A BCD6 35 80 1.0 3 Thr Ala 0.4 Thr Ala 30 0.8 60 4 0.3 2 25 0.6 40 20 0.2 0.4 1 2 20 intake(g)Food Body weight(g) 15 0.1 0.2 Serum TSH (ng/ml) T4 plasmaT4 levels (ng/ml) plasma levelsT3 (ng/ml) 1/V 10 0.0 0 0.0 0 5 10 15 Thr Ala Thr Ala Thr Ala -1.0 -0.5 0.5 1.0 1.5 2.0 Thr Ala Thr Ala Age (weeks) 1/[T4] (nM) dark light (fmols/min/mg ptn) (fmols/min/mg -1 Ala Thr IKMN O Femur 16.5 0.55 1100 0.24 E F 16.0 1050 0.22 G H 0.50 Thr92‐D2 15.5 1000 0.20 Ala92‐D2 0.45 15.0 950 0.18 (mgHAcm-3) Femur volume 0.40 14.5 900 0.16 Cortical thickness Femur length (mm) length Femur Bone MineralDensity 14.0 0.35 850 0.14 Thr Ala Thr Ala Thr Ala Thr Ala JLPQR Vertebrae 4.6 5.5 0.070 0.35 4.4 5.0 0.065 0.30 **P<0.01 0.060 4.2 *** 4.5 0.055 0.25 4.0 4.0 0.050 0.20 3.8 3.5 Trabecular number 0.045 Trabecular spacing Trabecular thickness Vertebral height (mm) height Vertebral 3.6 3.0 0.040 0.15 Thr Ala Thr Ala Thr Ala Thr Ala Figure S1: Additional phenotype of the Ala92‐Dio2 mouse. (A) Ala92‐Dio2 (Ala) mice exhibit normal growth curves without difference from Thr92‐Dio2 mice (Thr); (B) Ala92‐Dio2 mice have similar systemic thyroid hormone levels compared to Thr92‐Dio2 mice; (C) Lineweaver‐Burke ST U V plots of D2 activity as measured in cerebral cortex sonicates obtained from Thr92‐Dio2 and 22 80 120 450 Ala92‐Dio2 animals; both calculated Km(T4) are similar at 1.9nM; (D) food intake during light and 20 110 400 dark cycles in 24 h; (E‐H) digital x‐ray microradiography images of Ala92‐Dio2 femurs and 60 18 vertebrae show no gross differences compared to those from Thr92‐Dio2 mice; (I‐J) femoral bone 100 350 mineral content (BMC) was similar between genotypes and slightly increased in vertebrae from 16 40 90 300 Ala92‐Dio2 mice; (K‐L) femoral lengths and vertebrae heights were also quantified and while 14 20 there was no difference in length, a shorter vertebral height was observed in the Ala92‐Dio2 12 80 250 Tibial max force (N) mice; (M‐V) femoral bone volume and density, cortical thickness, trabecular number, thickness Vertebral max force(N) Tibial stiffness (Nmm-1) and spacing, and tibial and vertebral strength and stiffness showed no differences between the 10 0 70 Vertebral stiffness (Nmm-1) 200 Thr Ala Thr Ala Thr Ala Thr Ala genotypes; values are shown in a box and whiskers plot or mean±SEM; n=6‐10/group; in (J) Kolmogorov‐Smirnov test was used; ** p≤0.01 and ***p<0.001. Supplemental Figure 1 ABCDE Striatum Amygdala Prefrontal Cortex Hippocampus Cerebellum Ala Thr Ala Thr Ala Thr Ala Thr Ala Thr HTR2C NEFM MYCN LUZP1 NGF HR SEMA7A STARD4 IER5 RPE65 NGF LUZP1 PDP1 RPE65 ITGA7 FLYWCH2 PVALB KCNJ10 NEFM LUZP1 GLI1 ITGA7 CNTN2 NEFH FLYWCH2 genes SEMA7A PDP1 RPE65 KCNJ10 CNTN2 CNTN2 IER5 SEMA7A NGF PDP1 NEFM NEFH IER5 FLYWCH2 GLI1 ITGA7 MYCN FLYWCH2 MYCN NEFH LUZP1 HR LUZP1 HTR2C PVALB regulated ‐ RPE65 KCNJ10 NEFM HR HR T3 STARD4 STARD4 NEFH PVALB NEFM KCNJ10 HTR2C HR GLI1 MYCN PDP1 GLI1 ITIH3 STARD4 SEMA7A NEFH CNTN2 NGF PDP1 IER5 ITIH3 NGF ITGA7 SEMA7A ITIH3 Positively IER5 FLYWCH2 GLI1 ITIH3 KCNJ10 MYCN RPE65 HTR2C ITGA7 STARD4 PVALB ITIH3 PVALB CNTN2 HTR2C FDR q‐val=0.072 FDR q‐val= 0.028 FDR q‐val=0.081 FDR q‐val=0.153 FDR q‐val=0.164 NOM p‐val=0.042 NOM p‐val<0.001 NOM p‐val=0.049 NOM p‐val=0.133 NOM p‐val=0.142 COL6A1 SYCE2 SLC1A3 ODF4 ODF4 CXADR MAMDC2 AGBL3 CXADR CXADR genes CIRBP CXADR HMGCS2 HMGCS2 SYCE2 AGBL3 HAPLN1 CIRBP HAPLN1 CIRBP COL6A2 CIRBP MAMDC2 CIRBP MAMDC2 SLC1A3 DGKG HAPLN1 SLC1A3 MARCKSL1 ODF4 AGBL3 PCSK4 AGBL3 SLC1A3 regulated ‐ SYCE2 COL6A2 COL6A1 PCSK4 COL6A1 T3 HMGCS2 PCSK4 DGKG COL6A2 DGKG PCSK4 COL6A1 MARCKSL1 SYCE2 PCSK4 MARCKSL1 ODF4 CXADR MARCKSL1 HMGCS2 MAMDC2 MARCKSL1 SYCE2 DGKG HAPLN1 DGKG SLC1A3 ODF4 MAMDC2 AGBL3 HAPLN1 HMGCS2 COL6A2 COL6A1 COL6A2 Negatively FDR q‐val=0.03 FDR q‐val=0.533 FDR q‐val=0.414 FDR q‐val=0.239 FDR q‐val=0.154 NOM p‐val<0.001 NOM p‐val=0.481 NOM p‐val=0.446 NOM p‐val=0.216 NOM p‐val=0.127 Figure S2: Expression of positively or negatively T3‐regulated genes in 5 regions of the Ala92‐Dio2 and Thr92‐Dio2 brain. (A) Striatum; (B) Amygdala; (C) Prefrontal Cortex; (D) Hippocampus; (E) Cerebellum; upper heat maps feature 19 genes positively regulated by T3; lower heat maps feature 14 genes negatively regulated by T3; expression values are represented as colors (red: increased expression; blue decreased expression) with the degree of color saturation indicating level of expression; False Discovery Rate (FDR) q‐value and Nominal (NOM) p‐value are shown for each heat map; n=4/group. Supplemental Figure 2 Open Field A B 15 C 250 D 50 E 150 F 10 200 40 8 10 100 150 30 * * 6 100 20 4 5 # Rearing 50 # Grooming Immobility (s) Immobility Time Center (s) Time Center # Line Crossing 50 10 2 #Total center entries 0 0 0 0 0 Thr Ala Thr Ala Thr Ala Thr Ala Thr Ala Elevated Plus Maze G 300 H 300 I 30 J 30 K 30 30 * LM150 * ** 200 200 20 ** 20 20 100 20 100 100 10 10 p=0.07 # Rearing 10 10 p=0.08 50 Immobility (s) Immobility # Head-dipping # open arms entries # Closed arms entries Time in open ArmsTime (s) inopen Time in Closed Arms (s) * 0 0 0 0 0 0 0 Thr Ala Thr Ala Thr Ala Thr Ala Thr Ala Thr Ala Thr Ala Tail Suspension N O 300 60 ** 200 p=0.06 40 100 20 Latency (s) Immobility (s) Immobility 0 0 Thr Ala Thr Ala Figure S3: Additional behavioral tests in Thr92‐Dio2 (Thr) and Ala92‐Dio2 (Ala) mice. Open Field: (A) time in center; (B) total center entries; (C) total numbers of line crossing; (D) rearing; (E) immobility; (F) grooming. Elevated plus maze: (G) time spent inside closed arms or (H) open arms; (I) entries into closed arms or (J) open arms; (K) rearing; (L) immobility; (M) number of head‐dips. Tail suspension test: (N) time of immobility; (O) latency to the first immobility episode; values are shown in a box and whiskers plot; n=4‐6/group; Statistical analysis used was Mann‐Whitney U test; * p≤0.05 and ** p≤0.01. Supplemental Figure 3 Novel Object Recognition (Familiarization) ABCD EF 100 100 100 100 T 100 A 80 80 80 80 80 60 60 60 60 60 40 40 40 40 40 20 20 20 20 20 Preference index (%) Preference index(%) 0 0 0 0 0 RLRL RLRL RLRL RLRL RLRL Intact Intact+T3 Intact+4‐PBA Hypothyroid Hypothyroid+LT4 Hypothyroid+LT4+LT3 Figure S4: Familiarization session of NOR memory test displayed as preference index (%) of Thr92‐Dio2 (Thr) and Ala92‐Dio2 (Ala) mice. (A) intact animals; (B) intact + LT3 animals; (C) Intact + 4‐PBA animals; (D) hypothyroid animals; (E) hypothyroid+LT4 animals; (F) hypothyroid+LT4+LT3 animals; in each plot, R is the right object and L is the left object; values are shown in a box and whiskers plot indicating median and quartiles; n=5‐11/group; Statistical analysis used was Mann‐Whitney U test. Supplemental Figure 4 Social Interaction (Familiarization) ABCD EF 100 100 100 100 100 80 80 80 80 80 60 60 60 60 60 40 40 40 40 40 20 20 20 20 20 Preference indexPreference (%) Preference index (%) Preference index(%) 0 0 0 0 0 RLRL RLRL RLRL RLRL RLRL Intact Intact+T3 Intact+4‐PBA Hypothyroid Hypothyroid+LT4 Hypothyroid+LT4+LT3 Figure S5: Familiarization session of SI memory test displayed as preference index (%) of Thr92‐Dio2 (Thr) and Ala92‐Dio2 (Ala) mice. (A) intact animals; (B) intact + LT3 animals; (C) Intact + 4‐PBA animals; (D) hypothyroid animals; (E) hypothyroid+LT4 animals; (F) hypothyroid+LT4+LT3 animals; in each plot, R is the right subject and L is the left subject; values are shown in a box and whiskers plot indicating median and quartiles; n=5‐11/group; Statistical analysis used was Mann‐Whitney U test. Supplemental Figure 5 Table S1- ER stress-related genes mRNA in Thr92-D2HY and Ala92-D2HY cells treated with 100uM 4- PBA during 24h. Genes Thr92-D2HY vs Ala92-D2HY relative mRNA levels BIP 1.09±0.08 CHOP 1.07±0.20 sXBP1 0.99±0.08 ERGIC53 0.70±0.03** ATF4 1.08±0.08 Results are relative to bACTIN mRNA levels and normalized to Thr92-D2HY; **p≤ 0.01 vs. Thr92- D2HY-4PBA cells. values are the mean ± SEM of 4-6 independent samples; gene abbreviations are as indicated in the supplemental Tables S27. Table S2- Genes upregulated in the Amygdala of Ala92-Dio2 mice. (p<0.05) Fold Change ANOVA p- FDR p- Gene (linear) (AA value (AA value (AA Description Symbol Amg vs. TT Amg vs. TT Amg vs.