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3-Iodothyronamine (T1AM) is an endogenous high-affinity ligand of the trace amine-associated receptor 1 (TAAR1), detected in mammals in many organs, including the brain. Recent evidence indicates that pharmacological TAAR1 activation may offer a novel therapeutic option for the treatment of a wide range of neuropsychiatric and metabolic disorders. To assess potential neuroprotection by TAAR1 agonists, in the present work, we initially investigated whether T1AM and its corresponding 3-methylbiaryl-methane analog SG-2 can improve learning and memory when systemically administered to mice at submicromolar doses, and whether these effects are modified under conditions of MAO inhibition by clorgyline. Our results revealed that when i.p. injected to mice, both T1AM and SG-2 produced memory-enhancing and hyperalgesic effects, while increasing ERK1/2 phosphorylation and expression of transcription factor
Trace amine-associated receptors (TAARs) represent a novel class of G protein coupled receptors (GPCRs) identified in 2001 by two independent groups (
During the past two decades, trace amine-associated receptor 1 (TAAR1) has been the focus of extensive research, particularly because, in addition to TAs as principal binding ligands, it is also activated by a number of endogenous and exogenous molecules, including catecholamines, amphetamine and amphetamine-like compounds, ergot derivatives and several adrenergic ligands (
Chemical structures of thyronamines (T0AM, T1AM), their thyroacetic acid catabolites (TA0, TA1) and the corresponding synthetic analogs (SG1, SG2, SG5, and SG6)
Notably, the T1AM skeleton includes a β-phenylethylamine structure, a feature that may confer T1AM the ability of recognizing multiple cell targets including other G protein-coupled receptors, such as adrenergic receptors ADRα2A and ADRβ2 (
Pharmacological administration of T1AM affects reversibly and dose-dependently reversible effects on body temperature, cardiac function, energy metabolism, and neurological functions (
Collectively, these findings indicated that T1AM might represent a valuable tool to investigate the physiological role and pharmacological potentials of TAAR1, as well as a good starting point to advance development of new targets for potential therapeutic interventions in a wide array of pathological processes, including metabolic, endocrine, and neurological disorders. In this context, with the aim to ameliorate the number of selective TAAR1 agonists,
To assess the therapeutic potential of thyronamine derivatives for neuroprotection, in the present paper, we initially investigated whether T1AM and its corresponding 3-methylbiaryl-methane analog SG-2 can improve learning and memory when systemically administered to mice at submicromolar doses, and whether these effects are modified under conditions of MAO inhibition by clorgyline. In addition, several lines of evidence indicate that autophagy (ATG), the process by which cells digest their own cytoplasmic constituents within lysosomes, is key for neuronal plasticity (
Impairments of the autophagic process are associated with several neurodegenerative disorders, such as Alzheimer’s disease (AD), Parkinson’s disease, and Huntington’s disease, where a deficiency in the elimination of defective or aggregated proteins may lead to cellular stress, failure and death. As such, the induction of autophagy may be exploited as a strategy to assist neurons clearing abnormal protein aggregates and thus survive (
Therefore, to explore the potential neuroprotective effects of thyronamines, we also evaluated the ability of T1AM and newly developed analogs SG-1 and SG-2 to promote autophagy in U-87MG cells, while investigating the signaling pathway being involved.
3-Iodothyronamine was kindly provided by Prof. Thomas Scanlan (Portland, OR, United States) and was dissolved in 0.5% DMSO (Veh). SG-1, SG-2, and SG-6 analogs were synthesized by our group according a procedure previously described (
Experiments and animal care used procedures were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23, revised 1996). The experimental protocols were approved by the Animal Care Committee of the Department of Pharmacology, University of Florence, in compliance with the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes (ETS no. 123) and the European Communities Council Directive of 24 November 1986 (86/609/EEC). The authors further attest that all efforts were made to minimize the number of animals used and their suffering.
Male mice (CD1 strain; 20–30 g) from Envigo (Udine, Italy) were used. Five mice were housed per cage and the cages were placed in the experimental room 24 h before the test for adaptation. Animals were kept at 23 ± 1°C with a 12 h light–dark cycle (light on at 07:00 h) and were fed a standard laboratory diet with water
The passive avoidance paradigm test using the light–dark box apparatus was carried out using the step-through method by
Mice were injected i.p. with test compound (i.e., T1AM or SG-2; 1.32, 4, and 11 μg⋅kg-1) or vehicle (
In another set of experiments, additional mice were pretreated with i.p. clorgyline (2.5 mg⋅kg-1) or with the histamine H1 receptor antagonist pyrilamine (10 mg⋅kg-1) and after 30 min they received the test compound (i.e., T1AM or SG-2; 1.32, 4, and 4 μg⋅kg-1) or saline (i.p.). Measurements were performed 15 min after i.p. injections.
Mice were removed from the cage and injected i.p. with vehicle or test compound (i.e., T1AM or SG-2; 1.32, 4, and 11 μg⋅kg-1).
Animals were sacrificed at two different time points, 15 min or 5 h, after test compound or vehicle administration. The brain was excised and the forebrain was split into two sections: frontal cortex and diencephalon. The hippocampus, hypothalamus/thalamus were then quickly removed, and all tissues were flash-frozen in liquid nitrogen and stored at -80°C until use. In another set of experiments, mice were sacrificed 15 min after i.p. injection with vehicle or test compound (i.e., T1AM or SG-2; 1.32, 4, and 11 μg⋅kg-1). DRG were freshly isolated using a previously described procedure (
Brain and DRG samples were homogenized in homogenization buffer containing (in mM): 50 Tris-HCl at pH 7.5, 150 NaCl, 1 EDTA, 5 sodium pyrophosphate, 10 β-glycerophosphate, 1 Na3VO4, 0.2 PMSF, 25 μg⋅mL-1 leupeptin, 10 μg⋅mL-1 aprotinin and 0.1% SDS. To remove cell debris, homogenates were centrifuged at 1000 ×
Proteins (20 μg) isolated from selected brain regions and DRG homogenates were separated on 4–20% SDS-PAGE and transferred into PVDF membranes (60 min at 398 mA) using standard procedures. Membranes were blocked in PBST (PBS containing 0.1% Tween) containing 5% non-fat dry milk for 60 min. Blots were incubated overnight at 4°C with specific antibody against ERK1/2 phosphorylated on Thr202/Tyr204 (pERK1/2, Cell Signaling Technology) or c-fos (Sigma–Aldrich S.r.l, Milan, Italy). Primary antibodies were diluted in PTBS containing 3% albumin. After being washed with PBST, the membranes were incubated with polyclonal goat anti-rabbit HRP-conjugated secondary antisera (1:2,000, diluted in PTBS containing 5% non-fat dry milk) and left for 1 h at room temperature. Blots were then extensively washed and developed using an enhanced chemiluminescence detection system (Pierce Scientific, Rockford, IL, United States). Exposition and developing time were standardized for all blots. Densitometric analysis of scanned images was performed on a Macintosh iMac computer using the public domain NIH Image program. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Sigma–Aldrich S.r.l., Milan, Italy) or β-actin (Sigma–Aldrich, S.r.l., Milan, Italy) were used as loading control. Protein concentration was quantified using Bradford’s method (protein assay kit, Bio-Rad Laboratories, Segrate, Milan, Italy).
Data represent mean ± SEM of three different experiments. Results are expressed as arbitrary units (AU), consisting of the ratio between the expression levels of the protein of interest and that of the GAPDH.
U-87MG human glioma cell lines from Cell Bank (IRCC San Martino-IST, Genova) were cultured in standard DMEM-High Glucose medium (Sigma–Aldrich S.r.l., Milan, Italy) supplemented with 10% fetal bovine serum (FBS), 1% of MEM non-essential amino-acid (MEM-NEAA), penicillin (50 IU/mL) and 100 μg streptomycin (Sigma–Aldrich S.r.l., Milan, Italy). Cells were kept at 37°C in a humidified atmosphere with 5% CO2 and the medium was renewed two to three times per week.
For transmission electron microscopy (TEM) and Western blot (WB) assays, the cells were cultured at a density of 1 × 106 cells/well in a 6-well plate in a final volume of 2 ml/well. For confocal light microscopy (IF) assay 3 × 104 cells were seeded on cover- slips in 24-well plates in a final volume of 1 ml/well. In order to evaluate cytotoxicity (Trypan blue assay) and cell viability (MTT assay), 1 × 104 cells were seeded in 96-well plate in a final volume of 400 ml/well. Twenty-four hours after seeding, the cells were treated with test compounds (T1AM, SG1, and SG2) at the dose of 1 μM for different exposure times (30’, 4, 8, and 24 h). Dilutions of test compounds were obtained by a stock solution (1 mM in saline containing 10% DMSO).
For trypan blue staining, after incubation with the different drugs the cells were collected and centrifuged at 800 rpm for 5 min. The cell pellet was suspended in culture medium and 25 μl of the cellular suspension were added to a solution containing 1% of trypan blue (62.5 μl) and PBS (37.5 μl). The cells were incubated for 5 min at room temperature. Then, a volume of 10 μl of this solution was counted using a Bürker glass chamber and a light microscope. Viable and non-viable cells were recorded and the cell viability was expressed as a percentage of number of viable cells/number of total cells. The values represent the means of three independent cell counts.
Cell viability was measured by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent (
Glioblastoma U-87MG cells were collected using trypsin and centrifuged at 1,000 ×
For ultrastructural morphometry, non-serial sections were examined directly by TEM at 5,000× magnification. At least 10 cells per section were identified in each grid and several grids were examined to achieve a total of at least 30 cells per experimental group. Autophagy-like vacuoles, identified as double or multiple membranes (autophagosomes-like) containing cytoplasmic material and electrondense membranous structures (
Values were calculated using ImageJ software and expressed as density of autophagy-like vacuoles (number of autophagy-like vacuoles per surface unit).
U-87MG cells grown overnight on coverslips were washed twice with phosphate-buffered saline (PBS) and fixed with methanol at room temperature for 5 min. Retrieval of the antigen was achieved in 100 μM Tris-HCl, 5% urea at 95°C for 10 min. After washing with PBS, cells were permeabilized with 0.2% Triton X-100 for 10 min at room temperature, and subsequently blocked in PBST (PBS + 0.1% Tween-20), supplemented with 1% bovine serum albumin (BSA) and 22.52 mg/ml of glycine, for 30 min. The cells were then incubated overnight at 4°C in 1% BSA in PBST containing 1:50 anti-LC3 antibody (Abcam, Cambridge, United Kingdom). After extensive PBST washes, cells were incubated for 1 h at room temperature with a 1:200 dilution of goat anti-rabbit secondary antibody (Alexa 488, Molecular Probes, Life Technologies) in 1% BSA in PBST. Cells were then washed with PBS, mounted in Prolong Diamond Antifade Mountant (Molecular Probes, Life Technologies) and subsequently examined. The analysis was performed using a Leica TCS SP5 confocal laser-scanning microscope (Leica Microsystems, Mannheim, Germany) using a sequential scan procedure. Confocal images were collected every 400 nm intervals through the
1 × 106 U-87MG cells were seeded in 6-well plates in a final volume of 2 ml/well and grown to 80% of confluence with standard medium (DMEM-High Glucose). Cells were treated with vehicle (0.1% DMSO) or 1 μM test compounds (T1AM, SG-1, and SG-2) and incubated at 37°C for 30 min, 4, 8, and 24 h.
Treated cells were washed twice with PBS and lysed in Tris-buffered saline buffer-1% Triton-X100; NaCl 150 mM; Tris-HCl 20 mM; EDTA 1 mM; EGTA 1 mM; NaF 20 mM; Na4P2O7 25 mM; Na3VO4 1 mM; PMSF 1 mM; 8 μl/ml protein cocktail inhibitors (Sigma–Aldrich, Milan, Italy). Proteins (20–30 μg) were separated on Criterion TGXTM gel (4–20%) and transferred on Immuno-PVDF membrane (Bio-Rad, Milan, Italy) for 1 h. Blots were incubated for 12 h with diluted primary antibody [1:1000, LC3A/B; p62; Akt, p-Akt(Ser 473), β-actin, Cell Signaling] in 5% w/v BSA, 1X TBS and 0.1% Tween® 20 at 4°C under gentle shaking. Then, blots were washed three times for 10 min with 1X TBS, 0.1% Tween® 20 and incubated for 1 h with secondary antibody (peroxidase-coupled anti rabbit in 1X TBS, 0.1% Tween® 20). After washing three times for 10 min, the reactive signals were revealed by enhanced ECL Western Blotting analysis system (Amersham). Band densitometric analysis was performed using Image Lab Software (Bio-Rad, Milan, Italy).
Data are reported as the mean ± SEM. Statistical analysis was performed by one-way analysis of variance (ANOVA), followed by Student–Newman–Keuls multiple comparison
3-Iodothyronamine is known to induce pro-learning and anti-amnestic responses when administered i.c.v. at very low doses (1.32–4 μg⋅kg-1) to mice, but its effects on memory after systemic administration are currently unknown. To address this point, we first evaluated the behavior of CD-1 mice injected i.p. with T1AM or its 3-methylbiaryl-methane analog SG-2 (1.32, 4, and 11 μg⋅kg-1) in the passive avoidance test.
As described in the “Materials and Methods” section, retention sessions of the passive avoidance test were performed 1 and 24 h after T1AM or SG-2 injection. In the 1 h retention session, following administration of 4 μg⋅kg-1 T1AM or SG-2 the latency to enter the dark compartment was increased, but the difference versus the control group did not reach the threshold of statistical significance. However, when either of the two compounds was administered at the dose of 11 μg⋅kg-1, a significantly higher latency to enter the dark compartment was observed at 1 h (
SG-2 and T1AM modified learning in mice. Mice were injected i.p. either with SG-2 (1.32, 4, or 11 mg⋅kg-1), T1AM (1.32, 4, or 11 mg⋅kg-1) or with vehicle (Control) and subjected to the passive avoidance test as described in the “Materials and Methods” section. Results are expressed as mean ± SEM;
Since the effect on memory enhancement that was observed in the passive avoidance test following i.p. injection of T1AM or SG-2 may involve an analgesic action, we next checked whether T1AM and SG-2 had any effect on pain threshold. As shown in
SG-2 and T1AM reduce nociceptive threshold in mice and the effect is blunted by clorgyline pretreatment.
Oxidative deamination by amine oxidases, followed by aldehyde oxidation by the widely distributed enzyme aldehyde dehydrogenase, is known to be the main metabolic pathway for T1AM (
In addition, the pharmacokinetics of thyronamine-like synthetic analogs, such as SG-2, has not yet been completely described. Therefore, it seemed interesting to investigate whether the hyperalgesic effect observed after systemic administration of both T1AM and SG-2 was affected by the MAO inhibitor clorgyline, administered i.p. at the dose of 2.5 mg⋅kg-1.
In agreement with previous findings, the results of our pain threshold experiments revealed that the hyperalgesic effect produced by 11 μg⋅kg-1 T1AM or SG-2 was lost with clorgyline pretreatment (
Recent studies showed that i.c.v. injection in mice of equimolar doses of TA1 reproduced the pro-learning effects induced by T1AM. Notably, TA1 as well as T1AM pro-learning effects were modulated by histaminergic antagonists (
As shown in
Pretreatment with H1 receptor antagonist pyrilamine abolishes SG-2 or T1AM-induced hyperalgesia. The nociceptive threshold of mice pre-treated (s.c.) with the H1 receptor antagonist pyrilamine (10 mg kg-1) was determined by the hot plate test 15 min after SG-2 or T1AM i.p. injection (11 μg kg-1) (
In addition, i.p. injection in mice of equimolar doses of SG-6 (
SG-6 reduces nociceptive threshold in mice and the effect is blunted by pyrilamine pretreatment. Mice were injected i.p. with SG-6 (1.32, 4, or 11 μg⋅kg-1) or with vehicle (Control) and after 15 min their nociceptive threshold measured by the hot plate test. Experiments were repeated in mice pre-treated (s.c.) with the H1 receptor antagonist pyrilamine (10 mg kg-1) and exposed to the hot plate test 15 min after SG-6 i.p. injection (4 or 11 μg kg-1). Results are expressed as means ± SEM;
Notably, pyrilamine
Increased ERK1/2 phosphorylation and increased expression of transcription factor
T1AM and SG-2 induce p-ERK in CD-1 mice brain regions. Fifteen minutes after i.p. injection of either T1AM, SG-2 (4, 11 mg⋅kg-1) or vehicle (Control), mice (
T1AM and SG-2 induce
Spinal extracellular signal-regulated kinases (ERKs) have long been known as mediators of nociceptive plasticity (
T1AM and SG-2 induce p-ERK1/2 in dorsal root ganglia (DRG) from CD1 mice: the effect of pyrilamine pretreatment. DRG were isolated from mice randomly treated with saline (Control), T1AM (4, 11 μg kg-1) or SG-2 (4, 11 μg kg-1) with or without being exposed to pretreatment with pyrilamine (10 mg kg-1) and analyzed for p-ERK1/2 levels by Western blot as described in Methods.
Autophagy is a complex cellular lysosome-mediated process that eliminates misfolded proteins, protein complexes, or organelles through lysosomial degradation, and it is required for cellular homeostasis in cell survival, with neuronal cells representing one of the most studied systems (
With the aim to explore the role played by T1AM and recently developed thyronamine-analogs SG-1 and SG-2 in the autophagy process, we examined the formation of autophagosomes and the autophagic flux in human glioblastoma cell lines (U-87MG). U-87MG cells are commonly used as a model to study neurological diseases, characterized by an up-regulation of mTOR (
We then proceeded to elucidate which pathway was involved in ATG modulation by assessing our test compounds on the PI3K/AKT/mTOR signaling pathway (
As shown in representative pictures reported in
Transmission Electron Microscopy (TEM) shows time-dependent induction of autophagy in U-87MG cells by 1 μM SG-1, SG-2, or T1AM.
As shown in
Confocal microscopy confirms induction of autophagy in U-87MG cells treated with 1 μM SG-1, SG-2, or T1AM.
As shown in
T1AM, SG-1, or SG-2 increase LC3II/LC3I ratio in U-87MG cells.
Since ultrastructural studies (TEM and IF) and Western blot analysis have shown that T1AM and SG-1 are more effective than SG-2 at inducing autophagy in U-87MG cells, we next check the ability of T1AM and SG-1 to modulate the autophagic flux by examining their effect on p62 expression in U-87MG cells after 24 h incubation time. Consistently, a significant reduction of p62 expression was observed in U-87MG cells after treatment for 24 h with 1 μM T1AM and SG-1 (∗∗
Reduced p62 expression in U-87MG cells.
The PI3K/AKT/mTOR signaling pathway is an important regulator of autophagy, with mTOR playing a key modulatory role (
T1AM, SG-1 and SG-2 reduce the phosphorylation levels of Akt-Ser473 in U-87MG cells.
In addition, cellular viability was determined using the MTT colorimetric assay. No significant alterations of cell viability were observed in U-87MG cells treated for 24 h with 1 μM TAAR1 agonists (i.e., T1AM, SG-1, and SG-2) as compared to vehicle (0.1% DMSO) (
Cell viability assay. MTT assay showing U-87MG cells viability after 24 h treatment with tested compounds. Results are expressed as the mean ± SEM of 12 biological replicates.
Trace amine-associated receptor’s family (TAARs) represents a large class of receptors recently discovered that has become increasingly popular in Medicinal Chemistry as the focus of studies aimed at the discovery of new drugs (
Among TAAR1 ligands, the endogenous thyroid hormone (TH) derivative T1AM was firstly identified by Scanlan in rodent brain (
It is well known that thyroid hormone is fundamental for normal brain development and maintenance of optimal cognitive ability in different periods of life (
Several studies have explored the consequences of administering T1AM directly into the brain. Recently,
The study we report here demonstrated that T1AM behaves as a memory enhancer even when injected systemically into mice at doses comparable to those previously used in central administration experiments by
Recently, the thyronamine-like TAAR1 agonist SG-2, has been shown to produce a good mimic of T1AM functional effects in rodents (
Taken together, our results provide robust evidence that synthetic thyronamine-like analog SG-2 shares with T1AM the effectiveness on memory and pain, which seems to involve a common mechanism of action. Namely, both T1AM and SG-2 seem to rely on the action of ubiquitous enzymes MAO to produce the corresponding oxidative metabolites that are then able to activate the histaminergic system. Although our knowledge on the pharmacokinetic properties of SG-2 is still at a preliminary level, the oxidative deamination of SG-2 to generate the corresponding acid SG-6 has been observed
Alzheimer’s disease, accompanied by deterioration in memory and other cognitive functions, is the most frequent neurodegenerative disorder. Recent studies indicate that T1AM counteracts the effects of Aβ on LTP and restores recognition memory in a mouse model of AD (mhAPP mouse), but the underlying mechanisms are currently unknown (
Our data displayed that T1AM and thyronamine-like TAAR1 agonists SG-1 and SG-2 have neuroprotective properties, which also involve the induction of autophagy. This novel aspect requires further investigation. Future
Notably, neurodegenerative diseases are multifactorial debilitating disorders involving multiple pathways that, in addition to protein misfolding and aggregation, are characterized by several metabolic changes, such as mitochondrial dysfunction, oxidative stress, and phosphorylation impairment, all occurring concurrently (
GC, RZ, FF, and LRa designed and directed the project. LB, AL, MS, PL, AS, FB, and LRo carried out the experiments and analyzed the data. SS and SR prepared all the SG-compounds tested in the study. SR also contributed to design the project. All authors discussed the results and contributed to the final manuscript. GC wrote the manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The authors thank the COST action CA15135 (Multitarget Paradigm for Innovative Ligand Identification in the Drug Discovery Process MuTaLig) for support. They also thank Marco Tonelli for technical assistance, comments, and suggestions.