Mutation In Thyroid Hormone Receptor α1 Triggers Tau Hyperphosphorylation In Mice Cortex

Objective:Thyroid hormone（TH）is essential for normal brain development.The actions of thyroid hormones are mediated through nuclear thyroid hormone receptors（TRs,TRαandβ）and regulation of gene expression.Patients with mutations in THRA that lead to the development of RTHα.RTHαmanifested with some features of hypothyroidism.Clinical consequences of central nervous system including mental retardation,cognitive deficits,handwriting difficulties,ataxic gait,and seizures in one case.There is no effective cure for cognitive and fine motor skill defects.We have generated a new knock-in mouse model(Thra^E403X)that corresponds to the nonsense mutation found in the first RTHαpatient before.Our previous work indicated that the Thra ^E403X mice exhibited most of the phenotypic features of THRA^E403X/+patient including delayed postnatal growth and development.Notably,they exhibited severe neurological deficits.These results indicate that the Thra^E403X mouse is a faithful animal model of brain development in RTHα.To clarify the molecular mechanism underlying Thra ^E403Xmutation causes these neurocognitive deficits,quantitative proteomics and phosphoproteomics profiling were performed on the cortex tissue of Thra^E403X/E403Xand wild type（WT）mice at 3-weeks.Bioinformatics Analysis including Functional Classification and Pathway Enrichment were performed.GO enrichment analysis indicated that the“cytoskeleton organization”,“synapse”and“structural constituent of cytoskeleton”GO terms were significantly enriched.Regarding the KEGG pathways,“calcium signaling pathway”was significantly enriched.Therefore,we focused on phosphorylation of cytoskeleton and associated protein.We indicated that tau can be excessively phosphorylated at seven epitopes and mutation in thyroid hormone receptorα1 triggers tau hyperphosphorylation in mice cortex.The main objective of the present study is to investigate major molecular events lead to tau hyperphosphorylation in Thra^E403X mice.Methods:1.3 weeks-old Thra^E403X/E03Xmutant mice and WT mice were used in the present study,which were obtained by crossing 8-10 weeks-old heterozygous mutant mice.Primary culture neurons were prepared from the cortex of newborn postnatal Day 1mice and conducted experiments in vitro.2.Quantitative proteomics and phosphoproteomics profiling were performed on the cortex tissue of Thra^E403X/E03Xand WT mice.Bioinformatics Analysis including gene ontology（GO）annotations and Functional Enrichment were performed.3.HE staining was performed to observe the pathomorphological changes of the brain in Thra^E403X/E403X mice.4.Western blot experiments were performed to detect the expression of Cleaved caspase 3,in order to examine the effect of TRα1 mutation on apoptosis in cortical neurons.5.Myelin proteins expression were measured with Western blot analysis.6.Cortical sections were stained with the immunohistochemical method and Immunofluorescence methods for abnormal neurofibrillary tangle（anti-human PHF-Tau monoclonal antibody,AT8）.7.q PCR was performed to detect the expression of tau mRNA.8.Western blot experiments were performed to detect the expression of tau and phosphorylated tau at several phosphorylation sites of the cortex in Thra^E403X/E03X mice and WT mice.9.Western blot experiments were performed to detect the expression of key tau protein kinases and phosphatases.10.Western blot experiments were performed to detect the expression of phosphorylated CaMKⅡand tau in primary cortical neurons from Thra ^E403X/E03Xmutant mice and Thra^+/+mice.11.Treatment with CaMKII inhibitor KN93 blocked in primary cortical neurons and detected the expression of phosphorylated tau.12.The intracellular calcium concentration was determined by the fluorescent probe Fluo4-AM.13.q PCR and western blot experiments were performed to detect the mRNA and protein expression of plasma membrane Ca²⁺ATPase Atp2b2.14.Statistical significance was calculated by Student’s t test using Graph Pad Prism 8 software.Data was presented as the mean±SEM.Differences were considered statistically significant when P<0.05.Results:1.Phosphoproteomic studies revealed that Thra^E403Xmutation led to the 445differentially phosphorylated proteins and their 556 phosphorylation sites.Including 414upregulated and 142 downregulated differentially expressed phosphorylation proteins sites in 329 and 116 differentially expressed phosphorylation proteins,respectively.2.Apoptotic protein Cleaved caspase 3 was increased in Thra^E403X/E403X mice cortex（P<0.05）.3.Thra^E403X/E403X mice showed significantly decreased myelin protein expression in cortex（P<0.05）.4.The presence of neurofibrillary tangles in the cortex of3-week-old Thra^E403X/E03Xmice was assessed by Immunohistochemistry studies.5.An increased tau phosphorylation at multiple AD-related sites was detected in 3 weeks-old Thra^E403X/E03X mice,at Ser202,Thr231,Ser396,Ser404 epitope（P<0.05）.There was no significant difference in the mRNA and protein levels of tau.6.Phosphorylated CaMKII levels,a key tau kinase,were significantly higher in the 3-week-old Thra^E403X/E03Xmutant mice relative to the control mice（P<0.05）,other tau kinase or phosphatase protein levels were not significantly different from the control groups.Namely,kinase including p-GSK3β,p-CDK5,p-JNK,p-ERK,p-P38 and phosphatase p-PP2A,de Me-PP2A.7.Phosphorylated tau and CaMKII were also higher at primary cultures of Thra^E403X/E03Xmice cortical neurons compared with WT group.After treatment with CaMKII inhibitor KN93,the p-tau was not significantly increased at primary cultures of Thra^E403X/E03Xmice cortical neurons compared with the control group.8.The intracellular calcium concentration of primary cultured neurons in Thra^E403X/E03Xmice cortical neurons increased compared with the control group.9.The mRNA and protein levels of Atp2b2,a calcium pump in the cortical plasma membrane,were significantly downregulated in Thra^E403X/E03X mice.Conclusions:1.Thra^E403X mutation led to altered phosphorylation profiles in mouse cortex.2.Thra^E403Xmutation decreased of plasma membrane calcium pump Atp2b2,prevented Ca²⁺-ATPases from extruding Ca²⁺in cortical neurons and caused overload of intracellular calcium ions.Overload of intracellular calcium ions resulted in tau hyperphosphorylation via activation of CaMKII and subsequent the apoptosis of neurons.