Specific Expression And Function Of Ermin In Oligodendrocytes And Its Molecular Mechanism

【Background】Myelin sheath is the lipid membrane structure that surrounding the axons of myelinated nerve fibers.It not only ensures fast and directional transmission of nerve excitation along nerve fibers,but also plays an important role in providing nutrition and support for neuronal axons.Disruption and loss of myelin structure caused by imbalance of myelin homeostasis are common in clinic neurodegenerative diseases.The clinical symptoms of demyelination include changes in mental status,dysarthria,visual impairment,abnormal movement,etc.At present,the mechanism of demyelination and myelin regeneration is not clear,and there is no effective treatment for demyelination disease in central nervous system.Oligodendrocytes（OLs）are the main myelin-forming cells of the central nervous system.In the process of oligodendrocyte progenitor cells（OPCs）proliferation,migration and differentiation into mature OLs that form the myelin sheath,abnormalities in any step will cause myelin deficiency or failure of remyelination after damage of the myelin sheath.Cytoskeleton reorganization is a key process for OPCs and OLs to form the compact myelin sheath,in which various signaling pathways inside and outside cells play essential regulatory roles.However,there is still a lack of systematic and clear understanding of the specific molecular mechanisms of cytoskeleton regulation during OLs differentiation and myelination.Ermin was first reported as a new cytoskeleton-related oligodendrocyte-specific protein.Previous studies have indicated that Ermin promoted excessive process outgrowth in cultured oligodendrocytes through binding with F-actin.Moreover,Ermin was found to colocalize with 2’,3’-cyclic-nucleotide 3’-phosphodiesterase（CNP）,a known maker protein of oligodendroglial cells and expressed in the outermost layer of myelin.However,the detailed role of Ermin in myelin development and maintenace is not clear due to the lack of Ermn-deficient mouse models.In addition,the molecular mechanisms underlying Ermin regulation of oligodendrocyte differentiation are still unknown.【Objectives】1.To clarify the role of Ermin in oligodendrocyte maturation and myelin maintenance.2.To elucidate the influence of Ermn deficiency in the pathological process of demyelination.3.To explore the the molecular mechanism of cytoskeletal reorganization regulated by Ermin,which is expected to provide novel insights into understanding myelin dysfunction and identifying new targets for neurodegenerative diseases.【Methods and Results】1.The brain tissue of normal mice at different developmental stages was extracted and subject to Western blot analysis.The results showed that Ermin was expressed from postnatal day 14（P14）,peaked at P28 and remained stable until adulthood,which was synchronous with myelin proteins including MBP and MOG,but later than the emergence of CNP.Immunofluorescence staining showed that Ermin was mainly distributed in the corpus callosum of the brain,which was very similar to MBP and CNP.Subcellularly,Ermin was detected in the cytoplasm of oligodendrocytes.2.Ermn gene knockout mice were generated and the genotype of the offspring was identified by PCR.Western blot,immunohistochemistry and immunofluorescence furtherconfirmed the complete depletion of Ermin in the homozygous(Ermn^-/-,KO)but not wild-type(Ermn^+/+,WT)mice.In addition,immunofluorescence results showed that Ermin deficiency did not affect the distribution of MBP.3.Preliminary analysis of histological characteristics was performed on WT and KO mice.Mouse brain sections were prepared at ages of 1 to 12 months.Black-Gold II staining results showed Ermn knockout did not induce significant changes in myelination and morphology of the corpus callosum.Electron microscopy of the optic nerves and corpus callosum showed the breakdown of myelinated fibers,split myelin layers,peeling of the myelin from the axon in the old KO mice（over 9-month-old）.Consistently,g-ratios from old KO mice were significantly lower than those from age-matched WT mice.4.WT and KO mice were analyzed via electrophysiology and behavioral tests.Action potential conduction velocity assay indicated that conduction velocity of optic nerves were significantly reduced in 12-month-old KO mice compared with WT mice.Open field test that examines the ability of autonomous activity and elevated plus maze（EPM）test that evaluates anxiety displayed no difference at ages of 2 to 12 months in WT and KO mice.Rotarod test measuring the motor coordination ability showed that 12-month-old KO mice stayed for shorter time periods on the rotarod than age-matched wild-type mice.5.The cuprizone model for demyelination of WT and KO mice was established and histological characteristics were analyzed.Cuprizone induced comparable weight loss in WT and KO mice.Black-gold II staining showed that myelin sheath was lost time-dependently when fed 0.2%Cuprizone,and completely disrupted at week 5 of cuprizone diet.Moreover,myelin impairment was remarkably accelerated in KO mice compared with WT mice.Consistently,Western blot and immunohistochemistry results showed 0.2%Cuprizone diet significantly reduced the expression of MBP and upregulated the astrocyte marker GFAP especially in KO mice.6.Behavioral tests were performed in WT and KO mice of demyelination model weekly.Open field test showed that spontaneous locomotor activity did not differ in mice with 0.2%Cuprizone or normal diet.EPM test revealed a slight difference the level of anxiety between WT and KO mice treated with 0.2%Cuprizone at week 4 and 5.Rotarod test showed that mice exposed to cuprizone stayed less time on the rotarod than those with a normal diet,and KO mice displayed a decrease in the time spent on the rotarod after 4weeks with a 0.2%Cuprizone diet compared with the WT mice.However,mice with or without Ermn deficit showed no difference after 5 weeks of cuprizone diet.7.Ermin-overexpressing plasmid was transfected into COS-7,a cell line enriched in actin microfilaments.As a result,Ermin overexpression was found to induce the development of filopodia-like protrusions.Furthermore,the recombinant lentiviral vectors for full-length Ermin and truncated Ermin lacking the C-terminal F-actin binding region were constructed and used to infect the oligodendroglia cell line,OLN-93.Full-length Ermin in OLN-93 cells formed more intensive,mesh-like protrusions;C-terminal truncated Ermin also promoted outgrowth of the processes and higher degree of branches,but much less than the full-length Ermin.8.Mass spectrometry was used to screen for proteins co-precipitated with Ermin in the brain,and a cytoskeleton-associated protein,p116^RIP,was identified as a candidate Ermin-interacting protein.Co-immunoprecipitation experiments in cultured OLN-93 cells confirmed that p116^RIPbound to Ermin.Immunoprecipitation using prokaryotically expressed and purified proteins corroborated a direct interaction between Ermin and p116^RIP.Treatment of OLN-93 cells with a protein synthesis inhibitor,cycloheximide（CHX）,revealed that Ermin overexpression improved the stability of p116^RIP.Moreover,immunostaining showed that p116^RIPco-localized with Ermin in the cerebrum of adult mice.In addition,p116^RIPknockdown increased RhoA activity in OLN-93 cells,whereas p116^RIPoverexpression inhibited epithelial growth factor（EGF）-induced RhoA activation.Ermin repressed EGF-triggered RhoA activation,which was alleviated by knockdown of p116^RIP.Finally,p116^RIPwas involved in Ermin regulation of oligodendroglial differentiation.【Conclusion】1.Ermin is a myelin protein expressed exclusively in the cytoplasm of oligodendrocytes.2.The deletion of Ermn in older mice can lead to abnormal ultra-structure of myelin sheath,decrease in conduction velocity of optic nerve and corpus callosum and poor motor coordination ability.3.Ermn deficiency can accelerate demyelination process of central nervous system,aggravate myelin sheath lesions and cause abnormal behavioral in mice.4.Ermin can promote the growth of protrusion in COS-7 and OLN-93 cell lines,and truncated Ermin lacking the C-terminal F-actin binding region could still promote the protrusion formation and higher branches development.5.p116^RIPis a protein that interacts directly with Ermin in oligodendrocytes.6.Ermin can enhance the stability of p116^RIPin oligodendrocytes.7.Ermin/p116^RIPcan promote the protrusion formation though inhibiting the activity of RhoA in OLN-93 cells.