Study On The Regulation Of Myelin Stability By HB-EGF In The Central Nervous System

Heparin-binding epidermal growth factor is one of the ligands to Erb B receptors,which can specifically bind EGFR and Erb B4.Erb B signaling pathway can regulate cell proliferation,differentiation,migration,and apoptosis.Our laboratory has demonstrated that different Erb B receptors in oligodendroglia lineage coordinate to regulate myelin development and integrity.However,as a pan-ligand for Erb B receptors,HB-EGF’s role in the central nervous system is unclear.Previous studies in our laboratory found that HB-EGF is specifically expressed in deep excitatory neurons in the sensory cortex,with much higher expression in the caudal of the cerebrum than in the rostral.By cre-loxp system,we obtained Emx1-cre;HB-EGF^fl/fl mice which can specifically knockout HB-EGF in forebrain excitatory neurons.Electron microscopy results showed that the number of myelinated axons in the rostral and caudal corpus callosum were not altered in HB-EGF knockout mice at 3months old,but were reduced at the caudal corpus callosum in HB-EGF knockout mice at the 6 months old.These results suggested that HB-EGF regulates the myelin stability in the CNS.In the present study,we explored how HB-EGF regulates myelin stability by immunofluorescence,western blotting,RT-PCR and other techniques in the CNS.Immunofluorescence results showed that HB-EGF knockout didn’t change the number of OL at mature adult mice（3-6 months）.GFAP and cleaved-caspase3 staining showed OL didn’t have pathological changes in the caudal corpus callosum,indicating that HB-EGF regulates myelin stability independently of oligodendrocyte death.The staining results of myelin proteolipid protein（PLP）,myelin basic protein（MBP）and Fluoro Myelin（a lipophilic dye with a high selectivity for myelin）showed that the myelin structural protein didn’t change,but the myelin lipids reduced in HB-EGF knockout mice.The thin-layer chromatography results also showed lower lipid levels in the corpus callosum of HB-EGF knockout mice.All these results suggested that HB-EGF may control myelin integrity through regulating lipid metabolism.Further,by RT-PCR and western blotting,we found that the protein level of SREBP1,a transcription factor regulating lipid metabolism enzyme expression,was reduced in the corpus callosum of HB-EGF knockout mice,and the transcript levels of its regulated enzymes such as ACLY,FASN,ACC and other lipid synthesis-related enzymes were decreased.Inspiringly,only the protein levels of ACLY decreased in the corpus callosum,while the protein levels of ACC,FASN,and ACSS2 increased.We speculate that HB-EGF knockout may affect both the transcriptional and ubiquitination mechanisms of lipid synthesis enzymes in oligodendrocytes,and the decrease of ACLY should be responsible for the myelin lipid loss.By western blotting,we found that in HB-EGF knockout mice Akt was inhibited and the activity of m TORC1,m TORC2 was increased,suggesting that in mature oligodendrocytes,Akt directly regulates the transcriptional activity of SREBP and the protein level of ACLY,rather than through m TORC1 or m TORC2.Behavioral analysis of HB-EGF knockout mice showed that the loss of myelin in the caudal corpus callosum caused by the knockout of HB-EGF did not affect motor function,mechano-sensation,and visual function,but led to hearing deficits at mature adult mice.In addition,the results of PPI in normal hearing mice exhibited impaired sensory gating in HB-EGF knockout mice,suggesting that interhemispheric communications between bilateral sensory cortex through the corpus callosum is necessary for the screening and processing of sensory information.