Fig 2: Expression and correlation analysis of TTF1, TrkA, TrkB, and miR-204 in GN, well-differentiated NB, and undifferentiated NB(A) H&E staining of GN, well-differentiated NB, and undifferentiated NB tissues. Magnification, 20×; scale bar = 100 μm; magnification, 40×; scale bar = 50 μm.(B) TTF1, TrkA, and TrkB levels in GN, well-differentiated NB, and undifferentiated NB were determined using IHC. Magnification, 40×; scale bar = 50 μm.(C) MiR-204 expression in GN, well-differentiated NB, and undifferentiated NB tissue was detected using FISH. Magnification, 20 or 40 ×.(D) TTF1, miR-204, TrkA, and TrkB expression in GN, well-differentiated NB, and undifferentiated NB determined using RT-qPCR.(E) Correlation between miR-204 and TTF1, and miR-204 and TrkB.(F) TTF1, TrkA, and TrkB expression in GN, well-differentiated NB, and undifferentiated NB tissues determined using Western blot.(G–I) Predicted binding sites between TTF1 and the promoter of TrkA between TTF1 and the promoter of miR-204, and between miR-204 and 3'UTR of TrkB.(J) Direct target relationships between TTF1 and the promoter of TrkA, between TTF1 and the promoter of miR-204, and between miR-204 and 3'UTR of TrkB were determined using luciferase reporter assays.(K) RIP was conducted to verify the combination of miR-204 and TrkB. Data are represented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01 vs. GN; #p < 0.05, ##p < 0.01 vs. well-differentiated NB.

Fig 3: Effects of TTF1 on cell proliferation, apoptosis, and autophagy in SK-N-SH, SK-N-BE, and SH-SY5Y cells(A–D) SK-N-SH cells were transfected with si-TTF1 and si-NC, while SK-N-BE and SH-SY5Y cells were transfected with pcDNA 3.1-TTF1 (OE-TTF1) and pcDNA-NC (OE-NC), respectively. (A) TTF1, miR-204, TrkA, and TrkB mRNA expression was determined using RT-qPCR in transfected SK-N-SH, SK-N-BE, and SH-SY5Y cells. Cell proliferation was assessed using (B) CCK-8, (C) EdU, and (D) colony formation assays.(E) Apoptosis was detected and calculated using flow cytometry.(F) TTF1, TrkA, and TrkB protein expression were determined using Western blot.(G) Bcl-2, cleaved caspase-3 (C-caspase3), p53, LC3B-I, LC3B-II, Beclin1, and p62 levels were assessed using Western blot in the transfected SK-N-SH, SK-N-BE, and SH-SY5Y cells. Data are represented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01.

Fig 4: Expression of phospho-tropomyosin-related kinase A (p-TrkA) (a, b), phospho-extracellular signal-regulated kinase 1/2 (p-ERK1/2) (c, d) and phospho-cAMP response element-binding protein (p-CREB) (e, f) in the hippocampus of pups (N = 6, PND7). *p < 0.05 versus CON group, #p < 0.05 versus SCH group, &p < 0.05 versus OHT group. CON, control; E, gestational day; OHT, overt hypothyroidism; SCH, subclinical hypothyroidism

Fig 5: Enhanced D3 delivery and increased NGF protein levels in the basal forebrain following MRIgFUS-induced BBB opening.(A) Representative HPLC elution profile corresponding to D3 detection in the basal forebrain following D3/FUS treatment (blue line) relative to PBS/FUS control (black line). a.u., arbitrary units. (B) D3 levels were negligible in the brain after intravenous D3 injection without MRIgFUS (red bars). With MRIgFUS, D3 significantly entered the targeted MS/DBB and NBM in non-Tg and TgCRND8 mice (blue bars). (C) Plasma and serum D3 levels between unsonicated and sonicated animals for both genotypes were comparable. (D) Increases in D3 concentration, and (E) TrkA phosphorylation, were proportional to the contrast enhancement in MRIgFUS-treated regions for both genotypes. (F) NGF protein levels were increased in PBS/FUS- and D3/FUS-treated relative to PBS- and D3-treated animals, respectively. NGF protein levels were decreased in 6-month-old TgCRND8 mice compared to non-Tg littermates, but FUS-induced NGF protein levels were comparable between genotypes. (G) There was a positive correlation between the relative contrast enhancement and NGF levels measured. Statistics: Two-way ANOVA (B, C, and F). Significance: ^,†P < 0.05, **,^^P < 0.01; † indicates comparison with PBS-treated non-Tg mice (genotype effect); ^ indicates comparison with PBS-treated mice of the same genotype (FUS effect); * indicates comparison with D3-treated mice (i.e., intravenous D3, no MRIgFUS) of the same genotype (D3/FUS effect). (D, E, and G) Linear regression analysis. Dashed lines indicate a 95% confidence interval. Data represent means + SEM; n = 4 per group (B to D) and n = 6 per group (E to G).