| BackgroundCognitive dysfunction,also known as cognitive decline and cognitive impairment,are various degrees of mental health disorders that primarily affect cognitive abilities including learning,memory,perception,and problem solving.The endocrine hormones are highly effective bioactive substances released by internal glands or cells,regulating the physiological functions of target tissues or cells.Multiple studies have found that a variety of endocrine hormones are involved in regulating the structure and function of brain,and abnormal endocrine hormone levels will cause cognitive impairment.Among these endocrine hormones,thyroid hormones have been the primary focus for its crucial role in the nervous system.A large amount of clinical evidence suggests that thyroid dysfunction is closely related to cognitive impairment.As thyroid hormones play a central role in governing neurogenesis and gliogenesis from development to ageing,the relationship has been traditionally attributed to abnormal thyroid hormone levels.Interestingly,accumulating evidence has demonstrated cognitive impairment is associated with subclinical hyperthyroidism in recent years.Subclinical hyperthyroidism is a clinical condition characterized by thyroid stimulating hormone(TSH)is below the lower limit of the normal reference range while serum thyroid hormone levels are within the normal reference range.Subclinical hyperthyroidism is associated with multiple diseases such as cardiovascular disease,bone fracture and dementia.In terms of cognitive function,clinical systematic reviews have been substantial evidence in support of the relationship between subclinical hyperthyroidism and increased prevalence of cognitive impairment.Meanwhile,a meta-analysis of prospective cohort studies in population has found that subclinical hyperthyroidism is associated with an increased risk of dementia.Since the thyroid hormone levels remain normal,this association is more likely to be driven by other factors.As TSH is the characteristic indicator of subclinical hyperthyroidism,and multiple clinical studies have shown that low TSH level is associated with increased prevalence of cognitive impairment,it is possible that TSH is involved in the regulation of cognitive function.TSH,an endocrine hormone synthesized and secreted by the anterior pituitary gland,is a non-covalently linked glycoprotein heterodimer consists of an a-glycoprotein subunit and a specific ? subunit.TSH is the major ligand of thyroid stimulating hormone receptor(TSHR),a member of the G protein-coupled receptor superfamily of integral membrane proteins.Binding of TSH to the TSHR results in intracellular signaling via cyclic adenosine monophosphate(cAMP)and can induce both the phospholipase C and the protein kinase A signal transduction systems thereby exerting a variety of physiological functions.Although TSHR is of great importance for various functional aspects of thyroid gland,in recent years accumulating evidence has shown TSHR to be expressed and play roles in other organs,such as the liver,skin,and bone.As part of the limbic system,hippocampus is the core brain region for encoding,consolidating and extracting memories,and plays a crucial role in semantic episodic memory and new objects exploration as well as spatial navigation.In the central nervous system,TSHR expression has been detected in large neuronal cell bodies within all six limbic regions in the human brain.TSH has been found in brain and cerebrospinal fluid as well.Nevertheless,the role of TSHR signaling pathway on brain function remains unclear.To answer this question,this study started from the perspective of our population research and then utilized Tshr knockout mouse model to elucidate the role of TSHR signaling pathways on cognitive functions via behavioral tests,analysis of the synaptic structure and gene expression profiling.Our study provided new knowledge about the neurologic significance of TSHR signaling and may be helpful for the pathophysiology and clinical practice of cognitive impairment and thyroid disease.Objectives:1.To explore the relationship between TSH and cognitive impairment.2.To elucidate the role of TSHR signaling pathway in cognitive function,hippocampal structure and gene expression profile.3.To demonstrate the significant role of core synapse-related molecules in cognitive impairment caused by TSHR signaling pathway deficiency.Methods:1.Human subjectsThe present population-based study was part of the community-based REACTION study that aimed to investigate the epidemiology of metabolic diseases.All the participants underwent a standard examination in the morning including the following:a detailed medical history inquiry;Mini-Mental State Examination(MMSE);measurement of height,body weight,waist circumference and blood pressure;collection of fasting serum sample.Age,sex,smoking status and family history of Alzheimer's disease and other essential information were obtained via questionnaire.Subjects were fasted for at least 10 hours before serum sample collection at 7-8 AM.Thyroid function was assessed by serum levels of TSH,FT3 and FT4 using immunoassay analyzer.2.Animal model2.1 Tshr knockout mice:Tshr homozygous(Tshr-/-)mice and wild-type littermate control(Tshr+/+)mice were obtained by breeding Tshr heterozygous(Tshr+/-)mice.To maintain thyroid hormones within physiological levels,Tshr-/-mice were supplemented with thyroid hormone powder after birth.2.2 7,8-Dihydroxyflavone(7,8-DHF)administration model:Tshr homozygous(Tshr-/-)mice for experimentation were obtained from Tshr-/-mice breeding.To maintain thyroid hormones within physiological levels,Tshr-/-mice were supplemented with thyroid hormone powder after birth.Tshr-/-mice aged 3 months were given brain-derived neurotrophic factor(BDNF)mimics(7,8-DHF group)or solvent(Veh group)through drinking water for 8 weeks.3.Radioimmunoassay:serum levels of total triiodothyronine and total thyroxine were measured using radioimmunoassay kits.4.Behavioral tests:Morris water maze(including place navigation task and probe trial)and Y maze were performed to assess the spatial learning and memory of mice.5.Immunofluorescence:the expression and distribution of TSHR in the hippocampus were detected by immunofluorescence staining.6.Immunohistochemistry:the expression of various molecules(NEUN,FOS,EGR1)at protein level was measured by immunohistochemical staining.7.Nissl staining:Nissl bodies and hippocampal structure were detected by Nissl staining.8.Golgi staining:the structure of hippocampal pyramidal neurons and the number of dendritic spines were detected by Golgi staining.9.Transmission electron microscopy(TEM):synaptic density and ultrastructure in hippocampus were measured by TEM.10.RNA-sequencing(RNA-seq):RNA-seq was applied to obtain the gene expression profiles and differential genes of hippocampus for further bioinformatics analysis.11.RNA extraction and Real-time PCR were performed to detect the expression of various molecules at mRNA level:Actb,Arc,Bdnf,Cebpb,Fos,Egrl,Egr2,Grin2a,Homer1,Itprl,Junb,Kcnabl,Klk8,Npas4,Ppp3c?,Shank3,Tancl,Tshr.12.Protein extraction and immunoblotting were used to measure the expression of BDNF at protein level.13.Statistical analysis13.1 Clinical research:Quantitative variables for population data are expressed as mean±standard deviation,or median(inter-quartile range).Continuous variables were compared using one-way ANOVA test or Kruskall-Wallis test and categorical variables using Chi-squared test.Intergroup comparisons were performed using post-hoc Bonferroni test,and p<0.05 was considered significant.Binary logistic regression analysis was performed to study the relationship between TSH and cognitive impairment.TSH was defined as a dummy variable and the fourth quartile was the reference for other TSH quartiles.Statistical analyses were performed with SPSS v.22.0.13.2 Basic research:Experimental data are presented as mean ± SEM unless stated otherwise.Two-group comparisons were made using unpaired Student's t-test,and two-sided p<0.05 was considered significant.The experimental data was analyzed with GraphPad Prism 6.0.Results:1.Low serum TSH level may be a risk factor for cognitive impairmentIn our cross-sectional population study,the participants were classified into.4 groups according to their serum TSH levels.Binary logistic regression analysis was performed to study the relationship between TSH and cognitive impairment.Compared with the highest TSH quartile(Quartile 4),the lowest TSH quartile(Quartile 1)displayed approximately 2.1-fold increased risk for cognitive impairment.The results indicated that low serum TSH level may be a risk factor for the prevalence of cognitive impairment in human subjects.2.TSHR was shown to express in hippocampusThe result of immunofluorescence demonstrated the expression of TSHR in DG,CA1 and CA3 subfields of murine hippocampus.3.Tshr deficiency leads to pronounced deficits in learning and memory tasksSpatial learning and memory were examined by the place navigation task of Morris water maze.During acquisition training,Tshr-/-mice showed significantly impaired performance evidenced by remarkably increased swim path lengths.In the probe trial(no platform),Tshr-/-mice spent significantly less percentage of time in the target quadrant and had significantly fewer target crossings,indicating the spatial reference memory was defective.The swimming trace in the probe trial showed that Tshr-/-mice tended to swim aimlessly rather than across the former location of the platform repeatedly,suggesting their poor performance mainly resulted from hippocampal defects.To assess their spatial working memory,Y maze was subsequently performed.Tshr-/-mice showed significantly reduced percentage of spontaneous alteration,and no significant difference in the number of arm entries compared with wide-type littermates,indicating the spatial working memory was impaired as well.4.Tshr-/-mice displayed reduced excitatory synaptic density and altered synaptic structureNissl staining on serial brain sections showed that there was no alternation in the gross morphology of hippocampus in Tshr-/-mice and wide-type littermates at corresponding coronal levels.Moreover,neuron-specific antigen NeuN expression also revealed no significant difference in the number of NeuN+cells in DG,CA1,CA2/3 subfields of hippocampus.Golgi staining results showed that spine densities of apical and basal dendritic segments in hippocampal CA1 pyramidal neurons reduced dramatically in Tshr-/-mice when compared to their littermates.Further,the asymmetric(excitatory)synapses of hippocampus sections were directly observed by transmission electron microscopy.A significant reduction of excitatory synapse density and perforated synapses density was observed in the CA1 subfield of the hippocampus of Tshr-/-mice compared to their littermates.In addition,significant structural alterations(postsynaptic density width,synaptic cleft)were observed in Tshr-/-mice.5.Gene expression profile was altered in the Tshr-/-mice hippocampusThe hippocampal gene expression profile of Tshr-/-mice and wide-type littermates were examined by RNA-Seq.A total of 438 differentially expressed genes(DEGs)were revealed by the DESeq2,including 188 upregulated genes and 250 downregulated genes in the hippocampus of Tshr-/-mice compared to their littermates.Gene transcriptional profile of Tshr-/-mice were completely different from their littermates,as the two groups were well separated by the first three principal components.Bioinformatics analysis showed that the top of the significant terms identified in both KEGG pathway and GO enrichment analysis were synapse-related component and signaling.6.Tshr knockout induced changes in synapse-related gene expressionProtein-protein interactions and module analysis of DEGs were performed by STRING and Cytoscape,respectively.A deal of top 10 hub genes,the center of the distribution of node degrees,are synaptic plasticity-related genes.The changes of those DEGs were confirmed by real-time PCR.Consistent with RNA-Seq results,the mRNA levels of Egrl,Junb,Egr2,Arc,Fos,Bdnf and Naps4 were significantly downregulated in Tshr-/-mice hippocampus.Among the hub genes in our transcriptomic analysis,Bdnf,Egr1 and Fos genes were in the top-signaling network and required for learning and memory.We subsequently quantified the protein expression of BDNF and immediate early genes(IEGs).The expression of BDNF decreased significantly in Tshr-/-mice hippocampus.The immunohistochemistry of EGR1 and FOS demonstrated that the number of FOS-positive cells were significantly reduced in CA1,CA3,DG subfields,while the number of EGR1-positive cells showed a remarkable reduction in CA3,DG subfields.7.Small molecule mimic 7,8-DHF of BDNF could attenuate spatial learning and memory deficits of Tshr knockout miceBased on the core position of BDNF in the protein interaction and signaling network of DEGs,Tshr-/-mice aged 3 months old were administrated with BDNF mimic(7,8-DHF group)or solvent(Veh group)through drinking water for 8 weeks.In the place navigation task of Morris water maze,7,8-DHF group showed significantly improved performance evidenced by remarkably decreased latencies to target and swim path lengths during acquisition training.In the probe trial,7,8-DHF group had significantly more target crossings.Different from the Veh group,the swimming trace in the probe trial showed that 7,8-DHF group tended to swim across the former location of the platform rather than aimlessly.The results indicated that 7,8-DHF could attenuate spatial learning and memory deficits caused by TSHR signaling deficiency.Conclusion:1.Low serum TSH level is a risk factor for cognitive impairment2.TSHR signaling deficiency resulted in remarkably impaired spatial learning and memory,accompanied with alteration in synaptic structure and synapse-related gene expression profile of hippocampus in mice.3.The downregulation of BDNF is an important molecular mechanism of cognitive defects caused by TSHR signaling pathway. |
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