Tetracyclic Truncated Analogue of the Marine Toxin Gambierol

Subscriber access provided by University of Newcastle, Australia

Article

Tetracyclic truncated analog of the marine toxin gambierol modifies NMDA, tau and amyloid # expression in mice brains: implications in AD pathology. Eva Alonso, Andrés C. Vieira, Ines Rodriguez, Rebeca Alvariño, Sandra Gegunde, Haruhiko Fuwa, Yuto Suga, Makoto Sasaki, Amparo Alfonso, José Manuel Cifuentes, and Luis M. Botana ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00012 • Publication Date (Web): 26 Jan 2017 Downloaded from http://pubs.acs.org on January 27, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Chemical Neuroscience is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 29

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 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 54 55 56 57 58 59 60

ACS Chemical Neuroscience

Tetracyclic truncated analog of the marine toxin gambierol modifies NMDA, tau and amyloid β expression in mice brains: implications in AD pathology. Eva Alonso1, Andrés C Vieira1, Inés Rodriguez1, Rebeca Alvariño1, Sandra Gegunde1, Haruhiko Fuwa2, Yuto Suga2, Makoto Sasaki2, Amparo Alfonso1, José Manuel Cifuentes1 and Luis M Botana1* 1

Departamento de Farmacología, Facultad de Veterinaria, Universidad de Santiago de Compostela, Lugo,

Spain. 2Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai 9808577, Japan. Correspondence Author: Luis M Botana, Dpto de Farmacología, Facultad de Veterinaria, Universidad de Santiago de Compostela, 27002 Lugo, Spain. e-mail: [email protected], Phone/Fax: +34982822233.

ABSTRACT

Gambierol and its two, tetra and heptacyclic, analogs have been previously proved as promising molecules for the modulation of Alzheimer´s disease hallmarks in primary cortical neurons of 3xTg-AD fetuses. In this work, the effect of the tetracyclic analog of gambierol was tested in vivo in 3xTg-AD mice (10 months old) after 1 month of weekly treatment with 50 µg/kg. Adverse effects were not reported throughout the whole treatment period and no pathological signs were observed for the analyzed organs. The compound was found in brain samples after intraperitoneal injection. The tetracyclic analog of gambierol elicited a decrease of amyloid β1-42 levels and a dose-dependent inhibition of βsecretaseenzyme-1 activity. Moreover, this compound also reduced the phosphorylation of tau at the 181 and 159/163 residues with an increase of the inactive isoform of the glycogen synthase kinase-3β. In accordance with our in vitro neuronal model, this compound produced a reduction in the N2A subunit of the N-methyl-D-aspartate receptor. The combined effect of this compound on Amyloid β1-42 and tau phosphorylation represents a multitarget therapeutic approach for Alzheimer´s disease, which might be more effective for this multifactorial and complex neurodegenerative disease than the current treatments.

KEYWORDS:

Gambierol,

Gambierdiscus,

Alzheimer,

NMDA

ACS Paragon Plus Environment

receptor,

tau,

β-amyloid


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 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 54 55 56 57 58 59 60

1.

INTRODUCTION

Alzheimer´s disease (AD) is the most common dementia worldwide, particularly in developed countries (1). The pathological characteristics of AD include loss of neurons and synapses, deposition of amyloid β (Aβ) aggregates as plaques, and accumulation of hyperphosphorylated tau proteins to form neurofibrillar tangles in the cerebral cortex. Significant efforts have been made to develop effective therapeutics for this complex, devastating disease, focusing on the two main pathological hallmarks of AD, Aβ aggregation and tau hyperphosphorylation. However, no success has been achieved when these targets are pursued independently (2, 3).

Gambierol is a marine polycyclic ether originally identified from cultured cells of the ciguatera causative dinoflagellate Gambierdiscus toxicus (4). This natural product has been proved to have beneficial effect over AD pathology in an in vitro model obtained from 3xTg-AD mice (5). Although the biological activity of this natural product was initially elusive because of its scarce availability, chemical synthesis realized practical material supply and accelerated detailed biological investigations (6, 7). Its biological mode-of-action has been extensively studied in recent years, prompted by interest in its characteristic neurological symptoms similar to the observed ones in ciguatera intoxication. It has been proposed that this family of ladder-shaped polyether toxins targets transmembrane proteins within the lipid bilayer, as suggested for yessotoxin(8). Ciguatoxins are mainly strong and well known modulators of voltage gated sodium channels (Nav), although some effect in voltage gated potassium channels (Kv) channels has been reported (9-11). In contrast, gambierol does not block or affect Nav channels in the nanomolar range but, instead, it inhibits Kv in several cellular models (12-15) at nanomolar concentrations. In fact, Kopljar et al described that gambierol anchors the gating machinery of Kv in its resting state requiring stronger depolarizations for the channel opening, depolarizations above the physiological range (14). Among the wide Kv family, gambierol shows higher affinity for Kv3 subtypes than for example Kv2 or 4, with an IC50 value of only 1.2 nM for Kv3.1 (13). Kv3 subunits are widely expressed in the central nervous system including hippocampal pyramidal neurons and Purkinje cells where Kv3.1 and 3.2 have fast-spiking properties that allow the fast repolarization of the membrane and minimize the refractory period facilitating a high frequency firing (16). Recently, it has also been described that these compounds inhibited Kv1.3 currents in a low nanomolar range in human T lymphocytes (17). Investigations into the structure-activity relationship of gambierol have recently identified the minimal structure required for potent Kv inhibitory activity (5, 18). Contrary to what was previously thought, only the right wing domain of the polycyclic ether skeleton is necessary for the biological activity and not the whole structure. Specifically, the tetracyclic analog of gambierol inhibited Kv channels with nanomolar potency, largely maintaining the potent activity of the parent natural product. Moreover, gambierol and the tetracyclic analog induced significant reductions in the extra- and intracellular Aβ accumulation and in tau hyperphosphorylation in an in vitro model of 3xTg-AD mice. These findings open the possibility of designing potent analogs with improved characteristics at the cellular or biological level, such as reduced cellular toxicity or superior pharmacokinetic property to enhance its permeability through biological


Page 2 of 29

Page 3 of 29

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 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 54 55 56 57 58 59 60


barriers. In the present work, we take advantage of the previously synthesized tetracyclic analog to address the question whether, as we showed in vitro, this compound has beneficial effects over Alzheimer´s disease pathology in vivo.

2.

RESULTS AND DISCUSSION.

2.1 Histopathological effects of tetracyclic analog of gambierol.

To evaluate the possible damage produced by the chronic administration of gambierol tetracyclic analog in mice, we collected different organs to perform inmunohistological analyses. No damage was observed in neither of the samples analyzed (brain, heart, kidneys, liver, lungs, penis, small intestine and spleen). These results agree with the fact that no clinical symptoms were recorded through the whole treatment and, in contrast, point out that the tetracyclic analog did not cause damage in the organs targeted by its parent compound gambierol, at least in the concentration tested (19).

2.2 Tetracyclic analog-treatment decreases Aβ accumulation in vivo

We have previously demonstrated that gambierol and its hepta- and tetracyclic analogs decrease the levels of intracellular Aβ levels observed by confocal microscopy and Western blot assays and also the levels of extracellular Aβ expressed in a cellular model obtained from 3xTg-AD fetuses (5).

First, we examined the administration routes to assure that the tetracyclic analog reachs the central nervous system. Mice were administered with the deuterated tetracyclic analog (intraperitoneal) or tetracyclic (oral), and the presence of the compounds in brain, liver and fluids was analyzed by UPLC methods. A group of mice was administered simultaneously by both routes to guarantee that the technique efficiently differ the deuterated and non-deuterated compound. As can be observed in Table 3 and Fig 2, only the deuterated compound administered intraperitoneally was detected in liver samples at 15 min and in brain samples at 15 min and 1 h, pointing out that this compound is able to cross the brain blood barrier. The compound administered orally was not detected in any sample and any of the compounds was detected in fluid samples. Therefore, we choose the intraperitoneal route to check the in vivo effect of the tetracyclic in 3xTg-AD mice.

Aβ deposits are first detected at 6 months of age in hippocampus of 3xTg-AD mice and are strongly evident at 12 months showing an age-related appearance (20). Therefore, to verify the previously observed beneficial effect in vivo, mice brains were collected after tetracyclic ip administrations for 1-42 Aβ estimation by a sandwich ELISA kit. Hippocampus and cortex from each brain were dissected and lysed as described above in SDS or formic acid (FA) to obtain soluble and insoluble Aβ measurements. Aβ levels were markedly higher in hippocampal samples in both conditions, soluble and insoluble, than in cortex samples. Treatment with the tetracyclic analog reduced 1-42 Aβ levels in hippocampus in SDS and FA samples in greater than 80%, but in contrast, this reduction was not significant in cortex samples



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 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 54 55 56 57 58 59 60

where Aβ levels were maintained (Fig 3A and B). Immunohistochemical staining also confirmed significant differences between treated and control animals (Fig 3C). NonTg treated animals did not show any change in the levels of 1-42 Aβ, and the small amounts detected were constant in SDS and FA fractions in cortex and hippocampus with values in the range of 4-10 pg/mg of brain (data not shown).

Aβ production can be affected by the processing of amyloid precursor protein by several proteolytic enzymes, including β-secretase enzyme-1 (BACE1). 3xTg-AD mouse model harbor PS1M146V, APPSwe and tauP301L transgenes. The Alzheimer´s disease Swedish mutation (APPSwe) present in this model produces an abnormal BACE cleavage with the consequent overproduction of 1-42 Aβ. Thus, we studied with a fluorescence resonance energy transfer assay if the tetracyclic analog and the parent compound gambierol regulate Aβ levels by a direct interaction with the enzyme. As can be seen in Fig3d, both compounds display a dose dependent inhibition of BACE1 activity, falling to a 52.25 ± 9.17 % with gambierol and to a 58.04 ± 4.2 % with the tetracyclic analog (p < 0.05) at 10 µM.

2.3 Tetracyclic analog-treatment reduced Tau phosphorylation in vivo.

One of the main hallmarks of AD is characterized by tangle formation due to pathological changes in tau conformation and phosphorylation leading to the development of neurofibrilar tangles (21). To evaluate the effect of the tetracyclic analog on tau phosphorylation, Western blot and immunohistochemical analysis were performed using different antibodies that recognize several tau phosphorylated residues significantly altered in AD and widely used for this purpose. As with the above Aβ analysis, hippocampal and cortex samples were evaluated separately. In Fig4A, the effect of the compound treatment on the residues recognized by AT8, AT100, AT270 and HT7 antibodies in hippocampal samples is summarized. The tetracyclic analog did not affect the basal level of total tau proteins recognized by Tau 5 antibody (Fig4A) and also no effect was observed for the 199/202 and 212/216 residues recognized by AT8 and AT100 antibodies, respectively. In contrast, this compound produced a significant decrease of phosphorylation at the 181 and 159/163 residues, as detected by AT270 and HT7 antibodies, respectively (Fig4a). The immunohistochemical analysis was in line with the above results, revealing a reduced staining of hippocampus area with AT270 and HT7 antibodies (Fig 4B). No differences were detected with AT8 and AT100 antibodies (data not shown). Additionally, cortex samples were also evaluated and, in contrast to the Aβ results with changes only observed in hippocampal samples, the tetracyclic analog also produced tau changes in cortex. As in hippocampus, total tau levels recognized by Tau 5 antibody were not modify, but instead the expression of the aminoacids of the human isoforms recognized by HT7 was reduced by the compound treatment (Fig5). Notably, in this case, the phosphorylation level of the 212/216 residues was also reduced, while no change was observed for the phosphorylation at 181 (Fig5A). Once again, immunohistochemical assays support these data with a reduced staining with HT7 and AT100 antibodies (Fig5B).

2.4 Effect of tetracyclic analog-treatment over kinases involved in Tau and Aβ pathology.


Page 4 of 29

Page 5 of 29

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 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 54 55 56 57 58 59 60


To elucidate the mechanism underlying the reduction of these AD pathological hallmarks, we investigated if this compound affects glycogen kinase 3β (GSK-3β) expression, which is an important kinase related with tau phosphorylation. This kinase phosphorylates tau protein causing its detachment from microtubules and leading to their disorganization and to neurofibrillar tangle formation (22). We used an antibody for the inactive isoform of the enzyme phosphorylated at Ser9 (pGSK-3β) and another one that recognizes total GSK-3β (tGSK3-β). As can be seen in Fig6A, the ratio of pGSK-3β/tGSK-3β was not affected in the cortex samples but markedly increased in the hippocampal samples. Saline-treated samples of hippocampus have a ratio of 1.5 ± 0.5 versus the 4.4 ± 1 of tetracyclic analog-treated samples in the same zone (p = 0.02). This data pointed out that there was an enhancement of the inactive isoform of GSK-3β expression in the hippocampus of the animals treated with this gambierol analog.

2.5 Effect of tetracyclic analog-treatment over Kv and NMDA receptor expression Since gambierol and its analogs have been identified as Kv inhibitors (5, 23) and also since tetracyclic analog-treatment of primary neurons, obtained from 3xTg-AD fetuses, elicited a decreased expression of Kv 3.1 (5), we analyzed if the administration of this compound to 3xTg-AD mice changes the expression level of this channel. However, we observed by Western blot that the compound treatment did not affect Kv 3.1 expression level, neither in hippocampus nor in cortex samples (Fig 6B). In our previous work in primary cortical neurons, we observed that the AD hallmarks improvement was related to an effect over NMDA receptors, since NMDA blockage with APV ((2R)-amino-5phosphonovaleric acid; (2R)-amino-5-phosphonopentanoate) inhibited gambierol effects over tau pathology. Here, we observed that the treatment with the tetracyclic analog produced a diminishment of 56.5 ± 3.6 % (p = 0.001) in the expression of the NMDA receptor subunit N2A in the cortical samples. A similar decrease was also observed in the hippocampal samples with an inhibition of 67.3 ± 7 % (p = 0.02) (Fig6C). In contrast, the N2B subunit of the receptor was not affected (data not shown). These results are in agreement with the previous in vitro data where a five-day treatment with the tetracyclic analog at 5 µM reduced the N2A expression to 43.1±7.6% and no effect was shown over the N2B subunit (5).

Marine polycyclic ethers have fascinated the scientific community for their complex molecular structures and their potent and wide range of biological activities (24). Gambierol has evoked an important neurotoxicity in mammalian cells as well as an acute lethal toxicity in mice (50 µg/kg, ip) (25). Chemical synthesis has addressed its limited availability and enabled more studies on its specific molecular targets and possible therapeutic applications. Voltage-gated potassium channels have been defined as the primary cellular target of gambierol and its truncated analogs, where the heptacyclic truncated analog exhibit the most potent Kv inhibition and cellular toxicity in different potassium subunits and cellular models, respectively (12, 13). Gambierol has a high affinity for the resting channel conformation and shows nanomolar inhibitory activity, however, a higher concentration is needed to inhibit the channel in the open sate. Among the existing Kv subunits, gambierol elicits the highest affinity



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 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 54 55 56 57 58 59 60

for Kv1 and Kv3 subunits. This selectivity confers interesting characteristics on gambierol and its analogs for the development of new immunosupresants against autoimmune diseases (26). In fact, it has been recently showed that in human T lymphocytes cotreatment of cells with the tetracyclic gambierol analog and concanavalin results in a decrease of cytokines release through the inhibition of the Kv1.3 channels in these cells (17). In AD, microglia is the most important inflammatory cell. Recently, it has been settled that voltage-gated Kv1.3 channels play a key role in microglia function and that their expression is intimately related with Aβ plaques in animal models (27). Rangaraju et al proved in a study with human samples that Kv1.3 expression pattern of AD affected brains closely mimics that of human lymphocytes and that they are overexpressed in cortical AD tissues near Aβ plaques (28). Although Kv 3.1 expression is not altered by mice treatment, the tetracyclic analog could be modifying is functioning without affecting its expression levels, as happens in the in vitro studies (5). Moreover, it has been recently proved that memantine, one of the four currently available AD drugs that also target NMDA receptors, also inhibits Kv1.3 currents in lymphocytes from healthy and AD patient donors. This, altogether with the fact that it also repress lymphocyte migration, point out that memantine AD therapeutic effect can infer immunomodulation effects too(29). In this work we describe for the first time that the tetracyclic analog of gambierol is able to modulate tau and Aβ levels in 3xTg-AD after intraperitoneal administrations. These effects were related with a NMDA modulation as it was previously observed in vitro. Although the tetracyclic analog showed a dose dependent-inhibition of BACE-1 in vitro, this could not be the sole mechanism behind the Aβ reduction in this animal model, since other Aβ modulators, such as GSK-3β kinase, are also affected by this compound. Moreover, tau-related effects were associated with a reduction of the N2A subunit expression. It has been recently proved that N2A inhibition by a selective NMDA antagonist provoked tau phosphorylation inhibition at the 199/202 residues via theGSK-3β pathway in hippocampal slices (30), reinforcing the hypothesis that gambierol and their analogs AD beneficial effects are linked to NMDA modulation. Furthermore, other tau residues such as 181 are dephosphorylated directly by GSK-3β kinase inhibition, as can be also observed in this work (31, 32). Meanwhile, the human tau residues 159/163 recognized by HT7 antibody have been related with apoptosis phenomena in neurodegenerative diseases (33) and the reduction of HT7 expression observed could be pointing to a reduction of the normal and phosphorylated human tau isoforms provided by the transgene in these animals (34), whereas Tau 5 expression is not affected. NMDA modulation can also be responsible for the immunomodulation effect observed previously for gambierol in lymphocytes, since, as earlier described memantine is also able to suppress interleukin release in lymphocytes and to modulate neuroinflammation in rat brains after colchicines-induced neurodegeneration(35, 36). All of these confer to gambierol and its analogs a wide range of effects that positively alter different AD cellular damages.

Nowadays, AD is the most commonly acquired dementia in developed countries. Toward the development of effective AD therapeutics, the majority of efforts have been focused on either tau phosphorylation or Aβ aggregation, albeit without much success, and only palliative treatments are clinically available. The multifactorial and complex pathology of AD may need the development of new polypharmacological agents, such as those lowering both Aβ accumulation and tau hyperphosphorylation levels. We have shown that gambierol and its analogs have multiple targets, including Kv channel and β-


Page 6 of 29

Page 7 of 29

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 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 54 55 56 57 58 59 60


secretase, and simultaneously reduce Aβ and tau hyperphosphorylation in vitro and in vivo. The absence of collateral organ damage across the treatments in the present work is also noteworthy. The fact that this compound was able to cross the brain blood barrier makes it a valuable tool for further AD researches. However, the brain barrier cross after oral administrations cannot be dismiss since a unique dose was tested and the amount that can reach the brain by this route can be under the limit of the technique used in the present work.

Further studies about the relationship of NMDA and Kv modulation will be needed to fully understand the mechanism of action of these interesting molecules as well as the development of new analogs with more potency and improved pharmacokinetic characteristics.

3.

MATERIAL AND METHODS.

3.1 Tetracyclic and deuterated tetracyclic analogs.

The tetracyclic gambierol analog was synthesized as previously described (5). The synthetic analog was purified by HPLC prior to biological experiments. Dimethylsulfoxide (DMSO) was used for the preparation of stock solutions. For deuterated tetracyclic analog synthesis, the next scheme was followed (Fig 1):

Alcohol 2.To a solution of dibromoolefin1 (49.4 mg, 0.0831 mmol) (5) in benzene (1 mL) were added nBu3SnD (0.044 mL, 0.17 mmol) and Pd(PPh3)4 (9.6 mg, 0.0083 mmol), and the resultant mixture was stirred at room temperature until TLC showed disappearance of the starting material (ca. 2 h). The resultant mixture was concentrated under reduced pressure, and the residue was purified by flash column chromatography (silica gel, 8% Et2O/hexanes) to give a Z-vinyl bromide, which was used in the next reaction directly. To a solution of the above material in THF (1.5 mL) at 0 °C was added HF·pyridine (0.1 mL), and the resultant solution was stirred at 0 °C for 1 h. The reaction mixture was poured into saturated aqueous NaHCO3 solution at 0 °C, and the resultant mixture was extracted with EtOAc. The organic layer was washed with brine, dried (Na2SO4), filtered, and concentrated under reduced pressure. Purification of the residue by flash column chromatography (silica gel, 60% Et2O/hexanes) gave alcohol 2 (21.0 mg, 57% for two steps): 1H NMR (600 MHz, C6D6) δ5.93 (d, J = 7.8 Hz, 1H), 5.73 (dd, J = 12.6, 1.8 Hz, 1H), 5.69 (dd, J = 12.6, 2.4 Hz, 1H), 4.46 (d, J = 7.8 Hz, 1H), 4.33 (dt, J = 7.8, 1.8 Hz, 1H), 3.61 (ddd, J = 13.2, 6.6, 4.2 Hz, 1H), 3.48–3.37 (m, 3H), 2.99 (dd, J = 12.6, 3.6 Hz, 1H), 2.18 (ddd, J = 11.4, 4.8, 4.8 Hz, 1H), 2.15 (d, J = 13.2 Hz, 1H), 2.11 (d, J = 13.2 Hz, 1H), 1.91 (m, 1H), 1.76 (ddd, J = 12.6, 11.4, 11.4 Hz, 1H), 1.57 (m, 1H), 1.50–1.38 (m, 2H), 1.33–1.22 (m, 5H), 1.20 (s, 1H), 1.17 (s, 3H), 1.14 (s, 3H);

13

C

NMR (150 MHz, C6D6) δ138.9, 132.1, 131.6, 128.5, 86.4, 84.3, 82.2, 80.4, 76.1, 75.9, 72.4, 72.3, 64.1, 54.4, 32.5, 30.3, 29.5, 22.2, 21.4, 19.4, 15.8.



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 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 54 55 56 57 58 59 60

Deuterated gambierol analog 4.To a solution of alcohol 2 (21.0 mg, 0.0473 mmol) and Zvinylstannane3 (101 mg, 0.284 mmol) in THF/DMSO (1:1, v/v, 3.6 mL) were added CuCl (47 mg, 0.47 mmol), LiCl (24 mg, 0.57 mmol), and Pd(PPh3)4 (33 mg, 0.028 mmol), and the resultant mixture was stirred at 60 °C until TLC showed disappearance of the starting material (ca. 24 h). The reaction mixture was cooled to room temperature, and the reaction was quenched with 3% NH4OH solution. The resultant mixture was extracted with Et2O, and the organic layer was washed with 3% NH4OH solution and brine. The organic layer was dried (Na2SO4), filtered, and concentrated under reduced pressure. Purification of the residue by flash column chromatography (silica gel, 8% Et2O/hexanes) gave deuterated gambierol analog 4 (15.6 mg, 76%) as amorphous solid: 1H NMR (500 MHz, C6D6) δ 6.51 (dd, J = 11.5, 11.0 Hz, 1H), 5.94 (dd, J = 12.6, 2.4 Hz, 1H), 5.82 (dd, J = 12.6, 1.8 Hz, 1H), 5.67 (m, 1H), 5.51–5.44 (m, 2H), 5.03–4.92 (m, 2H), 4.50 (d, J = 7.8 Hz, 1H), 4.40 (m, 1H), 3.61 (ddd, J = 13.2, 6.6, 3.6 Hz, 1H), 3.48– 3.40 (m, 2H), 3.36 (ddd, J = 10.8, 9.0, 4.8 Hz, 1H), 3.03 (dd, J = 12.6, 3.6 Hz, 1H), 2.76–2.70 (m, 2H), 2.16–2.08 (m, 3H) 1.90 (m, 1H), 1.80 (ddd, J = 12.6, 11.4, 11.4 Hz, 1H), 1.55 (m, 1H), 1.48–1.35 (m, 2H), 1.33–1.21 (m, 8H), 1.17 (s, 3H);

13

C NMR (125 MHz, C6D6) δ 138.7, 136.2, 131.5, 131.4, 128.5,

128.4, 125.2, 115.4, 86.5, 83.1, 81.9, 80.5, 76.3, 76.1, 72.4, 72.2, 64.1, 54.4, 32.7, 31.8, 30.3, 29.5, 22.2, 21.9, 19.4, 15.9; HRMS (ESI) calcd for C26H37DO5Na [(M + Na)+] 454.2674, found 454.2671.This material was further purified by preparative reverse-phase HPLC using a SHISEIDO CAPCELL PAK C8 column (Ø20 mm × 250 mm, eluent: 80% CH3CN/H2O, flow rate: 0.5 mL/min, UV detection: 254 nm) prior to use.

3.2 Determination of tetracyclic analog presence in the brain.

3.2.1 Animals

Swiss mice of 20 g were used for tetracyclic analog detection. All protocols were approved by the Universidad de Santiago de Compostela Institutional animal care and use committee (process number: AE-LU-002-011/14) and followed the Guide for the Care and Use of Laboratory Animals published by the National Academy of Science (USA) and the EU Directive 2010/63/EU for animal experiments.

Toxin application was made by intraperitoneal injection (ip) and oral co-administration (gavage), dissolving the compound in saline solution in both cases. For the study and differentiation of both administration routes, we use tetracyclic analog for the oral route and deuterated-tetracyclic for the intraperitoneal one. This allows us to know which route have an optimal absorption profile while the use of animals is reduced. One unique dose of 50 µg/kg dissolved in 500 µl of saline solution and three different time points (15 min, 1 hour and 24 hours) were chosen to evaluate the tissue distribution in both administration routes. Non lethal dose 50 exist for this compound, but it has been previously reported non toxicity for this compound by ip injection at 50-80 µg/kg (23). At the specified time points after compound administration, animals were sacrificed and samples were collected for the following analysis. Whole brain, liver, blood, urine and faeces were extracted and weighed immediately.


Page 8 of 29

Page 9 of 29

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 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 54 55 56 57 58 59 60


3.2.2 Sample extraction

Whole blood samples were collected in tubes, stored at -20°C until analysis. Tissue samples (brain and liver) were stored in 0.5 mL of phosphate buffered saline 10 mM (PBS) and frozen at -20°C until analysis. In the case of feces and urine were collected in tubes and stored at -20°C. 40 µL of methanol (MeOH) were added for each 100 µL of blood and then were homogenized by vortex-mixed. Tissue samples and feces were homogenized in 1 mL PBS and MeOH (60:40) using a manual homogenizer and centrifuged at 10000xg for 10 min and the supernatant was separated from pellet. These samples, brain and liver, were concentrated and redissolved in 140 µL of MeOH. Urine samples were concentrated until dryness and redissolved in 1 mL of MeOH. Afterward, all samples were processed with the same method. To 140 µL of mixture 10 µL of 10% trichloroacetic acid were added and vortex-mixed for 30 s. Then, 50 µL of acetonitrile were added to the mixture and vortex-mixed for 1 min. Samples were centrifuged at 14500xg for 10 min, and the supernatant was filtered with 0.22 µm filter and injected 5 µL in the LC column.

3.2.3 LC-MS/MS analysis

For the analysis, a 1290 Infinity ultra-high-performance liquid chromatography (UHPLC) system coupled to a G6460C Triple Quadrupole mass spectrometer equipped with an Agilent Jet Stream ESI source (Agilent Technologies, Waldbronn, Germany). The nitrogen generator is a NITROMAT N-75 ECO from Worthington Creyssensac (Spain). Chromatographic separation was performed at 40°C, the injection volume was 5µL and the flow rate of 0.25 mL/min using a column Acquity UPLC BEH C18 (100x2.1 mm, 1.7 µm, Waters). Mobile phase A was water and mobile phase B was methanol: water (95:5), both containing 1 mM ammonium formate. The gradient program was started with 70%B for 1 minute and then a linear gradient to 100%B in 9 minutes. This condition was hold for 3 minutes and reducing afterward to 70%B over 0.5 minutes. This proportion was maintained for 2.5 minutes, until the next injection to equilibrate the system. The source conditions were optimized to achieve the best sensitivity: drying gas temperature 200°C and flow 7 L/min; sheath gas temperature 300°C and flow 10 L/min; the nebulizer gas pressure of 55 psi; the capillary voltage was set to 4000 V with a nozzle voltage of 500 V. The cell accelerator voltage was 1 for each compound in this method. All analyses were performed in multiple reaction monitoring (MRM) mode. The mass spectrometer was operated in positive mode. The collision energy (CE) and the fragmentor were optimized using MassHunter Optimizer software (Table 1).

For deuterated-tetracyclic gambierol analog and tetracyclic gambierol analog, an eight-point calibration curve among the range 250-1.95 ng/mL was done. The limit of detection (LOD) was 1.95 ng/mL and the limit of quantification (LOQ) was 6.5 ng/mL.

3.3-.Evaluation of the in vivo effect of tetracyclic analog.



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 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 54 55 56 57 58 59 60

Triple transgenic mice for Alzheimer´s disease (3xTg-AD) and wild type NonTg male and female mice were used. Males and females were distributed equally in all the groups, so the same number of each gender were located in each group: wild type (control and treated) 2 males and 2 females in each group and 3 males and 3 females in each group for 3xTg-AD animals (control and treated). 3xTg-AD animals were all of 11 months old whereas wild type animal were 20 month old. Mice were kept in the facilities of the animal care centre of Universidad de Santiago de Compostela. Food and water was supplied ad libitum. All protocols were approved by the Universidad de Santiago de Compostela Institutional animal care and use committee and followed the Guide for the Care and Use of Laboratory Animals published by the National Academy of Science (USA) and the EU Directive 2010/63/EU for animal experiments.

Tetracyclic application was made by intraperitoneal injection, dissolving the compound in saline solution. One unique dose (50 µg/Kg) was administered every week for a whole month. Control animals were administered with saline solution and the corresponding percentage of DMSO. Animal’s health was checked trough all the experiment and none of the animals showed any symptoms of pain or disease. Any of the animals lost more than the 5 % of the initial weight trough the whole month of treatment. At the end of the experiment animals were sacrificed and different samples were collected for analysis.

3.4.- .Histological damage evaluation

Samples of the following organs were collected after euthanasia of 3xTg-AD mice: brain, heart, kidneys, liver, lungs, penis, small intestine and spleen. After fixation by immersion in Bouin’s solution, all samples were embedded in paraffin according to standard laboratory procedures. Paraffin-embedded sections were cut 3-µm thick, mounted on silanized slides, and dried overnight at 37ºC. All tissue sections were stained with Mayer’s haematoxylin and eosin (H&E), while the brain was also stained with cresyl fast violet for Nissl substance (37), and examined under the light microscope for routine histochemical and morphological analyses.

3.5.- Protein extraction, Western blot and Immunohistochemistry analysis.

After mice sacrifice, brains were cut in half sagitally. One half was used for Western blot analysis and the other one for immunohistochemistry.

For Western blot analysis a dissection of hippocampus and cortex was done and samples were collected and lysed in 2 % SDS buffer with 0.7 mg/ml Pepstatin A, supplemented with phosphatase and protease inhibitor cocktails (Complete, MiniEDTA free and PhosSTOP, Roche). Samples were sonicated and centrifuged 1h at 15000 rpm and 4 ºC. This supernatant was stored as soluble fraction. The resulting pellet was homogenized again in 70 % formic acid and processed as previously mentioned to obtain the insoluble fraction. Total protein concentration of the soluble fraction was determined by Bradford assay and 20 µg of total protein were resolved in gel loading buffer (50 mM Tris-HCl, 100 mM dithiotreitol, 2% SDS, 20% glycerol, 0.05% bromophenol blue, pH 6.8) by SDS-PAGE and transferred onto PVDF


Page 10 of 29

Page 11 of 29

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 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 54 55 56 57 58 59 60


membranes (Millipore). The Snap i.d protein detection system was used for blocking and antibody incubation as previously described (38). Immunoreactive bands were detected using the Supersignal West Pico chemiluminiscent substrate (Pierce) and the Diversity 4 gel documentation and analysis system (Syngene, Cambridge, UK). Chemiluminiscence was measured with the Diversity GeneSnap software (Syngene). β-actin was used as control for lane loading and to normalize intensities values. Insoluble fraction was used for the measurement of insoluble βA by Elisa assay. All the antibodies used in the present work are compiled in Table 2.

For immunohystochemical analysis of cortex and hippocampal areas, brain sections were deparaffinized with xylene and rehydrated through a graded alcohol series. To block peroxidase activity and prevent non-specific staining, a pretreatment with Dako Real Peroxidase-Blocking Solution (Dako, Barcelona, Spain) was performed. After removal of blocking reagents, sections were rinsed 3 times in PBS with 0.005% Tween 20 and then incubated 2h at room temperature with the primary antibody. Thereafter, sections were rinsed 3 times in PBS-Tween 20 and incubated with the secondary antibody solution. Samples were washed 3 times with PBS-Tween 20 and revealed with diaminobenzidine. Finally, the slides were counterstained with haematoxylin, dehydrated, and permanently mounted in DPX. Blocking times, as well as dilution and incubation times of the different primary antibodies employed in our study are shown in Table 1.

3.6.- .Aβ Quantificacion by Elisa assay and BACE1 activity measurement.

Aβ 1-42 levels were measured in the soluble and insoluble fraction obtained above by an ultra sensitive sandwich Elisa (Human Ultra sensitive Aβ 412 Elisa Kit, Invitrogen, USA). Soluble fraction was added directly onto Elisa plate, whereas insoluble fraction samples were neutralized previously. Insoluble samples were diluted 1:20 in 1 M Tris Base, 0.5 M NaH2PO4 dibasic. The assay was done following manufacturer protocol.

Bace 1 (BACE1) activity was measured with a Bace 1 FRET assay kit (Invitrogen, USA). This assay utilizes fluorescence resonance energy transfer (FRET) technology to monitor the cleave of a peptide substrate. Enzyme activity is linearly related to the increase in fluorescence. This kit uses baculovirusexpressed Bace1, if the compounds tested inhibit Bace 1 activity, the enzyme cannot cleavage the FRET Bace 1 substrate and then a decrease in the fluorescence is observed.

3.7.- .Solvents and reactives.

Acetonitrile and methanol were supplied by Panreac (Barcelona, Spain). All solvents employed in this work were of HPLC or analytical grade, and the water was distilled and passed through a water purification system (Milli-Q, Millipore, Spain). Formic acid was purchased from Merck (Darmstadt, Germany). Ammonium acetate was from Fluka (Sigma-Aldrich, Spain). Trichloroacetic acid solution 6.1



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 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 54 55 56 57 58 59 60

Page 12 of 29

N was from Sigma-Aldrich (Spain). The composition of saline solution (PBS) 137 NaCl, 8.2 Na2HPO4, 1.5 KH2PO4, 3.2 KCl, and 2 EDTA (in millimolar) (Life Technologies). 3.8.- .Statistical analysis.

Values are expressed as means ± SEM of three or more experiments (each performed in duplicate). Statistical comparison was by one way ANOVA test with Dunnet´s posthoc analysis using the GraphPad Prism software. P values < 0.05 were considered statistically significant.

4.

CONFLICT OF INTEREST

The authors declare that they have no conflict of interest.

5.

AUTHOR CONTRIBUTIONS

Alonso, Eva; Vieira, Andrés; Rodriguez, Ines; Alvariño, Rebeca; Gegunde, Sandra, they all performed in vitro experiments. Fuwa, Haruhiko; Suga, Yuto; Sasaki, Makoto, they obtained by synthesis the molecules Cifuentes, José Manuel, did the morphological and microscopic studies Alfonso, Amparo; Botana, Luis, critical discussion and work design.

6.

ACKNOWLEDGEMENTS

The research leading to these results received funding from the following FEDER cofounded-grants. From CDTI and Technological Funds, supported by the Ministerio de Economía y Competitividad, AGL2012-40185-CO2-01, AGL2014-58210-R, and the Consellería de Cultura, Educación e Ordenación Universitaria, GRC2013-016, and through the Axencia Galega de Innovación, Spain, ITC-20133020 SINTOX. From CDTI under ISIP Programme, Spain, IDI-20130304 APTAFOOD. From the European Union’s Seventh Framework Programme managed by REA—Research Executive Agency (FP7/20072013) under grant agreement 312184 PHARMASEA. Grant-in-Aid for Scientific Research on Innovative Areas “Chemical Biology of Natural Products” (Grant Nos. 23102016 and 26102708) from MEXT, Japan and ERATO Murata Lipid Active Structure Project from Japan Science and Technology Agency (JST).

7.

FIGURE LEGENDS

Fig1. Scheme for deuterated tetracyclic gambierol synthesis.


Page 13 of 29

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 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 54 55 56 57 58 59 60


Fig2. Chromatograms in positive MRM mode of standard solution in methanol of tetracyclic gambierol (A) and deuterated tetracyclic gambierol (B). Chromatogram in positive mode of brain contaminated sample (C) and positive sample of brain at 15 minutes (D).

Fig 3. Tetracyclic analog treatment improves amyloid pathology in 3xTg-AD. Aβ1-42 levels measured in the soluble (A) and insoluble (B) fraction of brain lysates by quantitative Aβ ELISA. Cortical and hippocampal samples were measured independently. Each bar is expressed as mean ± SEM, * p

Tetracyclic Truncated Analogue of the Marine Toxin Gambierol

Recommend Documents