Research On Regulative Mechanism Of ATP7A In The Neuronal Morphogenesis

Copper is an essential micronutrient as its redox properties make a significant role in the body metabolism.Copper also functions as a cofactor within a host of cuproenzymes and copper-binding proteins that catalyze a wide range of biochemical reactions including cellular respiration,free radical defence,pigmentation,connective tissue formation,neuropeptide processing and iron transport in mammals.In addition to maintaining copper homeostasis,ATP7 A delivers copper to cuproenzymes and copper-binding proteins,the importance of which is illustrated by disorders of copper metabolism caused by mutations in the ATP7 A gene such as Menkes disease(MD).Dysfunction of ATP7 A often accompanies neurodegeneration.Current researches show expresson of ATP7 A is regulated spatially and temporally by nervous system development and may involve in neurodevelopment.To investigate the regulation of ATP7 A in the development of neurons,C57BL/6mice was selected as the research object,and expresson of ATP7 A was analyzed on the cell level in neural stem cells,astrocytes and hippocampal neuron cultured in vitro.Then using transdifferentiation of mouse embryo fibroblasts-one kind of induced pluripotent stem cells-to neurons,the difference of transdifferentiation before and after knock-out of Atp7 a was studied.The results as follows:1.Neural stem cells,astrocytes and hippocampal neurons were isolated from C57BL/6 mice embryo and newborn mice,and identified by immunofluorescence with purity above 90%.2.Mouse embryo fibroblasts(MEFs)were isolated from E13.5-14.5 mice embryo.The localization of ATP7 A in MEFs and other three kinds of cells was defined by immunofluorescence,showing that fluorescent signals were located between the nucleus and plasma membrane.3.Expression levels of ATP7 A mRNA and protein among four kinds of cells were compared through real-time QPCR and Western blot,showing that ATP7 A expression was decreased stepwise in MEF,NSC,astrocytes and hippocampal neuron.The result showed there may be a declining trend of ATP7 A during nervous system development,and the feasibility of the program inducting MEFs to neurons.4.The isolated embryo fibroblasts with Atp7afl/Y genotype(MEF7a+)was infected by recombinant adenovirus containing Cre gene in order to acquire MEF7a+·Cre+/-through Atp7 a knock-out.The efficience of knock-out determined by real-time QPCR and Western blot was about 70%.5.MEF7a+ or MEF7a+·Cre+/-were induced to neurons by recombinant adenovirus containing Ascl1,Brn2 and Ngn2(ABN)(ABN+ MEF7a+ and ABN+MEF7a+·Cre+/-)and by recombinant adenovirus containing ABN and Cre(ABNC+ MEF7a+).On the 7th day and 12 th day after differentiation,we identified differentiated neurons by immunofluorescense of ?-Tubulin III and computed a statistical analysis of neuronal morphogenesis including size of soma,total length of nerve fibers,length of the longest nerve fiber,number of nerve fibers from soma and number of braches.On the 7th day after differentiation,neuronal morphogenesis in experimental and control groups made little difference.The positive cells had a round,oval or triangle soma and elongated neurites extended from soma.The statistics on the 7th day showed that soma in ABN+MEF7a+·Cre+/-was smaller than other groups(*p<0.05),and compared to control group,somas in experimental groups sent forth fewer and shorter neurites(***p<0.001 and **p<0.01),as well as shorter axon(**p<0.01 and *p<0.05)and fewer branches(***p<0.001 and ***p<0.001).But on the 12 th day,there was no difference in the size of soma and silimar differences in other indicators.In conclusion,these experiments on the cell level prove that ATP7 A effects the maturation of nerve fibers in vitro.The cells absent of Atp7 a will have fewer and shorter neuritis and fewer branches.These results support a role for the regulation of ATP7 A on nerve development and may contribute to the severe neurodegeneration characteristic of copper-metabolic diseases caused by mutations in the ATP7 A gene.