Abstract
In the adult mammalian brain, the maintenance of the dynamic equilibrium between excitation and inhibition is necessary for normal neuronal function. Attenuation of synaptic inhibition can lead to over-excitation, mainly through NMDA-receptor over-activation, with subsequent excessive calcium influx and cell death. On the contrary, enhancement of synaptic inhibition due to GABAₐ-ergic system activation can prevent NMDA-induced cell death. GABAₐ receptor possesses several distinct and diverse binding sites for GABA, barbiturates, benzodiazepines, ethanol and endogenous neurosteroids, such as allopregnanolone (allo), dehydroepiandrosterone (DHEA) and its sulfate ester (DHEAS). Neurosteroids are produced in the central nervous system (CNS) de novo from cholesterol, secreted mainly by glia and neurons and accumulated in the brain, independently from the secretion of the peripheral steroidogenic glands. They represent a specific category of neuromodulators, which alter neuronal function pro ...
In the adult mammalian brain, the maintenance of the dynamic equilibrium between excitation and inhibition is necessary for normal neuronal function. Attenuation of synaptic inhibition can lead to over-excitation, mainly through NMDA-receptor over-activation, with subsequent excessive calcium influx and cell death. On the contrary, enhancement of synaptic inhibition due to GABAₐ-ergic system activation can prevent NMDA-induced cell death. GABAₐ receptor possesses several distinct and diverse binding sites for GABA, barbiturates, benzodiazepines, ethanol and endogenous neurosteroids, such as allopregnanolone (allo), dehydroepiandrosterone (DHEA) and its sulfate ester (DHEAS). Neurosteroids are produced in the central nervous system (CNS) de novo from cholesterol, secreted mainly by glia and neurons and accumulated in the brain, independently from the secretion of the peripheral steroidogenic glands. They represent a specific category of neuromodulators, which alter neuronal function probably through their direct, fast, non-genomic interaction with GABAₐ and NMDA receptors. The aim of the present study was to investigate the mechanisms of action of neurosteroids, which are associated with the enhancement of GABAₐ-ergic inhibition and the modulation of intracellular calcium homeostasis, in CNS neurons. We used two pluripotent embryonic cell lines, NT2 (human) and P19 (mouse), which in the presence of retinoic acid can be induced to differentiate into mature, post-mitotic neurons, which express neuronal markers and functional GABAₐ and NMDA receptors. NT2 and P19 precursors were successfully differentiated to NT2-N and P19-N neurons, respectively, expressing two neuron-specific markers, synaptophysin and β-ΙΙΙ tubulin. First, we assessed the effect of Ca²⁺ homeostasis perturbations on neuronal cell viability and apoptosis, as well as, the potent protective role of the neurosteroids allo, DHEA and DHEAS. Since the disturbance of Ca²⁺ homeostasis involves many cellular functions, we used agents that alter intracellular Ca²⁺ concentration through separate pathways: intracellular Ca²⁺ stores depletion focusing in the endoplasmic reticulum, non specific cytoplasmic Ca²⁺ overload, Ca²⁺ sequestration and increased Ca²⁺ influx through membrane receptors (L-glutamic acid and NMDA). Only the depletion of intracellular calcium stores caused excessive apoptotic neuronal death in NT2-N and P19-N neurons. This effect could not be reversed, since neither the presence of allo, DHEA or DHEAS, nor the presence of specific inhibitors of Ca²⁺ overload, such us Ruthenium Red and dantrolene, could abolish neuronal death. Therefore, neurosteroids most likely display specificity in their mechanism of action, which is not connected to massive and general alteration of cytoplasmic Ca²⁺ concentration. We next investigated the effects of elementary Ca²⁺ alterations that are generated from over-activation of NMDA receptor (in the presence of L-glutamate and NMDA). We observed that NT2-N cells were extremely resistant to NMDA- induced excitotoxicity, probably due to the presence of a large number of astrocytes in this particular cell system, which may protect neurons against glutamate toxicity. On the contrary, the presence of glutamate or NMDA resulted in excessive cell death of P19-N neurons. Particularly, NMDA treatment (1 mM, 60 min) resulted in apoptotic cell death, since pronounced DNA laddering and chromatin condensation was observed, phenomena which in the presence of allo or DHEA, were significantly attenuated. Similar experiments in primary cultures of hypothalamic neurons, further confirmed the protective role of neurosteroids on NMDA-induced excitotoxicity. In P19-N neurons NMDA-induced toxicity was probably the result of suppression of the PI3K→Akt pathway, which represents a central regulator of cell survival. Indeed, in the presence of NMDA, the levels of the active phosphorylated Akt kinase were reduced, while allo and DHEA increased significantly the levels of phospho-Akt, in the absence and in the presence of NMDA. The specificity of the neuroprotective effect of the above neurosteroids was confirmed by the use of wortmannin, the PI3K→Akt pathway inhibitor. Additionally, L-Glu or NMDA treatment on P19-N neurons, resulted in significant increase of calcium influx, while pre-treatment with allo or DHEA, inhibited this effect of L-Glu or NMDA. The role of calcium ions on NMDA-induced excitotoxicity was further confirmed by the observation that two calcium chelators, Bapta-AM and EGTA increased the viability of P19-N neurons and restored the levels of phospho-Akt, in the presence of NMDA. Excessive Ca²⁺ influx caused by NMDA, is probably responsible for the activation of the intrinsic mitochondrial apoptotic pathway, since NMDA treatment resulted in translocation of the pro-apoptotic protein Bax from the cytoplasm to the mitochondria, with subsequent release of cytochrome c to the cytoplasm, while no significant changes in the levels of the anti-apoptotic proteins Bcl-2 and Bcl-xL, were observed. These results indicate that cytochrome c / Bax mutual translocation is sufficient for triggering apoptosis, in P19-N neurons. Therefore, allo and DHEA inhibited NMDA-induced apoptosis, by directly activating the PI3K→Akt pathway, while preserving cytochrome c in the mitochondria and Bax in the cytoplasm. Similar protective effects were observed by three synthetic neurosteroids, suggesting a potential use of these neurosteroid analogues in excitotoxicity. Finally, we investigated the expression pattern of GABAₐ and NMDA receptor subunit mRNAs, during the differentiation of P19-N neurons and the effects of neurosteroids on GABAₐ receptor plasticity and function. Differentiated P19-N cells were found to express al and β2 subunit mRNAs of GABAₐ receptor and NR1 subunit mRNA of NMDA receptor and short-term treatment with allo or DHEA resulted in α1 and β2 subunit mRNAs upregulation, in a dose- and time- dependent pattern, while the levels of the NR1 subunit mRNA, were not changed significantly. Allo treatment resulted also in significant increase in the protein levels of α1 and β2 subunits of GABAₐR, which probably is accompanied by increased number of functional receptors in the membrane, and increased synaptic inhibition. In summary, our results support the hypothesis that neurosteroids represent a specific class of CNS modulators with potent neuroprotective action, which regulate neuronal function through concurrent influence on neuronal excitability and gene expression.
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