Study On The Function Of SMEK1 In The Cerebellar Development

SMEK1 is a regulatory subunit of protein phosphatase 4(PP4).also known as PP4R3a.Studies have shown that SMEK1 played an important role in neurogenesis.Previously,our group found that SMEK1 mutation could promote the progression of spinocerebellar ataxia III.but the mechanism remains unclear.Studies have shown that abnormal cerebellar development is closely related to spinocerebellar ataxia.In order to enrich the pathological mechanism of SMEK1 mutation promoting spinocerebellar ataxia,we constructed Smek1 knockout mice to study the function and mechanism of Smek1 on cerebellar development.Firstly,we detected the RNA and protein expression of SMEK1 in tissues of wild type mice at postnatal day 8(P8).and found that SMEK1 was highly expressed in the nervous system,especially in the cerebellum.Moreover,it was found that SMEK1 was highly expressed in the cerebellum at all periods tested.Immunohistochemical staining showed that SMEK1 was expressed in both granule cells and Purkinje cells in the cerebellum,with a higher expression level in purkinje cells,indicating that SMEK1 plays an important role in the development of the cerebellum.In addition,we found that the birth rate of Smekl KO mice was not in accord with Mendelian separation ratio.Therefore,we performed genotypic statistics of mice at different embryonic stage,and found that the proportion of KO fetus was normal at E12.5 and E16.5,while the proportion of KO fetus decreased at E18.5.These results demonstrated that deletion of Smekl leads to partially embryonic lethality,suggesting that SMEK1 is essential in mouse embryonic development.After verifying the knockout efficiency,we monitored the growth and development of KO mice.We found that KO mice were smaller and exhibited growth retardation.The body weight of KO mice was always lighter than WT and HET mice from P4.Therefore,Smekl is essential for normal growth and development of mice after birth.Next,we performed behaviour tests.Footprint analysis showed that the gait of KO mice showed ataxia and step-smaller phenomenon.We also tested the righting reflex of P8 pups.We found that KO mice took a longer time to flip over than WT and HET mice.These results indicated that Smek1 knockout affected the motor coordination ability of mice.Macrophenotypic examination revealed that the cerebellum of P8 P15 KO mice was significantly smaller than WT mice.The paraffin sections stained by H&E also revealed that the cerebellum of KO mice in development and adult stage was smaller than WT.Moreover,KO mice exhibited the atrophy of the cerebellar folia and the thinner external granule cell layer(EGL).These results indicated that Smek1 knockout affected the development of cerebellum.Through EdU labeling and immunofluorescence staining experiment,it was found that the proliferation of granule cell precursors(GCPs)in the EGL was reduced.GCPs of KO mice exited the cell cycle prematurely and entered the stage of differentiation.Through TUNEL and immunofluorescence staining experiments,we found that the cell apoptosis was normal.In addition,there were more glial cells and fewer unipolar brush cells in the cerebellum of KO mice.By immunofluorescence staining,we found that the number of Purkinje cells(PCs)decreased and the molecular layer became thinner in KO mice than that of WT.To further study the effect of SMEK1 on PCs,Golgi staining was performed.It was found that compared with WT mice,adult KO mice exhibited smaller PCs body,thinner diameter of the primary dendrite shafts,reduced dendrite branch complexity and decreased dendrite spine density.These results suggest that SMEK1 plays an important role in the process of development and maturation of Purkinje cells.In conclusion,SMEK1 plays an important role in cerebellar development.The deletion of Smek1 leads to abnormal cerebellum development and impaired motor coordination in mice.The developmental abnormality caused by SMEK1 mutation might aggravate the symptoms of spinocerebellar ataxia patients.