The Normal Intracellular Cholesterol Level Is Required For Maintaining The Activation Of IGF-1 Survival Signaling In Neuronal Cells

It is widely accepted that Alzheimer disease(AD)is associated with the abnormal cholesterol metabolism.Recent studies have shown that AD may represent a neuroendocrine disease in which substantial abnormalities arise due to impairments of insulin and insulin-like growth factor(IGF)signaling.We speculate that the reduced cholesterol level in neuron might contribute to the impairments of insulin/IGF-I signaling.3p hydroxy sterol △24-reductase(DHCR24)catalyzing the conversion of desmosterol to cholesterol,is a multiple function enzyme that possesses anti-apoptotic activities.Our previous study has demonstrated that DHCR24 could play an important role in insulin-Akt cell survival signaling through maintaining the cholesterol biosynthesis and normal structure and function of caveolae in mouse embryonic fibroblasts.In the present study we targeted DHCR24 through inhibiting its enzyme activity by the U18666A,a chemical inhibitor of DHCR24,and RNA interference to investigate the role of DHCR24 and cholesterol biosynthesis in IGF-I signaling in rat adrenal pheochromocytoma(PC 12)cells.The treatment of U18666A in serum free medium blocked the neuron-protective function of IGF-I,demonstrated by the 3-(4,5-Dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT)assay and in situs apoptotic assay.SiRNA targeting DHCR24 also shown the similar results.The treatment of U18666A in serum free medium also reduced the choleterol level and resulted in the accumulation of desmosterol by inhibiting DHCR24 activity in neuron,demonstrated by High Performance Liquid(HPLC).These results suggested that the reduced intracellular cholesterol level might contribute to the impairment of IGF-I function.Double immunocytochemical fluorescent analysis revealed that the IGF-I receptor is well co-localized in caveolae,a cholesterol-rich micro-domain on the plasma membrane of PC 12 when the cells are cultured in the growth medium.However,the treatment of U18666A in the serum free medium destroyed the location of IGF-1 receptor in caveolae.Consistent with this,western blot demonstrated that the phosphorylations of insulin receptor,insulin receptor substrate(IRS),Akt/protein kinase B and Bad induced by IGF-1 exposure were significantly inhibited in U18666A-treated cells,if comparing to that in control.Taken together,these results demonstrated that a normal intracellular cholesterol level and normal expression and function of DHCR24 are very important for maintaining the function of insulin/IGF-1-Akt survival cascade in neuronal cells.And these results are significant to clarify the molecular mechanism of Alzheimer disease,and to provid fundamental basis for the theory of "type 3 diabetes".