Perturbed Myelination And Differential Expression Of MicroRNAs In The Central Nerve System Of Niemann-pick Disease Type C1 Mice

BackgroundNiemann-Pick disease type C1(NPC1)is a neurodegenerative disease caused by genetic mutations of Npc1 gene,there is no effective cure.Study had found that NPC1 patients with severe myelination in the brain,if not timely and effective repair,not only affect the normal nerve conduction,will further lead to nerve axon degeneration and neuronal death,triggering irreversible neurological dysfunction.MicroRNAs(miRNAs)and their target genes have been constructed to play a key role in the formation and damage of myelination and involved in regulating the development of nervous system and neurodegenerative diseases.MiR-205-5p(miR-205)and its target genes,which are closely related to the formation of myelin,and to explore its molecular mechanism,and to understand the process of myelination,The treatment of the nervous system demyelinating disease provides new genes and drug targets,and ultimately provides important reference and basis for clinical early diagnosis and prevention.Objective1.Investigate distribution of MBP protein in Npc1 mice brain tissues of different parts over the growth;2.Analyse expression of microRNAs through illumina HiSeqTM2500/MiSeq highthroughput sequencing in Npc1-/-mice;3.Predict target genes and IPA analysis of the function and disease of differentially expressed microRNA,screening microRNA who related to myelination.Methods1.After genotyping at postnatal day 7,Npc1-/-and Npc1+/+ mice will be choosed for the following experiments;2.Mice of P9,P35,P60 days were taken for heart perfusion.The expression of MBP protein in different parts of brain tissue(olfactory bulb,cerebral cortex,hippocampus,cerebellum,midbrain and hindbrain)in mice at different stages was studied by fluorescence immunohistochemistry after immobilization,dehydration,embedding and cryopreservation;3.Western blotting was used to detect the expression of MBP protein in different tissues of P9,P35 and P60 mice,and gray scale and quantitative analysis were performed with Fiji / ImageJ.qPCR was used to detect the expression of MBP-related regulatory genes in cerebral cortex and hippocampus of P35 Npc1-/-and Npc1+/+ mice;4.The RNA of Npc1-/-and Npc1+/+ mice were analyzed by Small RNA sequencing,and the differentially expressed miRNAs were screened;5.Target gene prediction and IPA function and disease analysis of miRNAs differentially expressed in Npc1-/-mice;6.The expression of miR-205 and its target genes in the hippocampus of Npc1-/-mice was detected by qPCR.Results1.With the growth and development of mice,MBP protein expression in the brain showed a gradual increase trend.While the expression of MBP protein in the brain tissue of Npc1-/-mice was significantly decreased,and MBP in different parts showed different expression patterns;2.The expression of PLP,MAG,MOG and CNPase in the cerebral cortex and hippocampus of Npc1-/-and Npc1+/+ mice were detected by qPCR,and the expression levels of them were significantly decreased;3.Small RNA sequencing analysis showed that there were 59 miRNAs in Npc1-/-mice(Pvalue <0.05),among them,27 were up-regulated and 32 were down-regulated;4.The miRNAs target genes of differentially expressed mi RNAs were predicted and analysised the disease and function by IPA.The myelination and demyelination were significantly enriched,indicating that miRNAs were involved in the regulation of myelination in NPC1;5.QPCR analysis showed that the expression of miR-205 in the hippocampus of Npc1-/-mice was significantly increased,the expression of the target genes was decreased in different degrees,and the expression of MBP,PLP,MAG,MOG and CNPase proteins in Npc1-/-mice were significantly decreased,it is suggested that miR-205 was involved in the regulation of myelination through its target gene in the hippocampus of Npc1-/-mice.Conclusion1.The expression pattern of MBP protein of myelination marker in Npc1-/-mice has difference of time and space,and the expression of MBP protein was significantly down-regulated,revealing that the mutation of Npc1 gene leading to dysmyelination;2.Differentially expressed miRNAs in Npc1-/-mice brain are involved in the development of NPC1 disease;3.The up-regulation of miRNA-205 expression in the hippocampus of Npc1-/-mice resulted in a down-regulation of its target genes expression and a significant decrease of MBP,PLP and other proteins expression,which in the myelin marker,it is previously revealed that miRNA-205 regulates myelination through the target genes Csf1,Abcd1,Fam126 a and Lpar1 in the hippocampus of Npc1-/-mice.