3-iodothyronamine (T1AM) is an endogenous high-affinity ligand for the trace amine-associated receptor 1 (TAAR1) found circulating in mammals and accumulating in many tissues including the brain. Its functional effects are now known to include decrease in heart rate, cardiac output, body temperature, and metabolic rate, as well as stimulation of lipid vs carbohydrate catabolism and neurological effects, including prolearning and antiamnestic action, reduced pain threshold, and reduction of non-REM sleep. The close structural similarity with thyroid hormone (TH) induced to speculate that T1AM might be synthetized from TH through decarboxylation and deiodination, but this hypothesis still requires validation. Notably, in addition to the structural similarities between T1AM and TH, T1AM contains the same arylethylamine scaffold also found in monoamine neurotransmitters, implicating an intriguing role for T1AM as both a neuromodulator and a hormone-like molecule constituting a part of thyroid hormone action. Recent evidence indicates that targeting TAAR1 may provide a new therapeutic approach for the treatment of a range of neuropsychiatric and metabolic disorders. Consistently, T1AM and recently developed halogen free biaryl-methane thyronamine-like TAAR1 agonists, SG-1 and SG-2, have emerged as rapid modulators of behavior and metabolism, with SG-2 showing a potency almost comparable to that of T1AM. To assess the therapeutic potential of TAAR1 agonists for neuroprotection, in the present thesis we investigated whether T1AM and its corresponding halogen free synthetic analogue SG-2 improve learning and memory when systemically administered to mice at submicromolar doses. In addition, since autophagy is key for neuronal plasticity, T1AM and newly developed thyronamine-like TAAR1 agonists SG-1 and SG-2 were evaluated as autophagy inducers in cell lines. T1AM is known to induce pro-learning and anti-amnestic responses when administered icv at very low doses (1.32 to 4 μg.kg-1) to mice, but its effects on memory after systemic administration are currently unknown. To address this point, we evaluated the behavior of CD-1 mice injected i.p. with T1AM or SG-2 (1.32, 4 and 11 μg.kg-1), in the passive avoidance test, while measuring in specific brain areas the activation of typical signalling proteins involved in memory acquisition, including pERK and transcription factor c-fos. The passive avoidance test showed that when administered i.p. at 11 μg.kg-1 either T1AM or SG-2 induced significant memory enhancement. T1AM also significantly increased retention when administered at a lower dosage (4 μg.kg-1). Memory acquisition and storage are typically associated with increased ERK1/2 phosphorylation. After treatment with T1AM and SG-2 at doses of 1.32 μg·kg-1, pERK could be detected, but its levels were not significantly different from those found in vehicle injected mice. However, after treatment with T1AM and SG-2 at doses that proved to be effective on memory acquisition and retention (i.e. 4 and 11 μg·kg-1) pERK turned out to be significantly higher (p<0.01) than that observed in the vehicle group. Similar to pERK, c-fos was not significantly changed in any brain area by treatment with 1.32 μg·kg-1 of T1AM or SG-2. For both compounds, only when injected at the highest dose (i.e. 11μg·kg-1) moderate changes of c-fos expression were observed. At the dosages active on memory, T1AM and SG-2 also proved to have an hyperalgesic effect. In addition, our pain threshold experiments revealed that TA1 and SG-6, the oxidative metabolites of T1AM and SG-2, respectively, seem to play a critical role for the action of the corresponding amine. In fact, at our settings, pretreatment with clorgyline abolished almost completely the effect on pain of T1AM or SG-2 administered i.p. at the highest dosage (i.e., 11 μg/kg). The same effect was also shown to involve the histaminergic system, as it disappeared after pretreatment with the histamine H1-receptor antagonist, pyrilamine. Preliminary results from a LC/MS-MS pharmacokinetic study of TA1 bio-distribution after sistemic administration, indicated that there is a difference in the capacity of clearing the acid by different tissues, with the brain conserving TA1 levels for longer than plasma or liver. Therefore, the brain might represent one important target for TA1 action. Autophagy (ATG) is one of the most important mechanisms of neuroprotection. Inducing autophagy may represent a reasonable strategy to develop therapeutics for the treatment of neurodegenerative diseases, including Alzheimer's disease. Therefore, to explore the possible link between TAAR1 activation and neuroprotection, we investigated the ability of T1AM, SG-1 and SG-2 to induce autophagy in human glioblastoma cell lines (U-87MG) by examining the formation of autophagosomes and autophagic flux. During the process of autophagy, LC3I is converted to its lipidated form LC3II, which is one of the hallmarks of autophagy and essential for the formation of autophagosome. P62 is used as a marker of autophagic flux and inversely correlates with autophagic activity. It binds directly to LC3 and degrades during autophagy. In cultured U-87MG cells exposed to 1μM T1AM, SG-1, SG-2 (for 0.5, 4, 8 and 24h) transmission electron microscopy (TEM) and immunofluorescence (IF) showed a significant (p<0.01) time-dependent increase of autophagy vacuoles and microtubule-associated protein 1 light chain 3 (LC3), with T1AM and SG-1 being the most effective agents. Consistently, western blotting results showed that test compounds increased the conversion of LC3-I to LC3-II while decreasing the levels of P62 in an obvious time-dependent manner. We then proceeded to elucidate which pathway was involved in ATG induction by assessing the effect of our test compounds on the PI3K–AKT–mTOR pathway. We found that 1μM T1AM, SG-1 and SG-2 decreased pAKT/AKT ratio at 0.5 and 4h after treatment (p<0.01), suggesting that these compounds induce autophagy through a reduction of pAkt level. Taking into account the key role played by ATG and/or Ubiquitine-proteasome (UP) in all clearing pathways modulating cell survival and disease mechanisms, and considering their involvement in a wide range of disorders, including neurodegenerative diseases (NDDs), we were also able to prove the ability of T1AM, SG-1 and SG2 to modulate the UP system. Indeed, our TEM analysis showed increased 20S proteasome recruitment to autophagosome in U-87MG cells after treatment with 1μM T1AM, SG-1 and SG-2, suggesting that these compounds might modulate both ATG and UP protein clearing pathways within the autophagoproteasomes. A growing body of evidence has suggested the presence of a strong correlation between obesity and neurodegeneration. NDDs, such as Alzheimer Disease (AD) and Parkinson Disease (PD), are characterized by a progressive loss of memory and cognition, which can ultimately lead to death. This deterioration is majorly a result of inflammation due to aberrant protein deposition, oxidative stress and modification in lipid pathways. Recent studies demonstrated acute and chronic T1AM treatment to have potent effects on shifting whole body macro-nutrient metabolism in mammals. On the basis of these findings, we decided to explore whether T1AM, SG-2 and recently designed synthetic thyronamine/thyroid hormone-hybrid analogs IS25 and TG46 could promote lipolysis in HepG2 cells. Oil Red O staining of HepG2 revealed that all test compounds reduced total lipid accumulation into lipid droplets compared to the control. Moreover, the analysis of free glycerol release confirmed that decreased accumulation of lipids in HepG2 cells observed after Oil Red O staining was caused by increased lipolysis. The involvement of AMPK/ACC modulation was also confirmed by western blot analysis that showed an enhancement of both pAMPK/AMPK and pACC/ACC ratios. AMPK stimulation by test compounds led indeed to the phosphorylation and consequent inactivation of acetyl coenzyme A carboxylase (ACC), the major regulator of fatty acids synthesis. In conclusion, taken together our data displayed that T1AM and thyronamine-like compounds SG-1 and SG-2 have neuroprotective properties, which also involve the induction of autophagy and the regulation of Ubiquitine-Proteasome System. Indirectly, together with newly designed thyronamine/thyroid hormone- hybrid analogs, they could exert a protective action through lipolytic effects. This novel aspect requires further investigation.
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|Titolo:||Biochemical and functional effects of trace amine-associated receptor 1 (TAAR1) agonists in the central nervous system|
|Citazione:||Bellusci, L. (2019). Biochemical and functional effects of trace amine-associated receptor 1 (TAAR1) agonists in the central nervous system.|
|Appare nelle tipologie:||8.1 Tesi Dottorato|