Study On The Ataxia And Synaptic Plasticity In The Cerebellum Of Lgr4 Gene Trap Mice

Ataxia is a neurological disorder characterized by loss of balance and coordination and is often accompanied by neurological deficits such as unsteadiness in gait,dysmetria,asynergy,and nystagmus.Individuals suffering from ataxia at first are clumsy and unable to walk steadily,and have slurred speech.Patients eventually lose the ability to swallow and breathe in a coordinated fashion,which can be fatal.The origin of many forms of ataxia can be traced back to deficits in the cerebellum,which plays critical role in motor coordination and the fine adjustment of movements.It is widely assumed that Purkinje cell is regarded as the major location where cerebellar synaptic plasticity happens,for example long-term depression(LTD)at parallel fiber(PF)-Purkinje cell synapses,and its counterpart,long-term potentiation(LTP).Deficits in cerebellar synaptic plasticity,for example resulting from genetic knock-out or transgenic mice,have been shown to result in an impairment of cerebellar motor learning.G protein-coupled receptors(GPCRs)have been named based on their ability to recruit and regulate the activity of intracellular heterotrimeric GTP-binding proteins(G-proteins).GPCRs are numerous,widely expressed and have a role in various physiological functions.Lgr4 belongs to the leucine rich G protein couple receptor family(LGRs),and is highly conserved among different species.Human Lgr4 gene was detected from multiple tissues.The loss of Lgr4 results in developmental defects in many aspects,including intrauterine growth retardation associated with embryonic and perinatal lethality,abnormal renal development,defective postnatal development of the male reproductive tract,ocular anterior segment dysgenesis,bone formation and remodeling dysfunction,impaired hair placode formation,and defective development of the gall bladder and cystic ducts.Recent studies demonstrated that Lgr4 is required for Paneth cell differentiation and maintenance of intestinal stem cells.However,no data are available about the possible function of Lgr4 in cerebellum.Based on the Lgr4 gene trap mice mode,we performed a series of experiments to study the function of Lgr4 gene on balance and motor coordination,and cerebellar synapticity.The project can be divided into three parts:1.Lgr4-/-mice exhibit cerebellar ataxia phynotypeTo characterize the behavior of Lgr4-/-mice,we performed a series of behavioral tests between wild-type and mutant mice.Footprint analysis has been found that Lgr4 deficient mice weaved from side to side while moving through the tunnel,using a broader gait width in a gait that lacked a normal,uniform alternating left-right step pattern.Then Lgr4-/-mice showed sever deficits on the accelerating rotarod test and balance beam test.Furthermore,Lgr4-/-mice exhibit normal in other behavioral tests,e.g.strength and general motor performance.2.Purkinje cell loss in Lgr4-/-mice?-galactosidase histochemistry revealed prominent staining throughout the soma to dendrite of Purkinje cells.Moreover,number and morphology of Purkinje cells in adult Lgr4-/-mice was observed compared with control mice.Lgr4 mutant mice showed notable loss of Purkinje cells(loss?20%-30%)at each stage.But surviving Purkinje cells of Lgr4 deficient mice revealed no significant changes in dendritic arborization and spine density.Furthermore,we tested the development of cerebellum and number of of Purkinje cells in postnatal Lgr4-/-mice and wild-type mice.The results demonstrated that the cerebellar development was similar between two genotype mice.And Lgr4 gene was expressed in Purkinje cells from PI by?-galactosidase histochemistry.However,the loss of Purkinje cells in mutant mice was found from P1 to P10.3.Lgr4-/-mice showed defects in parallel fiber to Purkinje cell synaptic plasticityUsing field potential recordings,parallel fiber-PC synapse on Lgr4-/-cerebellum showed noral Input-output relationship but decreased PPF ratio compared with control mice.And climbing fibers-PC synapse showed normal synaptic transmission.Then we found that parallel fiber LTD and LTP were induced in Lgr4-/-mice,but significantly decreased compared with wild-type mice.By immunohistochemistry and immunofluorescence,phosphorylated CREB immunoreactivity appeared in the nuclei of rare Lgr4-/-Purkinje cells,which was expressed in almost whole Purkinje cells of wild-type mice.And the expression of ?-catenin and ERK1/2 showed no difference in those two genotype cerebellum.Subsequently,after application of forskolin,strong pCREB immunoreactivity was observed in calbindin-positive Purkinje cells of Lgr4-/-mice.And impaired long-term synaptic plasticity was rescued by application of forskolin using field potential recordings.In summary,this study is the first to demonstrate that Lgr4 is not only an essential gene for motor coordination and motor learning,but also is important for development of Purkinje cell and synaptic plasticity in cerebellum.Lgr4 is specifically expressed in the cerebellar Purkinje cells,and adult Lgr4 mutant mice displayed a reduction in the number of Purkinje cells by 20-30%,and an impairment of PF-PC LTD and LTP.Application of forskolin could reactivate pCREB and rescue impaired synaptic plasticity.Thus we suggest that Lgr4 plays a critical role in cerebellar plasticity through cAMP-CREB signaling pathway,and then takes part in the balance and motor coordination of cerebellum.