Pregnenolone enhances the proliferation of mouse neural stem cells and promotes oligodendrogenesis, together with Sox10, and neurogenesis, along with Notch1 and Pax6.

BACKGROUND: Pregnenolone is a precursor of various steroid hormones involved in osteoblast proliferation, microtubules polymerization and cell survival protection. Previous reports focused on the effects of pregnenolone metabolites on stem cell proliferation and differentiation; however, the effects of pregnenolone itself has not been well explored. The present study aimed to investigate the role of pregnenolone on NSC proliferation and to determine the doses required for NSC differentiation as well as the various genes involved in its mechanism of action. METHODS: NSCs were isolated from the embryonic cortex of E14 mice, incubated for 5 days, and then treated with pregnenolone doses of 2, 5, 10, 15 and 20 µM for another 5 days. The number of neurospheres and neurosphere derived cells were then counted. Flow cytometry was used to evaluate the differentiation of NSCs into oligodendrocytes, astrocytes, and neurons. The expression level of Notch1, Pax6 and Sox10 genes were also measured by Real Time PCR after 5 days of treatment. RESULTS: Our data suggest that treatment with 10 µM pregnenolone is optimal for NSC proliferation. In fact, this concentration caused the highest increase in the number of neurospheres and neurosphere derived cells, compared to the control group. In addition, treatment with low doses of pregnenolone (5 and 10 µM) caused a significant increase in NSC differentiation towards immature (Olig2+) and mature (MBP+) oligodendrocyte cell populations, compared to controls. However, NSC differentiation into neurons (beta III tubulin + cells) increased in all treatment groups, with the highest and most significant increase obtained at 15 µM concentration. It is worth noting that pregnenolone at the highest concentration of 15 µM decreased the number of astrocytes (GFAP+). Furthermore, there was an increase of Sox10 expression with low pregnenolone doses, leading to oligodendrogenesis, whereas Notch1 and Pax6 gene expression increased in pregnenolone groups with more neurogenesis. CONCLUSION: Pregnenolone regulates NSCs proliferation in vitro. Treatment with low doses of pregnenolone caused an increase in the differentiation of NSCs into mature oligodendrocytes while higher doses increased the differentiation of NSCs into neurons. Oligodendrogenesis was accompanied by Sox10 while neurogenesis occurred together with Notch1 and Pax6 expression.

Humanin Does Not Protect Against STZ-Induced Spatial Memory Impairment.

[Gly14]-Humanin (HNG) is a 24-amino acid peptide which was first identified in the brains of patients diagnosed with Alzheimer's disease (AD). In this region, some neurons were protected against cell damage occurring in this disease. Further studies suggested a neuroprotective role for humanin against Aß and some other insults. Intraventricularly administered streptozotocin (STZ) disrupts insulin signaling pathway which leads to behavioral and biochemical changes resemble to early signs of AD; therefore, STZ model has been proposed as a model for sporadic Alzheimer's disease (sAD). Regarding the reported beneficial effects of humanin in AD, this study was aimed to investigate if this peptide prevents spatial memory and hippocampal PI3/Akt signaling impairment induced by centrally injected STZ. Adult male Sprague-Dawely rats weighting 250-300 g were used, and cannuls were implanted bilaterally into lateral ventricles. STZ was administered on days 1 and 3 (3 mg/kg), and humanin (0.01, 0.05, 0.1, and 1 nmol) or saline were injected from day 4 and continued till day 14. The animal's learning and memory capability was assessed on days 15-18 using Morris water maze. After complement of behavioral studies, the hippocampi were isolated, and the level of phosphorylated Akt (pAkt) was assessed through Western blot analysis. The results showed that STZ significantly impaired spatial memory, and humanin in a wide range of doses (0.01, 0.05, 0.1, and 1 nmol) failed to restore STZ-induced deficit. It was also revealed that humanin was not efficient in restoring pAkt disruption. It seems that humanin is not capable in restoring memory deterioration that resulted from insulin signaling disruption.