Anticonvulsant and neuroprotective effects of the thyroid hormone metabolite 3-iodothyroacetic acid (TA1)
Annunziatina Laurino*1PhD, Elisa Landucci*2 PhD, Francesco Resta1 PhD, Gaetano De Siena2 PhD, Domenico E. Pellegrini-Giampietro2MD, Alessio Masi1,3 PhD, Guido Mannaioni1MD, and Laura Raimondi1 PhD
*These authors contributed equally.
1Department of Neurology, Psychology, Drug Sciences, and Child Health; Pharmacology Division, University of Florence, 50139 Florence, Italy
2Department of Health Sciences, Division of Clinical Pharmacology and Oncology, University of Florence, 50139 Florence, Italy
3School of Pharmacy, University of Camerino. Via Madonna delle Carceri 9, 62032 Camerino.
Running Head: TA1 and neuroprotection Corresponding Author:
Laura Raimondi
Department of Neurology, Psycology, Drug Sciences and Child Health; NEUROFARBA, Pharmacology Division
University of Florence Viale G. Pieraccini, 6 50139 Florence, Italy Tel: +390554271210 Fax: +390554271280 e-mail: laura.raimondi@unifi.it 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 3
Abstract Background
3-iodothyroacetic acid (TA1) is among thyroid hormone (T3) end metabolites acutely modifying mice behavior. We aimed to investigate whether TA1 was also able to reduce neuron hyper-excitability and protect from excitotoxic damage.
Methods
CD1 male mice were treated i.p. with saline solution or TA1 (4, 7, 11, or 33 g kg‧ -1) before receiving s.c. 90 mg kg‧ -1 pentylenetrazole(PTZ). The following parameters were measured: latency to first seizure onset, number of mice experiencing seizures, hippocampal levels of c-fos and IP3/AKT activation levels.
Organotypic Hippocampal slices were exposed to vehicle or to 5 M Kainic acid, (KA) in the absence or in the presence of 0.01-10 M TA. In another set of experiments, slices were exposed to vehicle or 5 M KA in the absence or in the presence of 10 M T3, 3,5,3’triiodothyroacetic acid (TRIAC), T1AM, thyronamine (T0AM) or thyroacetic acid (TA0). Neuronal cell death was measured fluorimetically.
The ability of TA1 and T3, TRIAC, T1AM, T0A, TA0 to activate the PI3K/AKT cascade was evaluated by Western blot.
The effect of TA1 on KA-induced currents in CA3 neurons was evaluated by patch clamp recordings on acute hippocampal slices.
Results
TA1 (7 and 11 g kg‧ -1) significantly reduced the number of mice showing convulsions and increased their latency of onset, restored PTZ-induced reduction of hippocampal c-fos levels, activated the PI3K/AKT and reduced GSK-3activity. In rat organotypic hippocampal slices, TA1 reduced KA-induced cell death by activating the PI3K/AKT cascade and increasing GSK-3phosphorylation levels. Protection against KA toxicity was also exerted by T3, and other T3 metabolites studied. TA1 did not interact at KA receptors. Both the anticonvulsant and neuroprotective effects of TA1 were abolished by 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53
pretreating mice or organotypic hippocampal slices with pyrilamine (PYR), an histamine type 1 receptor antagonist (10 mg/kg or 1 M respectively).
Conclusions
TA1 exerts anticonvulsant activity and is neuroprotective in vivo and in vitro. These findings extend the current knowledge on the pharmacological profile of TA1 and indicate possible novel clinical use for this T3 metabolite.
Keywords
3-iodothyroacetic acid; thyroid hormone metabolism; seizures; neuroprotection; histamine
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Introduction
Thyroid hormone (T3) critically regulates the development of the central nervous system but also neuronal excitability and plasticity after birth activating transcriptional and not-transcriptional mechanisms (1-3), including the pro-survival PI3K/AKT cascade (4). However, recent pharmacological evidence suggests that T3 metabolism does not terminate the effects of T3 on the central nervous system. In fact, some T3 metabolites, including iodothyronamine (T1AM) and its oxidative metabolite, the 3-iodothyroacetic acid (TA1), were described recently as having their own neuronal signaling capacity being able to modify certain behaviors when administered to rodents. In this respect, TA1 and T1AM acutely induce pro-learning and anti-amnesic effects (5, 6). Moreover, Doyle et al (2007) reported that high T1AM doses had neuroprotective action in a model of ischemia-reperfusion by reducing body temperature (7). In previous works, we reported that T1AM and TA1 behavioral effects depended on the activation of the histaminergic system (8, 9).
Aberrant neuronal discharge, secondary to increased brain levels of excitatory mediators, is among the causes of convulsions and a trigger for excitotoxic-induced cell death. From the therapeutic point of view, different classes of anticonvulsant drugs are available but it remains to be determined whether these drugs are effective in protecting neurons against excitotoxicity (10).
Considering that TA1 could potentially represent an active metabolite of T3 and of T1AM, we sought to investigate whether TA1 was also endowed of neuroprotective activity in respect of drug-induced convulsions and of excitotoxic neuron death. This aim was explored in the mouse treated with a single s.c. dose of pentylenetetrazole (PTZ), a selective antagonist at GABA-A receptor, a treatment which leads to neuronal hyper-excitability (11) and in organotypic hippocampal slices exposed to Kainic acid (KA), a well know model to study drug-induced neuroprotection (14).
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In the PTZ model we investigated whether TA1 treatment i) protected mice from drug-induced convulsions ii) activated signaling in mouse hippocampus including the PI3K/AKT cascade and conserved c-fos levels. In organotypic hippocampal slices, we asked whether TA1 reduced KA-induced neuronal death, if it activated survival mechanisms including the PI3K/AKT cascade and if TA1 effects were shared by other iodinated and not iodinated T3 metabolites, including T1AM. on excitotoxicity.
To investigate the involvement of the histamine system, the effect of TA1 was also studied in mice pre-treated with pyrilamine (PYR), a histamine type 1 receptor (H1R) antagonist, before receiving PTZ, and in organotypic hippocampal slices exposed to PYR before TA1 and before KA challenge.
Materials & Methods Animals
Male CD1 mice (weight: 20–30 g) purchased from ENVIGO (Italy) were used in the present study. Five mice were housed per cage. Cages were placed in the experimental room 24 h prior to testing to ensure adaptation. Animals were housed at 23±1°C under a 12 h light–dark cycle (lights on at 07:00) and were fed a standard laboratory diet with ad libitum access to water. Experiments and animal use 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 ethical Committee of the Italian Council of Health, 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.
Organotypic hippocampal slice cultures were prepared from male and female Wistar rat pups (7-9 days old) obtained from Charles River (Italy). Acute hippocampal slices were prepared from Wistar rats (15-28 days old), which were also obtained from Charles River. In vivo model 5 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 19
Male CD1 mice received an intraperitoneal (i.p.) injection of vehicle (saline solution) or TA1 (4, 7, 11, and 33 g kg‧ -1) and after 10 min 90 mg kg‧ -1PTZ s.c. Mice were then individually housed in transparent Plexiglas cages (25 × 15 × 10 cm) and observed for 30 min. The number of convulsing mice and the latency to first clonic seizure occurrence were recorded. For the purposes of this experiment, clonic seizure activity in mice was defined as clonus of the whole body lasting over 3 s, accompanied by loss of the righting reflex (16). At the end of the observation period, animals were then housed in cages, fed a standard laboratory diet with ad libitum water, and sacrificed 24 h later. The hippocampi of mice were removed and stored at -80° until Westernblot analysis. Additional mice were then treated i.p. with 7 gkg-1 TA1 or saline (Veh) and sacrificed 24 h later to remove the hippocampi for Western blot analysis.
In another set of experiments, PTZ (90 mgkg-1) was administered s.c. to mice that had received either vehicle or the H1R antagonist pyrilamine (PYR; 10 mgkg-1 i.p. ; Sigma-Aldrich SRL, Gallarate (MI), Italy) 20 min before the i.p. injection of vehicle or 7 gkg -1 TA The number of mice showing convulsions and the latency to first clonic seizure occurrence were recorded as previously described.
TA1 stock solution (10 mM) was prepared in 100%DMSO and then appropriate dilutions were made in saline solution (15).
Preparation of rat organotypic hippocampal slice cultures
Organotypic hippocampal cultures were prepared as previously described (13, 15). Briefly, hippocampi were removed from the brains of Wistar rat pups, transverse slices (420 μm) were prepared using a McIlwain tissue chopper and transferred onto 30-mm diameter semi-porous membrane inserts (Millicell-CM PICM03050; Millipore, Italy), which were placed in six-well tissue culture plates containing 1.2 ml of medium per well. The slice culture medium consisted of 50% Eagle's minimal essential medium, 25% heat-inactivated horse serum, 25% Hanks' balanced salt solution, 5 mg ml‧ -1 glucose, 2 mM L-glutamine, and 3.75 mg ml‧ -1 amphotericin B. Slices were incubated at 37 °C in an atmosphere of humidified air and 5% CO2 for two weeks. Prior to experimentation, all slices were screened for viability by incubating them for 30 min with the fluorescent dye propidium iodide (PI, 5 g ml‧ -1; Sigma-Aldrich SRL, Gallarate, Italy. Slices displaying signs of neurodegeneration were excluded from the study. 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152
Slices were exposed to vehicle or 10 M TA1 for 24 h. At the end of the incubation period, slices were collected for Western blot analysis to evaluate levels of AKT, p-GSK-3, p-mTOR, and phosho-p70S6K. In another set of experiments, slices were exposed for 24 h to 10 M T3, T1AM, 3,3’,5-triiodothyroacetic acid (TRIAC), thyronamine (T0AM), thyroacetic acid (TA0). At the end of the incubation period, slices were collected for Western blot analysis to evaluate levels of p-AKT.
Cell toxicity evaluation
Organotypic hippocampal slices are considered appropriate for in vitro analysis of the neuroprotective effects of drugs. A specific neuronal toxicity in the CA3 region is achieved by exposing slices to 5 M Kainic Acid (KA) for 24 h (Tocris, Bristol, United Kingdom) (14). This model was utilized for different experimental conditions, as described in the following section.
Slices were exposed to vehicle or increasing concentrations of TA1 (0.01, 0.1, 1 and 10) in the absence or presence of 5 M KA or vehicle. In another set of experiments, slices were exposed to 10 M T3, T1AM, TRIAC, T0AM, TA0 in the absence or presence of 5 M KA or vehicle.
Cell toxicity was evaluated 24 h later by measuring PI fluorescence (the fluorescence measured in KA-exposed slices in the CA3 region was reported as 100%).
Cell death in the presence of pharmacological modulation of PI3K/AKT and GSK-3 activities
Slices were preincubated for 5 min with vehicle or 10 M of LY294002 (a PI3K inhibitor; LY, Tocris, Bristol, United Kingdom), or 10 nM of the GSK-3β inhibitor TC-G (10 nM, Tocris, Bristol, United Kingdom) before adding TA1 (10 M) or vehicle. All slices were then exposed to 5 M KA and cell death evaluated 24 h later by measuring PI fluorescence
Cell death in the presence of pyrilamine
Slices were pre-incubated for 5 min with vehicle or 1 μM PYR, followed by TA1 (10 M). All slices were then exposed to 5 M KA. Cell death was assessed 24 h later by evaluating PI fluorescence. 7 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 27
Fluorescence was detected 30 min after the addition of PI using an inverted fluorescence microscope (Olympus IX-50; Solent Scientific, Segensworth, UK) equipped with a xenon-arc lamp, a low-power objective (4×), and a rhodamine filter. Images obtained using a CCD camera (Diagnostic Instruments Inc., Sterling Heights, MI, USA) were digitized using the accompanying software (InCyt Im1™; Intracellular Imaging Inc., Cincinnati, OH, USA) and subsequently analyzed using Image-Pro Plus morphometric analysis software (Media Cybernetics, Silver Spring, MD, USA). To quantify cell death, the CA3 hippocampal subfield was identified and encompassed in a frame using the drawing function in ImageJ (NIH, Bethesda, USA), and PI fluorescence was then evaluated in arbitrary units (AU). Morphological analysis revealed a linear correlation between CA3 PI fluorescence and the number of injured CA3 pyramidal cells.
Preparation of acute rat hippocampal slices and whole-cell recordings
Brains were removed from Wistar rat pups and mounted in the slicing chamber of a vibroslicer (Leica VT 1000S). Horizontal slices (250 µm in thickness) were cut from the hippocampi in chilled artificial cerebral spinal fluid (aCSF), composed of (in mM) NaCl, 130; KCl, 3.5; NaH2PO4, 1.25; NaHCO3 25; glucose, 10; CaCl2, 2; and MgSO4, 1; and saturated with a 95% O2 + 5% CO2 gas mixture. Hippocampal slices were allowed to recover in the same solution maintained at 34 °C with constant oxygenation for 1 h prior to the experiments. Slices were continuously perfused with the aCSF solution using a gravity-fed perfusion system. Whole-cell pipettes were filled with the following (in mM): K+ gluconate, 120; KCl, 15; HEPES, 10; EGTA, 5; MgCl2, 2; Na2phosphocreatine, 5; Na2GTP, 0.3; and MgATP, 2; resulting in a bath resistance of 3– 5 MΩ.
Voltage-clamp experiments were performed using a PC-505B amplifier (Warner, Handen, CT, USA), and the results were digitized using Digidata 1440 A and Clampex 10 (Axon, Sunnyvale, CA, USA). Pipettes were pulled from borosilicate capillaries (Harvard Apparatus, London, UK) using a Narishige PP830 vertical puller (Narishige International Ltd, London, UK). Pipette capacitance transients were cancelled and no whole-cell compensation was utilized. Cells were maintained at -60 mV, signals were sampled at 10 kHz and low-pass filtered at 1 kHz. All recordings were performed at 21-23 °C. 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213
KA (5 µM) was puff-applied for 10 s using a picospritzer (16). The combination of KA concentration and puff duration generated a non-desensitizing inward current. The KA puff was applied twice to exclude desensitization, then, 10 µM TA1 was bath perfused for 5 min. At the end of TA1 application, KA was puff-applied once again.
Peak amplitude (pA) and peak area (nA*s) were calculated using Clampfit software. Western blot analysis
Organotypic hippocampal slices treated as previously described were washed with cold 0.01 M phosphate-buffered saline (pH 7.4), blotted dry, and stored at -80°C until used in Western blot analysis.
Samples were gently transferred and dissolved in a lysis buffer tube containing 50 mM Tris HCl (pH 8), 150 mM NaCl, 1 mM EDTA, 0.1 % w/v SDS, and a protease and phosphatase inhibitor cocktail (Thermo Scientific, Monza, Italy). Total protein levels were quantified using the Pierce Protein Assay (Rockford, IL, USA)/BCA (bicinchoninic acid).
Proteins (20 μg) isolated from hippocampal slices or from mouse hippocampi were separated via 4-20% SDS-PAGE and transferred into PVDF membranes (60 min at 398 mA) using standard procedures. Blots were incubated overnight at 4 °C with specific antibodies against p-AKT S473 (AB_329825), p-GSK3β S9 (AB_2115201), p-mTOR S2448 (AB_330970), phosho-p70S6K T389 (AB_330944) (Cell Signalling Technology, Denver, MA, USA), c-fos (AB_259739) (Sigma-Aldrich SRL, Gallarate (MI), Italy), and -tubuline (AB_310035) (Merk-millipore, Darmstadt, Germany). Primary antibodies were diluted in PBS containing 1% albumin or 5% non-fat dry milk and 0.05% Tween. The antigen–antibody complexes were visualized using appropriate secondary antibodies (1:10 000, diluted in PBS containing 1% albumin or 5% non-fat dry milk and 0.05% Tween) and incubated for 1 h at room temperature. Blots were then extensively washed with PBS containing 0.1% Tween and developed using an enhanced chemiluminescence detection system (Pierce, Rodano, Italy). 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. Results are presented as the mean ± SEM of different gels and expressed as arbitrary units (AU), which depict the ratio between levels of target protein expression and -tubuline normalized to basal levels.
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Statistical analysis
Statistical analyses consisted of one- or two-way analyses of variance (ANOVA) followed by post hoc tests (Bonferroni test and Tukey test for two-way Anova, and One-was Anova test respectively) or unpaired t-test. When the experimental setting included only two groups, unpaired t-tests were used. Analyses of seizure occurrence were performed using χ2 tests with Yates correction. The threshold of statistical significance was set at P < 0.05. Data analysis was performed using GraphPad Prism 5.0 (GraphPad software, San Diego, CA, USA). Results are expressed as the mean ± SEM.
Results
TA1 protects mice from PTZ-induced convulsions
The potential efficacy of TA1 in reducing neuron hyper-excitability was examined in a model of drug-induced convulsions (i.e. single administration of 90 mgkg-1 PTZ). Our results indicated that 90 mgkg-1 PTZ s.c. induced convulsions in 100% of mice with a mean latency to first seizure of 548 ± 38.82 s. The χ2 test with Yates correction of results indicated that the treatment with 7 and 11 gkg-1 TA1 significantly reduced the number of mice experiencing convulsions (P=0.0183 and P= 0.0047 vs. vehicle, respectively, Fig. 1, panel A). Furthermore, the ANOVA test and the post hoc analysis indicated that 7 and 11 gkg-1 TA1 significantly increased (P<0.01 and P<0.05 vs. PTZ, respectively) the latency to first seizure (Fig. 1, panel B) with respect to PTZ plus vehicle treated mice.
TA1 activates hyppocampal PI3K/AKT pathway and increases the phosphorylation levels of GSK-3
We than evaluated whether TA1 was able to activate the PI3K/AKT cascade. To this aim, mice treated with 7 gkg-1 TA1 i.p. or with vehicle were sacrificed 24 h later to collect hippocampi for Western blotting analysis.
Our results show that, in mice treated with 7 gkg-1 TA1, hippocampal levels of p-AKT, p-GSK-3P=0.0491 and P=0.0157, respectively, unpaired t-test) but also of p-mTOR (P=0.0012, unpaired t-test; Fig. 2, panels A and B), were significantly higher than 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274
vehicle-treated mice. However, no changes in the phosphorylation levels of p70S6K, the downstream effector of the mTORC1 complex, were observed (data not shown).
TA1 restores hippocampal c-fos levels reduced by PTZ
It was reported that PTZ reduced hippocampal levels of c-fos (12, 13), an early gene product which is part of the pro-survival PI3K/AKT cascade.
Accordingly, we measured c-fos levels in hippocampi of mice treated i.p. with vehicle, PTZ or with PTZ plus 7 gkg-1 TA1 (PTZ+TA1). One-way ANOVA confirmed that PTZ treatment significantly reduced hippocampal c-fos levels compared to vehicle-injected mice (P<0.001)). Interestingly, c-fos levels in the hippocampus of mice that had received PTZ + TA1 were similar to levels measured in vehicle-treated mice (P<0.05) (Fig. 3, panels A and B).
TA1 does not induce cytotoxicity in organotypic hippocampal slices
Organotypic hippocampal slices are currently considered a model to study excitotoxic neuronal death (14, 17) and the associated signaling. Organotypic hippocampal slices exposed to vehicle or increasing concentrations of TA1 (0.01, 0.1, 1, and 10 M) for 24 h exhibited similar low levels of PI fluorescence, indicating that TA1 did not induce cell toxicity at any of the tested concentrations.
KA-induced toxicity in the CA3 region: the effect of TA1, T3 and other T3 metabolites
To mimic excitotoxicity, slices were exposed to vehicle or to 5 M KA for 24 h. Under such conditions, selective cell death induced by KA in the CA3 region (PI fluorescence in CA3: 100%) was noted. The exposure of slices to TA (0.01, 0.1, 1, and 10 M) and then to 5 M KA showed a lower cell death in the CA3 region than slices treated only with KA (5 M). The extent of cell death reduction became significant in slices exposed to 10 TA1 (P< 0.001; Fig. 4, panels A and B).
To verify whether this neuroprotective activity was shared also by other T3 metabolites with different iodide content, we exposed slices to 10 M T3, T1AM, TRIAC, T0AM. To verify whether neuroprotective activity was conserved by other T3 metabolites with different iodide content, we exposed slices to 10 M T3, T1AM, tri-iodothyroacetic acid (TRIAC), thyronamine (T0AM) and thyroacetic acid (TA0).
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Results indicated that all the compounds tested reduced KA-induced neuron cell death in the CA3 region (Fig. 4, panel C), thus suggesting that neuroprotection is likely a common feature of T3 metabolites.
TA1 activates the PI3/AKT cascade in hippocampal slices
We then examined whether TA1 was able to activate the PI3K/AKT and to increase GSK-3 phosphorylation also in organotypic hippocampal slices. Toward this aim, we exposed slices to 10 M TA1, a concentration found active in reducing KA-induced cell death, and we measured the phosphorylation levels of these kinases. Our results indicated that the exposure of slices to 10 µM TA1 for 24 h associated with a significant increase in p-AKT and p-GSK-3β levels relative to that observed in vehicle-treated slices (P=0.0016, P=0.0098 for p-AKT and p-GSK-3β, respectively, unpaired t-test; Fig. 5, panels A and B). As in the mice model, the activation of this cascade was associated with increased levels of p-mTOR but not of phosho-p70S6K (P< 0.0001 vs. vehicle unpaired t-test ; Fig. 5, panels A and B; data not shown).
Other T3 metabolites activate AKT
We then examined whether the other T3 metabolites studied activated the AKT pathway. Our results demonstrated that pAKT levels were higher in slices exposed to 10 M T3, TRIAC, T1AM, T0AM and TA0 for 24 h, than in slices exposed to vehicle (Fig. 6, panels A and B).
TA1-induced reduction of neuronal death depends on PI3K/AKT and on GSK-3β activity
We investigated whether activation of PI3K/AKT and inhibition of GSK-3 were involved in TA1-induced reduction of KA-induced cell death. To this aim, slices were pre-treated with vehicle or 10 M LY294002 or 10 nM TG-C, inhibitors of AKT and of GSK-3 respectively, before adding TA1 (10 M) and KA (5 M).
Our results indicated that: i)10 M LY294002 did not modify KA-induced neuronal toxicity (100%) but prevented the neuroprotective effects of 10 M TA, ii) 10 nM TC-G significantly reduced KA toxicity to 72.67 ± 4.2% (P<0.05; Fig. 7 panels A and B) iii), in the presence of 10 nM TC-G and of 10 M TA1, KA-induced cell death was reduced further (35.24 ± 9.62% ; Fig. 7, panels C and D).
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Anticonvulsant and neuroprotective effects of TA1 are prevented by pyrilamine
To investigate on histamine involvement,7 gkg-1 TA1or vehicle were administered to mice pre-treated with PYR (10 mgkg-1 i.p.)or vehicle(i.p.) and then they all received PTZ (90 mgkg-1 ) as described in “Methods”. As expected, in mice pre-treated with vehicle, 7 gkg-1 TA1 significantly increased seizure latency (P<0.001; vs. PTZ) and reduced the percentage of mice presenting convulsions (χ2 with Yates correction; P=0.0362 vs. vehicle; Fig. 8, panels A and B). It is worth of noting that PYR treatment abolished the protection offered by TA1 either on the number of convulsing mice or on the latency of first seizure onset.
The effect of PYR (1 M) on TA1-induced protection against KA-induced cell death was then evaluated in organotypic hippocampal slices. The ANOVA test of results indicated there was a significant effect of pre-treatment PYR (P<0.001), of treatment TA1(10 M; P<0.001), and a significant interaction of between pre-tretament and treatment (PYR +TA1; P<0.05) in reducing KA toxicity. Post-hoc test showed that PYR itself exerted no effect on KA-induced toxicity, but its presence completely prevented the neuroprotective effects of TA1 (P>0.05 vs. vehicle). Consistently, the levels of PI fluorescence in PYR+TA1+KA slices were not significantly different from those observed in slices exposed to KA alone (100%; Fig. 8, panels C and D).
TA1 does not interfere with KA-induced inward currents in acute brain slices
The effects of TA1 on ionotropic glutamate receptors were investigated by measuring KA-mediated currents in CA3 pyramidal neurons following treatment with TA1. Our results indicate that TA1 did not significantly modify the peak amplitude of KA-induced inward currents or peak area (Fig. 9 panels A and B).
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Discussion
Our findings indicate that TA1, a by-product of thyroid hormone metabolism, protects against PTZ-induced neuron hyper-reactivity, acting as an anticonvulsant, and activates pathways involved in protect neurons from excitotoxic insults. The anticonvulsant and neuroprotective effects of TA1 were prevented by pre-treating mice with PYR, and were independent of TA1 modulation on KA receptors. Interestingly, T3 and TETRAC, T1AM, T0AM and TA0 shared with TA1 the capacity to protect hippocampal neuron from excitotoxicity.
Neuron hyper-excitability was induced in mice by administering PTZ, a selective inhibitor of the GABAergic transmission (11). As a result, mice receiving PTZ were prone to seizures. Dong et al. (12) reported that PTZ-induced hyper-excitability can produce neurotoxic effects since this treatment associated with reduction of c-fos hippocampal levels. In fact, c-fos is an early gene product which controls the expression of neuroprotective factors including the BDNF. This latter peptide is, in turn, a target of AKT and GSK-3 pathways (18).
Under our experimental conditions, PTZ induced convulsions in 100% subjects. This number was significantly reduced when mice received 7 and 11 gkg-1 TA1. In addition, 7 and 11 gkg-1 TA1 also increased the latency to first seizure. The anticonvulsant effect of TA1 showed an inverted U-shaped dose-effect relationship, thus suggesting the a rapid desensitization (19). In agreement with previous findings, we now confirm that treatment with PTZ associated with the reduction of c-fos hippocampal levels. Interestingly, the reduction of c-fos expression was prevented by an effective anticonvulsant dose of TA1. Overall, TA1 acutely counteracts the effects of PTZ on neuronal hyper-excitability and c-fos transcription levels thus suggesting a neuroprotective potential of the acid. The data obtained in organotypic hippocampal slices confirmed TA1 is indeed neuroprotective from KA-induced cell death.
In fact, our results from organotypic hippocampal slices indicated that TA1 reduced KA-induced cell death in the CA3 region, the region which is known to contains histaminergic neurons (20). Our results also indicate that this kind of neuroprotection is also afforded by T3, and other T3 metabolites, although it remains undetermined whether or not these metabolites have anticonvulsant activity. Of note, effective TA1 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389
concentrations in vitro are higher than those effective in vivo. This is due to the well-established poor diffusion of drugs across organotypic slice thickness (14, 17, 21). In this respect, TA1 shows a trend to protection at 0.1M achieving statistical significance at 10M.
We explored the mechanism of TA1protection and found that the acid has the intrinsic capacity to activate a molecular “protective ground” which buffers the impact of possible negative insults (hyper-excitability or excitotoxicity) on neuron survival. In fact, we demonstrated that TA1 treatment of mice increased levels of pAKT and of GSK-3(kinase inhibition) in their hippocampus. This signaling pathway cascade was also found activated in organotypic hippocampal slices exposed to TA1 for 24 h. Furthermore, from in vitro studies, we can conclude that inhibition of AKT and of GSK-3 phosphorylation are necessary for TA1-induced neuroprotection, as TA1 neuroprotection was lost in slices pre-treated with LY294002, an inhibitor of PI3K. Consistently, the protective effect of TG-C, an inhibitor of GSK-3β (25), was further increased in the presence of TA1.
The PI3/AKT/GSK-3 cascade is reported as a fingerprint of the non-genomic neuroprotective effects of T3 (2-4). In line with this evidence, we demonstrated that T3 and other T3 metabolites activated AKT in organotypic hippocampal slices. Furthermore, like T3, TA1 signaling activity at hippocampus includes the activation of mTOR but, unlike T3, not that of phosho-p70S6K, the downstream effector of mTORC1 (4). Although the significance of mTOR activation by TA1 remains largely unknown, the absence of the phosho-p70S6K might indicate the involvement of TORC2 rather than TORC1 complexes. Overall, our evidence indicates that TA1-triggered signaling is maintained after its anticonvulsant action has decayed.
Overall, based on our in vivo and in vitro findings, we can state that TA1, directly or indirectly, not only activates pathways “preparing” neurons to fight against excitotoxicity but also that histamine is part of the neuroprotective program orchestrated by TA1. In fact, the dose-specific anticonvulsant and neuroprotective effect of TA1 against PTZ in vivo and KA in vitro were prevented by the H1 antagonist PYR. We chose PYR based on past experience. In fact, although it shows some affinity for cholinergic as well as non-H1 histamine receptors, PYR is one among few H1R antagonists showing central bioavailability following systemic administration. We 15 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 59
exclude non-histaminergic effects of PYR since we have already demonstrated that at the dose of 10 mgkg-1, PYR does not induce sedation, analgesia or amnesia in mice (8, 9,19). Furthermore we have previously demonstrated TA1 does not interact with muscarinic receptors (22).
Conclusions TA1 administration to mice exerts an immediate effect in reducing neuronal hyper-excitability (anticonvulsant effect) and initiates a protective, pro-survival, signaling cascade which is maintained for 24 h after administration. Such activation also occurs in hippocampal preparations and it is the mechanism, likely shared by other T3 metabolites, on which TA1 reduces neuron death by excitotoxicity. Our findings get further inside the pharmacological profile of TA1, and propose this acid as a novel anticonvulsant treatment which also attenuates the detrimental effects of excitotoxicity on neuron survival
Acknowledgments
This work was supported by a grant from University of Florence and from Ente Cassa di Risparmio di Firenze (2017)
Disclosure
The authors declare no conflict of interest. Corresponding author Laura Raimondi 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441
Figure Legends Fig. 1
3-iodothyroacetic acid (TA1) decreases the percentage of convulsant mice and increases seizure latency
Mice pre-treated with TA1 (4, 7, 11, and 33 g kg‧ -1, i.p. ) or vehicle (Veh.) were treated 10 min later with pentylenetetrazole (90 mg kg‧ -1; PTZ). Mice were observed for 30 min and the number of mice presenting convulsions and the latency to first clonic seizure occurrence were recorded.
Panel A: the percentage of mice showing convulsions over the total of mice treated was reported ( *P<0.05 and **P<0.01 vs. vehicle respectively; χ2 test with Yates correction). The number inside the bars indicate the number of mice showing convulsions in respect of the total number of mice treated.
Panel B: Post-hoc analysis (One-Way ANOVA test followed by Dunnet’s post hoc test) indicated that 7 and 11 g kg‧ -1 TA1 significantly increased seizure latency (*P<0.05 and **P<0.01 respectively vs. Veh;. n≥18 for each treatment).
Fig. 2
3-iodothyroacetic acid (TA1) activates the PI3K/AKT and increases phosphorylation levels of the GSK-3 in mouse hippocampus
Male CD1 mice received an intraperitoneal injection of either vehicle (Veh) or 7 g kg‧ -1 TA1. Hippocampi were isolated from mice sacrificed 24 h following treatments. Levels of p-AKT, p-GSK-3β, and p-mTOR were assessed via Western blotting.
Panel A: Levels of p-mTOR, p-AKT, p-GSK-3β in hippocampal protein lysates. A representative experiment is showed.
Panel B: Densitometric analysis of p-AKT, p-GSK-3β, p-mTOR levels in the hippocampus of CD1 mice. Results are presented as the mean ± SEM of two different gels (n=4 for each treatment); *P<0.05 and **P<0.01 vs. Veh. (Unpaired t test)
Fig. 3 17 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 67
3-iodothyroacetic acid (TA1) restores hippocampal c-fos levels reduced by penthylentetrazole (PTZ)
Hippocampi were isolated from mice treated 24 h before with vehicle (Veh; n=3), PTZ (90 mg kg‧ -1; n=3), or PTZ plus 7 g kg‧ -1TA1 (PTZ +TA1; n=3). c-fos levels of expression were analyzed via Western blotting. c-fos levels were significantly reduced by PTZ treatment.
Results from a representative experiment (Panel A) and densitometric analysis of results are showed as mean ± SEM of two different gels (n=4 for each treatment); ***P<0.001 vs. Veh; One-Way ANOVA test followed by Bonferroni’s test; Panel B). Fig. 4
3-iodothyroacetic acid (TA1) and other T3 metabolites reduce kainic acid (KA)-induced neuron death
Organotypic hippocampal slices were exposed to vehicle (Veh) or to 5 M kainic acid (KA) for 24 h in the absence (Veh) or in the presence of 0.01, 0.1, 1, or 10 M TA1. Cell death was evaluated by measuring propidium iodide (PI) levels of fluorescence 24 h after treatment.
Fluorescence levels measured in hippocampal slices exposed to KA were associated with neuronal death in the CA3 region. Drug effect was reported as a percentage of KA toxicity value (regarded as 100%) .
Panel A: Representative experiments are showed.
Panel B: Densitometric evaluation of PI fluorescence in slices exposed to KA (regarded as 100%) in the absence (Veh.) or in the presence of 0.01, 0.1, 1, or 10 M TA1; ***P<0.001 vs. Veh. One-Way ANOVA test followed by Tukey’s post hoc test (n≥6 for each condition).
Panel C: Densitometric evaluation of PI fluorescence in slices exposed to KA (regarded as 100%) in the absence (Veh) or in the presence of 10 M T3, TRIAC, T1AM, T0AM or T0A; *P<0.005 vs. Veh. (One-Way ANOVA test followed by Tukey’s post hoc test) (n≥12 for each condition).
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Fig. 5 3-iodothyroacetic acid (TA1) activates the IP3/AKT cascade in organotypic hippocampal slices
Slices were exposed to Veh or to 10 M TA1 for 24 h, following which levels of p-AKT, p-GSK-3β,and p-mTOR were evaluated via Western blot analysis.
Panel A: A representative experiment is showed.
Panel B: Densitometric analysis of p-AKT, p-GSK-3β, and p-mTOR levels in hippocampal slices. Results are presented as the mean ± SEM of three different gels (n=3 for each treatment); ***P< 0.001 and **P<0.01 vs. Veh. Unpaired t test
Fig. 6
Other T3 metabolites increase p-AKT hippocampal levels
Slices were exposed to Veh or to 10 M T3, TRIAC, T1AM, T0AM or T0A. for 24 h, levels of p-AKT, were evaluated via Western blot analysis 24 h after.
Panel A : A representative experiment is showed
Panel B: Densitometric analysis of p-AKT levels in hippocampal slices. Results are presented as the mean ± SEM of three different gels (n=3 for each treatment); ***P< 0.001, **P<0.01, *P<0.05 vs. Veh.
Fig. 7 3-iodothyroacetic acid (TA1)-induced reduction of neuronal death depends on the activation of PI3K/AKT and GSK-3β
Panel A: Cultured slices were exposed to vehicle (Veh.), or 10 M LY294002 (LY), or 10 nM TC-G. 10 M TA1 was then added followed by 5 M kainic acid (KA). Cell death was evaluated 24 h later by measuring PI fluorescence. The fluorescence measured in KA-exposed slices was reported as 100%. Fluorescence distribution in a representative experiment is showed.
Panel B: Densitometric analysis of PI fluorescence is reported. ***P<0.0001 and *p<0,05 vs. Veh; °P<0.05 vs. TC-G; ## P<0.01 vs. TA1 (Two-Way ANOVA test followed by Bonferroni post hoc test;. n≥7 for each condition).
Fig. 8 19 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 75
Anticonvulsant and neuroprotective effects of 3-iodothyroacetic acid (TA1) are prevented by pyrilamine (PYR).
Mice were pre-treated with 10 mg kg‧ -1 PYR, after which they received vehicle (Veh) or 7 g kg‧ -1 TA1 prior to treatment with pentylenetetrazole (90 mg kg‧ -1; PTZ).
Panel A: The latency to first seizure onset were evaluated. ***P<0.001 vs. Veh. (Two-Way ANOVA test followed by Bonferroni’s post hoc test).
Panel B the number of mice presenting convulsions is showed *P<0.05 vs. Veh. χ2 test with Yates correction
Cultured slices were exposed to 1 M PYR or to vehicle (Veh), then to 10 M TA1 prior to receive 5 M kainic acid (KA). Cell toxicity was evaluated for 24 h after by measuring propidium iodide (PI; 5 μg ml‧ -1) fluorescence.
Panel C: A representative experiment is shown.
Panel D: Densitometric analysis of PI fluorescence levels are reported as a percentage of KA toxicity (regarded as 100%). *P<0.05 and *** P<0.01 vs. Veh (Two-way ANOVA test followed by Bonferroni’s post hoc test, n≥12 for each treatment).
Fig. 9
3-iodothyroacetic acid (TA1) does not interfere with kainic acid (KA)-induced inward currents in acute brain slices
KA-induced currents were evaluated via voltage clamping of neurons from the CA3 region of acute hippocampal slices. KA (5 µM) was puff-applied for 10 s using a picospritzer.
Panel A: KA-mediated currents before and after the application of 10 M TA1 for 5 min in a representative experiment.
Panel B: Bar graphs showing the average peak amplitude and area of KA-induced inward currents before and after the application of TA1 10 µM for 5 min n=5 for each condition). 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549
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