3-iodothyronamine (T1AM) effects on glutamatergic postsynaptic signaling pathway

Thyroid hormones (TH), namely thyroxine (T4) and 3,5,3’-triiodothyronine (T3), are crucial regulators of multiple growth processes and control systems of energy metabolism. T4 and T3 undergo a complex metabolism in vivo, by several enzymes encompassing deiodinases, amine transferases, amine oxidases, decarboxylases and several classes of conjugating enzymes, particularly sulfotransferases and UDP-glucuronosyltransferases. T4 or T3 metabolites can produce significant functional effects when administered via interaction either with Thyroid Hormone Receptor (TR), or with other receptors. They are considered as chemical messengers further enriching TH signaling, and have become known as “novel thyroid hormones” or “active thyroid hormones metabolites”. These novel hormones include: T2; Thyronamines (TAMs), mostly 3-iodothyronamine (T1AM) and non-iodinated thyronamine (T0AM); thyroacetic acids, mostly 3,5,3’,5’-thyroacetic acid (TA4), 3,5,3’-thyroacetic acid (TA3), and 3-thyroacetic acid (TA1). Recently, it emerged that 3-iodothyronamine (T1AM), a derivative of decarboxylation and deiodination of thyroid hormones, has pro-learning and anti-amnestic effects, modulates pain threshold, sleep pattern and food intake. It also counteracts beta-amyloid toxicity in mice. Glutamatergic neurotransmission, the major excitatory system in the brain, plays a key role in regulating neuroplasticity, learning and memory, and it is often compromised in neurological disorders. T1AM reduced availability might results in some disorders associated with thyroid hormones. T1AM binds to the trace amine-associated receptor 1 (TAAR1) a G-protein coupled receptor with a putative role in neurotransmission. In the present work, firstly we characterized the gene expression profile of two different brain cell lines and then we evaluated the effects of T1AM on the expression of proteins involved in the glutamatergic postsynaptic pathway. A hybrid line of cancer cells of mouse neuroblastoma and rat glioma (NG 108-15) and a human glioblastoma cell line (U-87 MG) were used. We first characterized the in vitro model by analyzing gene expression of several proteins involved in the glutamatergic postsynaptic cascade by real time PCR (RT-PCR), and cellular uptake and metabolism of T1AM by HPLC coupled to mass spectrometry (HPLC MS-MS). The cell lines were then treated with T1AM, ranging from 0.1 to 10 μM, alone or in combination with 10 µM resveratrol (RSV) and/or 10 µM amyloid β peptide (25-35). Cell viability, glucose consumption, protein expression, cAMP production and calcium concentration in cell lysates were assessed. Our results indicated that both cell lines expressed receptors implicated in glutamatergic pathway, namely AMPA, NMDA and EphB2, but only U-87 MG cells expressed TAAR1 and they took up T1AM which was catabolized to TA1 and might be used as biochemical model to study its post synaptic signaling cascade. At micromolar concentration T1AM had a slightly but significant cytotoxic effect, that is completely blunted if incubated with RSV and it was able to induce different post-translational modification in neuronal cell lines. T1AM reduced glucose consumption and decreased intracellular calcium concentration in NG 108-15 cell line, while increased cAMP concentration, albeit at different doses. At pharmacological concentrations, the major effect highlighted in both cell lines was an increase in the phosphorylation of proteins involved in the glutamatergic postsynaptic signaling. In the NG 108-15 cells an increase in phosphorylation of ERK extracellular signal-regulated kinases (ERKs) (pERK/ total ERK) and CaMKII Ca-calmodulin-dependent protein kinase (CaMK) II (pCaMKII/total CaMKII). In U-87 MG cells, T1AM induced the phosphorylation of the transcriptional factor cAMP response element-binding protein (CREB) and increase the expression of cFOS. Expression or post-translational modifications of other proteins were not affected. We then extend investigation on the effects of 3-iodothyroacetic acid TA1, a catabolite of T1AM and of thyroid hormone, on brain cell lines focusing on the glutamatergic postsynaptic pathway that we explored by infusion with T1AM, assuming that TA1 may either strengthen T1AM effects or exert parallel actions, especially in brain tissue. First, we assessed uptake and metabolism of TA1. Cell lines were treated with TA1 for 24h, at concentration ranging from 0.1 to 10 μM. Uptake, cell viability, cAMP production and protein expression were assessed. TA1 was taken up by cells, even though only a slight reduction in medium concentration was recorded upon 24h of incubation. Cell viability was significantly increased by TA1 10 µM in U-87 MG cell line, while NG 108-15 cells were unaffected. Western blot analysis indicated that, upon infusion of pharmacological doses of TA1, neither the expression of Sirtuin 1, (p=NS) nor the post-translational modifications of ERK (pERK/total ERK, p=NS) were affected in U-87 MG. Instead TA1 induced the phosphorylation of the transcriptional factor cAMP response element-binding protein (CREB) (pCREB/total). In NG 108-15 cell line, preliminary analysis on protein expression and post-translational modification after TA1 infusion, indicated that no modifications of ERK (pERK/total ERK) were occurred. In conclusion our results indicated that NG 108-15 and U-87 MG cells express receptors implicated in the glutamatergic system and, at pharmacological concentrations, T1AM can affect glutamatergic signaling. Therefore, our preliminary results suggest that, in our experimental models, TA1 does not seem to mimic T1AM effects.