Automated Patch Clamp for the Detection of Tetrodotoxin in Pufferfish Samples.

Tetrodotoxin (TTX) is a marine toxin responsible for many intoxications around the world. Its presence in some pufferfish species and, as recently reported, in shellfish, poses a serious health concern. Although TTX is not routinely monitored, there is a need for fast, sensitive, reliable, and simple methods for its detection and quantification. In this work, we describe the use of an automated patch clamp (APC) system with Neuro-2a cells for the determination of TTX contents in pufferfish samples. The cells showed an IC50 of 6.4 nM for TTX and were not affected by the presence of muscle, skin, liver, and gonad tissues of a Sphoeroides pachygaster specimen (TTX-free) when analysed at 10 mg/mL. The LOD achieved with this technique was 0.05 mg TTX equiv./kg, which is far below the Japanese regulatory limit of 2 mg TTX equiv./kg. The APC system was applied to the analysis of extracts of a Lagocephalus sceleratus specimen, showing TTX contents that followed the trend of gonads > liver > skin > muscle. The APC system, providing an in vitro toxicological approach, offers the advantages of being sensitive, rapid, and reliable for the detection of TTX-like compounds in seafood.

Role of c-Jun N-Terminal Kinases (JNKs) in Epilepsy and Metabolic Cognitive Impairment.

Previous studies have reported that the regulatory function of the different c-Jun N-terminal kinases isoforms (JNK1, JNK2, and JNK3) play an essential role in neurological disorders, such as epilepsy and metabolic-cognitive alterations. Accordingly, JNKs have emerged as suitable therapeutic strategies. In fact, it has been demonstrated that some unspecific JNK inhibitors exert antidiabetic and neuroprotective effects, albeit they usually show high toxicity or lack therapeutic value. In this sense, natural specific JNK inhibitors, such as Licochalcone A, are promising candidates. Nonetheless, research on the understanding of the role of each of the JNKs remains mandatory in order to progress on the identification of new selective JNK isoform inhibitors. In the present review, a summary on the current gathered data on the role of JNKs in pathology is presented, as well as a discussion on their potential role in pathologies like epilepsy and metabolic-cognitive injury. Moreover, data on the effects of synthetic small molecule inhibitors that modulate JNK-dependent pathways in the brain and peripheral tissues is reviewed.

Towards a Novel Class of Multitarget-Directed Ligands: Dual P2X7-NMDA Receptor Antagonists.

Multi-target-directed ligands (MTDLs) offer new hope for the treatment of multifactorial complex diseases such as Alzheimer's Disease (AD). Herein, we present compounds aimed at targeting the NMDA and the P2X7 receptors, which embody a different approach to AD therapy. On one hand, we are seeking to delay neurodegeneration targeting the glutamatergic NMDA receptors; on the other hand, we also aim to reduce neuroinflammation, targeting P2X7 receptors. Although the NMDA receptor is a widely recognized therapeutic target in treating AD, the P2X7 receptor remains largely unexplored for this purpose; therefore, the dual inhibitor presented herein-which is open to further optimization-represents the first member of a new class of MTDLs.

High-fat diet-induced deregulation of hippocampal insulin signaling and mitochondrial homeostasis deficiences contribute to Alzheimer disease pathology in rodents.

Global obesity is a pandemic status, estimated to affect over 2 billion people, that has resulted in an enormous strain on healthcare systems worldwide. The situation is compounded by the fact that apart from the direct costs associated with overweight pathology, obesity presents itself with a number of comorbidities, including an increased risk for the development of neurodegenerative disorders. Alzheimer disease (AD), the main cause of senile dementia, is no exception. Spectacular failure of the pharmaceutical industry to come up with effective AD treatment strategies is forcing the broader scientific community to rethink the underlying molecular mechanisms leading to cognitive decline. To this end, the emphasis is once again placed on the experimental animal models of the disease. In the current study, we have focused on the effects of a high-fat diet (HFD) on hippocampal-dependent memory in C57/Bl6 Wild-type (WT) and APPswe/PS1dE9 (APP/PS1) mice, a well-established mouse model of familial AD. Our results indicate that the continuous HFD administration starting at the time of weaning is sufficient to produce ß-amyloid-independent, hippocampal-dependent memory deficits measured by a 2-object novel-object recognition test (NOR) in mice as early as 6months of age. Furthermore, the resulting metabolic syndrome appears to have direct effects on brain insulin regulation and mitochondrial function. We have observed pathological changes related to both the proximal and distal insulin signaling pathway in the brains of HFD-fed WT and APP/PS1 mice. These changes are accompanied by a significantly reduced OXPHOS metabolism, suggesting that mitochondria play an important role in hippocampus-dependent memory formation and retention in both the HFD-treated and AD-like rodents at a relatively young age.

Enantiopure Indolo[2,3-a]quinolizidines: Synthesis and Evaluation as NMDA Receptor Antagonists.

Enantiopure tryptophanol is easily obtained from the reduction of its parent natural amino acid trypthophan (available from the chiral pool), and can be used as chiral auxiliary/inductor to control the stereochemical course of a diastereoselective reaction. Furthermore, enantiopure tryptophanol is useful for the syntheses of natural products or biological active molecules containing the aminoalcohol functionality. In this communication, we report the development of a small library of indolo[2,3-a]quinolizidines and evaluation of their activity as N-Methyl d-Aspartate (NMDA) receptor antagonists. The indolo[2,3-a]quinolizidine scaffold was obtained using the following key steps: (i) a stereoselective cyclocondensation of (S)- or (R)-tryptophanol with appropriate racemic δ-oxoesters; (ii) a stereocontrolled cyclization on the indole nucleus. The synthesized enantiopure indolo[2,3-a]quinolizidines were evaluated as NMDA receptor antagonists and one compound was identified to be 2.9-fold more potent as NMDA receptor blocker than amantadine (used in the clinic for Parkinson's disease). This compound represents a hit compound for the development of novel NMDA receptor antagonists with potential applications in neurodegenerative disorders associated with overactivation of NMDA receptors.

Early alterations in energy metabolism in the hippocampus of APPswe/PS1dE9 mouse model of Alzheimer's disease.

The present study had focused on the behavioral phenotype and gene expression profile of molecules related to insulin receptor signaling in the hippocampus of 3 and 6 month-old APPswe/PS1dE9 (APP/PS1) transgenic mouse model of Alzheimer's disease (AD). Elevated levels of the insoluble Aß (1-42) were detected in the brain extracts of the transgenic animals as early as 3 months of age, prior to the Aß plaque formation (pre-plaque stage). By the early plaque stage (6 months) both the soluble and insoluble Aß (1-40) and Aß (1-42) peptides were detectable. We studied the expression of genes related to memory function (Arc, Fos), insulin signaling, including insulin receptor (Insr), Irs1 and Irs2, as well as genes involved in insulin growth factor pathways, such as Igf1, Igf2, Igfr and Igfbp2. We also examined the expression and protein levels of key molecules related to energy metabolism (PGC1-α, and AMPK) and mitochondrial functionality (OXPHOS, TFAM, NRF1 and NRF2). 6 month-old APP/PS1 mice demonstrated impaired cognitive ability, were glucose intolerant and showed a significant reduction in hippocampal Insr and Irs2 transcripts. Further observations also suggest alterations in key cellular energy sensors that regulate the activities of a number of metabolic enzymes through phosphorylation, such as a decrease in the Prkaa2 mRNA levels and in the pAMPK (Thr172)/Total APMK ratio. Moreover, mRNA and protein analysis reveals a significant downregulation of genes essential for mitochondrial replication and respiratory function, including PGC-1α in hippocampal extracts of APP/PS1 mice, compared to age-matched wild-type controls at 3 and 6 months of age. Overall, the findings of this study show early alterations in genes involved in insulin and energy metabolism pathways in an APP/PS1 model of AD. These changes affect the activity of key molecules like NRF1 and PGC-1α, which are involved in mitochondrial biogenesis. Our results reinforce the hypothesis that the impairments in both insulin signaling and energy metabolism precede the development of AD amyloidogenesis.

Ritter reaction-mediated syntheses of 2-oxaadamantan-5-amine, a novel amantadine analog.

Two alternative syntheses of 2-oxaadamantan-5-amine, a novel analog of the clinically approved drug amantadine, are reported. The compound has been tested as an anti-influenza A virus agent and as an NMDA receptor antagonist. While the compound was not antivirally active, it displayed moderate activity as an NMDA receptor antagonist.

Tryptophanol-derived oxazolopiperidone lactams: identification of a hit compound as NMDA receptor antagonist.

N-Methyl-D-aspartate receptors (NMDAR) exacerbated activation leads to neuron death through a phenomenon called excitotoxicity. These receptors are implicated in several neurological diseases (e.g., Alzheimer and Parkinson) and thus represent an important therapeutic target. We herein describe the study of enantiopure tryptophanol-derived oxazolopiperidone lactams as NMDA receptor antagonists. The most active hit exhibited an IC50 of 63.4 µM in cultured rat cerebellar granule neurons thus being 1.5 fold more active than clinically approved NMDA antagonist amantadine (IC50=92 µM).

Novel benzopolycyclic amines with NMDA receptor antagonist activity.

A new series of benzopolycyclic amines active as NMDA receptor antagonists were synthesized. Most of them exhibited increased activity compared with related analogues previously published. All the tested compounds were more potent than clinically approved amantadine and one of them displayed a lower IC50 value than memantine, an anti-Alzheimer's approved drug.

First record of paralytic shellfish toxins in marine pufferfish from the Spanish Mediterranean coast using cell-based assay, automated patch clamp and HPLC-FLD.

Pufferfish is one of the most poisonous marine organisms, responsible for numerous poisoning incidents and some human fatalities due to its capability to accumulate potent neurotoxins such as tetrodotoxins (TTXs) and paralytic shellfish toxins (PSTs). In this study, tissue extracts (muscle, skin, liver, intestinal tract and gonads) obtained from sixteen pufferfish specimens of the Lagocephalus lagocephalus and Sphoeroides pachygaster species, collected along the Spanish Mediterranean coast, were analysed for the presence of voltage-gated sodium channel (also known as Nav channel) blockers using cell-based assay (CBA) and automated patch clamp (APC). No toxicity was observed in any of the S. pachygaster specimens, but toxicity was detected in the liver of most L. lagocephalus specimens. Instrumental analysis of these specimens, as well as in one Lagocephalus sceleratus specimen, by high-performance liquid chromatography coupled to fluorescence detection (HPLC-FLD) was performed, which confirmed the presence of PSTs only in L. lagocephalus specimens. This analysis reported the presence of saxitoxin (STX) and decarbamoylsaxitoxin (dcSTX) in all positive samples, being dcSTX the major analogue. These results demonstrate the ability of this species to accumulate PSTs, being the first report of the presence of PSTs in Mediterranean L.lagocephalus specimens. Furthermore, the presence of high PSTs contents in all five tested tissues of one L. lagocephalus specimen pointed the risk that the presence of this toxic fish in the Mediterranean Sea may represent for seafood safety and human health in case of accidental consumption.

Toxicity Equivalency Factors for Tetrodotoxin Analogues Determined with Automated Patch Clamp on Voltage-Gated Sodium Channels in Neuro-2a Cells.

Tetrodotoxin (TTX) is a potent marine neurotoxin, responsible for numerous poisoning incidents and some human fatalities. To date, more than 30 TTX analogues have been identified, but their individual toxicities and roles in poisoning remain largely unknown. In this work, the toxicity equivalency factors (TEFs) of five TTX analogues were determined by assessing the blockade of voltage-gated sodium channels in Neuro-2a cells using automated patch clamp (APC). All TTX analogues were less toxic than TTX. The derived TEFs were applied to the individual TTX analogues concentrations measured in pufferfish samples, using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). A comparison of these results with those obtained from APC analysis demonstrated that TEFs can be effectively used to translate LC-MS/MS analytical data into meaningful toxicological information. This is the first study to utilize APC device for the toxicological assessment of TTX analogues, highlighting its potential as a bioanalytical tool for seafood safety management and human health protection.

SUCNR1 regulates insulin secretion and glucose elevates the succinate response in people with prediabetes.

Pancreatic ß-cell dysfunction is a key feature of type 2 diabetes, and novel regulators of insulin secretion are desirable. Here we report that the succinate receptor (SUCNR1) is expressed in ß-cells and is up-regulated in hyperglycemic states in mice and humans. We found that succinate acts as a hormone-like metabolite and stimulates insulin secretion via a SUCNR1-Gq-PKC-dependent mechanism in human ß-cells. Mice with ß-cell-specific Sucnr1 deficiency exhibit impaired glucose tolerance and insulin secretion on a high-fat diet, indicating that SUCNR1 is essential for preserving insulin secretion in diet-induced insulin resistance. Patients with impaired glucose tolerance show an enhanced nutritional-related succinate response, which correlates with the potentiation of insulin secretion during intravenous glucose administration. These data demonstrate that the succinate/SUCNR1 axis is activated by high glucose and identify a GPCR-mediated amplifying pathway for insulin secretion relevant to the hyperinsulinemia of prediabetic states.

Synthesis of benzopolycyclic cage amines: NMDA receptor antagonist, trypanocidal and antiviral activities.

The synthesis of several 6,7,8,9,10,11-hexahydro-9-methyl-5,7:9,11-dimethano-5H-benzocyclononen-7-amines is reported. Several of them display low micromolar NMDA receptor antagonist and/or trypanocidal activities. Two compounds are endowed with micromolar anti vesicular stomatitis virus activity, while only one compound shows micromolar anti-influenza activity. The anti-influenza activity of this compound does not seem to be mediated by blocking of the M2 protein.

Design, synthesis, and in vitro and in vivo characterization of new memantine analogs for Alzheimer's disease.

Currently, of the few accessible symptomatic therapies for Alzheimer's disease (AD), memantine is the only N-methyl-d-aspartate receptor (NMDAR) blocker approved by the FDA. This work further explores a series of memantine analogs featuring a benzohomoadamantane scaffold. Most of the newly synthesized compounds block NMDARs in the micromolar range, but with lower potency than previously reported hit IIc, results that were supported by molecular dynamics simulations. Subsequently, electrophysiological studies with the more potent compounds allowed classification of IIc, a low micromolar, uncompetitive, voltage-dependent, NMDAR blocker, as a memantine-like compound. The excellent in vitro DMPK properties of IIc made it a promising candidate for in vivo studies in Caenorhabditis elegans (C. elegans) and in the 5XFAD mouse model of AD. Administration of IIc or memantine improved locomotion and rescues chemotaxis behavior in C. elegans. Furthermore, both compounds enhanced working memory in 5XFAD mice and modified NMDAR and CREB signaling, which may prevent synaptic dysfunction and modulate neurodegenerative progression.

Sirtuin activators: designing molecules to extend life span.

Resveratrol (RESV) exerts important pharmacological effects on human health: in addition to its beneficial effects on type 2 diabetes and cardiovascular diseases, it also modulates neuronal energy homeostasis and shows antiaging properties. Although it clearly has free radical scavenger properties, the mechanisms involved in these beneficial effects are not fully understood. In this regard, one area of major interest concerns the effects of RESV on the activity of sirtuin 1 (SIRT1), an NAD(+)-dependent histone deacetylase that has been implicated in aging. Indeed, the role of SIRT1 is currently the subject of intense research due to the antiaging properties of RESV, which increases life span in various organisms ranging from yeast to rodents. In addition, when RESV is administered in experimental animal models of neurological disorders, it has similar beneficial effects to caloric restriction. SIRT1 activation could thus constitute a potential strategic target in neurodegenerative diseases and in disorders involving disturbances in glucose homeostasis, as well as in dyslipidaemias or cardiovascular diseases. Therefore, small SIRT1 activators such as SRT501, SRT2104, and SRT2379, which are currently undergoing clinical trials, could be potential drugs for the treatment of type 2 diabetes, obesity, and metabolic syndrome, among other disorders. This review summarises current knowledge about the biological functions of SIRT1 in aging and aging-associated diseases and discusses its potential as a pharmacological target.

Resveratrol inhibits proliferation and promotes apoptosis of neuroblastoma cells: role of sirtuin 1.

Resveratrol prolongs lifespan and prevent cancer formation; however, the mechanisms are not understood. Here we evaluated the cell-cycle inhibition and apoptosis of resveratrol in B65 neuroblastoma cells, and we also studied the effects of resveratrol on the mammalian silent information regulator 2 (SIRT1). Results show that resveratrol reduces cell viability and causes apoptosis at 24 h of treatment. Resveratrol partially blocked cell proliferation, and significantly increased the fraction of cells arrested in the S phase. The role of SIRT1 in cell-cycle effects mediated by resveratrol was studied through changes in the expression of SIRT1 using western blot. Exposure to resveratrol decreased SIRT1 content, concomitant with an increase in the acetylated form of sirtuin substrates p53 and NFκ-ß. Treatment of B65 neuroblastoma cells with resveratrol also reduced the content of the phosphorylated form of AKT. Exposure to the SIRT1 inhibitors nicotinamide and sirtinol altered neither cell viability nor the fraction of apoptotic cells. Furthermore, when cells were exposed simultaneously to resveratrol and nicotinamide or sirtinol, no changes were observed in the fraction of apoptotic cells. Our results show that a decrease in SIRT1 content, caused by exposure to resveratrol, does not appear to be involved in cell-cycle arrest or activation of apoptosis.

Activation of ataxia telangiectasia muted under experimental models and human Parkinson's disease.

In the present study we demonstrated that neurotoxin MPP(+)-induced DNA damage is followed by ataxia telangiectasia muted (ATM) activation either in cerebellar granule cells (CGC) or in B65 cell line. In CGC, the selective ATM inhibitor KU-55933 showed neuroprotective effects against MPP(+)-induced neuronal cell loss and apoptosis, lending support to the key role of ATM in experimental models of Parkinson's disease. Likewise, we showed that knockdown of ATM levels in neuroblastoma B65 cells using an ATM-specific siRNA attenuates the phosphorylation of retinoblastoma protein without affecting other cell-cycle proteins involved in the G(0)/G(1) cell-cycle phase. Moreover, we demonstrated DNA damage, in human brain samples of PD patients. These findings support a model in which MPP(+) leads to ATM activation with a subsequent DNA damage response and activation of pRb. Therefore, this study demonstrates a new link between DNA damage by MPP(+) and cell-cycle re-entry through retinoblastoma protein phosphorylation.

Pharmacological Strategies to Improve Dendritic Spines in Alzheimer's Disease.

To deeply understand late onset Alzheimer's disease (LOAD), it may be necessary to change the concept that it is a disease exclusively driven by aging processes. The onset of LOAD could be associated with a previous peripheral stress at the level of the gut (changes in the gut microbiota), obesity (metabolic stress), and infections, among other systemic/environmental stressors. The onset of LOAD, then, may result from the generation of mild peripheral inflammatory processes involving cytokine production associated with peripheral stressors that in a second step enter the brain and spread out the process causing a neuroinflammatory brain disease. This hypothesis could explain the potential efficacy of Sodium Oligomannate (GV-971), a mixture of acidic linear oligosaccharides that have shown to remodel gut microbiota and slowdown LOAD. However, regardless of the origin of the disease, the end goal of LOAD-related preventative or disease modifying therapies is to preserve dendritic spines and synaptic plasticity that underlay and support healthy cognition. Here we discuss how systemic/environmental stressors impact pathways associated with the regulation of spine morphogenesis and synaptic maintenance, including insulin receptor and the brain derived neurotrophic factor signaling. Spine structure remodeling is a plausible mechanism to maintain synapses and provide cognitive resilience in LOAD patients. Importantly, we also propose a combination of drugs targeting such stressors that may be able to modify the course of LOAD by acting on preventing dendritic spines and synapsis loss.

New oxapolycyclic cage amines with NMDA receptor antagonist and trypanocidal activities.

The synthesis of several (1,2,3,5,6,7-hexahydro-1,5:3,7-dimethano-4-benzoxonin-3-yl)amines and related compounds is reported. Several of them display very similar activity to memantine as NMDA receptor antagonists. Several derivatives showed a significant level of trypanocidal activity.

Gambierdiscus and Fukuyoa as potential indicators of ciguatera risk in the Balearic Islands.

Gambierdiscus and Fukuyoa are genera of toxic dinoflagellates which were mainly considered as endemic to marine intertropical areas, and that are well known as producers of ciguatoxins (CTXs) and maitotoxins (MTXs). Ciguatera poisoning (CP) is a human poisoning occurring after the consumption of fish or more rarely, shellfish containing CTXs. The presence of these microalgae in a coastal area is an indication of potential risk of CP. This study assesses the risk of CP in the Balearic Islands (Western Mediterranean Sea) according to the distribution of both microalgae genera, and the presence of CTX-like and MTX-like toxicity in microalgal cultures as determined by neuro-2a cell based-assay (neuro-2a CBA). Genetic identification of forty-three cultured microalgal strains isolated from 2016 to 2019 revealed that all of them belong to the species G. australes and F. paulensis. Both species were widely distributed in Formentera, Majorca and Minorca. Additionally, all strains of G. australes and two of F. paulensis exhibited signals of CTX-like toxicity ranging respectively between 1 and 380 and 8-16 fg CTX1B equivalents (equiv.) • cell-1. Four extracts of F. paulensis exhibited a novel toxicity response in neuro-2a cells consisting of the recovery of the cell viability in the presence of ouabain and veratridine. In addition, G. australes showed MTX-like toxicity while F. paulensis strains did not. Overall, the low CTX-like toxicities detected indicate that the potential risk of CP in the Balearic Islands is low, although, the presence of CTX-like and MTX-like toxicity in those strains reveal the necessity to monitor these genera in the Mediterranean Sea.