• adult-onset hypothyroidism
• neurocognitive alterations
• 3-iodothyronamine

Hypothyroidism is a common endocrine disorder. Symptoms include mood alterations, such as anxiety and depression, and impairment in neurocognition, that point to an underlying disruption of hippocampal function. Guidelines from international professional societies recommend Levothyroxine (L-T4) monotherapy as the treatment of choice to cure hypothyroidism, under the assumption that L-T4 is transformed to the active hormone triiodothyronine (T3) in target organs. Although L-T4 monotherapy is effective in most cases, there is a percentage of patients that keep showing hypothyroidism-related dysfunctions, even though euthyroidism is biochemically restored. 3-iodothyronamine (T1AM) is an active thyroid hormone metabolite showing pro-learning and anti-amnestic effects. Here, we aimed at investigating whether combining L-T4 with L-T3 or T1AM may have beneficial effects in the treatment of neurocognitive and associated neurobiological alterations in a mouse model of adult-onset hypothyroidism.
Hypothyroidism was induced in (n=80) six-week old C57BL/6J male mice by Methimazole (0.2 mg/g/die) and Potassium Perchlorate (0.3 mg/g/die) administration in drinking water for 49 days, while controls (n=17) received plain water. At day 21, mice were implanted with subcutaneous ALZET® osmotic pumps delivering replacement treatments for 28 days. The experimental groups were 6, as follows: (1) hypothyroid; (2) L-T4; (3) L-T4+L-T3; (4) L-T4+T1AM; (5) T1AM; (6) euthyroid. Mice were tested with behavioural assays to evaluate hippocampus-dependent memory (i.e., novel object recognition test), locomotion and anxiety (i.e., open field test and elevated plus maze test), and depression (i.e., tail suspension test). We analysed mRNA isolated from the hippocampi by quantitative real-time PCR analysis with the mouse PrimePCRTM collection panel (BIO-RAD), a predesigned 96-well panel of gene targets involved in neurogenesis pathway for use with SYBR® Green. To assess hippocampal neurogenesis in the subgranular zone (SGZ) of the dentate gyrus, we performed immunofluorescence for specific markers, such as Ki67 for cell proliferation and doublecortin (DCX) for new-born neuroblasts and immature neurons.
L-T4 serum concentrations, determined by high performance liquid chromatography coupled with tandem mass spectroscopy (HPLC-MS/MS), confirmed the validity of our model. At the behavioural level, hypothyroid mice showed significant impairment in hippocampus-dependent memory as compared to euthyroid mice. L-T4 monotherapy substantially improved the discrimination index. However, we observed the average best performances in mice treated with L-T4+T1AM. T1AM did not induce any effect per se. Hippocampus-dependent memory remained impaired in mice treated with L-T4+ L-T3. These findings were influenced neither by locomotor activity nor by anxiety- and depression-related behaviours, which remained unchanged. The real-time PCR analysis revealed consistent significant changes in the expression of genes related to neurogenesis. The ANOVA analysis revealed an upregulation in the expression of Dll1 in L-T4 + T1AM treated mice compared to hypothyroid mice, and an upregulation in the expression of Mapk1 and Mapk3 in L-T4+T1AM treated mice compared to L-T4 treated mice. Experiments of immunofluorescence preliminarily showed enhanced neurogenesis at the level of the SGZ of the hippocampus in mice treated with L-T4+T1AM and L-T4 +L-T3 relative to the other groups.
In our pharmacological mouse model of adult-onset hypothyroidism, L-T4+T1AM combination had beneficial effects on hypothyroidism-related neurocognitive and neurobiological alterations. Future studies should elucidate whether L-T4+T1AM combination may prevent the neurodegenerative consequences of hypothyroidism.