Abnormal Cerebellar Neurogenesis Is Involved In Movement Disorders Of Autistic BTBR Mice

BackgroundAutism spectrum disorder（ASD）is characterized by impaired social interaction and restrictive or repetitive stereotypical behaviors,with increasing prevalence in recent years.Movement disorders are the most common complications of ASD,including tremble,ataxia,akinesia,dystonia and so on.For those ASD patients who already have badly affected social communication and social adjustment,movement disorders further limit their independence and life quality.In addition,motor ability affects the higher cognitive function,and plays important roles in language communication,emotion,execution and motor control.It is supposed that movement disorders may contribute to the pathogenesis of ASD.Cerebellum is an important encephalic region behind the hemicerebrum which involved in the regulation of motor function.Structural abnormality of cerebellum and motor dysfunctions are commonly reported in ASD patients or autistic animal models,suggesting that cerebellum contribute to pathogenesis of ASD.To study the relationship between the pathological process underlying the movement disorders and the occurred abnormal cerebellar neurogenesis would interpret the underlying mechanismof ASD.The cerebellum is characterized by a typical three-layer lamellar structure,consisting of the molecular layer（ML）,the Purkinje cell layer（PCL）and the inner granule layer（IGL）.The development of cerebellum is mainly accomplished during the postnatal 2 years in human beings and in 2 weeks after birth in the mouse.During the cerebellar development,billions of granule neurons are produced from granule cell precursors（GCPs）in the external granule layer（EGL）,and then migrate inward to the IGL through the radial fibers of Bergmann glia.In this process,granule neurons extend“T-shape”fibers parallel to the cortical surface,and connect to the dendrite of Purkinje cell in the ML.The Purkinje cells then send axon to contact the deep nucleus in the cerebellum and then project to wider brain regions.When all the granule neurons migrate to the destination,the EGL disappear and development of Purkinje cells is accomplished,thus the three-layer lamellar structure of cerebellum are formed.In addition,during the lamellar structure formation,GCPs proliferate faster and by the anchoring traction of Purkinje axon,the cerebellar cortex fold inward in this field.With the expansion of cerebellar cortex,the lobular structures are gradually formed.During the critical time of cerebellar development,impairments in the key cytological events would disturb the lobular and lamellar structure formation,which further affect the neural network formation and lead to abnormal neural activity.Purkinje cell is the sole efferent neurons in the cerebellum.Pathological alteration and dysfunction of Purkinje cells are found in autism individuals and autistic model mice,which are highly associated with the movement disorders in autism,while the underlying mechanism is still not clear.BTBR T⁺Itpr3^tf/J（BTBR）mouse is a widely used autistic model mouse at present,which carries the mutants of Disc1^del、T⁺、Itpr3^tf、Cox7a2l^l,and can perfectly mimic the core symptoms of ASD such as social dysfunction and repeated stereotypical behaviors.Recently,BTBR mice are reported to display poor sensorimotor and spatial impairment,and BTBR mice are found to be hyperactivity and impulsiveness.In addition,some studies have reported that immunity and oxidative stress were altered in the BTBR mouse cerebellum.What’s more,Haijie Yang found the increased lobes in BTBR mouse cerebellum.However,how the movement disorders develop and how it affected the autistic behaviors are unknown,and the abnormal cerebellar development involved in the motor dysfunction should be further illuminated.In this study,we further explored and confirmed that BTBR mice displayed dystonia-like behaviors and attention deficit-like behaviors.At the same time,we found abnormal postnatal development of BTBR mice cerebellum,which affected the lobular formation and development of Purkinje cell.These abnormal cytological events may be involved in the motor dysfunction of BTBR mice.MethodsDystonic behaviors were elicited in BTBR mice and wide type（WT）mice using tail suspension test during postnatal day 3（P3）to 150（P150）.The coordination ability and the ability to grasp were compared between BTBR and WT mice using grid hang test during P14to P150.The ladder rung walking test,accelerating rotarod test,and open field test were used to observe and compare the fine motor skills,motor learning ability,movement coordination and activity between BTBR and WT mice when they were 8 weeks old.The cerebellar samples of mice were collected at P3,P7,P14 and P90.Gross morphological changes and development process of cerebellum were observed in BTBR and WT mice using HE-staining.BrdU and Ki67 immunofluorescent staining were used to observe the proliferation of GCPs in BTBR and WT mice at P3 and P7.NeuN staining,Nissl staining and HE-staining were used to observe the migration of granule neurons and evaluate whether there exited ectopic mature granule neurons in BTBR or WT mice.The Bergmann glia development in the mouse cerebella was detected using its special makers of GFAP and S100βat P7,and the development of Purkinje neurons was observed with calbindin immunofluorescent staining,and the calbindin protein level was analyzed with western blotting.Golgi staining of P14 cerebella from BTBR and WT mice was performed to analyze the dendritic morphology,the number and classification of dendritic spines of Purkinje neurons.GAD67 and vGlut1 immunofluorescent staining and western blot experiments for adult BTBR and WT mouse cerebellum were used to detect the expression of excitatory or inhibitory neurotransmitter in the cerebellum.Finally,the fresh cerebellar samples of BTBR and WT mice were collected at P14 and then directly used for the complete transcriptome sequencing and real-time fluorescent quantitative PCR.When the differential expressed genes of cerebella of BTBR and WT mice at P14 were achieved,pathway enrichment analysis and protein interaction analysis were performed for further exploration in the possible mechanisms of abnormal cerebellar development and movement disorders of BTBR mice.Results1.Movement disorders in BTBR mice（1）In tail suspension test,adult BTBR mice exhibited severe dystonia-like behaviors.Compared to the WT mice,BTBR mice displayed distinct hyperflexion,hindlimb clasping or fore-and hindlimb clasping,severe trunk twisting and hyperextension.（2）In the tracing tail suspension test,the dystonic movement of BTBR mice presented firstly at P10.The incidence rate increased and 90%of the BTBR mice exhibited dystonia-like behaviors at P14,then the morbidity maintained and last into adulthood.（3）In the Grid hang test,the latency to fall was significantly decreased in BTBR mice at P21 compared to the WT mice,and it continued to decline and remained significantly shorter in BTBR mice at P150 compared to WT mice.（4）In the ladder rung walking test,compared to the WT mice,limb falls were significantly increased both in regular pattern and irregular pattern in the adult BTBR mice.The time spent crossing the ladder was decreased in BTBR mice both in two patterns.（5）In the open field test,distances in all zones and the central zone were both significantly increased in the adult BTBR mice compared to the WT mice.Average velocity of the first 5-minutes bin of BTBR mice was significantly increased compared to WT mice.（6）In the accelerating rotarod test,the adult WT mice showed significant promotion in day 2,compared to day1,and the promotion was more evident in the following days.While adult BTBR mice displayed significant promotion until day 5.The motor learning ability of BTBR mice showed significant worsen compared to the WT mice,and BTBR mice displayed attention deficit-like behaviors in the accelerating rotarod motor learning.2.Abnormal cerebellar neurogenesis of autistic BTBR mice in postnatal period（1）In the DAPI staining of cerebellar section of adult mice,the sagittal section area of BTBR mice cerebellum was significant larger compared to the WT mice,and the enlarged portion was mainly in the IGL and the ML.The number of cerebellar lobules was increased in BTBR mice compared to the WT mice.The weight of cerebella in BTBR mice was increased significantly at P14 and the mass ratio of cerebellum to the brain was increased in BTBR mice.In HE-staining,number of lobules was comparable between BTBR and WT mice at P3,but it was significantly increased in BTBR mice at P7 and P14.The sagittal section area of cerebella was larger in BTBR mice both at P3,P7 and P14 compared to the WT mice.The average sagittal section perimeter was elongated in BTBR mice both at P3,P7 and P14,compared to the WT mice.（2）GCPs in the cerebellum rapidly proliferate and differentiate into granule neurons in the first postnatal week and BrdU and Ki67 were used to detect the proliferation of GCPs.In BrdU immunostaining of cerebellar sections from mice at P3,the EGL thickness of BTBR mice was much thicker compared to WT mice,and the density of,total number of and percentage of BrdU positive cells in EGL was significantly increased in BTBR mice.In Ki67immunostaining of cerebella at P3 and P7,total number of positive cells was significantly increased in BTBR mice compared to the WT mice.（3）NeuN immunostaining of cerebella at P7 showed that NeuN positive neurons were mainly distributed in the IGL,with no obvious ectopic mature granule neurons both in BTBR and WT mice,and the EGL thickness was comparable between BTBR and WT mice.HE-staining of P7 cerebella showed that the number of migrating granule neurons with slim nuclei in the ML was increased in BTBR mice compared to the WT mice,but there was no difference in the migrating rate of granule neurons between BTBR mice and WT mice.Radial fibers of Bergmann glia contribute to the migration of granule neurons and S100βor GFAP immunostaining can be used to detect its soma or fiber,respectively.GFAP and S100βimmunostaining of cerebella at P7 showed that neither the number of the soma nor the fibers of Bergmann glia were different between BTBR and WT mice.Nissl staining of adult cerebella displayed that the distribution and number of mature granule neurons in the ML and IGL were comparable between groups.These results indicate that the migration of granule neurons and development of Bergmann glia were not affected in the BTBR mice.（4）Calbindin（CB）immunostaining of cerebella at P14 showed that the somas of Purkinje cells were monolayer arranged in PCL,and their dendrites extended into the ML.The density of Purkinje neurons in each lobes was comparable between BTBR and WT mice,but the Purkinje neurons in BTBR mice exhibited significant hypotrophy with decreased soma area compared to WT mice,in lobeⅣ/Ⅴ、Ⅵ/Ⅶ、Ⅷ、Ⅸ、Ⅹ.Western blot of cerebella at P14showed lower CB protein expression in BTBR mice compared to WT mice.CB immunostaining of adult cerebella showed that the density of Purkinje neurons in lobeⅧ、Ⅸ、Ⅹof BTBR mice were significantly decreased than that of WT mice.（5）Golgi staining of cerebella at P14 showed that there were no differences in the extended areas of dendrites,primary dendrite length or the complexity of the dendrite arborization of Purkinje neurons between BTBR and WT mice.While the dendritic spine density was significantly increased in BTBR mice,and type classification of spines showed that the percentage of thin form was increased but percentages of stubby and the matured mushroom forms were decreased in BTBR mice.（6）Double labeled immunofluorescent staining of GAD67 and CB of adult cerebella showed that GAD67 protein were mainly distributed in cytoplasm of Purkinje cell,and GAD67 were obviously overlapped with CB.Western blot further confirmed that expression of GAD67 protein was significantly decreased in BTBR mice compared to WT mice.Double labeled immunofluorescent staining of vGlut1 and CB of adult cerebella showed that the vGlut1 were mainly distributed in parallel fibers in ML and granule neurons in IGL,and the expression of vGlut1 protein was increased in BTBR mice.3.Regulation of gene expression in the developmental cerebella of autistic BTBR mice（1）Quality inspection of P14 cerebellar samples were fine in BTBR and WT mice.The correlation of the in-group samples was good,and there exist differences in gene expression patterns between groups.（2）The complete transcriptome sequencing of cerebella at P14 showed that there were3992 differential expressed genes between BTBR and WT mice,including 1858 up-regulated genes and 2134 down-regulated genes in BTBR mice.（3）KEGG pathway enrichment analysis showed that there were 23 changed pathways in BTBR mice compared to the WT mice,and most were involved in various types of transmitter synaptic pathways,such as glutamatergic synapse,cholinergic synapse,dopaminergic synapse,GABAergic synapse,serotonergic synapse and so on.（4）GO pathway enrichment analysis showed that there were 16 changed pathways in biological process of BTBR mice than WT mice,and most of them were pathways involved in neurogenesis,including negative regulation of neuron differentiation,negative regulation of nervous system development,negative regulation of neurogenesis,regulation of cell morphogenesis involved in differentiation,negative regulation of cell development and so on.There were 50 changed pathways in cellular component between groups,with the most are involved in synaptic components,such as synaptic membrane,axon,presynapse,postsynapse,dendritic shaft,dendritic spine and so on.（5）The protein-protein interaction（PPI）analysis showed the critical genes such as the down-regulated genes of Dynlt1b,Draxin,Rbpj and the up-regulated genes of Nog,Trpc6,Bdnf.Real-time fluorescent quantitative PCR showed that all these genes’significant alterations were confirmed.Among these,Trpc protein family paly an important role in the cerebellar development and the expressions of gene Trpc4 and CamkⅣ（downstream of Trpc6）were significantly up-regulated while gene Trpc3 was down-regulated in BTBR mice compared to WT mice.ConclusionBTBR mice exhibited distinct dystonia-like behaviors,attention deficit-like behaviors,hyperactivity,and motor impairments such as impaired motor coordination,fine motor impairment and impaired motor learning.These movement disorders might be due to the abnormal cerebellar neurogenesis,including disturbed lobular formation,impaired cellular development of granule neurons,Purkinje neurons,and the wrong synaptic connections formation.While the further mechanism might be the affected regulation of gene expression pattern in BTBR mice which disturbed the cerebellar neurogenesis during the critical developmental period.