Decreased oxidative stress during glycolytic inhibition enables maintenance of ATP production and astrocytic survival.

Depressed energy metabolism and oxidative stress are common features in many pathological situations in the brain, including stroke. In order to investigate astrocytic responses to such stress, we induced metabolic depression in cultured rat astrocytes. Iodoacetate (IA), an inhibitor of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used and resulted in a rapid inhibition of GAPDH activity. After 1h of GAPDH inhibition the ATP levels started to decrease and were completely abolished at 4h. In parallel, the activity of reactive oxygen species (ROS) was significantly increased, followed by extensive cell death involving flipping of phosphatidylserine and translocation of apoptosis-inducing factor, but not caspase-3 activation. When IA was combined with azide, a respiratory chain complex IV inhibitor, the ATP levels decreased immediately. Interestingly, with azide present, the ROS activity remained low and the astrocytes remained viable even at very low ATP levels. Addition of exogenous ROS-scavengers prevented the IA-induced ROS activity, the ATP levels were maintained and cell death was prevented. Similar protection could be obtained when astrocytes, prior to addition of IA, were incubated with substances known to activate the nuclear factor erythroid 2-related factor 2 (Nrf2)-regulated endogenous antioxidant system. When IA was washed out, after a relatively moderate ATP depression, massive cell death occurred. This was efficiently prevented by addition of azide or ROS scavengers during the IA treatment or by pre-activation of the Nrf2 system. Our results demonstrate that astrocytes in culture can endure and recover from glycolytic inhibition if the ROS activity remained at a low level and suggest that oxidative stress can be an important component for astrocytic cell death following metabolic stress.

Trauma-induced reactive gliosis is reduced after treatment with octanol and carbenoxolone.

BACKGROUND: Reactive gliosis and scar formation after brain injury can inhibit the recovery process. As many glial cells utilize gap junctions for intercellular signaling, this study investigated whether two commonly used gap junction blockers, octanol and carbenoxolone, could attenuate reactive gliosis following a minor traumatic brain injury. METHODS: Octanol (710 mg/kg) or carbenoxolone (90 mg/kg) was administered 30 minutes before or after a needle track injury in adult male Sprague-Dawley rats. To mark dividing cells, animals were injected with bromodeoxyuridine (BrdU; 150 mg/kg) intraperitoneally two times per day, 8 hours apart and killed 2 days later. Immunohistochemistry for BrdU and markers for reactive glial cells [glial fibrillary acidic protein (GFAP), ED1, and NG2] were investigated using immunohistochemistry and western blot techniques. RESULTS: Two days after injury, increased cellular proliferation, activated astrocytes and microglia, and upregulation of NG2 expression were observed surrounding the injury site. Octanol and carbenoxolone administrated prior to injury significantly decreased cell proliferation by 60 and 70% respectively. The distance of GFAP immunoreactive astrocytes from the wound margin was decreased by 32 and 18% when octanol was administrated prior to or post injury respectively. Treatment with octanol also decreased the number of reactive microglia by 55% and, when administrated prior to injury, octanol reduced the distance of NG2 expression from the wound by 48%. CONCLUSION: The present study demonstrates that two important components of reactive gliosis, cellular activation and proliferation, can be attenuated by octanol and carbenoxolone.

Repeated transient sulforaphane stimulation in astrocytes leads to prolonged Nrf2-mediated gene expression and protection from superoxide-induced damage.

Oxidative stress is a major contributor to slowly developing diseases like Parkinson's disease, Alzheimer's disease and cancer and one of the main causes of tissue damage following ischemic insults in the brain. Nrf2 is a transcription factor responsible for much of the inducible cellular defense against oxidative stress. Nrf2 can also be activated by xenobiotics like sulforaphane, a component highly enriched in cruciferous vegetables such as broccoli. Ingestion of broccoli or sulforaphane results in long-term protection against radical damage, although absorbed sulforaphane is cleared from the body within a few hours. Here we have examined whether the prolonged protection induced by sulforaphane is explained by a slow down regulation of the Nrf2 response. Furthermore, to simulate daily ingestion of sulforaphane, we examined the hypothesis that repeated transient sulforaphane stimulation results in an accumulation of Nrf2-mediated gene expression and an increased protection against oxidative damage. The kinetics of sulforaphane-induced Nrf2 response was studied in astrocytes, a cell type known to be highly involved in the defense against oxidative stress in the brain. Sulforaphane stimulation for 4 h induced an Nrf2-dependent increase of Nqo1 and Hmox1 mRNA that remained elevated for 24 h, and the corresponding proteins remained elevated for over 48 h. In addition, peroxide-clearing activity and the levels of glutathione were elevated for more than 20 h after stimulation for 4 h with sulforaphane, resulting in an increased resistance to superoxide-induced cell damage. Repeated sulforaphane stimulation resulted in an accumulation of mRNA and protein levels of Nqo1 and a persistent cell protection against oxidative damage. These findings indicate that brief stimulation of the Nrf2 pathway by sulforaphane results in long-lasting elevation of endogenous antioxidants in astrocytes. The findings also demonstrate that part of this response can be built up by repeated transient stimulation, possibly explaining how intermittent intake of sulforaphane can result in long-term protection from radical-induced disease.

Enhanced glutathione efflux from astrocytes in culture by low extracellular Ca2+ and curcumin.

Efflux of glutathione (GSH) from astrocytes has been suggested as a key factor for neuroprotection by astrocytes. Here we evaluated if the Nrf2 activator curcumin affects basal and stimulated (Ca(2+) omission) GSH efflux from cultures of astroglial cells. Stimulated efflux of GSH was observed at medium concentration of 0, 0.1 mM Ca(2+), but not at 0.2 or 0.3 mM Ca(2+). Astroglia treated with 30 microM curcumin increased the cellular content of GSH in parallel with elevated basal and stimulated efflux. Conversely treatment with buthionine sulfoximine lowered efflux of GSH. The efflux stimulated by Ca(2+)- omission was not affected by the P2X7-receptor antagonist Blue Brilliant G (100 nM) or the pannexin mimetic/blocking peptide (10)Panx1 but inhibited by the gap junction blocker carbenoxolone (100 microM) and a hemichannel blocker Gap26 (300 microM). RNAi directed against Nrf2 partly inhibited the effect of curcumin. The results show that elevated cellular GSH by curcumin treatment enhance efflux from astroglial cells, a process which appear to be a prerequisite for astroglial mediated neuroprotection.

Protective role of reactive astrocytes in brain ischemia.

Reactive astrocytes are thought to protect the penumbra during brain ischemia, but direct evidence has been lacking due to the absence of suitable experimental models. Previously, we generated mice deficient in two intermediate filament (IF) proteins, glial fibrillary acidic protein (GFAP) and vimentin, whose upregulation is the hallmark of reactive astrocytes. GFAP(-/-)Vim(-/-) mice exhibit attenuated posttraumatic reactive gliosis, improved integration of neural grafts, and posttraumatic regeneration. Seven days after middle cerebral artery (MCA) transection, infarct volume was 210 to 350% higher in GFAP(-/-)Vim(-/-) than in wild-type (WT) mice; GFAP(-/-), Vim(-/-) and WT mice had the same infarct volume. Endothelin B receptor (ET(B)R) immunoreactivity was strong on cultured astrocytes and reactive astrocytes around infarct in WT mice but undetectable in GFAP(-/-)Vim(-/-) astrocytes. In WT astrocytes, ET(B)R colocalized extensively with bundles of IFs. GFAP(-/-)Vim(-/-) astrocytes showed attenuated endothelin-3-induced blockage of gap junctions. Total and glutamate transporter-1 (GLT-1)-mediated glutamate transport was lower in GFAP(-/-)Vim(-/-) than in WT mice. DNA array analysis and quantitative real-time PCR showed downregulation of plasminogen activator inhibitor-1 (PAI-1), an inhibitor of tissue plasminogen activator. Thus, reactive astrocytes have a protective role in brain ischemia, and the absence of astrocyte IFs is linked to changes in glutamate transport, ET(B)R-mediated control of gap junctions, and PAI-1 expression.

Gap junction blockage limits intercellular spreading of astrocytic apoptosis induced by metabolic depression.

Astrocytes are highly coupled by gap junction channels, which allow transfer of intracellular signalling molecules and metabolites between connected cells. Astrocytic gap junctions remain open during ischemic conditions as previously demonstrated in vitro and in situ. In this study, we investigated the effect of gap junction blockage on iodoacetate-induced ATP depression and cell death progression in astrocytes in primary rat hippocampal cultures. We demonstrated that blockage of gap junctions during iodoacetate-induced inhibition of the glycolysis induced an earlier onset of the ATP depression. Moreover, initiation of apoptotic processes, demonstrated by binding of Annexin V, was critically dependent on the ATP levels. The apoptotic event was also shown to spread and involve neighbouring cells, a process that was inhibited by blockage of gap junction communication. Chelating intracellular calcium using BAPTA-AM decelerated the iodoacetate-induced ATP depression. The chelation also decelerated the spreading of apoptotic processes. Inhibition of caspases did not alter the expansion of cell groups being Annexin V positive. However, the proportion of Annexin V positive cells also being propidium iodide positive was increased after caspase inhibition. The results show that inhibition of gap junctions during cellular metabolic depression interferes with the metabolic status and cell death progression in astrocytes.

Cys2,7EtalphaCGRP is a potent agonist for CGRP1 receptors in SK-N-MC cells.

The present study reveals that cystein2,7 ethyl-amidealphaCGRP (Cys2,7EtalphaCGRP), an advertised calcitonin gene-related peptide 2 (CGRP2) receptor subtype-selective agonist, is also a potent agonist for the calcitonin gene-related peptide 1 (CGRP1) receptors natively expressed in the SK-N-MC human neuroblastoma cell line. Cys2,7EtalphaCGRP and alpha calcitonin gene-related peptide (alphaCGRP) promote cyclic AMP accumulation in intact SK-N-MC cells to the same extent with EC50 of 1.6+/-0.2 and 0.4+/-0.08 nM, respectively. The antagonist alpha calcitonin gene-related peptide-8-37 (alphaCGRP-(8-37)) produces a concentration-dependent rightward shift of the alphaCGRP- and Cys2,7EtalphaCGRP concentration-response curves with KB-values (71+/-33 and 47+/-21 nM, respectively). The competitive antagonism by alphaCGRP-(8-37) and the similar KB-values suggests that alphaCGRP and Cys2,7EtalphaCGRP stimulate the same receptor. In competition binding studies with [125I]-alphaCGRP on SK-N-MC cell membranes, Cys2,7EtalphaCGRP and alphaCGRP-(8-37) display high affinity for the majority of the binding sites with Ki-values of 0.030+/-0.013 and 0.60+/-0.013 nM, respectively. The present findings are at odds with the proclaimed utilization of Cys2,7EtalphaCGRP as a CGRP2 receptor-selective pharmacological tool. Differences between the agonistic profile of this ligand in this and other experimental systems might be species--or even cell type--dependent.