Functional Characteristics Of AQP9 In The Yak Brain

Hypoxia is one of the potent inducing factors of the occurrence and development to many diseases,especially brain diseases.In recent years,the physiological and genetic mechanisms of the plateau animal adaptation to the extremely hypoxia environment of the Qinghai-Tibet Plateau have drawn great attention.The yak(Bos grunniens)is the only large mammal that is currently known to have adapted to the extremely hypoxia environment in the Qinghai-Tibet Plateau(>4,000 m above sea level).Aquaporin 9(AQP9)as the ?neuron channel? participates in the transportation of water and energy substrates(glycerol,urea,adenine,uracil,lactic acid and ?-hydroxybutyrate),and also is involved in p53-associated signal pathways in cell apoptosis and energy metabolism.The functional characteristics of the yak brain AQP9 adaptation to hypoxia,however,is unknown.Therefore,in order to study the molecular mechanism of the animal adapted to their extreme low oxygen habitat in the Tinghai-Tibet Plateau.In this paper,the yak brain as the research object,the structure,expression and function characteristics of AQP9 as the breakthrough point,the functional characteristics of AQP9 in the low oxygen habitat were analyzed systematically using gene cloning,cell culture,gene over-expression,and in situ hybridization and RT-PCR,immunohistochemistry,immunofluorescence,and Western blotting technology.Result2.The results of in situ hybridization positioning analysis showed that the AQP9 mRNA positive reaction products was distributed in the molecular layer,granular layer,lateral pyramidal cell layer,the pyramidal cell layer,internal granular layer and polymorphic layer of the yak cerebral cortex,the dyeing of the molecular and granular layers was the deepest,the dyeing of the polymorphic layer is the lightest,which was similar with those of the cattle,but,overall,the dyeing of the yak was less than that of the cattle;The positive products was distributed in the cortex and dentate gyrus of the yak hippocampus,the dyeing of the granular layer of the dentate gyrus was the deepest,which was similar with those of the cattle,but,overall,the dyeing of the yak was less than that of the cattle,too.The results of western blotting analysis showed that the AQP9 protein in the cerebral cortex and hippocampus of the yak were lower than those of the cattle(Pcerebral cortex<0.05;Phippocampus>0.05).3.The functional test analysis showed that,(1)The expression of green fluorescence were the lowest in the N2 a cells though transfecting the yak PEGFP-AQP9 plasmid,compared with the transfection of the mouse and human;(2)Compared with the transfection of the mouse and human,the cell body of the primary culture neuron of the rat though transfecting the yak PEGFP-AQP9 plasmid was the smallest,the growth of the axons and dendrites obviously is inhibited,most of the neurons did not form a normal connection,and more significantly promoted the apoptosis,especially under low oxygen stress;(3)Compared with the transfection of the mouse and human,under the normal oxygen condition,the transfection of the yak CAGTAG-AQP9 plasmid was more likely to promote apoptosis of N2 a cells,however,under the low oxygen stress,the transfection of the yak CAGTAG-AQP9 plasmid was more likely to inhibit apoptosis of N2 a cells;(4)Under the normal oxygen,the overexpression of the yak,mouse and human AQP9 down-regulated the expression of caspase-3,Bcl-2 and c-fos,and than up-regulated the expression of Bax and p53,however,under the low oxygen stress,the overexpression of the yak,mouse and human AQP9 up-regulated the expression of caspase-3,Bcl-2 and c-fos,and than down-regulated the expression of Bax and p53,but the yaks had the least influence under the two states.Conclusion:1.In the plateau hypoxia adaptation,the unique amino acid structure characteristics(the 273 th of the yak and Tibetan antelope are same high hydrophobicity leucine,and than that of the low altitude species is the low hydrophobic proline;The deficiency 29 amino acids fragments in the yak's;the higher folded two-dimensional structure and three-dimensional conformation of differentiation)are the important mechanisms of the yak brain AQP9 adaptation to its environment.But,its specific features still need to be deeply explored.2.In the plateau hypoxia adaptation,the low expression of AQP9 in the yak brain is one of the important mechanisms of the gene adaptation to its environment.But,the regulation mechanism needs to be deeply explored.3.In the plateau hypoxia adaptation,the low expression of AQP9 may maintain its normal function through directly or indirectly involving in the regulation of the p53,Bcl-2/Bax value,c-fos and caspase-3 inhibiting the low oxygen induced apoptosis in the yak brain.