Developmental differences in HO-induced oligodendrocyte cell death: role of glutathione, mitogen-activated protein kinases and caspase 3.

The molecular mechanisms underlying H(2)O(2)-induced toxicity were characterized in rat oligodendrocyte cultures. While progenitor cells were more sensitive than mature oligodendrocytes to H(2)O(2), the antioxidant, N-acetyl-L-cysteine, blocked toxicity at both stages of development. Differentiated oligodendrocytes contained more glutathione than did progenitors and were less susceptible to decreases in glutathione concentration induced by H(2)O(2) stress. As free radicals have been considered to serve as second messengers, we examined the effect of H(2)O(2) on activation of the mitogen-activated protein kinases (MAPK), extracellular signal-regulated kinases (ERK) 1/2 and p38. H(2)O(2) caused a time- and concentration-dependent increase in MAPK phosphorylation, an effect that was totally blocked by N-acetyl-L-cysteine. Further exploration of potential mechanisms involved in oligodendrocyte cell death showed that H(2)O(2) treatment caused DNA condensation and fragmentation at both stages of development, whereas caspase 3 activation and poly (ADP-ribose) polymerase cleavage were significantly increased only in oligodendrocyte progenitors. The pan-caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone, blocked DNA fragmentation in progenitors and produced a small but significant level of protection from H(2)O(2) toxicity in progenitors and mature oligodendrocytes. In contrast, inhibitors of both p38 and MEK reduced H(2)O(2)-induced death most significantly in oligodendrocytes. The poly (ADP-ribose) polymerase inhibitor, PJ34, reduced H(2)O(2)-induced toxicity on its own but was most effective when combined with benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone or PD169316. The finding that molecular mechanisms conferring resistance to reactive oxygen species toxicity are regulated during oligodendrocyte differentiation may be of importance in designing therapies for certain neurological diseases affecting white matter.

Regulation of muscarinic receptor function in developing oligodendrocytes by agonist exposure.

1 Oligodendrocytes, the myelin forming cells in the CNS, express muscarinic acetylcholine receptors (mAChR), primarily M3, coupled to various signal transduction pathways. 2 In the present study we have investigated whether mAChR undergo functional agonist-induced regulation in cultured oligodendrocyte progenitors and differentiated oligodendrocytes. 3 The muscarinic agonist, carbachol (CCh) caused a time-dependent desensitization of phosphoinositide (PI) hydrolysis, and the internalization and down-regulation of receptors. Short-time desensitization (5 min) of PI hydrolysis occurred without receptor internalization and reached 54% by 1 h. The same treatment decreased cell surface receptors labelled with the non-permeable ligand [(3)H]-NMS by 47%, while total receptor density ([(3)H]-scopolamine binding) decreased by 30%. Longer CCh treatment down-regulated receptors by 70% and desensitized the PI response by 80%. 4 Although protein kinase C (PKC) activation desensitized mAChR, CCh-mediated desensitization was independent of PKC. 5 Inhibition of receptor endocytosis by low temperature during the pre-stimulation period or in the presence of hyperosmotic sucrose (0.5 M) blocked desensitization, receptor internalization and down-regulation. 6 Recovery of surface mAChR and their functional activity following down-regulation was slow, returning to control levels by 24 h after agonist removal. In progenitor cells, dose-response curves for CCh-mediated PI hydrolysis and c-fos mRNA expression showed that newly synthesized mAChR were supersensitive after recovery. 7 Overall, the present results provide evidence of functional agonist-mediated mAChR regulation in brain oligodendroglial cells.

Catecholamine-induced oligodendrocyte cell death in culture is developmentally regulated and involves free radical generation and differential activation of caspase-3.

Oligodendrocyte cultures were used to study the toxic effects of catecholamines. Our results showed that catecholamine-induced toxicity was dependent on the dose of dopamine or norepinephrine used and on the developmental stage of the cultures, with oligodendrocyte progenitors being more vulnerable. A role for oxidative stress and apoptosis on the mechanism of action of catecholamines on oligodendrocyte cell death was next assessed. Catecholamines caused a reduction in intracellular glutathione levels, an accumulation in reactive oxygen species and in heme oxygenase-1, the 32 kDa stress-induced protein. All these changes were prevented by N-acetyl-L-cysteine, a thiocompound with antioxidant activity and a precursor of glutathione, and were more pronounced in progenitors than mature cells, which could contribute to their higher susceptibility. Apoptotic cell death, as assessed by activation of caspase-9 and -3 and cleavage of poly(ADP-ribose) polymerase (a substrate of caspase-3), was only observed in oligodendrocyte progenitors. Pretreatment with zVAD, a general caspase inhibitor, prevented activation of caspase-9 and -3, DNA fragmentation, and decreased progenitors cell death. Furthermore, the expression levels of procaspase-3 and the ratio of the proapoptotic protein bax to antiapoptotic protein bcl-xl were several folds higher in immature than mature oligodendrocytes. Taken together, these results strongly suggest that the catecholamine-induced cytotoxicity in oligodendrocytes is developmentally regulated, mediated by oxidative stress, and have characteristics of apoptosis in progenitor cells.

Developmental regulation of alpha 1A-adrenoceptor function in rat brain oligodendrocyte cultures.

In this study, we examined the effect of norepinephrine (NE) on phosphatidylinositol-4,5-bisphosphate (PI) hydrolysis in progenitors and differentiated oligodendrocytes. NE caused a time- and concentration-dependent increase in total inositol phosphate (IP(t)) formation. The magnitude of this response increased as oligodendrocytes matured and was accompanied with an increase in alpha(1)-adrenoceptor (alpha(1)-AR) levels. To pharmacologically characterize the alpha(1)-AR subtype mediating PI hydrolysis in 12-day differentiated oligodendrocytes, various selective antagonists were used. Prazosin, the non-selective 1-AR antagonist, blocked NE-mediated IP(t) formation. Similarly, the alpha(1A)-AR selective competitive antagonists, 5-methyl urapidil (5-MU) and WB4104, were potent blockers of NE-mediated IP(t) formation. In contrast, the alpha(1B)- and alpha(1D)-AR antagonist, chloroethylclonidine and the alpha(1D)-AR antagonist, BMY 7378, had no effect. These results suggest that NE-induced PI hydrolysis in differentiated oligodendrocytes was mediated through the alpha(1A)-AR. Furthermore, this response was prevented by EGTA and CdCl(2), suggesting a requirement for extracellular calcium. The presence of alpha(1)-AR subtypes in oligodendrocytes was confirmed by reverse transcriptase coupled polymerase chain reaction and by immunoprecipitation, with subtype specific antibodies. The results indicated that mRNA and protein for the alpha(1A)-, alpha(1B)- and alpha(1D)-AR subtypes were expressed. In conclusion, our findings show that oligodendrocytes express all three alpha(1)-AR subtypes but that only the alpha(1A)-AR was involved in NE-mediated IP(t) formation.