Effects of cholestanol on neuronal cell death

脑腱黄瘤病（CTX）是一种遗传性脂质储存疾病，以高胆固醇血症、小脑共济失调、黄瘤和白内障为特征。我们推测CTX患者血清中的胆固醇可能诱导小脑神经元细胞死亡，最终导致小脑性共济失调。为了阐明这一假说，我们通过喂食胆固醇建立了高胆固醇血症大鼠。以苏丹黑B染色显示，仅饲喂胆固醇的大鼠的神经元细胞，尤其是小脑浦肯野细胞，可见胆固醇在小脑内沉积。以体外培养的小脑神经元细胞为研究对象，研究了胆固醇的体外作用。胆固醇浓度升高，细胞活力下降。采用末端脱氧核苷酸转移酶（TdT） dUTP缺口末端标记（TUNEL）方法，在胆固醇培养的细胞中检测到细胞凋亡的频率高于对照细胞。用胆固醇培养细胞后，白细胞介素-1β -多肽转换酶（ICE）和CPP32蛋白酶活性升高。我们进一步研究了胆固醇对体外诱导角膜内皮细胞和晶状体上皮细胞死亡的影响。10 μg/ml胆固醇培养的角膜内皮细胞和晶状体上皮细胞均明显丧失活力。与10 μg/ml胆固醇或1%乙醇相比，10 μg/ml胆固醇培养的细胞细胞核染色频率更高。凋亡DNA片段的定量分析证实，胆固醇诱导这些细胞凋亡具有时间依赖性。10 μg/ml胆固醇培养细胞的ICE和CPP32蛋白酶活性显著高于对照细胞。在体外，胆固醇被角膜内皮细胞和晶状体上皮细胞吸收，导致这些细胞凋亡。提示胆固醇可诱导小脑神经元细胞、角膜内皮细胞和晶状体上皮细胞凋亡。本研究结果可能解释CTX患者小脑性共济失调与白内障的发病机制。少

Cerebrotendinous xanthomatosis (CTX) is a hereditary lipid storage disease characterized by hyper-cholestanolemia, crebellar ataxia, xanthoma, and cataract. We hypothesized that cholestanol in serum of CTX patients might induce neuronal cell death in cerebellum and eventually lead to cerebellar ataxia. To elucidate this hypothesis we have established hyper-cholestanolemia rats by feeding cholestanol. Neuronal cells, especially Purkinje cells in the cerebellum were stained by Sudan black B only in cholestanol-fed rats, indicating the deposit of cholestanol in cerebellum. To examine effects of cholestanol in vitro, cerebellar neuronal cells were cultured with cholestanol. The cholestanol concentration increased and the viability decreased in cells cultured with cholestanol. Apoptosis was detected in cells cultured with cholestanol more frequently than in control cells using terminal deoxynucleotidyl transferase (TdT) dUTP nick end-labeling (TUNEL) method. The activities of interleukin-1β … More -converting enzyme (ICE) and CPP32 protease were increased in cells cultured with cholestanol. We further studied the effect of cholestanol on induce cornea endothelial and lens epithelial cell death in vitro. Both cornea endothelial and lens epithelial cells cultured with 10 μg/ml cholestanol showed a significant loss of viability. The nuclei of these cells cultured with 10 μg/ml cholestanol were more frequently stained than those exposed to 10 u」/ml cholesterol or 1 % ethanol. Quantitative analysis of apoptotic DNA fragmentation confirmed that the cholestanol induced apoptosis of these cells in a time-dependent manner. The activities of ICE and CPP32 proteases for cells cultured with 10 μg/ml cholestanol were significantly higher than those observed in control cells. In vitro, cholestanol was taken up by cornea endothelial cells and lens epithelial cells, an event which led to apoptosis of these cells.The data suggest that cholestanol induced apoptosis of cerebellar neuronal cells, cornea endothelial cells and lens epithelial cells. The results in the present study may explain the mechanism of cerebellar ataxia and cataract of CTX patients. Less