Function And Mechanisms Of Myelination In Hypoxia Induced Neurological Dysfunction

White matter injury(WMI)is a type of neonatal brain injury,affecting ~5-10% of preterm infants and about 50% low-birth-weight preterm survivors(<1500g).WMI can be diagnosed by the presence of a number of neurological deficits,and MRI(Magnetic Resonance Imaging)can reveal abnormal signals in the white matter.According to the data from WHO(World Health Organization),the incidence of WMI keeps increasing,accompanying with the increasing preterm birth rate.Accumulating evidence has shown that WMI may lead to long-term neurological dysfunction,including motor deficits,neurosensory impairments and cognitive delay.The neurological dysfunction may persist into adulthood,even throughout lifetime,which brings heavy burden to patients and the society.A number of factors,including hypoxia,cerebral hemorrhage,brain trauma in neonates,have been considered as risky factors that may be involved in WMI.Though pre-treatment of magnesium sulfate to expectant mothers may improve neurofunction injury in preterm newborns,the pathogenic mechanisms remain unclear.With that being said,clinical therapies for WMI are still unavailable up to date.White matter is mainly composed of myelinated axons.Myelin sheaths are generated by oligodendrocytes(OLs)in CNS.The processes of OL concentrically and repeatedly wrap axons and the myelin segments distribute intermittently along axons.Neighboring myelin sheaths are segregated with very short unmyelinated axons,so-called Node of Ranvier,which are enriched with sodium channels and so that action potential can be transmitted rapidly and efficiently by the Nodes.Recent studies have shown that OL could transport lactic acid to axon through monocarboxylate transporter 1(MCT1)on myelin membrane to provide metabolic support for axons.Myelination in human brain is initiated from embryonic stage,reaching the peak during adolescence.The myelinating OLs differentiate from oligodendrocyte precursor cells(OPCs),which distribute evenly throughout CNS.In the WMI lesions,hypomyelination is evident,as well as other pathological changes,including neuroinflammation,activated glia and apoptosis of neuron.Roles of these pathological changes were unknown.In addition,the number of CC1 positive mature OLs was found decreased but Olig2(Oligodendroglia transcription factor 2)positive cells was increased in the WMI lesions,suggesting that arrested OPC differentiation.In this thesis,we focused on the change and functional significance of myelination in WMI,aiming to understand the relationship between hypomyelination and neurofunctional deficits,the potential mechanisms of hypomyelination in functional deficits and whether enhancing myelination represents a promising approach for WMI.To address these issues,we carried out our research as following four aspects.(1).Establish chronic hypoxia mouse model to mimic hypomyelination and long-term neuronal dysfunctions in WMI.(2).Knockout Olig2 specifically in oligodendroglia to uncouple hypomyelination and understand the consequence of impaired myelination on neurological function and potential mechanisms.(3).Deletion of muscarinic receptor 1(M1R),which negatively regulates OPCs differentiation,specifically in OPCs to examine whether promoting myelination could rescue synaptic and functional deficits in the hypoxic mice.(4).Evaluate whether the drug-based strategy myelination as an approach to promote functional recovery against WMI.In this study,immunofluorescent staining and transmission electron microscope(TEM)were used to identify histological changes of myelin and synapse development;meanwhile,electrophysiology and behavioral test were used to detect synaptic transmission and neurofunctions.Main results were listed below:1.Chronic hypoxia impairs myelination,synaptogenesis and long-term neurological function.Newborn mice were subjected to chronic hypoxia model in 10% oxygen concentration environment from P3(postnatal day 3)to P10.Immunostaining and TEM showed arrested OPCs differentiation and hypomyelination,without significant neuron apoptosis and axon degeneration.Immunostaining for synaptic markers and electrophysiology showed decreased expression of special synaptic protein and impaired neuronal transduction,which suggested that hypoxia inhibited synaptogenesis structurally and functionally.Myelin and synapse development in hypoxic mice did not catch up even on P40.The modified beam walking test showed impaired motor coordination,suggesting that hypoxic mice mirror prolonged neuronal dysfunction.These results indicate that chronic hypoxia model is valid for WMI research.2.Delayed myelination disrupts synaptogenesis and impairs neuronal function.The CNP-Cre mouse line and Olig2 fl/fl mouse line were crossed to obtain CNP-Cre;Olig2 fl/fl,in which Olig2 was specifically deleted in oligodendroglia.Immunostaining and TEM showed persistent hypomyelination in the Olig2 cKO brains that is similar with the hypoxic mice,suggesting that Olig2 cKO mice contribute to understand functional importance of hypomyelination in synaptogenesis and neurofunction.Strikingly,immunostaining showed less synaptogenesis and electrophysiology showed decreased frequency of miniature excitatory postsynaptic currents(mEPSCs).Abnormal motor functions were also detected by behavioral tests in the adolescent Olig2 cKO mice.These results indicate that hypomyelination alone is sufficient to inhibit synaptogenesis and impair neurofunction.3.Enhancing myelination rescues synaptic deficits and improves functional recovery in hypoxic mice.To enhance myelination in a genetic way,we generated NG2-CreERT;M1R fl/fl mouse line.Treated with tamoxifen to induce deletion of M1 R in OPCs,the newborns were subjected to chronic hypoxia.Immunostaining and TEM showed the M1 R deletion could promote OPCs differentiation and enhance myelination in hypoxic brains.Consistently,the synaptic development detected by immunostaining and mEPSCs detected by electrophysiology were largely rescued by enhanced myelination in the M1 R cKO mice.As expected,neurofunctional recovery was observed in M1 R cKO mice by the beam walking test.These results reveal that enhancing myelination can improve the recovery of synaptic and neurofunctional impairments in hypoxic mice.4.Drug-based myelination improves synaptogenesis and neurofunctional injury.Clemastine and(±)U-50488 are two drugs that have been proved to be effective in promoting OPC differentiation in vitro and remyelination.We treated hypoxic mice with Clemastine or(±)U-50488 and the MBP expression and synaptic markers were increased revealed by immuostaining.The behavioral test showed drug treatment improved motor coordination and cognitive function.Interestingly,drug treatment also enhanced myelination and improved neurofunction,post hypoxia exposure in mice.These results suggested that drug-based strategy may represent a promising approach in clinical for structural and functional recovery in WMI.In conclusion,we established the chronic hypoxia model that can mimic histological and functional injury of WMI.We found that hypomyelination can directly impair synaptic development and neurofunctions by using Olig2 conditional knockout mice.We further identified that specific deletion of M1 R in OPCs or drug-based myelination can improve the recovery of synaptic deficits and functional damage in hypoxic mice,suggesting enhancing myelination is a promising strategy for WMI treatment.Taking together,our study found myelination is an important regulator for synaptogenesis in early developmental stage and enhancing myelination represents a promising strategy for structural and functional recovery in WMI.