Polymeric 3-alkylpyridinium Salts Research Articles

Polymeric alkylpyridinium salts permit intracellular delivery of human Tau in rat hippocampal neurons: requirement of Tau phosphorylation for functional deficits.

Patients suffering from tauopathies including frontotemporal dementia (FTD) and Alzheimer's disease (AD) present with intra-neuronal aggregation of microtubule-associated protein Tau. During the disease process, Tau undergoes excessive phosphorylation, dissociates from microtubules and aggregates into insoluble neurofibrillary tangles (NFTs), accumulating in the soma. While many aspects of the disease pathology have been replicated in transgenic mouse models, a region-specific non-transgenic expression model is missing. Complementing existing models, we here report a novel region-specific approach to modelling Tau pathology. Local co-administration of the pore-former polymeric 1,3-alkylpyridinium salts (Poly-APS) extracted from marine sponges, and synthetic full-length 4R recombinant human Tau (hTau) was performed in vitro and in vivo. At low doses, Poly-APS was non-toxic and cultured cells exposed to Poly-APS (0.5 µg/ml) and hTau (1 µg/ml; ~22 µM) had normal input resistance, resting-state membrane potentials and Ca(2+) transients induced either by glutamate or KCl, as did cells exposed to a low concentration of the phosphatase inhibitor Okadaic acid (OA; 1 nM, 24 h). Combined hTau loading and phosphatase inhibition resulted in a collapse of the membrane potential, suppressed excitation and diminished glutamate and KCl-stimulated Ca(2+) transients. Stereotaxic infusions of Poly-APS (0.005 µg/ml) and hTau (1 µg/ml) bilaterally into the dorsal hippocampus at multiple sites resulted in hTau loading of neurons in rats. A separate cohort received an additional 7-day minipump infusion of OA (1.2 nM) intrahippocampally. When tested 2 weeks after surgery, rats treated with Poly-APS+hTau+OA presented with subtle learning deficits, but were also impaired in cognitive flexibility and recall. Hippocampal plasticity recorded from slices ex vivo was diminished in Poly-APS+hTau+OA subjects, but not in other treatment groups. Histological sections confirmed the intracellular accumulation of hTau in CA1 pyramidal cells and along their processes; phosphorylated Tau was present only within somata. This study demonstrates that cognitive, physiological and pathological symptoms reminiscent of tauopathies can be induced following non-mutant hTau delivery into CA1 in rats, but functional consequences hinge on increased Tau phosphorylation. Collectively, these data validate a novel model of locally infused recombinant hTau protein as an inducer of Tau pathology in the hippocampus of normal rats; future studies will provide insights into the pathological spread and maturation of Tau pathology.

Pathophysiological effects of synthetic derivatives of polymeric alkylpyridinium salts from the marine sponge, Reniera sarai.

Polymeric 3-alkylpyridinium salts (poly-APS) are among the most studied natural bioactive compounds extracted from the marine sponge, Reniera sarai. They exhibit a wide range of biological activities, and the most prominent among them are the anti-acetylcholinesterase and membrane-damaging activity. Due to their membrane activity, sAPS can induce the lysis of various cells and cell lines and inhibit the growth of bacteria and fungi. Because of their bioactivity, poly-APS are possible candidates for use in the fields of medicine, pharmacy and industry. Due to the small amounts of naturally occurring poly-APS, methods for the synthesis of analogues have been developed. They differ in chemical properties, such as the degree of polymerization, the length of the alkyl chains (from three to 12 carbon atoms) and in the counter ions present in their structures. Such structurally defined analogues with different chemical properties and degrees of polymerization possess different levels of biological activity. We review the current knowledge of the biological activity and toxicity of synthetic poly-APS analogues, with particular emphasis on the mechanisms of their physiological and pharmacological effects and, in particular, the mechanisms of toxicity of two analogues, APS12-2 and APS3, in vivo and in vitro.

Antifouling Activity of Synthetic Alkylpyridinium Polymers Using the Barnacle Model

Polymeric alkylpyridinium salts (poly-APS) isolated from the Mediterranean marine sponge, Haliclona (Rhizoniera) sarai, effectively inhibit barnacle larva settlement and natural marine biofilm formation through a non-toxic and reversible mechanism. Potential use of poly-APS-like compounds as antifouling agents led to the chemical synthesis of monomeric and oligomeric 3-alkylpyridinium analogues. However, these are less efficient in settlement assays and have greater toxicity than the natural polymers. Recently, a new chemical synthesis method enabled the production of poly-APS analogues with antibacterial, antifungal and anti-acetylcholinesterase activities. The present study examines the antifouling properties and toxicity of six of these synthetic poly-APS using the barnacle (Amphibalanus amphitrite) as a model (cyprids and II stage nauplii larvae) in settlement, acute and sub-acute toxicity assays. Two compounds, APS8 and APS12-3, show antifouling effects very similar to natural poly-APS, with an anti-settlement effective concentration that inhibits 50% of the cyprid population settlement (EC50) after 24 h of 0.32 mg/L and 0.89 mg/L, respectively. The toxicity of APS8 is negligible, while APS12-3 is three-fold more toxic (24-h LC50: nauplii, 11.60 mg/L; cyprids, 61.13 mg/L) than natural poly-APS. This toxicity of APS12-3 towards nauplii is, however, 60-fold and 1200-fold lower than that of the common co-biocides, Zn- and Cu-pyrithione, respectively. Additionally, exposure to APS12-3 for 24 and 48 h inhibits the naupliar swimming ability with respective IC50 of 4.83 and 1.86 mg/L.

Effects of synthetic analogues of poly-APS on contractile response of porcine coronary arteries

APS12-2 and APS3 are synthetic analogues of polymeric alkylpyridinium salts (poly-APS) isolated from the marine sponge Reniera sarai. The aim of the present study was to determine the possible direct contractile effects of these two synthetic molecules on coronary arteries, in order partly to explain hemodynamic and cardiotoxic effects of APS12-2 previously observed in in vivo studies and to reveal possible adverse effects on the organism in the case of their clinical use. In contrast to APS3, APS12-2 caused a concentration-dependent vascular smooth muscle contraction of isolated porcine coronary ring preparations in a concentration-range from 1.36 to 13.60μM. Lanthanum chloride (5mM) and verapamil (10μM) completely abolished the APS12-2 evoked contraction of the coronary rings. Pre-incubation with indomethacin (10μM) had no effect on the contractile responses of coronary ring preparations. These results indicate that APS12-2 contracts vascular smooth muscle in a concentration-dependent manner, due to an increase of Ca2+ influx through the voltage-gated Ca2+ channels. Our data show for the first time that APS12-2 induces concentration-dependent contraction of coronary ring preparations, which may contribute to the cardiotoxic effects of APS12-2, in addition to hyperkalemia.

The non-competitive acetylcholinesterase inhibitor APS12-2 is a potent antagonist of skeletal muscle nicotinic acetylcholine receptors

APS12-2, a non-competitive acetylcholinesterase inhibitor, is one of the synthetic analogs of polymeric alkylpyridinium salts (poly-APS) isolated from the marine sponge Reniera sarai. In the present work the effects of APS12-2 were studied on isolated mouse phrenic nerve–hemidiaphragm muscle preparations, using twitch tension measurements and electrophysiological recordings. APS12-2 in a concentration-dependent manner blocked nerve-evoked isometric muscle contraction (IC50=0.74μM), without affecting directly-elicited twitch tension up to 2.72μM. The compound (0.007–3.40μM) decreased the amplitude of miniature endplate potentials until a complete block by concentrations higher than 0.68μM, without affecting their frequency. Full size endplate potentials, recorded after blocking voltage-gated muscle sodium channels, were inhibited by APS12-2 in a concentration-dependent manner (IC50=0.36μM) without significant change in the resting membrane potential of the muscle fibers up to 3.40μM. The compound also blocked acetylcholine-evoked inward currents in Xenopus oocytes in which Torpedo (α12β1γδ) muscle-type nicotinic acetylcholine receptors (nAChRs) have been incorporated (IC50=0.0005μM), indicating a higher affinity of the compound for Torpedo (α12β1γδ) than for the mouse (α12β1γε) nAChR. Our data show for the first time that APS12-2 blocks neuromuscular transmission by a non-depolarizing mechanism through an action on postsynaptic nAChRs of the skeletal neuromuscular junction.

Cardiovascular effects induced by polymeric 3-alkylpyridinium salts from the marine sponge Reniera sarai

Water-soluble polymeric 3-alkylpyridinum salts (poly-APS), isolated from the marine sponge Reniera sarai, are natural products with promising biomedical applications. However, their ability to form non-specific cell membrane pores raises safety issues. Therefore, the aim of the present study was to investigate the direct toxic effects of poly-APS on the cardiovascular system. To study the impact of poly-APS toxicodynamics on vascular function, the relaxation and contraction responses of isolated rat thoracic aortas incubated in poly-APS solutions (0.01–10 μM) were tested. In addition, cardiac toxicity was studied by measuring coronary flow, lactate dehydrogenase release rate, left ventricular pressure, heart rate, and the duration of arrhythmias in isolated rat hearts perfused with poly-APS (0.001–1 μM). Poly-APS diminished endothelium-dependent relaxation and contraction in a concentration- and time-dependent manner. Endothelial function was affected earlier and to a greater extent than contractile responses. Likewise, in isolated hearts the most evident cardiotoxic effects were observed after perfusion with the highest concentration (1 μM) of poly-APS: compared to the control group the coronary flow and heart rate were diminished by 2.2- and 1.8-fold, while lactate dehydrogenase release rate and left ventricular pressure were increased by 7.8- and 2.2-fold (all P < 0.001). Further, poly-APS had evident proarrhythmogenic activity in a concentration-dependent manner. However, in the low concentration range (1–10 nM) poly-APS showed only minor toxicity. Our results confirmed the direct toxic effects of poly-APS on the rat cardiovascular system. Therefore, it seems reasonable to conclude that the use of poly-APS as therapeutic adjuvants has limited safety margins.

In vivo toxic and lethal cardiovascular effects of a synthetic polymeric 1,3-dodecylpyridinium salt in rodents

APS12-2 is one in a series of synthetic analogs of the polymeric alkylpyridinium salts isolated from the marine sponge Reniera sarai. As it is a potential candidate for treating non small cell lung cancer (NSCLC), we have studied its possible toxic and lethal effects in vivo. The median lethal dose (LD 50) of APS12-2 in mice was determined to be 11.5 mg/kg. Electrocardiograms, arterial blood pressure and respiratory activity were recorded under general anesthesia in untreated, pharmacologically vagotomized and artificially ventilated rats injected with APS12-2. In one group, the in vivo effects of APS12-2 were studied on nerve-evoked muscle contraction. Administration of APS12-2 at a dose of 8 mg/kg caused a progressive reduction of arterial blood pressure to a mid-circulatory value, accompanied by bradycardia, myocardial ischemia, ventricular extrasystoles, and second degree atrio-ventricular block. Similar electrocardiogram and arterial blood pressure changes caused by APS12-2 (8 mg/kg) were observed in animals pretreated with atropine and in artificially ventilated animals, indicating that hypoxia and cholinergic effects do not play a crucial role in the toxicity of APS12-2. Application of APS12-2 at sublethal doses (4 and 5.5 mg/kg) caused a decrease of arterial blood pressure, followed by an increase slightly above control values. We found that APS12-2 causes lysis of rat erythrocytes in vitro, therefore it is reasonable to expect the same effect in vivo. Indeed, hyperkalemia was observed in the blood of experimental animals. Hyperkalemia probably plays an important role in APS12-2 cardiotoxicity since no evident changes in histopathology of the heart were found. However, acute lesions were observed in the pulmonary vessels of rats after application of 8 mg/kg APS12-2. Predominant effects were dilation of interalveolar blood vessels and lysis of aggregated erythrocytes within their lumina.

Cardiotoxic activity of polymeric 3-alkylpyridinium salts from the marine sponge Reniera sarai

Background Marine sponges are a rich source of novel biologically active substances such as haemolytic proteins, hemagglutinins and proteins with antimicrobial, antiviral and antifungal activity. Toxic water-soluble polymeric 3alkylpyridinum salts (poly-APS) have been isolated from the marine sponge Reniera sarai. Poly-APS has pore-forming properties expressed in a dose-dependent manner. These interactions with cell membranes results in increased Ca2+ permeability. In in vivo experiments performed in rats, poly-APS caused transient bradycardia, prolongation of expirium, lowered blood pressure, formed numerous thrombocyte aggregates and caused death. The aim of the present study was to investigate the direct cardiotoxic effects of poly-APS on isolated rat hearts.

Induction of fruiting in oyster mushroom ( Pleurotus ostreatus) by polymeric 3-alkylpyridinium salts

Polymeric 3-alkylpyridinium salts (poly-APS), surface-active compounds from the marine sponge Reniera sarai, have been shown to stimulate the fruit body formation from Pleurotus ostreatus mycelium. In nutrient media supplemented with poly-APS (≥0.01 μg ml −1), the formation of primordia and development of fruit bodies were detected approximately 10 d earlier than in the absence of poly-APS, and also led to a considerably larger quantity of young mushrooms. This effect appears to be specific, as other surface-active compounds, lysophospholipids and fatty acids, showed no induction of fruiting.

Influence of polymeric 3-alkylpyridinium salts from the marine sponge Reniera sarai on the growth of algae and wood decay fungi

Polymeric alkylpyridinium salts (poly-APS) isolated from the marine sponge Reniera sarai act as antifouling and anticholinesterase agents. They also show moderate haemolytic and cytotoxic activities against different cell lines. The haemolytic activity of poly-APS is due to their detergent-like structure and behaviour in aqueous solutions. In this work, the lytic activity of poly-APS against freshwater and marine algae, and inhibitory effects on wood decay fungi, were investigated. The results show that poly-APS inhibit the proliferation and movements of susceptible algae. Effects of poly-APS were time- and concentration-dependent and differed between various algal species. No growth inhibition effects were observed towards the examined wood fungi.

Mechanisms of Toxicity of 3-Alkylpyridinium Polymers from Marine Sponge Reniera sarai

Polymeric 3-alkylpyridinium salts (poly-APS) present in the marine sponge Reniera sarai show a broad spectrum of biological activities. They are lytic to erythrocytes and various other mammalian cells, enabling the transfection of the latter with alien DNA. Furthermore, they show inhibitory effects to marine bacteria and can inhibit fouling of micro- and macroorganisms to submerged surfaces. Finally, poly-APS act as potent cholinesterase inhibitors. The kinetics of acetylcholinesterase inhibition by poly-APS in vitro is complex and comprises several successive phases ending in irreversible inhibition of the enzyme. The latter is accounted for by aggregation and precipitation of the enzyme-inhibitor complexes. Poly-APS are lethal to rats in concentrations above 2.7 mg/kg. Monitoring of the basic vital functions and histopathological analysis showed that the effects directly ascribable to acetylcholinesterase inhibition are only observed after application of lower concentrations of poly-APS. At higher concentrations, such effects were masked by other, more pronounced and faster developing lethal effects of the toxin, such as haemolysis and platelet aggregation.

Mechanisms of Toxicity of 3-Alkylpyridinium Polymers from Marine Sponge Reniera sarai

Polymeric 3-alkylpyridinium salts (poly-APS) present in the marine spongeReniera sarai show a broad spectrum of biological activities. They are lytic to erythrocytesand various other mammalian cells, enabling the transfection of the latter with alien DNA.Furthermore, they show inhibitory effects to marine bacteria and can inhibit fouling ofmicro- and macroorganisms to submerged surfaces. Finally, poly-APS act as potentcholinesterase inhibitors. The kinetics of acetylcholinesterase inhibition by poly-APS invitro is complex and comprises several successive phases ending in irreversible inhibitionof the enzyme. The latter is accounted for by aggregation and precipitation of the enzyme-inhibitor complexes. Poly-APS are lethal to rats in concentrations above 2.7 mg/kg.Monitoring of the basic vital functions and histopathological analysis showed that theeffects directly ascribable to acetylcholinesterase inhibition are only observed afterapplication of lower concentrations of poly-APS. At higher concentrations, such effectswere masked by other, more pronounced and faster developing lethal effects of the toxin,such as haemolysis and platelet aggregation.

Marine sponge-derived polymeric alkylpyridinium salts as a novel tumor chemotherapeutic targeting the cholinergic system in lung tumors

Previous studies have shown that the cholinergic system plays a pivotal rule in small cell lung cancer (SCLC) cell growth through an autocrine loop that activates the nicotinic cholinergic receptor, which together with the activation of this receptor by nicotine links SCLC evolution with tobacco use. Non-small cell lung cancer (NSCLC) is the most common form of lung cancer and is also linked to tobacco use. Here we describe the presence of molecules of the cholinergic system in NSCLC samples and cell lines and investigate the implications of the cholinergic system in cell growth regulation. Cholino-acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT) and acetylcholinesterase (AChE) were observed in NSCLC tumor biopsies and in NSCLC cell lines. Polymeric alkylpyridinium salts (poly-APS) are AChE inhibitors isolated from the crude extract of the marine sponge, Reniera sarai. These metabolites were characterized as a mixture of two polymers of 3-octylpyridinium, including 29 and 99 monomeric units. Exposure of normal lung fibroblast and NSCLC cell lines to poly-APS revealed a selective cytotoxicity for cancer cells as compared to the normal fibroblast cell lines. FACS analysis indicated poly-APS induced apoptosis in NSCLC cells but not in normal lymphocytes. Non-toxic doses of poly-APS also potently reduced NSCLC cell-cell adhesion in suspension cultures. The limited toxicity of poly-APS on normal cells was confirmed by injection in the caudal vein of mice. No overt effects on health parameters, such as weight gain and physical behavior, were observed, and histological analysis of major organs did not reveal differences between the treated animals as compared to controls. These data demonstrate that NSCLC cells express cholinergic molecules that may be involved in cell growth regulation and that the cholinesterase inhibitor, poly-APS, shows selective toxicity toward NSCLC cells while having no apparent toxicity towards normal cells and tissue in vitro and in vivo.

Comparative antibacterial activity of polymeric 3-alkylpyridinium salts isolated from the Mediterranean sponge Reniera sarai and their synthetic analogues

Metabolites from marine sponges are considered a promising alternative to heavy metals in antifouling coatings. Water-soluble polymeric 3-alkylpyridinium salts and 14 related synthetic analogues showed considerable antibacterial activity against marine biofilm bacteria and may represent good candidates as natural biocides for marine technology applications.

Non-toxic Antifouling Activity of Polymeric 3-alkylpyridinium Salts from the Mediterranean Sponge Reniera sarai (Pulitzer-Finali)

The antifouling activity and toxicity of polymeric 3-alkylpyridinium salts (poly-APS) isolated from the Mediterranean sponge Reniera sarai were studied. The activity of these natural products was compared to that of zinc and copper complexes of pyrithione, two non-persistent booster biocides successfully used in current antifouling coatings. Larvae of Balanus amphitrite (cyprids and nauplii) were used to monitor settlement inhibition and the extent to which inhibition was due to toxicity. The microalga Tetraselmis suecica and larvae of the mussel Mytilus galloprovincialis were used in toxicity bioassays. Compared to the booster biocides, poly-APS were less effective at inhibiting cyprid settlement, but their effects were non toxic and reversible, with very low toxicity against the organisms used in the toxicity bioassays. Although encouraging, these results are not enough to warrant the use of poly-APS as a potential commercial antifoulant. They however justify possible future efforts to chemically synthesize poly-APS analogues for further tests.

Toxic effects of head-to-tail 3-alkylpyridinium polymers isolated from the marine sponge Reniera sarai in rat

Toxic water soluble polymeric 3-alkylpyridinium salts (poly APS; MW 18900 and 5520Da) were isolated from the marine sponge Raniera sarai. In vitro they strongly inhibited acetylcholinesterase. In order to evaluate the role of acetylcholinesterase inhibition in toxin lethality, and to assess other possible lethal effects, in vivo experiments were performed on male Wistar rats, and ECG, blood pressure and breathing pattern were monitored. The results showed that none of the animals died due to the acetylcholinesterase inhibitory action of poly-APS. Doses lower than 1mg/kg caused only transient bradycardia and transient prolongation of expirium. At doses above 2.7mg/kg of poly-APS all treated animals died, but signs were not typical of acetylcholinesterase inhibition. Arterial blood pressure fell to mid-circulatory pressure, and breathing stopped after a few breaths with an increase of the residual volume. Autopsy of the experimental animals that died due to the effects of the toxin revealed that mid-size and small sized blood vessels in the heart and lungs were filled with granular brownish material with inclusions of red blood cells and platelets. Data obtained on blood samples from animals treated with poly-APS also revealed numerous thrombocyte aggregates. In vitro poly-APS induced thrombocyte aggregation in a dose dependent manner. The acetylcholinesterase-inhibitory effects were most pronounced only at lower doses of poly-APS. With higher doses those effects were masked or covered by other, more pronounced and faster developing lethal effects of the toxin such as platelet aggregation. Therefore it is reasonable to assume that acetylcholinesterase inhibitory effects are not responsible for the lethal activity of the toxin.

In vivo effects of head-to-tail 3-alkylpiridinium polymers isolated from the marine sponge Raniera sarai

Water soluble polymeric 3-alkylpyridinium salts (poly APS; MW 18900 and 5520 Da) were isolated from the marine sponge Raniera sarai. In vitro it strongly inhibited acetyl cholinesterase (AChE) from different species (electric eel, horse serum, human erythrocytes). In our experiments the importance of anti AChE activity in the toxin lethality was evaluated. In vivo experiments were performed on male Wistar rats and ECG, blood pressure and breathing pattern were monitored. After i.v. application of lethal doses of the toxin ECG showed signs of hypo perfusion. Arterial blood pressure fell to mid-circulatory pressure, and breathing stopped after a few breaths At sublethal doses the toxin caused an increase of residual volume, prolongation of expiration, and bradycardia. Patho-anatomical examination revealed that the plugs in lung circulation may cause the death of experimental animals due to cardiorespiratory failure.

AFM imaging of surface adsorbed polymeric 3-alkylpyridinium salts from the marine sponge Reniera sarai

Bioactive 3-alkylpyridinium polymers (poly-APS) have recently been isolated from the marine sponge Reniera sarai. Previous results have shown that these molecules in aqueous solutions form supramolecular aggregates with an average hydrodynamic radius of 23±2 nm. To obtain additional evidences about the shape and the dimensions of poly-APS aggregates, we used atomic force microscopy (AFM) operating in tapping mode. The images clearly showed adsorbed aggregates with a lateral dimension of ≈40 nm and a thickness of the order of ≈1 nm. The distribution of volumes of the adsorbed aggregates is very similar to the distribution of hydrodynamic radii as obtained from the dynamic light scattering experiments. The volume distribution of these aggregates shows a maximum at 1750 nm 3, which corresponds to a sphere with a radius of 7.5 nm.

Characterization of hemolytic activity of 3-alkylpyridinium polymers from the marine sponge Reniera sarai

Polymeric alkylpyridinium salts (poly-APS) isolated from the marine sponge Reniera sarai act as potent anticholinesterase agents; in addition they show moderate hemolytic and cytotoxic activities. The hemolytic activity of poly-APS is due to their detergent-like structure and behavior in aqueous solutions. In this work, the hemolytic activity of poly-APS is analyzed and compared to that of structurally-related monomeric cationic surfactants. The influence of different divalent cations and lipids on poly-APS induced hemolysis is discussed. The dimensions of lesions caused by poly-APS in erythrocyte membranes are determined by the use of osmotic protectants. Finally, the possible role of poly-APS in their natural environment is proposed.

Inhibition of acetylcholinesterase by an alkylpyridinium polymer from the marine sponge, Reniera sarai

Large polymeric 3-alkylpyridinium salts have been isolated from the marine sponge Reniera sarai. They are composed of N-butyl(3-butylpyridinium) repeating subunits, polymerized head-to-tail, and exist as a mixture of two main polymers with molecular weights without counterion of about 5520 and 18 900. The monomer analogue of the inhibitor, N-butyl-3-butylpyridinium iodide has been synthesized. This molecule shows mixed reversible inhibition of acetylcholinesterase. The polymers also act as acetylcholinesterase inhibitors and show an unusual inhibition pattern. We tentatively describe it as quick initial reversible binding, followed by slow binding or irreversible inhibition of the enzyme. This kinetics suggests that there are several affinity binding sites on the acetylcholinesterase molecule where the polymer can bind. The first binding favors binding to other sites which leads to an apparently irreversibly linked enzyme-inhibitor complex.