4'-Chlorodiazepam modulates the development of primary hippocampal neurons in a sex-dependent manner.

The translocator protein 18kDa (TSPO) is located in the outer mitochondrial membrane and is involved in the cholesterol transport into the mitochondria and in the regulation of steroidogenesis and other mitochondrial functions. It is known that steroid hormones, such as estradiol, testosterone and dihydrotestosterone are neuroprotective and regulate neuritogenesis in the CNS by different mechanisms. However, the developmental effects of TSPO ligands in the CNS are not known. Therefore, the aim of this study was to identify the developmental effects of 4'-chlorodiazepam (4'-CD), a TSPO ligand, in primary cultures of male and female mouse hippocampal neurons. We observed that female neurons showed an advanced neuritogenesis compared to male neurons after 2days in vitro. Moreover, it was shown that 4'-CD administration accelerated the maturation of male hippocampal neurons, without changing the development of female neurons. These findings, showing that 4'-CD modulates the development of hippocampal neurons in a sex-dependent manner, suggest that TSPO may be involved in the regulation of neuritogenesis.

4'-Chlorodiazepam is neuroprotective against amyloid-beta through the modulation of survivin and bax protein expression in vitro.

The translocator protein of 18kDa (TSPO) is located in the outer mitochondrial membrane and is involved in the cholesterol transport into the mitochondria and in the regulation of steroidogenesis, mitochondrial permeability transition pore opening and apoptosis. TSPO ligands have been investigated as therapeutic agents that promote neuroprotective effects in experimental models of brain injury and neurodegenerative diseases. The aim of this study was to identify the neuroprotective effects of 4'-chlorodiazepam (4'-CD), a ligand of TSPO, against amyloid-beta (Aß) in SHSY-5Y neuroblastoma cells and its mechanisms of action. Aß decreased the viability of SHSY-5Y neuroblastoma cells, while 4'-CD had a neuroprotective effect at the doses of 1nM and 10nM. The neuroprotective effects of 4'-CD against Aß were associated with the inhibition of Aß-induced upregulation of Bax and downregulation of survivin. In summary, our findings indicate that 4'-CD is neuroprotective against Aß-induced neurotoxicity by a mechanism that may involve the regulation of Bax and survivin expression.

Notch/neurogenin 3 signalling is involved in the neuritogenic actions of oestradiol in developing hippocampal neurones.

The ovarian hormone oestradiol promotes neuritic outgrowth in different neuronal types, by mechanisms that remain elusive. Recent studies have shown that the Notch-regulated transcription factor neurogenin 3 controls neuritogenesis. In the present study, we assessed whether oestradiol regulates neurogenin 3 in primary hippocampal neurones. As expected, neuritogenesis was increased in the cultures treated with oestradiol. However, the neuritogenic action of oestradiol was not prevented by ICI 182,780, an antagonist of classical oestrogen receptors (ERs). Oestradiol decreased the expression of Hairy and Enhancer of Split-1, a Notch-regulated gene that negatively controls the expression on neurogenin 3. Furthermore, oestradiol increased the expression of neurogenin 3 and regulated its distribution between the neuronal cell nucleus and the cytoplasm. The effect of oestradiol on neurogenin 3 expression was not blocked by antagonists of classical nuclear ER-mediated transcription and was not imitated by selective agonists of nuclear ERs. By contrast, G1, a ligand of G protein receptor 30/G protein-coupled ER, fully reproduced the effect of oestradiol on neuritogenesis, neurogenin 3 expression and neurogenin 3 subcellular localisation. Moreover, knockdown of neurogenin 3 in neurones by transfection with small interference RNA for neurogenin 3 completely abrogated the neuritogenic actions of oestradiol and G1. These results suggest that oestradiol regulates neurogenin 3 in primary hippocampal neurones by a nonclassical steroid signalling mechanism, which involves the down-regulation of Notch activity and the activation of G protein receptor 30/G protein-coupled ER or of other unknown G1 targets. In addition, our findings indicate that neurogenin 3 participates in the neuritogenic mechanisms of oestradiol in hippocampal neurones.