| Backgrouds(1)Hereditary ataxias are classified as autosomal dominant,autosomal recessive,X-linked,and mitochondrial based on their mode of inheritance.Autosomal recessive cerebellar ataxias(ARCA)are also a heterogeneous group of disorders that primarily include Friedreich’s ataxia,ataxia telangiectasia,ataxia with vitamin E deficiency,autosomal recessive spastic ataxia of Charlevoix-Saguenay(ARSACS),abetalipoproteinemia,and ataxia with oculomotor apraxia types 1 and 2.(2)Hereditary ataxias with clinical and genetic heterogeneity,blong to progressive neurodegenerative diseases,can cause ataxia gait,dysarthria and other neurological signs,including the pyramidal signs,extrapyramidal signs,eye movement disorders and cognitive dysfunction.The most common cause of the pathogenesis is the amplification of the trinucleotide CAG repeats,which can cause the accumulation of the corresponding protein encoded polyglutamine chain in the cell;the secondary cause is the non-coding nucleotide sequence amplification.This type includes more than 20 different pathogenic genes.Even so,there are still 40%of the causes of hereditary ataxias are not clear.Therefore,the research of hereditary ataxia etiology and neurobiology will help to find a new target for the treatment of such diseases and to improve the quality of human health.Objective(1)To investigate the effect of Ankfy1 mutation on the behavior of the mouse model and to explore the new mechanism of ataxia.(2)To investigate the role of Ankfyl in the function of Purkinje cells,revealing the role of Purkinje cell degeneration in the pathogenesis of ataxia.(3)To explore the mechanisms of Purkinje cell loss and provide a theoretical basis for the development of new drμgs for ataxia.Methods(1)Ankfyl expression of central nervous system(CNS)in wild-type(WT)mice:The expression of Ankfyl mRNA in CNS and other organs of WT mice was detected by in situ hybridization and real-time quantitative polymerase chain reaction(RT-qPCR).The expression of Ankfyl in the cerebellum Purkinje cells was detected by immunofluorescence.Using western blot and the quantitative method to detect the expression of Ankfyl protein in different tissues and organs of adult WT mice.(2)Ataxia mouse model and the behavioral analysis:The animal model of transgenic mice was made by gene capture technique.Using PCR to identify the genotype of WT and the transgenic mice.In order to futher explore the expression level of Ankfyl protein and mRNA in transgenic mice,the western blot method and RT-qPCR method were used.The behavioral changes of mice were analyzed by rotarod test and gait analysis.(3)Histopathological analysis:The expression of Ankfyl and Calbindin-28k in mouse cerebellum were detected by immunofluorescence.Meanwhile,the number of Purkinje cells was quantified by H&E staining of cerebellar tissue.The expression of glial fibrillary acidic protein(GFAP)in cerebellum,substantia nigra and vestibular nucleus was observed.The myelin sheaths of cerebellum,spinal cord and corpus callosum were stained with Eriochrome cyanine staining,and the thickness of myelin sheaths was quantified.Further more,the RT-qPCR method was used to test myelin basic protein(MBP)and RORa mRNA expression.Also,brain-derived neurotrophic factor(BDNF),mesencephalic astrocyte-derived neurotrophic factor(MANF)and neurotrophin-3(NT-3)of cerebellum in P7,P14,P21 and P30 mice were detected by RT-qPCR.(4)Primary cultured Purkinje cells:Purkinje cells were cultured in vitro and stained with immunofluorescence of Calbindin-28k and Ankfyl.The morphology of Purkinje cells were analyzed by Sholl analysis software,Bonfire program,NeuronJ plμgin of ImageJ,NeuronStudio and MATLAB(MathWorks)software.(5)The mechanism of Purkinje cell loss:The TUNEL assay to detect the DNA break in cell was used in cerebellum of WT and Ankfy1/+ mice.In order to detect whether the apoptosis pathway take part in the ARSACS,we detected the caspase 3 and cleaved caspase 3 in cerebellum of adult WT and heterogeneous mice.Besides,we used the shRNA method to reduce the Ankfyl level in A172 cells and SH-SY5Y cells.The expression of protein and mRNA of Ankfyl in A172 and SH-SY5Y cells were detected by western blot and RT-qPCR.The expression of caspase protein,Akt and p-Akt in A172 cells was detected by western blot.Results(1)Ankfyl mRNA was mainly expressed in the spinal cord,hippocampus and cerebellum by in situ hybridization.Using immunofluorescence of anti-Ankfyl and anti-Calbindin to detect the expression mode in vitro cultured Purkinje cells,we found that Ankfyl specific expressed in dendrites,axons and cell body.Ankfy1/+ transgenic mice were obtained by gene capture technique,but no homozygous was found.However,the proportion of heterozygous and WT were matched with Mendelian law,indicating that Ankfyl knockout mice was embryonic lethal.The expression of Ankfyl was detected by RT-qPCR and western blot.The mRNA and protein expression of Ankfy1/+ transgenic mice were decreased.Early movement coordination dysfunction and dystonia were found in heterozygous.(2)Ankfy1/+ mice were severely lost from the 60th day of cerebellar Purkinje cells,and the aberrant dysplasia abnormalities of the transgenic mice appeared early and did not show a large loss of Purkinje cells.Ankfy1/+ mouse glial expression may be a protective mechanism for the loss of Purjinje cells.There was no significant difference in the expression of MBP between Ankfy1/+ mice and WT mice,indicating that there was no demyelination in Ankfy1/+ mice.Ankfyl mutations affect the expression of BDNF,NT-3 and MANF in mice.The decrease of expression of Ankfy1 reduces the expression of MANF and BDNF in animal models of ARSACS disease,which may be involved in the maintenance of Purkinje cells.(3)Purkinje cells of Ankfy1/+ Transgenic mouse showed axonal swelling,which was more susceptible than WT mouse cells.The number of dendritic branches of Purkinje cells in Ankfy1/+ transgenic mice was less than WT Purkinje cells at the same position from the cell body.The total length and total number of dendritic dendrites in Ankfy1/+ Purkinje cells were significantly lower than those of WT.There was no significant difference in the length of dendritic root between Ankfy1/+ Purkinje cells and WT Purkinje cells,while the middle and terminal parts of dendrites of Ankfy1/+ Purkinje cells were significantly less than WT mice.(4)Ankfy1/+ transgenic mouse Purkinje cells undergo apoptosis.The caspase 3 and cleaved caspase 3 expression level were increased.Besides,apoptosis had happend in A172 cells whose Ankfyl were inhibited by shRNA.Further more,cleaved caspase 3 and caspase 8 expression levels were increased in those cells.Downregulation of Ankfyl in A172 cells and SH-SY5Y cells had no effect on cell proliferation.Conclusions(1)Ankfyl is widely expressed in mice,mainly in the spinal cord,hippocampus and cerebellum,and specifically expressed in the cerebellum Purkinje cells.(2)The mice manifest disorders of movement coordination after down-regulation of Ankfyl level.(3)Abnormal phenotypes in Ankfy1/+ mice is associated with Purkinje cells loss.(4)In vitro cultured Ankfy1/+ Purkinje cells,the dendritic length and number of branchs are reduced.Besides,axonal swelling has happened in those cells which occurre before Purkinje cells loss.(5)The loss of Purkinje cells is associated with apoptotic signaling pathways.(6)The expression of neurotrophic factor in Ankfy1/+ mice is abnormal and regulated by PI3K/Akt signaling pathway. |
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