Characterization Of A New Resistance To Thyroid Hormone Mouse Model And Effects Of Thrαl^E403x Mutation On The Development Of Cerebral Cortex

Objective:Thyroid hormones（THs）play an essential role in neurodevelopment.They mainly regulated the transcription of target genes by binding to thyroid hormone receptors（THRs）and played their biological functions.In this study,a Thrα1^E403X403X mouse model was constructed based on the mutation site（THRA-E403X）of the first reported patient with a mutation in the THRA gene.The whole body phenotype of mutant mice was analyzed,and the mechanism of the effect of Thrα1^E403X403X mutation on the morphology and function of mouse cerebral cortex was emphatically discussed.Methods:Using the homologous recombination principle and the homologous DNA fragment replacement target gene fragment technology,the base at the 403 site of THRA gene was subjected to a site-specific change（G-T）,and the corresponding amino acid sequence was changed（403E mutation was 403X）,so as to prepare THRα1^E403X403X mice with specific site mutation.The experimental mice were aged 3 weeks,6 weeks and 16weeks after birth,which were equivalent to human infancy,adolescence and adulthood.Research contents:（1）.Mouse phenotypic analysis was performed at 3 weeks,6 weeks and 16 weeks after birth.1.Mice in the same litter were monitored for 42 weeks,including body weight,body length and tail length.The changes of body weight and growth of mice in different periods were observed.2.Radioimmunoassay was used to detect thyroid hormone levels in mice.3.Micro-CT detector and DXA scanning were used to analyze bone development,femur and skull development,and changes in femur microstructure（bone mass and trabecular bone）.4.Morris water maze experiment,Rota-rod experiment and gear experiment were carried out at the age of 6 weeks and 16weeks to evaluate the learning and memory ability and motor coordination ability of the nervous system of mice at different periods.5.The grip experiment was used to evaluate the development of muscles in the limbs of mice.6.The changes of heart function of mice in different periods were analyzed by mouse heart ultrasonic detector.The changes of heart weight in different periods were measured.7.The development of adipose system（white fat,brown fat）and gastrointestinal system（small intestine and colorectum）in mice was measured at different stages.8.The changes of anus temperature in mice were detected by mouse body temperature detector.（2）.RNAscope?in situ hybridization（ISH）technique was used to observe the expression of THRα1 in the cerebral cortex of mice at different developmental stages.The experimental time points were:embryo（E）11.5,E13.5,E15.5,E17.5,postnatal（P）0,P3,P5,P7,P14,P56.（3）.To investigate the effect of Thrα1 mutation on the morphological structure of cerebral cortex in mice.Experimental time points:P0,P10,3 weeks,6 weeks and 16 weeks after birth,and the development of neurons in each layer of the cerebral cortex were observed by Nissl staining.（4）.TMT quantitative proteome expression profile analysis was performed on cerebral cortex tissues of 3-week-old male WT mice and Thrα1^E403X/E430X403X/E430X mice.The amino group of specific labeled polypeptide was analyzed by mass spectrometry to screen the differential proteins related to the development and function of cerebral cortex,explore the changes of functional proteins affected by THRA gene mutation in mouse cerebral cortex and the changes of related pathways,and further verify the screened differential proteins.Results:（1）.Phenotypic analysis 1.The growth and development results showed that compared with WT mice,the growth and development of Thrα1^E403X/+mice in infancy and adolescence were significantly delayed,showing low body weight and short stature.Adult Thrα1^E403X/+mice showed improvement in growth,but did not fully recover.Thrα1^E403X/E403X403X/E403X mice had more severe phenotype,showing survival time of no more than30 days,severe growth retardation,and significant reduction in body weight,body length and tail length.2.The results of thyroid function indicated that Thrα1^E403X/+mice showed thyroid hormone resistance,which showed serum TT3 level elevated,TT4 level decreased or normal,TT4/TT3 level decreased,rT3 level decreased,and serum TSH level showed no difference.3.Skeletal system:DXA results showed that,compared with WT mice,Thrα1^E403X/+mice had short and delayed bone development in adolescence,and there was no significant difference in limb bone length in adulthood.Thrα1^E403X/E403X403X/E403X mice had severe bone development disorders.Micro-CT results showed that the growth of epiphyseal of Thrα1^E403X/+mice and Thrα1^E403X/E403X403X/E403X mice were delayed in juvenile stage,and the bone mass,trabecular number,trabecular separation and cortical thickness of bone were significantly reduced.The abnormality of Thrα1^E403X/E403X403X/E403X mice was more obvious,suggesting that the mutant mice suffered from bone dysplasia,osteoporosis and delayed ossification during this period.In adolescence,the femur of Thrα1^E403X/+mice was still short.Although there was no difference in femur bone mass of mice with Thrα1^E403X/+during this period,the bone cortex was still slightly thin,the number of trabeculae began to increase,and the separation degree of trabeculae decreased,suggesting that bone development was still delayed,but there was a spontaneous recovery trend.In the adult stage,Thrα1^E403X/+mice still had slightly thinner bone cortex,significantly increased trabecular number and decreased trabecular separation,indicating the manifestation of bone sclerosis.4.The results of Morris water maze experiment on the nervous system showed that the average escape latency of Thrα1^E403X/+mice was significantly longer than that of WT mice,and the frequency of crossing the target quadrant and platform was significantly lower than that of WT mice.The results suggested that Thrα1^E403X/+mice in adolescence and adulthood had impaired nervous system and reduced ability of learning and memory.5.The results of the Rota-rod experiment and gear experiment showed that Thrα1^E403X/+mice exercise duration and exercise ability were significantly lower than that of WT mice.The results suggested that the ability of motor balance in Thrα1^E403X/+mice was impaired and muscle strength was decreased in adolescence and adulthood.6.The results showed that Thrα1^E403X/+mice had significantly decreased muscle strength.7.In different periods,the heart weight of Thrα1^E403X/+mice was significantly reduced.The results of cardiac ultrasound indicated that the ventricular wall thickness and ventricular volume of Thrα1^E403X/+mice were significantly lower than that WT mice.8.White adipose tissue（female and male）and brown adipose tissue（female only）were significantly decreased in the juvenile stage of Thrα1^E403X/+mice,while omentum adipose tissue and gonadal adipose tissue were significantly decreased in the adolescent stage of Thrα1^E403X/+mice,suggesting that the effect of Thrα1^E403X403X mutation on adipose tissue production in mice was mainly before adolescence.9.At the age of juvenile,the small intestine of Thrα1^E403X/+mice was short and thin,but the length of the small intestine was increased significantly in adulthood.10.In different periods,the anal temperature of Thrα1^E403X/+mice were significantly decreased,approximately decreased 1℃,suggesting that Thrα1^E403X/+mice had persistent hypothermia.（2）.RNAscope?analysis of in-situ hybridization（ISH）revealed that Thrα1 was expressed in the cerebral cortex of mice at different developmental stages from the beginning of neural tube development in early embryonic stage to the maturation of the mouse.（3）.The morphological results of the cerebral cortex showed that during P0,the I,V and VI layers of the cerebral cortex of WT mice had been formed,but the I and VI layers of the cerebral cortex of Thrα1^E403X/+mice only formed,and the neuronal migration delayed.Thrα1^E403X/E403X403X/E403X mice had significantly reduced brain tissue,slower neuronal migration,and only layer 1 of the cerebral cortex was formed.During P7,the six-layer structure of the cerebral cortex of WT mice had been completely formed,and the neurons had migrated and the structure of each layer was clear and stratified.Thrα1^E403X/+mice cerebral cortex neuron migration was not completed,only the I and VI layers were formed,in which the II,III,IV and V layers had fuzzy structures and unclear boundaries,showing the appearance of neuron invasion in adjacent layers.The brain tissue of Thrα1^E403X/E403X403X/E403X mice was still significantly reduced,with all layers of neurons arranged in a disorderly manner.During P21,the cortical neuron migration Thrα1^E403X/+mice was completed,but the stratification of layer II,III and IV was not obvious,and the location of neurons was still unclear.The cerebral cortex of Thrα1^E403X/E403X403X/E403X mice was still not stratified,and the structure of each layer of neurons was not clear.The experimental results suggested that during the critical period of the development of the cerebral cortex of the nervous system,Thrα1^E403X/+mice and Thrα1^E403X/E403Xmice showed significant abnormalities of the nervous system,delayed development of the cerebral cortex,delayed neuronal migration in the cerebral cortex and decreased neuronal localization ability.（4）.The results of TMT quantitative proteomics analysis of cerebral cortex tissues showed that in the difference expression change greater than 1.3 times,159proteins were up-regulated and 177 proteins were down-regulated in the cerebral cortex tissues of Thrα1^E403X/E403X403X/E403X mice.The differentially expressed proteins include a variety of proteins involved in the proliferation,development,migration,synapse formation,and myelin formation of mouse cortical neurons.The proteins involved in the amyotrophic lateral sclerosis pathway in the cerebral cortex of Thrα1^E403X/E403X403X/E403X mice were significantly down-regulated,and the proteins involved in the prion diseases pathway were significantly up-regulated.Conclusion:1、Thrα1^E403X/+mice exhibited most of the clinical phenotypes of patients with RTHα,and was an ideal animal model for studying the function of TRα1.2、Preliminary phenotypic analysis showed that Thrα1^E403X403Xmutation caused growth and development disorders and homeostasis imbalances in the nervous,body temperature,bone,muscle,blood,heart,intestinal tract,fat,and female reproductive system in mice.3、The spatio-temporal specific expression of Thra1 gene in the cerebral cortex of mice was detected by RNAscope?in situ hybridization,suggesting that TRa1 may be involved in the proliferation,migration and maturation of precursors of neurons in the cerebral cortex.4、Thrα1^E403X/E403X403X/E403X mice showed obviously cortical development disorders,including reduced brain volume,thinning of cortical thickness,delayed migration of neurons,decreased number of neurons,and cortical structural disorders.Thrα1^E403X/+mice developed delayed neuronal migration and cortical structural disorders that persisted into adulthood.5、The Thrα1^E403X403X mutation significantly affected the expression of proteins related to neuron migration,synaptic formation,and myelin formation in mouse cerebral cortex.6、Some of the differential proteins caused by mutation of Thrα1^E403X403X mutation were enriched in the amyotrophic lateral sclerosis（ALS）and prion diseases pathways,suggesting that mutation of Thrα1 may be involved in the occurrence of amyotrophic lateral sclerosis and prion diseases.