Effect Of PI3K Pathway On Myelin Sheath And Body Weight In Mice

Background:2’,3’-cyclic nucleotide-3,-phosphodiesterase（CNP）has long been considered as a specific myelin protein,being mainly present in the nervous system of vertebrates.CNP has two isoforms,CNP1 and CNP2.CNP1 is mostly expressed in nerve cells.CNP2 has much broad expression in many different types of cells.At present,functions of CNP are not fully understood.Experiments have shown that CNP plays an important role in the formation of myelin sheath,maintenance of cell surface morphology,and participation in the metabolism of 2’,3’-cyclic adenosine phosphate（cAMP）.CNP-Cre mice are mutant mice expressing Cre recombinase under the control of endogenous Cnp promoter.Since Klaus-Armin Nave et al successfully constructed CNP-Cre mice in 2003,it has been widely used to study the effect of genes on myelin sheath and axon.Conditional P110αknockout mice(CNP-cre:P110α^flox/flox)were obtained by crossing CNP-Cre mice with P1110α^flox/floxlox/flox mice to study the effect of PI3K pathway on myelination and remyelination.Our previous study has found that at postnatal day 7 levels of MBP mRNA is decrease in CNP-cre:P110α^flox/floxlox/flox mice.No significant abnormality was found at other time points.We have also found that the weight of CNP-cre:P110α^flox/floxlox/flox mice decreased compared to control mice and this weight loss is likely related to FGF21 overexpression in the liver.In this study,we further studied on the mouse in order to thoroughly understand the myelin phenotype of the transgenic mice and the mechanism of weight loss in CNP-cre:P110α^flox/flox.Objective:1)To study whether the myelination disorder of CNP-cre:P110α^flox/floxlox/flox mice is related to weight loss;2)To study the effect of conditional knockout of P110αon remyelination in mice;3)To explore the cellular biological basis of CNP-cre:P110αflox/flox mice with weight loss（increased expression of FGF21）.Methods:1)CNP-cre mice were crossbred with P110α^flox/floxlox/flox and td-Tomato transgenic mice respectively to obtain CNP-cre:P110α^flox/floxlox/flox and CNP-cre:td-Tomato mice for subsequent experiments;2)The mice were fed with cuprizone（CPZ）to cause demyelination in the mice.After successful demyelination,the mice returned to the normal diet for remyelination;3)The myelination and remyelination of mice was observed by chrome cyanide staining and immunofluorescence;4)Detection of MBP mRNA expression in mouse P7 by in situ hybridization;5)CNP was double-stained with CD31/CD34;6)After the liver two-step perfusion method,the liver parenchyma cells were obtained by differential centrifugation,and the expression level of FGF21 was detected;7)Flow cytometry was used to detect the proportion of CNP positive cells in hepatocytes and endothelial cells;8)After treatment of hepatic cell lines and endothelial cell lines with P110αinhibitor,the expression of FGF21 was measured by Q-PCR.Results:1)The weight of CNP-cre:P110α^flox/floxlox/flox mice decreased significantly,while the weight of OLIG1-cre:P110α^flox/floxlox/flox mice had no significant effect,but the level of MBP mRNA in spinal cord of CNP-cre:P110α^flox/floxlox/flox mice and OLIG1-cre:P110α^flox/floxlox/flox mice decreased significantly at P7.2)In CNP-cre:P110α^flox/floxlox/flox mice,MBP mRNA and APC protein in spinal cord decreased significantly only at P7,but no significant changes were found at other time points.3)After CNP-cre:P110α^flox/floxlox/flox mice successfully established demyelination model,the remyelination was significantly impaired.4)In the frozen section of the liver of td-Tomato transgenic mice,CNP overlapped with endothelial cell markers CD31 and CD34 was found.5)Flow cytometry showed that CNP+cells in hepatic non-parenchymal cells were basically hepatic endothelial cells,and about 36%of hepatic endothelial cells were CNP+cells,but only about 5%of hepatic parenchymal cells were CNP+cells.6)After primary isolation of hepatic parenchymal cells,the expression of FGF21 was detected.The results showed that the expression of FGF21 in hepatic parenchymal cells of CNP-cre:P110α^flox/floxlox/flox mice decreased significantly.7)Inhibiting P110αdid not increase the level of FGF21 in hepatocyte lines,but the expression of FGF21 in endothelial cell lines increased significantly.Conclusion:1)Myelination disorder in CNP-cre:P110α^flox/floxlox/flox mice was not associated with weight loss in mice.2)Conditional knockout of P110αhas a temporary effect on myelination in mice.3)P110αpromotes the remyelination in mice.4)FGF21 is expressed in endothelial cells,and the weight loss（increased expression of FGF21）in P110αknockout mice may be related to endothelial cells.