The Identification And Function Investigation Of Ethanol Response Genes In Neuron Cell

Aim:Ethanol is documented to involve in many diseases concerned with energy and substrate metabolism, e.g., arteriosclerosis, dementia, diabetes, and conditions associated with Zn metabolism. High consumption of ethanolic beverages facilitates neurodegeneration. It has been reported that ethanol exposure leads to impairments of the cerebra and cerebellum in different areas, and triggers widespread apoptotic neurodegeneration in infant rat and mouse brain. The neuron alteration of function and pathology elicited by ethanol consumption was attributed to the facts as follows: extratoxicity, deficiency of Vitamin B1, abnormality of glial cell, growth factors change, cell apoptosis, oxidative stress and disturbance of energe metabolism. The sensitivity of neoron cells to the impairment elicited by ethanol increases significantly in the period of development, e.g.: 1) Using a dual mechanism, which is blockade of N- methyl-D-aspartate (NMDA) glutamate receptors and excessive activation of GABA (A) receptors, ethanol can trigger widespread apoptotic neurodegeneration in the developing rat forebrain,especially in the period of synaptogenesis; Ethanol causes millions of nerve cells to commit death by suppressing neuronal activity through altering glutamate and GABA transmission in the developing rodent brain;2) Ethanol selectively decreases Purkinje cell expression of tyrosine-kinase B (TrkB) and tyrosine-kinase C (TrkC) receptors following exposures within postnatal days of 4-6; 3) In humans, vulnerability coincides with the period of synaptogenesis and extends from the sixth month of gestation to several years after birth. This pro-apoptotic effect of ethanol provides a likely explanation for the diminished brain size and lifelong neurobehavioral disturbances associated with the human fetal ethanol syndrome. The oxidative stress induced by ethanol is considered to facilitate neurodegeneration. As a major risk in senile people, neurodegeneration is appeared as forms of dementia, brain shrinking, Parkinson's disease and act as one of the main contributors for elderly people death in modern society. Based on the facts that the pathology elicited by ethanol remains to be elusive,considerable research attention has been focused on identifying the endogenous and environmental factors that contribute to its etiology. To investigate the molecular mechanism of impairment elicited by ethanol in neuron cell, we plan to identify ethanol response genes and investigate function-concerned matters subsequently. Methods:With suppressed subtractive hybridization (SSH), ethanol response genes were identified in B104 cells and confirmed subsequently by reverse dot blot. The time-dependent gene expressions were investigated by Northern blot. The characterization and function of identified gene were investigated by the approaches as follows: 5'RACE (full-length gene preparation), Northern blot (tissue distribution detection, differentiation detection), recombinant plsmids construction (up-regulation: full length gene construction; down-regulation: RNAi sequences construction), transient transfection, and two-hybrid of yeast (protein interaction). Results:With SSH and Reverse dot blot approach, seven ethanol response genes were identified, in which three genes were up-regulated (aquaporin 1, NW₀47693; prohibitin, XM₅79541; SCIRP10, NW₀47402) and fours including two new genes were down-regulated by ethanol (zinc finger protein 341, XM₀01074788; Cab45a, MMU45977). One of them, Cab45, exhibited time-dependent down regulation by ethanol, was chosen for investigation further. The full-length sequence of Cab45 was determined for the first time in rat and exhibited high conservation among different species. With Northern blot analysis, this gene was characterized by ubiquitous expression in different tissues of rat and differentiation-concerned down-regulation in NGF trested PC12 cells as well as in different postnatal cerebellum tissues of rat. With transient transfection of recomnient plasmids in PC12 cells, Cab45 were up-regulated and down-regulated respectively dependent on different plasmids and confirmed by RT-PCR. Up-regulation of this gene can attenuate the impairment elicited by ethanol and UV exposure in PC12 cells, and down-regulation can intensify the impairment elicited by UV exposure. A transactivity was found in Cab45 when we tried to screen its interaction protein with the approach of two-hybrid of yeast and failed to identify its interaction protein with this method. Conclusion:With SSH approach, we identified seven ethanol response genes in B104 cells and one of them termed Cab45 was investigated further. As a calcium binding protein, Cab45 with full-length sequence was prepared for the first time in rat and characterized subsequently, which were high conservation, ubiquitous expression, and differentiation-concerned down-regulation. The up-regulation of Cab45 could attenuate the impairment elicited by ethanol and UV exposure in PC12 cells, and accordingly, down-regulation of Cab45 could intensify this impairment. The highlights of this study were summarized as follows: 1) many ethanol response genes were identified in B104 and one of them, Cab45, exhibited a time dependent down-regulation manner; 2) full-length sequence of Cab45 gene was prepared for the first time in rat and exhibited a high conservation in different species with its deduced amino acids; 3) the ubiquitous expression of Cab45 was investigated; 4) with the models of NGF treated PC12 cells and rat cerebellums, Cab45 were characterized by differentiation-concerned down-regulation; 5)the over expression of Cab45 could attenuate the impairment elicited by ethanol treatment and UV exposure; 6)Cab45 was identified for its transactivity by the approach of two-hybrid of yeast. 7) many transcription factor binding elements were identified in the upstream of transcription start position of human Cab45.Cab45 belong to new calcium binding protein family (Cab45, reticulocalbin, ERC-55 and calumenin). The common features in this family include six EF-hand motifs and secretory-pathway localization. The other members in this family are localized in endoplasmic reticulum, whereas Cab45 is localized in Golgi lumen. Based on the fact that calcium homeostasis plays an important role in the process of cell proliferation and differentiation and apoptosis, as calcium binding protein, Cab45, was considered to involve in the precise regulation of calcium and probably determine retention of soluble proteins in the Golgi compartment through calcium regulation. The aberrant expression of Cab45 could result in many neuron-concerned disease. This hypothesis was consolidated by recent report that a Golgi-localized protein Pmr1p, a counterpart of Cab45 in yeast, played an important role in calcium homeostasis. As a promising molecule, Cab45 will be a good target for development of neuron-concerned drug by investigating further.