution of histamine H
receptors on glial cells we further investigated their presence in cultured astrocytes from different brain regions. Real-time PCR was performed to examine the expression of native histamine H
receptor in cultured rat astrocytes from cortex, cerebellum, hippocampus and striatum. Double-antigen immunofluorescence staining and
HN-α-methylhistamine (
HNαMH) binding studies were utilized to specifically identify and characterize receptor binding sites in astrocytes. Histamine H
receptor mRNA was detected in rat astrocytes from all the regions under investigation with the highest levels in striatal astrocytes followed by hippocampal astrocytes and approximately equal levels in cerebellar and cortical astrocytes. Double-antigen immunofluorescence confirmed the presence of histamine H
receptors on the membrane of all examined astroglial populations.
HNαMH bound with high affinity and specificity to an apparently single class of saturable sites on cortical astrocytic membranes (K
=4.55±0.46nM; B
=5.63±0.21fmol/mg protein) and competition assays with selective agonists and antagonists were consistent with labeling of histamine H
receptor (range of pKi values 7.50-8.87). Our study confirmed the ability of cultured astrocytes from different rat brain regions to express histamine H
receptors. The observed diverse distribution of the receptors within various astrocytic populations possibly mirrors their heterogeneity in the brain and indicates their active involvement in histamine-mediated effects.
PDF