Preclinical Analyses of the Therapeutic Potential of Allopregnanolone to Promote Neurogenesis In Vitro and In Vivo in Transgenic Mouse Model of Alzheimers Disease
Roberta D. Brinton and Jun M. Wang
Affiliation: Department of MolecularPharmacology and Toxicology, Pharmaceutical Sciences Center, Universityof Southern California, 1985 Zonal Avenue, Los Angeles, CA 90089-9121,USA.
Herein, we present data to support a preclinical proof of concept for the therapeutic potential of allopregnanolone to promote neurogenesis. Our recent work has demonstrated that the neuroactive progesterone metabolite, allopregnanolone (3α-hydroxy-5α-pregnan-20-one), (APα) induced, in a dose dependent manner, a significant increase in proliferation of neuroprogenitor cells (NPCs) derived from the rat hippocampus and human neural stem cells (hNSM) derived from the cerebral cortex . Proliferative efficacy was determined by incorporation of BrdU and 3H-thymidine, FACS analysis of MuLV-GFP-labeled mitotic NPCs and quantification of total cell number. Allopregnanolone-induced proliferation was isomer and steroid specific, in that the stereoisomer 3β-hydroxy-5β-pregnan-20-one and related steroids did not increase 3H-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 RT-PCR and western blot validation revealed that allopregnanolone increased the expression of genes which 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 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 L-type calcium channel. Preliminary in vivo data indicate that APα for 24 hrs significantly increased neurogenesis in dentate gyrus, as determined by unbiased stereological analysis of BrdU positive cells, of 3-month-old male triple transgenic Alzheimers disease mice. The in vitro and in vivo neurogenic properties of APα coupled with a low molecular weight, easy penetration of the blood brain barrier and lack of toxicity, are key elements required for developing APα as a neurogenic / regenerative therapeutic for restoration of neurons in victims of Alzheimers disease.
Keywords: Allopregnanolone, neurogenesis, hippocampus, cell cycle genes, L-type calcium channel, therapeutics
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