An Experimental Study About The Deletion Of Dicer In Astrocyte Inhibiting Myelination And Demyelination

Increasing evidence has shown that astrocyte is implicated in regulating oligodendrocyte myelination,but most of the underlying mechanisms remain unknown.mi RNA is an indispensable kind of non-coding RNA in the development of cell.The research of the regulation role of mi RNA in astrocyte on oligodendrocyte differentiation and myelination can help us understand the relationship between astrocyte and myelin.Dicer is a kind of ribonuclease,which plays important roles in the mature of mi RNA.To understand whether mi RNA in astrocyte function in regulating oligodendroglial differentiation and myelination in developing and adult CNS,we generated inducible astrocyte-specific Dicer conditional knockout mice(hGFAP-Cre ERT;Dicer fl/fl).Adding a reporter gene by crossing with a reporter mouse line(m T/m G),we induced the recombination of astrocyte by tamoxifen treatment from postnatal day 3(P3)-P7.We performed the following studies :(1)Observing the efficiency and specificity of astrocyte recombination and the astrogliosis in CNS of different ages by immunofluorescent staining;(2)Using transmission electronic microscope and immunostaining to count the myelination in mice and comparing the capacities of motor coordination by behavior test;(3)Observing the influence of Dicer deletion in astrocyte on remylination by inducing demylination,The results are listed as following:1.Astrocyte-specific recombination driven by hGFAP-Cre ERTTo verify if the hGFAP-Cre ERT line can induce astrocyte-specific recombination in postnatal CNS,we carried out immunostaining for GFAP,brain lipid binding protein(BLBP,astrocytes),Neu N(neurons),CC1(OLs),Iba1(microglia)and NG2(OPCs)on the hGFAP-Cre ERT;m T/m G brain sections.Our results indicated the m GFP positive cells were exclusively co-localized with astroglial markers,GFAP or BLBP,and not overlapping with CC1,Iba1,Neu N or NG2 expression.It is noticeable that about 85% GFAP and 60% BLBP positive cells express m GFP in the cortex and corpus callosum respectively,indicating that the hGFAP-Cre ERT line is reliable in inducing astrocyte-specific recombination in the gray and white matters of developing CNS.2.Dicer deletion in astrocyte inhibits oligodendrocyte myelinationWe examined myelination by using TEM,and the myelinated axon density was greatly decreased in the P7 spinal cord white matter of Dicer c KO mice,manifested as higher g-ratio,suggesting Dicer deletion in astrocytes inhibits myelination developing CNS.In support with this notion,the MBP positive myelin area and CC1 positive mature OL number were greatly decreased in the spinal cords of the P10 Dicer c KO mice as compared to wildtype controls.Consistently,MBP expression and CC1 positive cells were found also significantly decreased in the brains of P10 Dicer c KO mice.Furthermore,to investigate whether Dicer deletion in astrocyte could affect neural function development,we examined the behavioral changes in adulthood at P60 with induction of recombination at P3.The Dicer c KO mice display impaired motor coordination.3.Dicer deletion in astrocyte delays remyelination after lysolecithin induced demyelinationTo investigate if Dicer deletion in astrocytes could change myelin regeneration in lesions,we induced recombination at P 90 and performed lysolecithin injection into corpus callosum 7d after the recombination.Demyelination induced by lysolecithin injection triggers an automatic myelin reparative process in CNS,that allows for observing remyelination kinetics in the lesions.The MBP immunostaining results indicated that remyelination in corpus callosum was greatly decreased 14 d post injury,suggesting that Dicer deletion in astrocytes also inhibits remyelination after lysolecithin lesions.Conclusions:Dicer deletion in astrocyte will not only impair demylination but also inhabit remyelination,and Dicer conditional knock out in young mice will impair the coordination capacity of adult mice.