Seipin Deletion Inhibits Oligodendroglial Differentiation And Myelination In Spinal Cord

Objective:The myelin sheath of spinal cord is a lipid-rich multi-layer spiral membrane structure formed by oligodendrocyte progenitor cells（OPCs）gradually differentiating into oligodendrocytes（OLs）during development.OLs processes repeatedly wrap axons,which not only ensures the efficient conduction of nerve impulses,but also provides nutritional support for ascending and descending axons.Clinically,abnormal myelin sheath of spinal cord can lead to axonal conduction obstruction,local inflammation and other pathological changes,resulting in spasmodic gait,quadriplegia and other symptoms^[1].The nonsense mutation of Bscl2 gene encoding lipid metabolism molecule Seipin can lead to the most serious congenital generalized lipodystrophy type 2（CGL2）,with fat loss,systemic lipid metabolism disorder,and neurodegenerative symptoms such as spasmodic gait and dystonic quadriplegia in order to clarify that mechanism of CGL2 neurodegeneration,we investigated the effect of Seipin dysfunction on the differentiation and myelination of OLs in the spinal cord of Seipin knockout mice,in order to find the possible mechanism of CGL2 neurodegeneration induced by Seipin dysfunction.Methods:1.In order to determine that expression of Seipin in spinal cord and oligodendrocytes,the expression of Seipin in spinal cord tissue of Seipin heterozygous(Seipin^+/-)mice was detect byβ-gal staining.the spinal cord of wild type（WT）mice were double stain with Seipin,platelet derived growth factor Receptor alpha（PDGFRα）,a mark of OPC,and adenomatous polyposis coli protein（CC1）,a marker of mature OLs.2.In order to clarify the effect of Seipin deficiency on myelination of spinal cord and its mechanism,the motor function of 1-month-old Seipin^-/-mice was measured by rotarod test,beam walking test and inverted screen test.The white matter of spinal cord was collected at P7,P14,P21,P30.The content of myelin basic protein（MBP）was detected by Western Blot and immunofluorescence staining.The number of OLs（OLIG2⁺）cells,OPC（OLIG2⁺PDGFRα⁺）cells and mature OLs（OLIG2⁺PDGFRα^-）cells per unit area in the white matter of the dorsal column of the spinal cord were counted by double immunofluorescence staining of OLIG2（Oligodendrocyte transcription factor 2）and OPC（PDGFRα）.3.To investigate whether the effect of Seipin deficiency on myelin development persists into adulthood.The motor function of adult 5-month-old Seipin^-/-mice was tested.The myelin protein and lipid levels were detected by Western blot,Fluoro Myelin immunofluorescence staining and immunohistochemistry.The myelin sheath microstructure was observed by transmission electron microscope.4.To investigate whether rosiglitazone,a PPARγagonist,can rescue spinal myelination disorder in Seipin^-/-mice and its mechanism.The in vivo PPARγactivation test of rosiglitazone in 5-month-old Seipin^-/-mice was conducted,and the experiment was divided into three groups:in WT group,Seipin^-/-group and Seipin^-/-mice rosiglitazone group(Seipin^-/-+RG group),q PCR was use to detect that m RNA expression level of lipid metabolism molecules downstream of PPARγin the white matt of spinal cord of Seipin^-/-mice after rosiglitazone administration.The cervical transection of spinal cord of the three groups of mice were collected for double immunofluorescence staining of OLIG2 and PDGFRα,and the number of OLs,mature OLs and OPC per unit area of gray matter and white matter were counted;The levels of myelin protein and lipid were detected by Western blot and immunofluorescence staining.Results:1.Seipin was expressed in spinal cord and OLs.Seipin was highly expressed in spinal cord tissue;The PDGFRα⁺OPCs and CC1⁺OLs in the gray and white matter of the spinal cord expressed Seipin.2.Seipin deletion affects myelination by inhibiting differentiation and matu ration of OPC.Compared with WT group,1-month-old Seipin^-/-mice showed de creased motor function in all motor function tests.From P7 to P30,the expres sion of MBP in the myelin sheath of WT mice increased gradually,but the ex pression of MBP in Seipin^-/-mice at P14,P21 and P30 was significantly lower than that in WT mice.The densities of OL lineages,OPCs and OL cells inWT spinal white matter were highest at P7,and decreased with age.Compared with WT mice,the number of OL lineages and mature OLs per unit area of s pinal white matter in Seipin^-/-group mice decreased significantly from P14,but the number of OPCs did not differ significantly.3.Seipin deficiency can affect myelin development in the spinal cord until adulthood,and lead to aggravation of myelin loss.Compared with WT group at the same age,the motor function of adult 5-month-old Seipin^-/-mice was still significantly decreased.The myelin lipid level of Seipin-/-mice decreased significantly Notably,MBP expression in the spinal cord white matter of Seipin^-/-mice decreased by 20-30%at 1 month of age at the end of myelination,and by 50-70%at adulthood;The g-ratio of myelin sheath in the white matter of spinal cord in Seipin^-/-mice was significantly lower than that in WT mice,and the thickness of myelin sheath was increased but the structure was loose.4.Rosiglitazone,a PPARγagonist,can promote the proliferation and differentiation of OPC and partially rescue the myelination disorder of spinal cord in Seipin^-/-mice.Compared with Seipin^-/-group,the expression level of lipid metabolism related molecules downstream of PPARγin myelin sheath of Seipin^-/-+RG group was up-regulated;Compared with WT group,the total number of OLs,mature OLs and white matter OPCs per unit area in the gray matter of spinal cord were significantly decreased in Seipin^-/-group,while the number of gray matter OPCs was not significantly changed.The myelin protein expression and myelin lipid level were significantly decreased in Seipin^-/-group.Compared with Seipin^-/-group,Seipin^-/-+RG group significantly increased the total number of OLs,mature OLs and white matter OPCs per unit area of spinal cord gray matter,but had no significant change in gray matter OPCs.The myelin protein expression and lipid level were significantly increased,and the motor function of mice was improved.Conclusion:1.Seipin was expressed in spinal OLs.2.Seipin deletion inhibited the differentiation and maturation of OPC,affected the myelin development of spinal cord,and caused the myelin loss in adult mice.3.PPAR agonist rosiglitazone promotes the proliferation and differentiation of OPC and partially rescues the myelination disorder of spinal cord in Seipin^-/-mice.