New Myelin Deposition In Regulating Visual Function In Diabetes Mellitus

As a common chronic and metabolic disease,diabetes mellitus（DM）has an increasing incidence year by year.Long-term hyperglycemia can cause damage to various target organs,lead to various complications,bring heavy mental pressure and economic burden to patients,endanger human physical and mental health,and even endanger life in severe cases.The main ocular complications of DM patients include diabetic optic neuropathy mainly caused by optic nerve damage,diabetic retinopathy mainly caused by retinal tiny blood vessel damage,and diabetic cataract mainly caused by crystal turbidity.All of these are the main factors causing visual impairment and even blindness in DM patients.Although studies on the treatment of DM ocular complications have made some progress in recent years,currently available clinical treatment schemes are only suitable for complications at a later stage,such as diabetic retinopathy,and there are few intervention measures for early lesions.Relevant clinical studies and rodent studies have successively demonstrated that in the course of DM,retinal ganglion cells（RGCs）and retinal microvascular damage predate optic nerve dysfunction and histological changes.Diabetic optic neuropathy,characterized by optic neuropathology,is considered to be an important cause of early DM visual dysfunction.Therefore,to further investigate the pathological changes and pathogenesis of optic neuropathy caused by DM is an important basis for exploring effective targets for the treatment of early diabetic visual impairment.The optic nerve starts from the RGCs in the inner retina,emanates from the orbit,and terminates at optic chiasm（OC）through the optic nerve canal,which is a segment of highly myelinated white matter tissue.Oligodendrocytes（OLs）differentiate from oligodendrocyte precursor cells（OPCs）and form myelin in the central nervous system.It can form membranes that repeatedly wrap around the axons of neurons to form myelin sheaths.The myelin sheath on the optic nerve is segmented and insulates the RGC axon.The exposed axon membrane in the adjacent segments of the myelin sheath is rich in sodium ion channels,which can generate action potentials called nodes of Ranvier.In addition to the efficient and rapid conduction of action potential between nodes of Ranvier,myelin sheath can also regulate ion levels in the environment and supplement energy requirements by metabolites to support the normal physiological function of RGC axons.In recent years,imaging and histological evidence has shown that with the progression of DM,the axons and myelin of the optic nerve show obvious pathological changes,and visual function is also decreased.It is worth noting that almost all pathological changes of optic neuropathy in DM patients occur in adulthood,while the development of the myelin sheath of human optic nerve starts from the embryonic period,reaches a peak after birth to adolescence,and maintains a stable and highly myelinated state in adulthood.The above evidence suggests that pathological changes may occur in the myelin sheath of the optic nerve in early diabetes,but the role of such changes in early diabetic visual dysfunction remains unclear.Recent studies have shown that myelin in the central nervous system is not static in adulthood,on the contrary,myelin changes throughout life,which is myelin turnover.Although the degree of myelination of adult optic nerve axons is as high as 90%,researchers have observed that OPCs in the optic nerve continue to differentiate and form new OLs and new myelin,suggesting the existence of highly active myelin replacement in adult optic nerve.Widely distributed in the central nervous system,OPCs still have active proliferative ability after adulthood,which is thought to be beneficial for adult individuals to adapt to new environment,acquire new skills or cope with neurodegenerative changes.Previous studies of our team found that reduced formation of new myelin may be an important cause of decreased visual function in aging mouse models,and promoting myelin formation can alleviate age-related visual dysfunction.At the same time,our team also revealed the role of dynamic brain myelin changes in the mouse model of Alzheimer’s disease,and proposed therapeutic strategies to improve Alzheimer’s disease-related cognitive impairment by promoting myelination.In addition,experimental data from other researchers have revealed that the formation of new myelin in adulthood also plays an important role in the progression of fear and spatial memory.Based on the above findings,we speculate that the deposition of new myelin of optic nerve may also play an important role in early DM visual impairment.The highly myelinated feature of the adult optic nerve increases the difficulty in observing the formation of new myelin by morphology.To solve this problem,our team successfully constructed inducible cell-specific fluorescent reporter transgenic mice that can label the new myelin sheath,and screened out small molecule drugs that act on myelin-related receptors and promote myelin formation.Therefore,with the help of the existing technical advantages of the research group,this study plans to explore the specific changes and functional significance of the new myelin in early DM visual dysfunction from the following three aspects:1.RNA-seq assay was used to explore the correlation between visual dysfunction and the pathology of optic nerve myelin in early DM mice.2.NG2-Cre ERt;Tau-m GFP and NG2-Cre ERt;Tau-m GFP;Olig2fl/fl reporter mice were combined with fluorescence staining to determine the relationship between visual dysfunction and new myelin deposition of optic nerve in early DM mice.3.To observe whether pro-myelinating drug treatment can increase myelination in DM mice,and to explore the possibility of pro-myelinating drug treatment of improving visual dysfunction in early DM mice.In this study,we involved mouse line with different reporter gene,combined with experimental methods such as RNA-seq,visual electrophysiology,immunofluorescence staining,electron microscopy,RT-PCR,etc.,to systematically observe the myelin alteration associated with early DM and the effects of myelin formation promotion on visual dysfunction of early DM.The main results are as follows:1.Visual dysfunction in early DM mice was highly correlated with pathological changes of optic myelin sheath.DM mice models were induced by administering STZ to C57BL/6 mice.Fasting blood glucose（FBG）testing,weight monitoring,electroretinogram（ERG）and visual evoked potential（VEP）and immunofluorescence staining were employed to identify the changes of blood glucose and body weight,early retinal function,visual conduction function and retinal neurons alteration in the vehicle and DM mice.It was found that delayed visual conduction occurred before the loss of retinal neurons,and 28 days after STZ injection（STZ-D28）was determined as the timepoint of early DM.The results of electron microscopy and staining showed that the proportion of myelinated axons decreased and the structure of myelin was damaged in the optic nerve of DM mice,suggesting that pathological myelin changes existed in the optic nerve of DM mice.The acutely isolated optic nerves of vehicle and DM mice were used for RNA-seq.GO analysis revealed enrichment in the cellular component related to myelin-related structures.To validate these changes,we tested optic nerve mitochondrial RNA levels in the vehicle and DM mice and confirmed the decrease in several myelin-related transcripts,indicating that pathological alteration of myelin of the DM optic nerves were highly correlated with the visual impairment.2.OPC differentiation was impaired in the optic nerves of DM mice,resulting in insufficient deposition of new myelin and visual dysfunction.Immunofluorescence staining results revealed a significant reduction of mature OLs and no obvious change of OPCs,indicating OPC differentiation disorder may exist in the DM optic nerve.Pathway enrichment analysis revealed that non-canonical Notch signaling pathway related differential genes were most significantly enriched in the OPC of the optic nerve of DM mice,which again verified the OPC differentiation disorder in the optic nerve of DM mice.We used the NG2-Cre ERt;Tau-m GFP mice to label newly formed myelin sheaths in adult mice.Noticeably,numerous m GFP-positive myelin sheaths were seen in the optic nerve of adult mice at 28 days after recombination,implying a robust new myelin deposition within one month of young-adult age.In contrast,m GFP-positive myelin sheaths were sparsely distributed in the optic nerve of diabetic mice,demonstrating that diabetic insult impaired new myelin deposition in the optic nerve.The number of new OL（CC1⁺/Sox10⁺/Ed U⁺cells）was significantly decreased in the optic nerves of diabetic mice on D28.We mimicked the absence of Olig2 in diabetic mice by deleting olig2 in OPCs and concurrently labeling newly formed myelin by using NG2-Cre ERt;Tau-m GFP mice.Ablating Olig2 during this specific time of diabetic insult induced a significant decrease of myelinogenesis.The function test of Olig2 c KO mice exhibit delayed signal transmission.Overall,these results are direct evidence that the deposition of new myelin,which is essential for maintaining the function of optic nerve,is impaired for a short interval after a diabetic insult.3.Pro-myelinating drug treatment promotes functional recovery of optic nerve in diabetic mice.Immunofluorescence staining results show that after treatment of Clemastine,the number of mature OL and new myelin deposition rebounded,indicating pro-myelinating drug can promote the new myelin formation.Subsequent visual electrophysiological function tests showed that the clemastine-treated mice showed decreased latency of P2 wave than the vehicle-treated mice,indicating that clemastine restored signal transmission in diabetic optic nerves.In conclusion,we reported a reduced number of myelinated axons,impaired optic nerve transmission,and retinal dysfunction in streptozotocin（STZ）-induced diabetic mice.Using NG2Cre ERt;Tau-m GFP mice to label newly generated myelin sheaths,we observed significantly decreased new myelin generation in the optic nerve during the short time window of diabetic insult.Then,using NG2Cre ERt;Tau-m GFP;Olig2^fl/fl mice to mimic the condition of dampened newly generated myelin,we observed optic nerve dysfunction at the same time.Furthermore,enhancing new myelin deposition by clemastine effectively improved the functional outcome of deficient optic nerve in diabetic mice.Together,our results clearly demonstrate that a deficit of new myelin deposition and OLs contribute to optic neuropathy under diabetic insult and that enhancing new myelin deposition may be a promising approach for functional recovery of damaged optic nerves.