The neurosteroid allopregnanolone promotes proliferation of rodent and human neural progenitor cells and regulates cell-cycle gene and protein expression

Our previous research demonstrated that the neuroactive progesterone metabolite allopregnanolone ( 3 alpha- hydroxy- 5 alpha- pregnan- 20- one) rapidly induced hippocampal neuron neurite regression ( Brinton, 1994). We hypothesized that allopregnanolone- induced neurite regression was a prelude to mitogenesis initiated by a rise in intracellular calcium. Supporting this hypothesis, the current data demonstrate that allopregnanolone, in a dose- dependent manner, induces a significant increase in proliferation of neuroprogenitor cells ( NPCs) derived from the rat hippocampus and human neural stem cells ( hNSCs) derived from the cerebral cortex. Proliferation was determined by incorporation of bromodeoxyuridine and [ H-3] thymidine, fluorescence- activated cell sorter analysis of murine leukemia virus - green fluorescent protein- labeled mitotic NPCs, and total cell number counting. Allopregnanolone- induced proliferation was isomer and steroid specific, in that the stereoisomer 3 beta- hydroxy- 5 beta- pregnan- 20- one and related steroids did not increase [ H-3] thymidine uptake. Immunofluorescent analyses for the NPC markers nestin and Tuj1 indicated that newly formed cells were of neuronal lineage. Furthermore, microarray analysis of cell- cycle genes and real- time reverse transcription- PCR and Western blot validation revealed that allopregnanolone increased the expression of genes that promote mitosis and inhibited the expression of genes that repress cell proliferation. Allopregnanolone- induced proliferation was antagonized by the voltage- gated L- type calcium channel ( VGLCC) blocker nifedipine, consistent with the finding that allopregnanolone induces a rapid increase in intracellular calcium in hippocampal neurons via a GABA type A receptor- activated VGLCC ( Son et al., 2002). These data demonstrate that allopregnanolone significantly increased rat NPC and hNSC proliferation with concomitant regulation in mitotic cell- cycle genes via a VGLCC mechanism. The therapeutic potential of allopregnanolone as a neurogenic molecule is discussed.