1，25（OH）_2D_3缺乏对成年神经发生的影响及其调控机制的研究
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摘要
研究背景
1,25-二羟基维生素D3(1α,25-dihydroxyvitamin D3,1,25(OH)2D3)是维生素D3(Vitamin D3,VD3)最重要的活性形式。2007年的统计资料表明,成年人VD3缺乏的发生率高达24%,而妊娠期妇女和老年人群的发生率比预计的更高。1,25(OH)2D3能调节钙磷代谢,维持骨骼矿化,预防佝偻病和骨质疏松的发生。近年的研究发现,胚胎期的1,25(OH)2D3缺乏能导致中枢神经系统发育异常。临床资料已提示,老年人群的1,25(OH)2D3缺乏与认知功能减退和老年性痴呆病的发生相关。阿尔茨海默病患者发生1,25(OH)2D3缺乏的比例显著高于同龄健康人群,并且1,25(OH)2D3受体(Vitamin D receptor,VDR)的表达有下调趋势。1,25(OH)2D3治疗能减缓老年大鼠海马脑区的萎缩。但是,有关1,25(OH)2D3缺乏是否影响认知功能迄今仍未见有明确的报道。
在成年啮齿类和某些哺乳动物脑内,存在神经干细胞和神经前体细胞,这类细胞具有终生自我更新的能力,可以分化为神经细胞—成年神经发生(Neurogenesis)。在海马齿状回(Dentat Gyrus,DG),这些新生神经元显示了与成熟颗粒细胞相同的形态和功能特征,能与CA3区神经元和内嗅皮层的传入纤维建立突触联系,诱发突触传递和可塑性。特别是DG区的神经发生已被证实与空间认知功能密切相关,如新生神经元数量增多能提高学习记忆功能,而阻碍成年神经发生则造成记忆功能减退。本研究室的近期研究已证明,孕酮通过促进新生神经元的存活和成熟能增强空间认知功能,而β-淀粉肽通过阻碍新生神经细胞的分化和存活导致空间认知障碍。因此,成年的神经发生被认为能取代和修复由于自然老化或病变造成的神经元缺失,最大限度地维护脑的结构和功能的完整性。
近年来,1,25(OH)2D3对多种肿瘤细胞的抗增殖、促凋亡和促分化作用引起了神经科学研究领域的广泛关注。妊娠期1,25(OH)2D3缺乏对脑发育的影响在近期已有较多的研究报道。1,25(OH)2D3缺乏能增加细胞周期调控蛋白如细胞周期素D1、细胞周期素B1和视网膜母细胞瘤蛋白表达,促进细胞的有丝分裂。在脑发育阶段,1,25(OH)2D3缺乏促进神经干细胞增殖,同时减少神经细胞死亡,从而导致脑的发育异常。1,25(OH)2D3缺乏引起体外培养室管膜下区神经元的神经球数量明显增加。但有关1,25(OH)2D3缺乏对成年神经发生的调节作用及其分子机制目前尚未见有报道。
研究目的
选用靶向敲除1α-羟化酶基因(1α(OH)ase-/-)小鼠作为成年1,25(OH)2D3缺乏的实验动物模型,研究1,25(OH)2D3缺乏对成年海马DG区神经发生的调节作用及其相关的分子机制,并探讨1,25(OH)2D3缺乏引起成年神经发生的改变对空间记忆功能的影响。
实验方法
1.生后7周龄的1α(OH)ase-/-小鼠血液循环中1,25(OH)2D3浓度已低到检测不出,将生后8周龄的1α(OH)ase-/-小鼠作为成年1,25(OH)2D3缺乏的实验动物模型。用剪尾提取DNA进行PCR扩增的方法来鉴定基因型。
2.腹腔注射5-溴-2’-脱氧核苷尿嘧啶(5-bromo-2’-deoxyuridine,BrdU)标记有丝分裂细胞。分别在BrdU末次给药后24小时,第7天和第28天用免疫组织学技术检测BrdU标记的阳性细胞,以评价海马DG区新生神经细胞的增殖(24小时-BrdU+细胞)、存活(7天-BrdU+细胞)和分化(28天-BrdU+细胞)。
3.用增殖细胞核抗原(Proliferating cell nuclear antigen,PCNA)染色法,检测海马DG区神经干细胞的增殖情况。分别用神经元特异性核蛋白(Neuron-specific nuclear protein,NeuN)或胶质纤维酸性蛋白(Glial fibrillary acidic protein,GFAP)与BrdU双标记免疫荧光染色法,检测海马DG区新生神经细胞的分化。用TUNEL细胞凋亡免疫染色法,检测海马DG区神经细胞的凋亡。用甲苯胺蓝染色法检测海马DG区的成熟颗粒细胞。
4.分别用1,25(OH)2D3替代治疗、高钙高磷“补救饲料”喂养、或L-型电压门控钙通道(L-type voltage-gated calcium channel,L-VGCC)阻断剂尼非地平给药,并结合L-VGCC蛋白免疫荧光染色,来探讨1,25(OH)2D3缺乏调节成年海马DG区神经发生的分子机制。
5.用Morris水迷宫实验法检测空间记忆功能。
实验结果
1.与同窝或同周龄的野生型小鼠相比,1α(OH)ase-/-小鼠海马DG区24小时-BrdU+细胞数和PCNA+细胞数都增加了近2倍,而7天-和28天-BrdU+细胞数均减少了大约50%。但是,1α(OH)ase-/-小鼠的BrdU+/NeuN+和BrdU+/GFAP+细胞占整个BrdU+细胞数的比值与同周龄野生型小鼠相比无统计学差异。
2.补充1,25(OH)2D3能完全纠正1α(OH)ase-/-小鼠的24小时-BrdU+细胞增多和28天-BrdU+细胞减少。但是,喂养“补救饲料”恢复正常血钙磷水平对1α(OH)ase-/-小鼠的神经发生改变没有作用。
3.与同周龄的野生型小鼠相比,成年1α(OH)ase-/-小鼠海马DG区的L-VGCC表达增多。L-VGCC阻断剂尼非地平给药能阻止成年1α(OH)ase-/-小鼠的24小时-BrdU+细胞增多,但不能改善7天-BrdU+细胞减少。
4.与同周龄的野生型小鼠相比,8周龄1α(OH)ase-/-小鼠海马DG区发生凋亡的神经细胞增多,12周龄1α(OH)ase-/-小鼠海马DG区的成熟颗粒细胞数减少大约10%。
5. Morris水迷宫行为学结果显示,12周龄1α(OH)ase-/-小鼠登台潜伏期比同周龄的野生型小鼠明显延长,喂养“补救饲料”恢复正常血钙磷水平对1α(OH)ase-/-小鼠的登台潜伏期延长没有作用,而补充1,25(OH)2D3能明显缩短1α(OH)ase-/-小鼠的登台潜伏期。
结论
1. 1,25(OH)2D3缺乏促进海马DG区神经干细胞增殖,但又阻碍新生神经细胞的存活,并不影响神经前体细胞的分化。
2. 1,25(OH)2D3缺乏是成年神经发生异常的主要原因,低钙血症和低磷血症并不参与1,25(OH)2D3缺乏引起神经发生改变的过程。1,25(OH)2D3缺乏可能是通过上调L-VGCC表达和增加钙内流,促进神经干细胞增殖。
3. 1,25(OH)2D3缺乏通过损伤成年神经发生导致海马DG区的颗粒细胞数减少,是空间记忆功能减退的原因之一。
我们的研究结果提示,1,25(OH)2D3缺乏能损伤成年神经发生,导致认知功能减退和促进老年性痴呆的发生。补充1,25(OH)2D3,而不是调节血钙和血磷水平,能更有效地预防和治疗由VD3缺乏所导致的成年神经发生障碍,并有可能改善老年人群和脑损伤患者的认知功能。
INTRODUCTION
Vitamin 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.
OBJECTIVE
In 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.
METHODS
a) 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.
RESULTS
1. 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.
CONCLUSION
1. 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 SIGNIFICANCE
The 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.
1,25-二羟基维生素D3(1α,25-dihydroxyvitamin D3,1,25(OH)2D3)是维生素D3(Vitamin D3,VD3)最重要的活性形式。2007年的统计资料表明,成年人VD3缺乏的发生率高达24%,而妊娠期妇女和老年人群的发生率比预计的更高。1,25(OH)2D3能调节钙磷代谢,维持骨骼矿化,预防佝偻病和骨质疏松的发生。近年的研究发现,胚胎期的1,25(OH)2D3缺乏能导致中枢神经系统发育异常。临床资料已提示,老年人群的1,25(OH)2D3缺乏与认知功能减退和老年性痴呆病的发生相关。阿尔茨海默病患者发生1,25(OH)2D3缺乏的比例显著高于同龄健康人群,并且1,25(OH)2D3受体(Vitamin D receptor,VDR)的表达有下调趋势。1,25(OH)2D3治疗能减缓老年大鼠海马脑区的萎缩。但是,有关1,25(OH)2D3缺乏是否影响认知功能迄今仍未见有明确的报道。
在成年啮齿类和某些哺乳动物脑内,存在神经干细胞和神经前体细胞,这类细胞具有终生自我更新的能力,可以分化为神经细胞—成年神经发生(Neurogenesis)。在海马齿状回(Dentat Gyrus,DG),这些新生神经元显示了与成熟颗粒细胞相同的形态和功能特征,能与CA3区神经元和内嗅皮层的传入纤维建立突触联系,诱发突触传递和可塑性。特别是DG区的神经发生已被证实与空间认知功能密切相关,如新生神经元数量增多能提高学习记忆功能,而阻碍成年神经发生则造成记忆功能减退。本研究室的近期研究已证明,孕酮通过促进新生神经元的存活和成熟能增强空间认知功能,而β-淀粉肽通过阻碍新生神经细胞的分化和存活导致空间认知障碍。因此,成年的神经发生被认为能取代和修复由于自然老化或病变造成的神经元缺失,最大限度地维护脑的结构和功能的完整性。
近年来,1,25(OH)2D3对多种肿瘤细胞的抗增殖、促凋亡和促分化作用引起了神经科学研究领域的广泛关注。妊娠期1,25(OH)2D3缺乏对脑发育的影响在近期已有较多的研究报道。1,25(OH)2D3缺乏能增加细胞周期调控蛋白如细胞周期素D1、细胞周期素B1和视网膜母细胞瘤蛋白表达,促进细胞的有丝分裂。在脑发育阶段,1,25(OH)2D3缺乏促进神经干细胞增殖,同时减少神经细胞死亡,从而导致脑的发育异常。1,25(OH)2D3缺乏引起体外培养室管膜下区神经元的神经球数量明显增加。但有关1,25(OH)2D3缺乏对成年神经发生的调节作用及其分子机制目前尚未见有报道。
研究目的
选用靶向敲除1α-羟化酶基因(1α(OH)ase-/-)小鼠作为成年1,25(OH)2D3缺乏的实验动物模型,研究1,25(OH)2D3缺乏对成年海马DG区神经发生的调节作用及其相关的分子机制,并探讨1,25(OH)2D3缺乏引起成年神经发生的改变对空间记忆功能的影响。
实验方法
1.生后7周龄的1α(OH)ase-/-小鼠血液循环中1,25(OH)2D3浓度已低到检测不出,将生后8周龄的1α(OH)ase-/-小鼠作为成年1,25(OH)2D3缺乏的实验动物模型。用剪尾提取DNA进行PCR扩增的方法来鉴定基因型。
2.腹腔注射5-溴-2’-脱氧核苷尿嘧啶(5-bromo-2’-deoxyuridine,BrdU)标记有丝分裂细胞。分别在BrdU末次给药后24小时,第7天和第28天用免疫组织学技术检测BrdU标记的阳性细胞,以评价海马DG区新生神经细胞的增殖(24小时-BrdU+细胞)、存活(7天-BrdU+细胞)和分化(28天-BrdU+细胞)。
3.用增殖细胞核抗原(Proliferating cell nuclear antigen,PCNA)染色法,检测海马DG区神经干细胞的增殖情况。分别用神经元特异性核蛋白(Neuron-specific nuclear protein,NeuN)或胶质纤维酸性蛋白(Glial fibrillary acidic protein,GFAP)与BrdU双标记免疫荧光染色法,检测海马DG区新生神经细胞的分化。用TUNEL细胞凋亡免疫染色法,检测海马DG区神经细胞的凋亡。用甲苯胺蓝染色法检测海马DG区的成熟颗粒细胞。
4.分别用1,25(OH)2D3替代治疗、高钙高磷“补救饲料”喂养、或L-型电压门控钙通道(L-type voltage-gated calcium channel,L-VGCC)阻断剂尼非地平给药,并结合L-VGCC蛋白免疫荧光染色,来探讨1,25(OH)2D3缺乏调节成年海马DG区神经发生的分子机制。
5.用Morris水迷宫实验法检测空间记忆功能。
实验结果
1.与同窝或同周龄的野生型小鼠相比,1α(OH)ase-/-小鼠海马DG区24小时-BrdU+细胞数和PCNA+细胞数都增加了近2倍,而7天-和28天-BrdU+细胞数均减少了大约50%。但是,1α(OH)ase-/-小鼠的BrdU+/NeuN+和BrdU+/GFAP+细胞占整个BrdU+细胞数的比值与同周龄野生型小鼠相比无统计学差异。
2.补充1,25(OH)2D3能完全纠正1α(OH)ase-/-小鼠的24小时-BrdU+细胞增多和28天-BrdU+细胞减少。但是,喂养“补救饲料”恢复正常血钙磷水平对1α(OH)ase-/-小鼠的神经发生改变没有作用。
3.与同周龄的野生型小鼠相比,成年1α(OH)ase-/-小鼠海马DG区的L-VGCC表达增多。L-VGCC阻断剂尼非地平给药能阻止成年1α(OH)ase-/-小鼠的24小时-BrdU+细胞增多,但不能改善7天-BrdU+细胞减少。
4.与同周龄的野生型小鼠相比,8周龄1α(OH)ase-/-小鼠海马DG区发生凋亡的神经细胞增多,12周龄1α(OH)ase-/-小鼠海马DG区的成熟颗粒细胞数减少大约10%。
5. Morris水迷宫行为学结果显示,12周龄1α(OH)ase-/-小鼠登台潜伏期比同周龄的野生型小鼠明显延长,喂养“补救饲料”恢复正常血钙磷水平对1α(OH)ase-/-小鼠的登台潜伏期延长没有作用,而补充1,25(OH)2D3能明显缩短1α(OH)ase-/-小鼠的登台潜伏期。
结论
1. 1,25(OH)2D3缺乏促进海马DG区神经干细胞增殖,但又阻碍新生神经细胞的存活,并不影响神经前体细胞的分化。
2. 1,25(OH)2D3缺乏是成年神经发生异常的主要原因,低钙血症和低磷血症并不参与1,25(OH)2D3缺乏引起神经发生改变的过程。1,25(OH)2D3缺乏可能是通过上调L-VGCC表达和增加钙内流,促进神经干细胞增殖。
3. 1,25(OH)2D3缺乏通过损伤成年神经发生导致海马DG区的颗粒细胞数减少,是空间记忆功能减退的原因之一。
我们的研究结果提示,1,25(OH)2D3缺乏能损伤成年神经发生,导致认知功能减退和促进老年性痴呆的发生。补充1,25(OH)2D3,而不是调节血钙和血磷水平,能更有效地预防和治疗由VD3缺乏所导致的成年神经发生障碍,并有可能改善老年人群和脑损伤患者的认知功能。
INTRODUCTION
Vitamin 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.
OBJECTIVE
In 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.
METHODS
a) 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.
RESULTS
1. 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.
CONCLUSION
1. 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 SIGNIFICANCE
The 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.
引文
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33. Burne TH, Féron F, Brown J, Eyles DW, McGrath JJ, Mackay-Sim A. Combined prenatal and chronic postnatal vitamin D deficiency in rats impairs prepulse inhibition of acoustic startle. Physiol Behav, 2004,81(4):651-655.
34. Morris JC. The Clinical Dementia Rating (CDR): current version and scoring rules. Neurology, 1993,43(11):2412-2414.
35. Katzman, R, Brown, T. Validation of a short orientation-memory-concentration test of cognitive impairment. Am. J. Psychiatr, 1983,140(6):734-739.
36. Sutherland MK, Somerville MJ, Yoong LK, Bergeron C, Haussler MR, McLachlan DR. Reduction of vitamin D hormone receptor mRNA levels in Alzheimer as compared to Huntington hippocampus: correlation with calbindin- 28k mRNA levels. Brain Res. Mol, 1992,13:239-250.
37. Gezen-Ak D, Dursun E, Ertan T, Hana?asi H, Gürvit H, Emre M, Eker E, Oztürk M, Engin F, Yilmazer S. Association between vitamin D receptor gene polymorphism and Alzheimer’s disease. Tohoku J. Exp. Med, 2007,212:275-282.
38. Landfield PW, Cadwallader-Neal L. Long-term treatment with calcitriol (1,25(OH)2vitD3) retards a biomarker of hippocampal aging in rats. Neurobiol. Aging, 1998,19:469-477.
39. Buell JS, Dawson-Hughes B. Vitamin D and neurocognitive dysfunction: Preventing‘‘D”ecline? Molecular Aspects of Medicine, 2008,29(6):415-422.
40. Wang JY, Wu JN, Cherng TL, Hoffer BJ, Chen HH, Borlongan CV, Wang Y. Vitamin D(3) attenuates 6-hydroxydopamine-induced neurotoxicity in rats. Brain Res, 2001,904(1):67-75.
41. Dekkers J, Bayley P, Dick JR, Schwaller B, Berchtold MW, Greensmith L. Over-expression of parvalbumin in transgenic mice rescues motoneurons from injury-induced cell death. Neuroscience, 2004,123(2):459-466.
42. G. Pirianov, K.W. Colston, Interaction of vitamin D analogs with signaling pathways leading to active cell death in breast cancer cells. Steroids, 2001,66: 309-318.
43. B.L. Lokeshwar, G.G. Schwartz, M.G. Selzer, K.L. Burnstein, S.H, Zhuang, N.L. Block, L. Binderup. Inhibition of prostate cancer metastasis in vivo: a comparison of 1,25-dihydroxyvitamin D (calcitriol) and EB 1089. Cancer Epidemiology, Biomarkers and Prevention, 1999,8:241-248.
44. G.D. Diaz, C. Paraskeva, M.G. Thomas, L. Binderup, A. Hague. Apoptosis is induced by the active metabolite of vitamin D3 and its analogue EB1089 in colorectal adenoma and carcinoma cells: possible implications for prevention and therapy. Cancer Research, 2000,60:2304-2312.
45. Baudet C, Chevalier G, Chassevent A, Canova C, Filmon R, Larra F, Brachet P, Wion D. 1,25-Dihydroxyvitamin D3 induces programmed cell death in a rat glioma cell line. J Neurosci Res, 1996,46(5):540-550.
46. Gumireddy K, Ikegaki N, Phillips PC, Sutton LN, Reddy CD. Effect of20-epi-1alpha,25-dihydroxyvitamin D3 on the proliferation of human neuroblastoma: role of cell cycle regulators and the Myc-Id2 pathway. Biochem Pharmacol, 2003,65(12):1943-1955.
47. Furman I, Baudet C, Brachet P. Differential expression of M-CSF, LIF, and TNF-αgenes in normal and malignant rat glial cells: regulation by lipopolysaccharide and vitamin D. J. Neurosci. Res, 1996,46:360-366.
48. Neveu I, Naveilhan P, Menaa C, Wion D, Brachet P, Garabédian M. Synthesis of 1,25-dihydroxyvitamin D3 by rat brain macrophages in vitro. J. Neurosci. Res, 1994,38:214-220.
49. Lemire JM, Archer DC. 1,25-dihydroxyvitamin D3 prevents the in vivo induction of murine experimental autoimmune encephalomyelitis. J Clin Invest, 1991,87(3):1103-1107.
50. Garcion E, Sindji L, Nataf S, Brachet P, Darcy F, Montero-Menei CN. Treatment of experimental autoimmune encephalomyelitis in rat by 1,25-dihydroxyvitamin D3 leads to early effects within the central nervous system. Acta Neuropathol, 2003,105(5):438-448.
51. Spach KM, Pedersen LB, Nashold FE, Kayo T, Yandell BS, Prolla TA, Hayes CE. Gene expression analysis suggests that 1,25-dihydroxyvitamin D3 reverses experimental autoimmune encephalomyelitis by stimulating inflammatory cell apoptosis. Physiol Genomics, 2004,18(2):141-151.
52. Cai W, Zhu Y, Furuya K, Li Z, Sokabe M, Chen L. Two different molecular mechanisms underlying progesterone neuroprotection against ischemic brain damage. Neuropharmacology, 2008,55:127-138.
53. Losem-Heinrichs E, G?rg B, Schleicher A, Redecker C, Witte OW, Zilles K, Bidmon HJ. A combined treatment with 1alpha,25-dihydroxy-vitamin D3 and 17beta-estradiol reduces the expression of heat shock protein-32 (HSP-32) following cerebral cortical ischemia. J Steroid Biochem Mol Biol, 2004,(1-5): 371-374.
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30. Becker A, Eyles DW, McGrath JJ, Grecksch G. Transient prenatal vitamin D deficiency is associated with subtle alterations in learning and memory functions in adult rats. Behav Brain Res, 2005,161(2):306-312.
31. Burne TH, Becker A, Brown J, Eyles DW, Mackay-Sim A, McGrath JJ. Transient prenatal Vitamin D deficiency is associated with hyperlocomotion in adult rats. Behav Brain Res, 2004,154(2):549-555.
32. Kesby JP, Burne TH, McGrath JJ, Eyles DW. Developmental vitamin D deficiency alters MK 801-induced hyperlocomotion in the adult rat: An animal model of schizophrenia. Biol Psychiatry, 2006,60(6):591-596.
33. Burne TH, Féron F, Brown J, Eyles DW, McGrath JJ, Mackay-Sim A. Combined prenatal and chronic postnatal vitamin D deficiency in rats impairs prepulse inhibition of acoustic startle. Physiol Behav, 2004,81(4):651-655.
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36. Sutherland MK, Somerville MJ, Yoong LK, Bergeron C, Haussler MR, McLachlan DR. Reduction of vitamin D hormone receptor mRNA levels in Alzheimer as compared to Huntington hippocampus: correlation with calbindin- 28k mRNA levels. Brain Res. Mol, 1992,13:239-250.
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38. Landfield PW, Cadwallader-Neal L. Long-term treatment with calcitriol (1,25(OH)2vitD3) retards a biomarker of hippocampal aging in rats. Neurobiol. Aging, 1998,19:469-477.
39. Buell JS, Dawson-Hughes B. Vitamin D and neurocognitive dysfunction: Preventing‘‘D”ecline? Molecular Aspects of Medicine, 2008,29(6):415-422.
40. Wang JY, Wu JN, Cherng TL, Hoffer BJ, Chen HH, Borlongan CV, Wang Y. Vitamin D(3) attenuates 6-hydroxydopamine-induced neurotoxicity in rats. Brain Res, 2001,904(1):67-75.
41. Dekkers J, Bayley P, Dick JR, Schwaller B, Berchtold MW, Greensmith L. Over-expression of parvalbumin in transgenic mice rescues motoneurons from injury-induced cell death. Neuroscience, 2004,123(2):459-466.
42. G. Pirianov, K.W. Colston, Interaction of vitamin D analogs with signaling pathways leading to active cell death in breast cancer cells. Steroids, 2001,66: 309-318.
43. B.L. Lokeshwar, G.G. Schwartz, M.G. Selzer, K.L. Burnstein, S.H, Zhuang, N.L. Block, L. Binderup. Inhibition of prostate cancer metastasis in vivo: a comparison of 1,25-dihydroxyvitamin D (calcitriol) and EB 1089. Cancer Epidemiology, Biomarkers and Prevention, 1999,8:241-248.
44. G.D. Diaz, C. Paraskeva, M.G. Thomas, L. Binderup, A. Hague. Apoptosis is induced by the active metabolite of vitamin D3 and its analogue EB1089 in colorectal adenoma and carcinoma cells: possible implications for prevention and therapy. Cancer Research, 2000,60:2304-2312.
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46. Gumireddy K, Ikegaki N, Phillips PC, Sutton LN, Reddy CD. Effect of20-epi-1alpha,25-dihydroxyvitamin D3 on the proliferation of human neuroblastoma: role of cell cycle regulators and the Myc-Id2 pathway. Biochem Pharmacol, 2003,65(12):1943-1955.
47. Furman I, Baudet C, Brachet P. Differential expression of M-CSF, LIF, and TNF-αgenes in normal and malignant rat glial cells: regulation by lipopolysaccharide and vitamin D. J. Neurosci. Res, 1996,46:360-366.
48. Neveu I, Naveilhan P, Menaa C, Wion D, Brachet P, Garabédian M. Synthesis of 1,25-dihydroxyvitamin D3 by rat brain macrophages in vitro. J. Neurosci. Res, 1994,38:214-220.
49. Lemire JM, Archer DC. 1,25-dihydroxyvitamin D3 prevents the in vivo induction of murine experimental autoimmune encephalomyelitis. J Clin Invest, 1991,87(3):1103-1107.
50. Garcion E, Sindji L, Nataf S, Brachet P, Darcy F, Montero-Menei CN. Treatment of experimental autoimmune encephalomyelitis in rat by 1,25-dihydroxyvitamin D3 leads to early effects within the central nervous system. Acta Neuropathol, 2003,105(5):438-448.
51. Spach KM, Pedersen LB, Nashold FE, Kayo T, Yandell BS, Prolla TA, Hayes CE. Gene expression analysis suggests that 1,25-dihydroxyvitamin D3 reverses experimental autoimmune encephalomyelitis by stimulating inflammatory cell apoptosis. Physiol Genomics, 2004,18(2):141-151.
52. Cai W, Zhu Y, Furuya K, Li Z, Sokabe M, Chen L. Two different molecular mechanisms underlying progesterone neuroprotection against ischemic brain damage. Neuropharmacology, 2008,55:127-138.
53. Losem-Heinrichs E, G?rg B, Schleicher A, Redecker C, Witte OW, Zilles K, Bidmon HJ. A combined treatment with 1alpha,25-dihydroxy-vitamin D3 and 17beta-estradiol reduces the expression of heat shock protein-32 (HSP-32) following cerebral cortical ischemia. J Steroid Biochem Mol Biol, 2004,(1-5): 371-374.