Effects Of Thyroid Hormone On The Neuronal Differentiation Of Neural Stem Cells And The Underlying Mechanisms

Objectives:Thyroid gland is one of the major target organs of occupational hazards.Exposure to specific hazards during pregnancy can cause maternal TH deficiency andimpair the neurodevelopment of offspring. Thyroid hormone (TH) is essential to braindevelopment. TH deficiency during development may influence the process of neuriteoutgrowth, synapses formation, myelinization, and cell migration, causingneurodevelopmental disorders. Considerable researches have been carried out focused onthe functions of THs during brain development during the past decades. However, themajority of the previous demonstrated effects of T3occur during in late gestation or afterbirth; its effects on the development of embryonic neural stem cells (NSCs) during the earlyto middle gestation period are still unknown. It has been established that THs appear in thefetal brain very early in gestation, and TRs were detected in the fetal brain much earlierthan THs. These facts indicate that TH may participate in the early brain developmentprogram. Based on these facts, the following study will be carried out to detect the effectsof maternal TH deficiency on the embryonic neurogenesis and the learning and memory ofoffspring. In addition, the effects of TH and TR on the neuronal differentiation of NSCswill be investigated in the cultured NSCs in vitro. Finally, the signaling transductionmechanism of TH and TR will be examined on NSCs by detecting the involvement of theMAPK(Erk1/2), the mTOR(Akt), and the JAK/STAT3signaling pathway.Methods:①Timed pregnancy Balb/c mice were used in all the experiments.Hyperthyroid was induced by PTU treatment administered orally for21days combinedwith intraperitoneal injection given at day0and day7.②The telencephalon neurogenesiswas detected by the Tuj1mRNA expression and the number of Tuj1+cells in thetelencephalon. The mRNA expression of Tuj1was detected by Real time PCR, and thenumber of Tuj1+cells was detected by immunohistochemical staining.③The blood serum of the pregnancy mice was obtained on the13th day of PTU treated, and the serumof the offspring was obtained1M after birth, and then serum FT3and FT4concentrationwere determined by radioimmunoassay. Morris water maze was used to determine thelearning and memory of2M old offspring.④NSCs were obtained from E13.5mousetelencephalon. The cultured NSCs were treated by a physiological level of T3(0.3μＭ), andthen the maintenance, proliferation, apoptosis, and differentiation of NSCs was detected byNeurosphere assay, CCK-8assay, TUNEL, and Real time PCR, respectively. The neuriteoutgrowth of differentiated neurons was detected by immunocytochemical stainingcombined with quatitation with Image J.⑤The mRNA and protein expression of TRs wasdetected by RT-PCR and ICF both in vivo and in vitro, respectively. The role of TRα1inregulating the fate decision of NSCs was investigated by silencing the TRα1proteinexpression using a specific TRα1-targeting-siRNA.⑥The effects of T3on the Erk1/2,Akt, and STAT3protein expression and phosphorylation were detected by Western blot.The alteration of STAT3-DNA binding activity induced by T3was detected by EMSA.⑦The role of STAT3signaling in regulation the neuronal differentiation of NSCs wasdetected using the STAT3specific inhibitor Stattic. The cultured NSCs were treated withStattic(5μＭ), and then the neuronal differentiation, proliferation, and maintenance ofNSCs were determined as previous described.⑧To detect the role of STAT3signaling inmediating the effects of T3on NSCs, the STAT3was overexpressed on NSCs, and then theneuronal differentiation, proliferation, and maintenance of NSCs were determined asprevious described.⑨The interactions between TRα1and STAT3was detected using aspecific TRα1-targeting-siRNA. The protein expression of TRα1was silenced by thesiRNA, and then the phosphorylation and DNA binding activity of STAT3was determinedas previously described.⑩All statistical tests were conducted with SPSS softwarepackage (version13.0).Results:①After treatment with PTU for13days, a significant reduction in the serumlevels of FT3and FT4was observed in the pregnancy mice. However, the FT3and FT4levels in the serum of the offspring were not affected by PTU as detected one month afterbirth. The number of Tuj1-positive cells in the VZ/SVZ of the telencephalon was muchlower in PTU-treated mice when compared with control mice. Tuj1mRNA expression inthe telencephalon also decreased significantly after PTU treatment. In Morris water maze, the escape latencies in the PTU-treated group remained much longer than in the controlgroup. During the probe trail, the mice in the control group spent a longer time in thetarget zone than the PTU-treated mice and showed a stronger spatial bias for the area of thepool where the platform was located during training.②Physiological concentration of T3treatment for6days increased the percentage of Tuj1-positive cells. Tuj1mRNA levelswere robustly up-regulated by T3. T3increased the number of Tuj1-positive neuriteextensions as detected by inducing single NSCs differentiation, and the T3-induceddifferentiated neurons appeared with bulging cytoplasm and longer neurites. However, theGFAP-positive cells were markedly decreased after T3treatment and GFAP mRNA levelswere significantly down-regulated after T3treatment. After T3treated for9days, theCNPase-positive cells and CNPase mRNA levels were up-regulated significantly. Inaddition, we also found that T3treatment decrease the absorbance in the CCK-8assay, themRNA expression of the NSC-specific marker Sox2, and the number of primary andsecondary neurospheres formation. However, T3has no effects on cell apoptosis.③TheTRα transcript was detected both in cultured NSCs and in the E13.5telencephalon. Incontrast, TRβ was not detected. Further studies found that TRα1protein is present in E13.5NSCs both in vivo and in vitro. The TRα1-specific siRNA abolished the T3-inducedalterations in the expression of Tuj1, GFAP and CNPase mRNA. TRα1silencing alsoeliminated the effects of T3on the neurite outgrowth. In addition, the siRNA against TRα1blocked the T3-induced decrease in NSC proliferation. The T3-induced reduction insecondary neurosphere formation and Sox2mRNA expression was also attenuated by theTRα1-specific siRNA.④After T3treatment for1,2, and3days, neither the proteinexpression nor the phosphorylation of Erk1/2and Akt were altered. However, the treatmentof NSCs with T3resulted in a marked decrease in the tyrosine phosphorylation of STAT3,although total STAT3protein expression was not affected. T3treatment also resulted in asignificant decrease in the intensity of the band representing the STAT3–DNA bindingcomplex.⑤To assess the involvement of TRα1in regulating STAT3phosphorylation, aTRα1-specific siRNA was first used to silence TRα1expression. Western blot analysisrevealed that the TRα1-specific siRNA blocked the effects of T3on STAT3phosphorylation.The EMSA experiments revealed that the inhibitory effects of T3on the STAT3-DNAbinding activity were attenuated by TRα1-specific siRNA.⑥After Stattic treatment, the percentage of Tuj1-positive cells and Tuj1mRNA level were markedly increased, while thepercentage of GFAP-positive cells and GFAP mRNA level were significantly decreased. Inaddition, we also found that treatment with Stattic inhibited NSC proliferation andmaintenance.⑦After overexpressed STAT3in NSCs by transfecting the pEX_EF1_STAT3-YFP plasmid before T3treatment, the alterations in the expression of Tuj1andGFAP mRNA induced by T3were eliminated. The results from the CCK-8and neurosphereassays indicated that STAT3overexpression attenuated the inhibitory effects of T3on NSCproliferation and maintenance.Conclusions:①Maternal hypothyroidism during pregnancy in the mouse inhibitsneurogenesis in the embryonic telencephalon and causes learning and memory impairmentin the offspring.②T3promoted neuronal differentiation of NSCs, while inhibitedastrocytic differentiation. The proliferation and maintenance of NSCs were also inhibitedby T3.③Furthermore, the TH receptor alpha1(TRα1) was detected in the NSCs both invivo and in vitro. Silencing TRα1protein expression with specific siRNA eliminated theeffects of T3on NSCs.④T3decreased STAT3phosphorylation and STAT3-DNA bindingactivity through TRα1.⑤Inhibiting STAT3signaling activity differentiated NSCs intoneurons. The over expression of STAT3attenuated the promotive effects of T3on theneuronal differentiation of NSCs. Taken together, these results suggest that T3promotes theneuronal differentiation of NSCs by inhibiting STAT3signaling activity through TRα1andcontributes to early neurogenesis in the embryonic telencephalon.