Ahi1 Over-expression Causes Abnormal Cerebellar Development And Movement Dysfunction In Mice

Objective:Ahi1 is associated with several psychiatric diseases including Jourbert,depression,autism spectrum disorders and schizophrenia.Our previous studies have also shown that Ahi1 deficiency causes cerebellum middle fusion defect and depressive behaviors in mice.In this study,we used the Ahi1 over-expression(Ahi1 OE)mice to examine whether over-expression of Ahi1 has effect on cerebellum development and whether over-expression of Ahi1 has anti-depressive effect,to further elucidate the role of Ahi1 protein in the brain development.Methods:Q-PCR was used to measure mRNA levels of Ahi1 in brain of Ahi1 OE mice and WT mice.Western blot analysis was used to detect the expression levels of Ahi1 protein at different time points after birth and the expression levels of Ahi1 protein in different brain regions.Balance beam test,Paw reaching for food test and Food intake test were used to determine whether Ahi1 OE mice have deficits in motor coordination.We detected whether Ahi1 OE mice have learning and memory disorders by Morris maze test.Behavioral experiments such as Tail suspension test(TST),Forced swimming test(FST).Elevated plus maze test and Open field test were used to observe whether Ahi1 OE mice have anti-depressive and anti-anxiety phenotypes under normal conditions and stress conditions.The development of cerebellum in Ahi1 OE mice was detected by BrdU labeling and immunofluorescence staining.By immunoprecipitation(CO-IP)and Western blot analysis,the interaction between Ahi1 and ?-catenin was detected.Cytoplasmic and nuclear proteins in cerebellum were separated by nuclear plasma separation kit.The expression levels of ?-catenin protein in cytoplasm and nuclear were detected by Western blot analysis.Q-PCR and Western blot analysis was used to detect the changes of downstream genes and proteins in Ahi1 OE mice.Results:The result of Western blot analysis showed that Ahi1 protein had a highest expression level at the first postnatal week and then maintained a stable level for a long period in Ahil OE mice,as well as WT mice.The results of Western blot analysis showed that Ahi1 protein increased only in the cerebellum and the cortex,but not in the hippocampus,the hypothalamus and the brainstem.Ahi1 OE mice did not show anti-depressive and anti-anxiety phenotypes under normal or stress condition;Ahi1 OE mice did not show Learning and memory dysfunction;however,Ahi1 OE mice demonstrated the deficits in motor coordination in the balance beam test,the food intake test,and the paw reaching for food test.Immunofluorescent staining showed that the number of Purkinje cells was significantly increased and the Radial granule cell migration was delayed in cerebellum of postnatal day 7 Ahil OE mice.The results of Nuclear and cytoplasmic protein extraction and Western blot analysis showed that total?-catenin level was not changed in the cerebellum of Ahi1 OE mice;however,nuclear?-catenin level was significantly increased,indicating Ahi1 promoted the nuclear translocation of ?-catenin.The results of Q-PCR showed that the downstream signaling of ?-catenin including Lef1,Tcf1,and Cyclin D1 were altered by Ahi1 over-expression that are related to neuronal proliferation.As well,Cyclin D1 protein was increased.Conclusions:Our findings suggest that normal Ahi1 protein level is required for cerebellar development.The number of Purkinje cells was increased and Radial granule cells migration was delayed in cerebellum of Ahi1 OE mice.Ahi1 over-expression affected cerebellar development by impairing ?-catenin signaling and resulted in the deficits in balance and fine movements.