Molecular Pharmacology of Gelsemium Alkaloids on Inhibitory Receptors.

Indole alkaloids are the main bioactive molecules of the Gelsemium genus plants. Diverse reports have shown the beneficial actions of Gelsemium alkaloids on the pathological states of the central nervous system (CNS). Nevertheless, Gelsemium alkaloids are toxic for mammals. To date, the molecular targets underlying the biological actions of Gelsemium alkaloids at the CNS remain poorly defined. Functional studies have determined that gelsemine is a modulator of glycine receptors (GlyRs) and GABAA receptors (GABAARs), which are ligand-gated ion channels of the CNS. The molecular and physicochemical determinants involved in the interactions between Gelsemium alkaloids and these channels are still undefined. We used electrophysiological recordings and bioinformatic approaches to determine the pharmacological profile and the molecular interactions between koumine, gelsemine, gelsevirine, and humantenmine and these ion channels. GlyRs composed of α1 subunits were inhibited by koumine and gelsevirine (IC50 of 31.5 ± 1.7 and 40.6 ± 8.2 µM, respectively), while humantenmine did not display any detectable activity. The examination of GlyRs composed of α2 and α3 subunits showed similar results. Likewise, GABAARs were inhibited by koumine and were insensitive to humantenmine. Further assays with chimeric and mutated GlyRs showed that the extracellular domain and residues within the orthosteric site were critical for the alkaloid effects, while the pharmacophore modeling revealed the physicochemical features of the alkaloids for the functional modulation. Our study provides novel information about the molecular determinants and functional actions of four major Gelsemium indole alkaloids on inhibitory receptors, expanding our knowledge regarding the interaction of these types of compounds with protein targets of the CNS.

Elephant Black Garlic's Beneficial Properties for Hippocampal Neuronal Network, Chemical Characterization and Biological Evaluation.

Garlic has been used for decades as an important food and additionally for its beneficial properties in terms of nutrition and ancestral therapeutics. In this work, we compare the properties of fresh (WG) and aged (BG) extract obtained from elephant garlic, harvested on Chiloe Island, Chile. BG was prepared from WG with a 20-day aging process under controlled temperature and humidity conditions. We observed that in BG, compounds such as diallyl disulfide decrease, and compounds of interest such as 5-hydroxymethylfurfural (69%), diallyl sulfide (17%), 3H-1,2-Dithiole (22%) and 4-Methyl-1,2,3-trithiolane (16%) were shown to be increased. Using 2,2-diphenyl-1-picrylhydrazyl (DPPH, BG: 51 ± 5.7%, WG: 12 ± 2.6%) and 2,20-azino-bis-(3-ethylbenzothiazoline-6 sulfonate) diammonium salt (ABTS, BG: 69.4 ± 2.3%, WG: 21 ± 3.9%) assays, we observed that BG possesses significantly higher antioxidant activity than WG and increased cell viability in hippocampal slices (41 ± 9%). The effects of WG and BG were shown to improve the neuronal function through an increased in intracellular calcium transients (189 ± 4%). In parallel, BG induced an increase in synaptic vesicle protein 2 (SV-2; 75 ± 12%) and brain-derived neurotrophic factor (BDNF; 32 ± 12%) levels. Thus, our study provides the initial scientific bases to foster the use of BG from Chiloe Island as a functional food containing a mixture of bioactive compounds that may contribute to brain health and well-being.

New Benzodihydrofuran Derivatives Alter the Amyloid ß Peptide Aggregation: Strategies To Develop New Anti-Alzheimer Drugs.

Alzheimer's disease is a neurodegenerative disorder that is the leading cause of dementia in elderly patients. Amyloid-ß peptide (1-42 oligomers) has been identified as a neurotoxic factor, triggering many neuropathologic events. In this study, 15 chalcones were synthesized employing the Claisen-Schmidt condensation reaction, starting from a compound derived from fomannoxine, a natural benzodihydrofuran whose neuroprotective activity has been proven and reported, and methyl aromatic ketones with diverse patterns of halogenated substitution. As a result, chalcones were obtained, with good to excellent reaction yields from 50 to 98%. Cytotoxicity of the compounds was assessed, and their cytoprotective effect against the toxicity associated with Aß was evaluated on PC-12 cells. Out of the 15 chalcones obtained, only the 4-bromo substituted was cytotoxic at most tested concentrations. Three synthesized chalcones showed a cytoprotective effect against Aß toxicity (over 37%). The 2,4,5-trifluoro substituted chalcone was the most promising series since it showed a cytoprotective impact with more than 60 ± 5% of recovery of cellular viability; however, 3-fluoro substituted compound also exhibited important values of recovery (50 ± 6%). The fluorine substitution pattern was shown to be more effective for cytoprotective activity. Specifically, substitution with fluorine in the 3,5-positions turned out to be particularly effective for cytoprotection. Furthermore, fluorinated compounds inhibited the aggregation rate of Aß, suggesting a dual effect that can be the starting point of new molecules with therapeutic potential.

Partial Inhibition of Complex I Restores Mitochondrial Morphology and Mitochondria-ER Communication in Hippocampus of APP/PS1 Mice.

Alzheimer's disease (AD) has no cure. Earlier, we showed that partial inhibition of mitochondrial complex I (MCI) with the small molecule CP2 induces an adaptive stress response, activating multiple neuroprotective mechanisms. Chronic treatment reduced inflammation, Aß and pTau accumulation, improved synaptic and mitochondrial functions, and blocked neurodegeneration in symptomatic APP/PS1 mice, a translational model of AD. Here, using serial block-face scanning electron microscopy (SBFSEM) and three-dimensional (3D) EM reconstructions combined with Western blot analysis and next-generation RNA sequencing, we demonstrate that CP2 treatment also restores mitochondrial morphology and mitochondria-endoplasmic reticulum (ER) communication, reducing ER and unfolded protein response (UPR) stress in the APP/PS1 mouse brain. Using 3D EM volume reconstructions, we show that in the hippocampus of APP/PS1 mice, dendritic mitochondria primarily exist as mitochondria-on-a-string (MOAS). Compared to other morphological phenotypes, MOAS have extensive interaction with the ER membranes, forming multiple mitochondria-ER contact sites (MERCS) known to facilitate abnormal lipid and calcium homeostasis, accumulation of Aß and pTau, abnormal mitochondrial dynamics, and apoptosis. CP2 treatment reduced MOAS formation, consistent with improved energy homeostasis in the brain, with concomitant reductions in MERCS, ER/UPR stress, and improved lipid homeostasis. These data provide novel information on the MOAS-ER interaction in AD and additional support for the further development of partial MCI inhibitors as a disease-modifying strategy for AD.

TG2 promotes amyloid beta aggregates: Impact on ER-mitochondria crosstalk, calcium homeostasis and synaptic function in Alzheimer's disease.

Alzheimer's Disease (AD) is a neurodegenerative disorder characterized by cognitive impairment that increasingly affects the elderly. AD's main features have been related to cellular and molecular events, including the aberrant aggregation of the amyloid beta peptide (Aß), Ca2+ dyshomeostasis, and increased mitochondria-associated membranes (MAMs). Transglutaminase type 2 (TG2) is a ubiquitous enzyme whose primary role is the Ca2+-dependent proteins transamidation, including the Aß peptide. TG2 activity has been closely related to cellular damage and death. We detected increased TG2 levels in neuronal cells treated with Aß oligomers (AßOs) and hippocampal slices from J20 mice using cellular and molecular approaches. In this work, we characterized the capacity of TG2 to interact and promote Aß toxic aggregates (AßTG2). AßTG2 induced an acute increase in intracellular Ca2+, miniature currents, and hiperexcitability, consistent with an increased mitochondrial Ca2+ overload, IP3R-VDAC tethering, and mitochondria-endoplasmic reticulum contacts (MERCs). AßTG2 also decreased neuronal viability and excitatory postsynaptic currents, reinforcing the idea of synaptic failure associated with MAMs dysregulation mediated by TG2. Z-DON treatment, TG2 inhibitor, reduced calcium overload, mitochondrial membrane potential loss, and synaptic failure, indicating an involvement of TG2 in a toxic cycle which increases Aß aggregation, Ca2+ overload, and MAMs upregulation. These data provide novel information regarding the role TG2 plays in synaptic function and contribute additional evidence to support the further development of TG2 inhibitors as a disease-modifying strategy for AD.

Understanding the Role of ATP Release through Connexins Hemichannels during Neurulation.

Neurulation is a crucial process in the formation of the central nervous system (CNS), which begins with the folding and fusion of the neural plate, leading to the generation of the neural tube and subsequent development of the brain and spinal cord. Environmental and genetic factors that interfere with the neurulation process promote neural tube defects (NTDs). Connexins (Cxs) are transmembrane proteins that form gap junctions (GJs) and hemichannels (HCs) in vertebrates, allowing cell-cell (GJ) or paracrine (HCs) communication through the release of ATP, glutamate, and NAD+; regulating processes such as cell migration and synaptic transmission. Changes in the state of phosphorylation and/or the intracellular redox potential activate the opening of HCs in different cell types. Cxs such as Cx43 and Cx32 have been associated with proliferation and migration at different stages of CNS development. Here, using molecular and cellular biology techniques (permeability), we demonstrate the expression and functionality of HCs-Cxs, including Cx46 and Cx32, which are associated with the release of ATP during the neurulation process in Xenopus laevis. Furthermore, applications of FGF2 and/or changes in intracellular redox potentials (DTT), well known HCs-Cxs modulators, transiently regulated the ATP release in our model. Importantly, the blockade of HCs-Cxs by carbenoxolone (CBX) and enoxolone (ENX) reduced ATP release with a concomitant formation of NTDs. We propose two possible and highly conserved binding sites (N and E) in Cx46 that may mediate the pharmacological effect of CBX and ENX on the formation of NTDs. In summary, our results highlight the importance of ATP release mediated by HCs-Cxs during neurulation.

Neuroprotective Properties of Eudesmin on a Cellular Model of Amyloid-ß Peptide Toxicity.

BACKGROUND: Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive cognitive impairment and memory loss. One of the hallmarks in AD is amyloid-ß peptide (Aß) accumulation, where the soluble oligomers of Aß (AßOs) are the most toxic species, deteriorating the synaptic function, membrane integrity, and neuronal structures, which ultimately lead to apoptosis. Currently, there are no drugs to arrest AD progression, and current scientific efforts are focused on searching for novel leads to control this disease. Lignans are compounds extracted from conifers and have several medicinal properties. Eudesmin (Eu) is an extractable lignan from the wood of Araucaria araucana, a native tree from Chile. This metabolite has shown a range of biological properties, including the ability to control inflammation and antibacterial effects. OBJECTIVE: In this study, the neuroprotective abilities of Eu on synaptic failure induced by AßOs were analyzed. METHODS: Using neuronal models, PC12 cells, and in silico simulations we evaluated the neuroprotective effect of Eu (30 nM) against the toxicity induced by AßOs. RESULTS: In primary cultures from mouse hippocampus, Eu preserved the synaptic structure against AßOs toxicity, maintaining stable levels of the presynaptic protein SV2 at the same concentration. Eu also averted synapsis failure from the AßOs toxicity by sustaining the frequencies of cytosolic Ca2+ transients. Finally, we found that Eu (30 nM) interacts with the Aß aggregation process inducing a decrease in AßOs toxicity, suggesting an alternative mechanism to explain the neuroprotective activity of Eu. CONCLUSION: We believe that Eu represents a novel lead that reduces the Aß toxicity, opening new research venues for lignans as neuroprotective agents.

Antiseizure medication in early nervous system development. Ion channels and synaptic proteins as principal targets.

The main strategy for the treatment of epilepsy is the use of pharmacological agents known as antiseizure medication (ASM). These drugs control the seizure onset and improves the life expectancy and quality of life of patients. Several ASMs are contraindicated during pregnancy, due to a potential teratogen risk. For this reason, the pharmacological treatments of the pregnant Women with Epilepsy (WWE) need comprehensive analyses to reduce fetal risk during the first trimester of pregnancy. The mechanisms by which ASM are teratogens are still under study and scientists in the field, propose different hypotheses. One of them, which will be addressed in this review, corresponds to the potential alteration of ASM on ion channels and proteins involved in relevant signaling and cellular responses (i.e., migration, differentiation) during embryonic development. The actual information related to the action of ASM and its possible targets it is poorly understood. In this review, we will focus on describing the eventual presence of some ion channels and synaptic proteins of the neurotransmitter signaling pathways present during early neural development, which could potentially interacting as targets of ASM. This information leads to elucidate whether these drugs would have the ability to affect critical signaling during periods of neural development that in turn could explain the fetal malformations observed by the use of ASM during pregnancy.

Metabolic profile and antioxidant capacity of five Berberis leaves species: A comprehensive study to determine their potential as natural food or ingredient.

A comprehensive study of bioactive compounds was carried out in the leaves of the main Berberis species growing in Chile (Berberis microphylla, Berberis darwinii, Berberis empetrifolia, Berberis trigona, and the introduced Berberis vulgaris). We identified 117 compounds, by a detailed analysis of each molecule, including phenolic acids, alkaloids, flavonols, and other compounds, using high-performance liquid chromatography and high-resolution mass spectrometry. Quantitative analysis of main compound was also included for all species. Hydroxycinnamic acid derivatives were the main compounds in all the studied leaves, with the highest concentration in Berberis microphylla. Quercetin derivatives were the most relevant flavonols in all species, except in Berberis vulgaris, in which isorhamnetin-3-rutinoside was the most concentrated. The fatty acid profile, determined by gas chromatography mass spectrometry revealed the presence of linoleic and linolenic acids in all species studied. Berberis vulgaris showed higher levels of these fatty acids. The antioxidant capacity, explored by three in vitro methods, showed high values for all studied Berberis species. The obtained levels are higher than those of other prominent foods. The findings will inform novel approaches for the valorization of these leaves as natural food or ingredient.

Microglial Activation Modulated by P2X4R in Ischemia and Repercussions in Alzheimer's Disease.

There are over 80 million people currently living who have had a stroke. The ischemic injury in the brain starts a cascade of events that lead to neuronal death, inducing neurodegeneration which could lead to Alzheimer's disease (AD). Cerebrovascular diseases have been suggested to contribute to AD neuropathological changes, including brain atrophy and accumulation of abnormal proteins such as amyloid beta (Aß). In patients older than 60 years, the incidence of dementia a year after stroke was significantly increased. Nevertheless, the molecular links between stroke and dementia are not clearly understood but could be related to neuroinflammation. Considering that activated microglia has a central role, there are brain-resident innate immune cells and are about 10-15% of glial cells in the adult brain. Their phagocytic activity is essential for synaptic homeostasis in different areas, such as the hippocampus. These cells polarize into phenotypes or subtypes: the pro-inflammatory M1 phenotype, or the immunosuppressive M2 phenotype. Phenotype M1 is induced by classical activation, where microglia secrete a high level of pro- inflammatory factors which can cause damage to the surrounding neuronal cells. Otherwise, M2 phenotype is the major effector cell with the potential to counteract pro-inflammatory reactions and promote repair genes expression. Moreover, after the classical activation, an anti-inflammatory and a repair phase are initiated to achieve tissue homeostasis. Recently it has been described the concepts of homeostatic and reactive microglia and they had been related to major AD risk, linking to a multifunctional microglial response to Aß plaques and pathophysiology markers related, such as intracellular increased calcium. The upregulation and increased activity of purinergic receptors activated by ADP/ATP, specially P2X4R, which has a high permeability to calcium and is mainly expressed in microglial cells, is observed in diseases related to neuroinflammation, such as neuropathic pain and stroke. Thus, P2X4R is associated with microglial activation. P2X4R activation drives microglia motility via the phosphatidylinositol-3-kinase (PI3K)/Akt pathway. Also, these receptors are involved in inflammatory-mediated prostaglandin E2 (PGE2) production and induce a secretion and increase the expression of BDNF and TNF-α which could be a link between pathologies related to aging and neuroinflammation.

Deciphering the role of PGC-1α in neurological disorders: from mitochondrial dysfunction to synaptic failure.

The onset and mechanisms underlying neurodegenerative diseases remain uncertain. The main features of neurodegenerative diseases have been related with cellular and molecular events like neuronal loss, mitochondrial dysfunction and aberrant accumulation of misfolded proteins or peptides in specific areas of the brain. The most prevalent neurodegenerative diseases belonging to age-related pathologies are Alzheimer's disease, Huntington's disease, Parkinson's disease and amyotrophic lateral sclerosis. Interestingly, mitochondrial dysfunction has been observed to occur during the early onset of several neuropathological events associated to neurodegenerative diseases. The master regulator of mitochondrial quality control and energetic metabolism is the transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α). Additionally, it has been observed that PGC-1α appears to be a key factor in maintaining neuronal survival and synaptic transmission. In fact, PGC-1α downregulation in different brain areas (hippocampus, substantia nigra, cortex, striatum and spinal cord) that occurs in function of neurological damage including oxidative stress, neuronal loss, and motor disorders has been seen in several animal and cellular models of neurodegenerative diseases. Current evidence indicates that PGC-1α upregulation may serve as a potent therapeutic approach against development and progression of neuronal damage. Remarkably, increasing evidence shows that PGC-1α deficient mice have neurodegenerative diseases-like features, as well as neurological abnormalities. Finally, we discuss recent studies showing novel specific PGC-1α isoforms in the central nervous system that appear to exert a key role in the age of onset of neurodegenerative diseases and have a neuroprotective function in the central nervous system, thus opening a new molecular strategy for treatment of neurodegenerative diseases. The purpose of this review is to provide an up-to-date overview of the PGC-1α role in the physiopathology of neurodegenerative diseases, as well as establish the importance of PGC-1α function in synaptic transmission and neuronal survival.

Modulation of GABAA receptors and of GABAergic synapses by the natural alkaloid gelsemine.

The Gelsemium elegans plant preparations have shown beneficial activity against common diseases, including chronic pain and anxiety. Nevertheless, their clinical uses are limited by their toxicity. Gelsemine, one of the most abundant alkaloids in the Gelsemium plants, have replicated these therapeutic and toxic actions in experimental behavioral models. However, the molecular targets underlying these biological effects remain unclear. The behavioral activity profile of gelsemine suggests the involvement of GABAA receptors (GABAARs), which are the main biological targets of benzodiazepines (BDZs), a group of drugs with anxiolytic, hypnotic, and analgesic properties. Here, we aim to define the modulation of GABAARs by gelsemine, with a special focus on the subtypes involved in the BDZ actions. The gelsemine actions were determined by electrophysiological recordings of recombinant GABAARs expressed in HEK293 cells, and of native receptors in cortical neurons. Gelsemine inhibited the agonist-evoked currents of recombinant and native receptors. The functional inhibition was not associated with the BDZ binding site. We determined in addition that gelsemine diminished the frequency of GABAergic synaptic events, likely through a presynaptic modulation. Our findings establish gelsemine as a negative modulator of GABAARs and of GABAergic synaptic function. These pharmacological features discard direct anxiolytic or analgesic actions of gelsemine through GABAARs but support a role of GABAARs on the alkaloid induced toxicity. On the other hand, the presynaptic effects of the alkaloid provide an additional mechanism to explain their beneficial effects. Collectively, our results contribute novel information to improve understanding of gelsemine actions in the mammalian nervous system.

PrP C as a Transducer of Physiological and Pathological Signals.

After the discovery of prion phenomenon, the physiological role of the cellular prion protein (PrP C ) remained elusive. In the past decades, molecular and cellular analysis has shed some light regarding interactions and functions of PrP C in health and disease. PrP C , which is located mainly at the plasma membrane of neuronal cells attached by a glycosylphosphatidylinositol (GPI) anchor, can act as a receptor or transducer from external signaling. Although the precise role of PrP C remains elusive, a variety of functions have been proposed for this protein, namely, neuronal excitability and viability. Although many issues must be solved to clearly define the role of PrP C , its connection to the central nervous system (CNS) and to several misfolding-associated diseases makes PrP C an interesting pharmacological target. In a physiological context, several reports have proposed that PrP C modulates synaptic transmission, interacting with various proteins, namely, ion pumps, channels, and metabotropic receptors. PrP C has also been implicated in the pathophysiological cell signaling induced by ß-amyloid peptide that leads to synaptic dysfunction in the context of Alzheimer's disease (AD), as a mediator of Aß-induced cell toxicity. Additionally, it has been implicated in other proteinopathies as well. In this review, we aimed to analyze the role of PrP C as a transducer of physiological and pathological signaling.

Phenolic Compounds in Calafate Berries Encapsulated by Spray Drying: Neuroprotection Potential into the Ingredient.

Calafate is a berry rich in anthocyanins that presents higher content of polyphenols than other fruits. Its compounds have been described previously, however, the potential thereof in preventing and treating degenerative disorders has not yet been studied. Due to its astringency, the consumption of this berry in its natural state is limited. To profit from the aforementioned properties and reduce palatability issues, calafate berry extracts were microencapsulated by spray drying, a rapid, cost-effective and scalable process, and were then compared with freeze drying as a control. The stability of its contents and its in-vitro potential, with respect to AChE activity and neuroprotection, were measured from the obtained microcapsules, resulting from temperature treatments and different encapsulant contents. The results indicated that the spray-dried powders were stable, despite high temperatures, and their encapsulation exhibited nearly 50% efficiency. The highest quantity of polyphenols and 3-O-glycosylated anthocyanins was obtained from encapsulation with 20% maltodextrin, at 120 °C. Temperature did not affect the microcapsules' biological action, as demonstrated by their antioxidant activities. The prevention of Aß peptide cytotoxicity in PC12 cells (20%) revealed that encapsulated calafate can confer neuroprotection. We conclude that spray-drying is an appropriate technique for scaling-up and producing new value-added calafate formulations with anti-neurodegenerative effects and vivid colors.

Increased P2×2 receptors induced by amyloid-ß peptide participates in the neurotoxicity in alzheimer's disease.

Amyloid beta peptide (Aß) is tightly associated with the physiopathology of Alzheimer's Disease (AD) as one of the most important factors in the evolution of the pathology. In this context, we previously reported that Aß increases the expression of ionotropic purinergic receptor 2 (P2×2R). However, its role on the cellular and molecular Aß toxicity is unknown, especially in human brain of AD patients. Using cellular and molecular approaches in hippocampal neurons, PC12 cells, and human brain samples of patients with AD, we evaluated the participation of P2×2R in the physiopathology of AD. Here, we reported that Aß oligomers (Aßo) increased P2×2 levels in mice hippocampal neurons, and that this receptor increases at late Braak stages of AD patients. Aßo also increases the colocalization of APP with Rab5, an early endosomes marker, and decreased the nuclear/cytoplasmic ratio of Fe65 and PGC-1α immunoreactivity. The overexpression in PC12 cells of P2×2a, but not P2×2b, replicated these changes in Fe65 and PGC-1α; however, both overexpressed isoforms increased levels of Aß. Taken together, these data suggest that P2×2 is upregulated in AD and it could be a key potentiator of the physiopathology of Aß. Our results point to a possible participation in a toxic cycle that increases Aß production, Ca2+ overload, and a decrease of PGC-1α. These novel findings put the P2×2R as a key novel pharmacological target to develop new therapeutic strategies to treat Alzheimer's Disease.

The Administration of the Synbiotic Lactobacillus bulgaricus 6c3 Strain, Inulin and Fructooligosaccharide Decreases the Concentrations of Indoxyl Sulfate and Kidney Damage in a Rat Model.

Indoxyl sulfate (IS) is involved in the progression of chronic kidney disease (CKD) and in its cardiovascular complications. One of the approaches proposed to decrease IS is the administration of synbiotics. This work aimed to search for a probiotic strain capable to decrease serum IS levels and mix it with two prebiotics (inulin and fructooligosaccharide (FOS)) to produce a putative synbiotic and test it in a rat CKD model. Two groups of Sprague-Dawley rats were nephrectomized. One group (Lac) received the mixture for 16 weeks in drinking water and the other no (Nef). A control group (C) included sham-nephrectomized rats. Serum creatinine and IS concentrations were measured using high-performance liquid chromatography with diode array detector (HPLC-DAD). Optical microscopy and two-photon excitation microscopy was used to study kidney and heart samples. The Lac group, which received the synbiotic, reduced IS by 0.8% while the Nef group increased it by 38.8%. Histological analysis of kidneys showed that the Lac group increased fibrotic areas by 12% and the Nef group did it by 25%. The synbiotic did not reduce cardiac fibrosis. Therefore, the putative synbiotic showed that function reducing IS and the progression of CKD in a rat model, but no heart protection was observed.

Stereospecific Inhibition of Ethanol Potentiation on Glycine Receptor by M554 Stereoisomers.

Blocking the interaction between the Gßγ protein and the glycine receptor (GlyR) has emerged as a promising pharmacological strategy to treat acute alcohol intoxication by inhibiting ethanol potentiation on GlyR. M554 is a recently discovered small molecule capable of binding to Gßγ with potent in vitro and in vivo inhibitory activity. This compound has been tested as a mixture of diastereomers, and no information is available concerning the stereospecific activity of each species, which is critical to pursue efforts on lead optimization and drug development. In this work, we explored the differential activity of four M554 stereoisomers by in silico molecular dynamics simulations and electrophysiological experiments. Our results revealed that the (R,R)-M554 stereoisomer is a promising lead compound that inhibits ethanol potentiation of GlyR.

Inhibition of the Glycine Receptor alpha 3 Function by Colchicine.

Colchicine is a plant alkaloid that is widely used as a therapeutic agent. It is widely accepted that colchicine reduces the production of inflammatory mediators mainly by altering cytoskeleton dynamics due to its microtubule polymerization inhibitory activity. However, other lines of evidence have shown that colchicine exerts direct actions on the function of ion channels, which are independent of cytoskeleton alterations. Colchicine is able to modify the function of several pentameric ligand-gated ion channels, including glycine receptors (GlyRs). Previous electrophysiological studies have shown that colchicine act as an antagonist of GlyRs composed by the α 1 subunit. In addition, it was recently demonstrated that colchicine directly bind to the α 3 subunit of GlyRs. Interestingly, other studies have shown a main role of α 3GlyRs on chronic inflammatory pain. Nevertheless, the functional effects of colchicine on the α 3GlyR function are still unknown. Here, by using electrophysiological techniques and bioinformatics, we show that colchicine inhibited the function of the α 3GlyRs. Colchicine elicited concentration-dependent inhibitory effects on α 3GlyRs at micromolar range and decreased the apparent affinity for glycine. Single-channel recordings show that the colchicine inhibition is associated with a decrease in the open probability of the ion channel. Molecular docking assays suggest that colchicine preferentially bind to the orthosteric site in the closed state of the ion channel. Altogether, our results suggest that colchicine is a competitive antagonist of the α 3GlyRs.

Altered Glutaminase 1 Activity During Neurulation and Its Potential Implications in Neural Tube Defects.

The neurulation process is regulated by a large amount of genetic and environmental factors that determine the establishment, folding, and fusion of the neural plate to form the neural tube, which develops into the main structure of the central nervous system. A recently described factor involved in this process is glutamate. Through NMDA ionotropic receptor, glutamate modifies intracellular Ca2+ dynamics allowing the oriented cell migration and proliferation, essentials processes in neurulation. Glutamate synthesis depends on the mitochondrial enzyme known as glutaminase 1 (GLS1) that is widely expressed in brain and kidney. The participation of GLS 1 in prenatal neurogenic processes and in the adult brain has been experimentally established, however, its participation in early stages of embryonic development has not been described. The present investigation describes for the first time the presence and functionality of GLS1 in Xenopus laevis embryos during neurulation. Although protein expression levels remains constant, the catalytic activity of GLS1 increases significantly (~66%) between early (stage 12) and middle to late (stages 14-19) neurulation process. Additionally, the use of 6-diazo-5-oxo-L-norleucine (L-DON, competitive inhibitor of glutamine-depend enzymes), reduced significantly the GLS1 specific activity during neurulation (~36%) and induce the occurrence of neural tube defects involving its possible participation in the neural tube closure in Xenopus laevis embryos.

Changes in PGC-1α/SIRT1 Signaling Impact on Mitochondrial Homeostasis in Amyloid-Beta Peptide Toxicity Model.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by cognitive impairment that increasingly afflicts the elderly population. Soluble oligomers (AßOs) has been implicated in AD pathogenesis: however, the molecular events underlying a role for Aß are not well understood. We studied the effects of AßOs on mitochondrial function and on key proteins that regulate mitochondrial dynamics and biogenesis in hippocampal neurons and PC-12 cells. We find that AßOs treatment caused a reduction in total Mfn1 after a 2 h exposure (42 ± 11%); while DRP1 increased at 1 and 2 h (205 ± 22% and 198 ± 27%, respectively), correlating to changes in mitochondrial morphology. We also observed that SIRT1 levels were reduced after acute and chronic AßOs treatment (68 ± 7% and 77 ± 6%, respectively); while PGC-1α levels were reduced with the same time treatments (68 ± 8% and 67 ± 7%, respectively). Interestingly, we found that chronic treatment with AßOs increased the levels of pSIRT1 (24 h: 157 ± 18%), and we observed changes in the PGC-1α and p-SIRT1 nucleus/cytosol ratio and SIRT1-PGC-1α interaction pattern after chronic exposure to AßOs. Our data suggest that AßOs induce important changes in the level and localization of mitochondrial proteins related with the loss of mitochondrial function that are mediated by a fast and sustained SIRT1/PGC-1α complex disruption promoting a "non-return point" to an irreversible synaptic failure and neuronal network disconnection.