| INTRODUCTIONVitamin D metabolite 1,25-dihydroxyvitamin D (1,25(OH)2D3) remains the most potent active form of Vitamin D known to date. 1,25(OH)2D3 acts via a member of the nuclear hormone receptor family (VDR) to directly regulate gene transcription. 1,25(OH)2D3 has long been known for its important role in regulating body levels of calcium and phosphorus, and bone mineralization. There is now accumulating evidence that activation of VDR induces the expression of NGF and GDNF. Physiological and pharmacological actions of 1,25(OH)2D3 in various systems, along with detection of VDR in various target cells, suggest potential therapeutic applications of VDR ligands in osteoporosis, cancer, secondary hyperparathyroidism, and autoimmune diseases.Biosynthetic and degradative pathways of1,25(OH)2D3 have been well defined in human brain. Clinical data show a linkage between low levels of 1,25(OH)2D3 and cognitive deterioration and dementia, particularly in Alzheimer's disease patients. Circulating 25(OH)2D3 less than 30 ng/ml is commonly related to substantial cognitive impairment. The treatment with 1,25(OH)2D3 has been shown to improve cognitive deterioration and dementia in Alzheimer's disease. In addition, it has been shown that mRNA levels of vitamin D receptor (VDR) in hippocampal CA1 and CA2 regions in Alzheimer's brains are reduced. To date, however, the specific effect of 1,25(OH)2D3 deficiency on cognitive performances has not yet been fully elucidated.The hippocampus contains stem cells and neural progenitor cells that retain the ability to proliferate and many daughter cells develop into neurons throughout life in primates and some mammalian species—neurogenesis. The continuously formed new granule cells in the adult brain, similar to established ones, are electrically active and make connections to hippocampal CA3 field. The functional integration of the newly formed neurons have been demonstrated to be correlative with cognitive behavior. This is reinforced by evidence that increased survival of newborn neurons is strongly correlated with hippocampus-dependent memory, while the inhibition of neurogenesis has adverse effects on hippocampus-dependent behaviors, thus implying that hippocampal neurogenesis contributes, at least in part, to learning and memory. However, little is known about the factors that regulate the processes of adult neurogenesis including the proliferation of neural progenitor cells and the survival of newborn cells.Apart from the classical regulatory function on calcium and phosphorus metabolism of 1,25(OH)2D3 as a pleiotropic hormone in numerous cell types, its anti-proliferative, pro-apoptotic and pro-differentiation functions has recently attracted much attention. 1,25(OH)2D3 treatment leads to the accumulation of human cancer cell in G0/G1 phase of cell cycle. At birth of rats, 1,25(OH)2D3 deficiency significantly increases mitosis and impairs apoptosis of cells. In brain development, vitamin D3 deprivation disrupts the normal sequence of apoptotic and mitotic activity. Thus, it would be interesting to explore whether the 1,25(OH)2D3 deficiency influences the process of adult neurogenesis. OBJECTIVEIn order to fully deplete 1,25(OH)2D3 in adult brain, in this study we applied an established adult mice model, the 1α-hydroxylase knockout mice (1α(OH)ase-/- mice) by targeted ablation of the hormone-binding and heme-binding domains in 1α-hydroxylase gene. In the current study, we evaluated the role of 1,25(OH)2D3 deficiency on the production of newborn cells, the neuronal differentiation (neuron or glia) and the survival of newborn neural cells (newborn cells that survive to maturity) in the hippocampal DG in mice. The knockout of 1α-hydroxylase gene leads to hypocalcaemia and hypophosphatemia in 1α(OH)ase-/- mice. By supplying 1,25(OH)2D3 or adjusting serum calcium and phosphorus concentrations in 1α(OH)ase-/- mice, we explored the molecular mechanisms underlying the abnormal adult neurogenesis by 1,25(OH)2D3 deficiency and its effects on hippocampus- dependent spatial memory.METHODSa) Homozyous 1α(OH)ase-/- mice were obtained through breeding of heterozygous mice and were identified by PCR using genomic DNA from mice tail.b) Bromodeoxyuridine (BrdU) was used for mitotic labeling. At 48 hr and 28th day after the first injection of BrdU, BrdU-positive cells by BrdU-staining in the subgranular zone (SGZ) of the hippocapal DG were examined to investigate the effects of 1,25(OH)2D3 deficiency on cell proliferation and the survival of newborn neurons. We applied the proliferating cell nuclear antigen (PCNA) staining to further confirm the status of cells proliferation in the DG. Effect of 1,25(OH)2D3 deficiency on the differentiation of progenitor cells was examined by the double staining with BrdU and NeuN or GFAP on 28th day after the last BrdU-injection. DNA fragmentation (TUNEL) staining was used to examine the apoptosis. Coronal sections were prepared from dorsal hippocampus and then were stained with toluidine blue.c) By 1,25(OH)2D3 treatment,'rescue diet'to recover the level of serum calcium and phosphorus in 1α(OH)ase-/- mice, L-VGCC inhibitor nifedipine administrated, the molecular mechanisms underlying the effects of 1,25(OH)2D3 deficiency on adult neurogenesis was examined.d) Animals were trained in the standard Morris water maze task to examine the spatial memory function.RESULTS1. The number of both 24-hr-old BrdU+ cells and proliferating cell nuclear antigen positive cells in 8-week-old 1α(OH)ase-/- mice increased approximately 2-fold compared to wild-type littermates. In contrast, the number of 7- and 28-day-old BrdU+ cells in 1α(OH)ase-/- mice decreased by 50% compared to wild-type mice, while the proportion of BrdU+/NeuN+ cells in 28-day-old BrdU+ population showed no difference between 1α(OH)ase-/- and wild-type mice. Apoptotic cells in the DG markedly increased in 1α(OH)ase-/- mice.2. Replenishment of 1,25(OH)2D3, but not correction of serum calcium and phosphorus levels, prevented the above mentioned changes in the neurogenesis in 1α(OH)ase-/- mice.3. Level of L-VGCC protein expression in the DG of 8-week-old 1α(OH)ase-/- mice significantly increased compared to age-matched wild-type mice. The treatment with L-VGCC inhibitor nifedipine blocked the enhanced cell proliferations in 1α(OH)ase-/-, but it did not influence the suppression of newborn cell survival.4. The abnormal neurogenesis in 1α(OH)ase-/- mice caused 10% reduction of granule cells in the DG, which was accompanied by a significant prolongation of the escape-latency to the hidden-platform in 12-week-old 1α(OH)ase-/- mice compared to age-matched wild-type mice.CONCLUSION1. 1,25(OH)2D3 deficiency enhances the proliferation of neural progenitor cells, while depresses the survival of newborn cells without affecting the differentiation of progenitor cells in adult hippocampal DG.2. The 1,25(OH)2D3 deficiency-induced abnormal neurogenesis is hypocalcaemia and hypophosphatemia-independent,, whereas partially is related with the increase in L-VGCC expression.3. The abnormal neurogenesis by 1,25(OH)2D3 deficiency leads to 10% reduction of granule cells in the DG, which is accompanied by deficits in spatial learning and memory.CLINIC SIGNIFICANCEThe present study, for the first time, provides in vivo evidence that the 1,25(OH)2D3 deficiency interrupts the adult neurogenesis, which may help us to clarify the role of 1,25(OH)2D3 deficiency in cognitive deterioration and dementia in Alzheimer's disease. Thus, the complementarity of 1,25(OH)2D3 might prevent or treat cognitive deterioration and dementia, particularly in elder and Alzheimer's disease patients. |
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