Interactions Between the Ubiquitin-Proteasome System, Nrf2, and the Cannabinoidome as Protective Strategies to Combat Neurodegeneration: Review on Experimental Evidence.

Neurodegenerative disorders are chronic brain diseases that affect humans worldwide. Although many different factors are thought to be involved in the pathogenesis of these disorders, alterations in several key elements such as the ubiquitin-proteasome system (UPS), the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway, and the endocannabinoid system (ECS or endocannabinoidome) have been implicated in their etiology. Impairment of these elements has been linked to the origin and progression of neurodegenerative disorders, while their potentiation is thought to promote neuronal survival and overall neuroprotection, as proved with several experimental models. These key neuroprotective pathways can interact and indirectly activate each other. In this review, we summarize the neuroprotective potential of the UPS, ECS, and Nrf2 signaling, both separately and combined, pinpointing their role as a potential therapeutic approach against several hallmarks of neurodegeneration.

Crosstalk between Exercise-Derived Endocannabinoidome and Kynurenines: Potential Target Therapies for Obesity and Depression Symptoms.

The kynurenine pathway (KP) and the endocannabinoid system (ECS) are known to be deregulated in depression and obesity; however, it has been recognized that acute physical exercise has an important modulating role inducing changes in the mobilization of their respective metabolites-endocannabinoids (eCBs) and kynurenines (KYNs)-which overlap at some points, acting as important antidepressant, anti-nociceptive, anti-inflammatory, and antioxidant biomarkers. Therefore, the aim of this review is to analyze and discuss some recently performed studies to investigate the potential interactions between both systems, particularly those related to exercise-derived endocannabinoidome and kynurenine mechanisms, and to elucidate how prescription of physical exercise could represent a new approach for the clinical management of these two conditions.

Monoacylglycerol Lipase Inhibition Prevents Short-Term Mitochondrial Dysfunction and Oxidative Damage in Rat Brain Synaptosomal/Mitochondrial Fractions and Cortical Slices: Role of Cannabinoid Receptors.

Inhibition of enzymes responsible for endocannabinoid hydrolysis represents an invaluable emerging tool for the potential treatment of neurodegenerative disorders. Monoacylglycerol lipase (MAGL) is the enzyme responsible for degrading 2-arachydonoylglycerol (2-AG), the most abundant endocannabinoid in the central nervous system (CNS). Here, we tested the effects of the selective MAGL inhibitor JZL184 on the 3-nitropropinic acid (3-NP)-induced short-term loss of mitochondrial reductive capacity/viability and oxidative damage in rat brain synaptosomal/mitochondrial fractions and cortical slices. In synaptosomes, while 3-NP decreased mitochondrial function and increased lipid peroxidation, JZL184 attenuated both markers. The protective effects evoked by JZL184 on the 3-NP-induced mitochondrial dysfunction were primarily mediated by activation of cannabinoid receptor 2 (CB2R), as evidenced by their inhibition by the selective CB2R inverse agonist JTE907. The cannabinoid receptor 1 (CB1R) also participated in this effect in a lesser extent, as evidenced by the CB1R antagonist/inverse agonist AM281. In contrast, activation of CB1R, but not CB2R, was responsible for the protective effects of JZL184 on the 3-NP-iduced lipid peroxidation. Protective effects of JZL184 were confirmed in other toxic models involving excitotoxicity and oxidative damage as internal controls. In cortical slices, JZL184 ameliorated the 3-NP-induced loss of mitochondrial function, the increase in lipid peroxidation, and the inhibition of succinate dehydrogenase (mitochondrial complex II) activity, and these effects were independent on CB1R and CB2R, as evidenced by the lack of effects of AM281 and JTE907, respectively. Our novel results provide experimental evidence that the differential protective effects exerted by JZL184 on the early toxic effects induced by 3-NP in brain synaptosomes and cortical slices involve MAGL inhibition, and possibly the subsequent accumulation of 2-AG. These effects involve pro-energetic and redox modulatory mechanisms that may be either dependent or independent of cannabinoid receptors' activation.

Thallium-induced DNA damage, genetic, and epigenetic alterations.

Thallium (Tl) is a toxic heavy metal responsible for noxious effects in living organisms. As a pollutant, Tl can be found in the environment at high concentrations, especially in industrial areas. Systemic toxicity induced by this toxic metal can affect cell metabolism, including redox alterations, mitochondrial dysfunction, and activation of apoptotic signaling pathways. Recent focus on Tl toxicity has been devoted to the characterization of its effects at the nuclear level, with emphasis on DNA, which, in turn, may be responsible for cytogenetic damage, mutations, and epigenetic changes. In this work, we review and discuss past and recent evidence on the toxic effects of Tl at the systemic level and its effects on DNA. We also address Tl's role in cancer and its control.

The Endocannabinoid System in Caenorhabditis elegans.

The existence of a formal Endocannabinoid System in C. elegans has been questioned due to data showing the absence of typical cannabinoid receptors in the worm; however, the presence of a full metabolism for endocannabinoids, alternative ligands, and receptors for these agents and a considerable number of orthologous and homologous genes regulating physiological cannabinoid-like signals and responses - several of which are similar to those of mammals - demonstrates a well-structured and functional complex system in nematodes. In this review, we describe and compare similarities and differences between the Endocannabinoid System in mammals and nematodes, highlighting the basis for the integral study of this novel system in the worm.

Amyloid Beta Peptide-Mediated Alterations in Mitochondrial Dynamics and its Implications for Alzheimer's Disease.

Alzheimer's disease (AD) is considered the most frequent neurodegenerative disorder worldwide, compromising cognitive function in patients, with an average incidence of 1-3% in the open population. Protein aggregation into amyloidogenic plaques and neurofibrillary tangles, as well as neurodegeneration in the hippocampal and cortical areas, represent the neuropathological hallmarks of this disorder. Mechanisms involved in neurodegeneration include protein misfolding, augmented apoptosis, disrupted molecular signaling pathways and axonal transport, oxidative stress, inflammation, and mitochondrial dysfunction, among others. It is precisely through a disrupted energy metabolism that neural cells trigger toxic mechanisms leading to cell death. In this regard, the study of mitochondrial dynamics constitutes a relevant topic to decipher the role of mitochondrial dysfunction in neurological disorders, especially when considering that amyloid-beta peptides can target mitochondria. Specifically, the amyloid beta (Aß) peptide, known to accumulate in the brain of AD patients, has been shown to disrupt overall mitochondrial metabolism by impairing energy production, mitochondrial redox activity, and calcium homeostasis, thus highlighting its key role in the AD pathogenesis. In this work, we review and discuss recent evidence supporting the concept that mitochondrial dysfunction mediated by amyloid peptides contributes to the development of AD.

Modulatory Activity of the Endocannabinoid System in the Development and Proliferation of Cells in the CNS.

The Endocannabinoid System (ECS, also known as Endocannabinoidome) plays a key role in the function of the Central Nervous System, though the participation of this system on the early development - specifically in neuroprotection and proliferation of nerve cells - has been poorly studied. Here, we collect and describe evidence regarding how cannabinoid receptors CB1R and CB2R regulate several cell markers related to proliferation. While CB1R participates in the modulation of neuronal and glial proliferation, CB2R is involved in the proliferation of glial cells. The endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) exert significant effects on nerve cell proliferation. AEA generated during embryogenesis induces major effects on the differentiation of neuronal progenitor cells, whereas 2-AG participates in modulating cell migration events rather than affecting the neural proliferation rate. However, although the ECS has been demonstrated to participate in neuroprotection, more characterization on its role in neuronal and glial proliferation and differentiation is needed, especially in brain areas with recognized high neurogenesis rates. This has encouraged scientists to elucidate and propose specific mechanisms related with these cell proliferation mechanisms to better understand some neurodegenerative disorders such as Parkinson, Huntington and Alzheimer diseases, in which neuronal loss and poor neurogenesis are crucial factors for their onset and progression. In this review, we collect and present recent evidence published pointing to an active role of the ECS in the development and proliferation of nerve cells.

Oleamide Reduces Mitochondrial Dysfunction and Toxicity in Rat Cortical Slices Through the Combined Action of Cannabinoid Receptors Activation and Induction of Antioxidant Activity.

The potential treatment of neurodegenerative disorders requires the development of novel pharmacological strategies at the experimental level, such as the endocannabinoid-based therapies. The effects of oleamide (OEA), a fatty acid primary amide with activity on cannabinoid receptors, was tested against mitochondrial toxicity induced by the electron transport chain complex II inhibitor, 3-nitropropionic acid (3-NP), in rat cortical slices. OEA prevented the 3-NP-induced loss of mitochondrial function/cell viability at a concentration range of 5 nM-25 µM, and this protective effect was observed only when the amide was administered as pretreatment, but not as post-treatment. The preservation of mitochondrial function/cell viability induced by OEA in the toxic model induced by 3-NP was lost when the slices were pre-incubated with the cannabinoid receptor 1 (CB1R) selective inhibitor, AM281, or the cannabinoid receptor 2 (CB2R) selective inhibitor, JTE-907. The 3-NP-induced inhibition of succinate dehydrogenase (mitochondrial Complex II) activity was recovered by 25 nM OEA. The amide also prevented the increased lipid peroxidation and the changes in reduced/oxidized glutathione (GSH/GSSG) ratio induced by 3-NP. The cell damage induced by 3-NP, assessed as incorporation of cellular propidium iodide, was mitigated by OEA. Our novel findings suggest that the neuroprotective properties displayed by OEA during the early stages of damage to cortical cells involve the converging activation of CB1R and CB2R and the increase in antioxidant activity, which combined may emerge from the preservation of the functional integrity of mitochondria.

Marine Cyanobacteria as Sources of Lead Anticancer Compounds: A Review of Families of Metabolites with Cytotoxic, Antiproliferative, and Antineoplastic Effects.

The marine environment is highly diverse, each living creature fighting to establish and proliferate. Among marine organisms, cyanobacteria are astounding secondary metabolite producers representing a wonderful source of biologically active molecules aimed to communicate, defend from predators, or compete. Studies on these molecules' origins and activities have been systematic, although much is still to be discovered. Their broad chemical diversity results from integrating peptide and polyketide synthetases and synthases, along with cascades of biosynthetic transformations resulting in new chemical structures. Cyanobacteria are glycolipid, macrolide, peptide, and polyketide producers, and to date, hundreds of these molecules have been isolated and tested. Many of these compounds have demonstrated important bioactivities such as cytotoxicity, antineoplastic, and antiproliferative activity with potential pharmacological uses. Some are currently under clinical investigation. Additionally, conventional chemotherapeutic treatments include drugs with a well-known range of side effects, making anticancer drug research from new sources, such as marine cyanobacteria, necessary. This review is focused on the anticancer bioactivities of metabolites produced by marine cyanobacteria, emphasizing the identification of each variant of the metabolite family, their chemical structures, and the mechanisms of action underlying their biological and pharmacological activities.

NOTCH expression variability and relapse of breast cancer in high-risk groups.

BACKGROUND: In regards to breast cancer (BC), survival or disease-free periods are still compromised mainly in Triple Negative (TN) and HER2 tumors. The participation of estrogen receptor (ER) has been reported as crucial in the signaling pathways, including the NOTCH pathway. The study was aimed to evaluate the expression of NOTCH1 and NOTCH3 in BC and its relationship with the presence of ER, as well as with relapses. METHODS: NOTCH1 and NOTCH3 expression was evaluated in BC using Oncomine database, Breast Cancer Gene Expression Miner database and Kaplan Meier Plotter. Subsequently, detection of NOTCH1 and NOTCH3 in 100 paraffin-embedded BC samples from Mexican patients was achieved by immunohistochemistry (IHC) and RT-qPCR, a group of benign breast tumors were included as controls. Relapses were evaluated by BC subtypes and their relationship with NOTCH1 and NOTCH3 expression, as well as with ER expression. RESULTS: The analyses from public databases of TN and HER2 groups, which are estrogen receptor-negative (ERN), revealed NOTCH1 and NOTCH3 expression variability. The overexpression was associated with lower relapse-free survival (P = 0.00019). These data were concordant with results from tumor samples of patients included in this study, which showed overexpression of NOTCH1 and NOTCH3 in ERN tumors, as well as lower relapse-free survival (P < 0.0001). CONCLUSIONS: NOTCH1 and NOTCH3 were found to be overexpressed mainly in ERN tumors. HER2 and TN groups, are related to higher relapse rates. Therefore, anti-NOTCH therapy could be justified and implemented in conventional treatments of high-risk BC groups.

Thallium Induces Antiproliferative and Cytotoxic Activity in Glioblastoma C6 and U373 Cell Cultures via Apoptosis and Changes in Cell Cycle.

Thallium (Tl+) is a heavy metal that causes toxicity in several organs, including the brain. Its cytotoxic profile, combined with its affinity for tumor cells when used as a radioligand for labeling these cells, suggests its potential use as antitumor therapy. In this study, glioblastoma cell lines C6 (from rat) and U373 (from human) were exposed to increased concentrations of thallium(I) acetate (5, 10, 50, 100, or 200 µM) and several toxic endpoints were evaluated, including loss of confluence and morphological changes, loss of cell viability, changes in cell cycle, and apoptosis. Tl+ was detected in cells exposed to thallium(I) acetate, demonstrating efficient uptake mechanism. Confluence in both cell lines decreased in a concentration-dependent manner (50-200 µM), while morphological changes (cell shrinkage and decreased cell volume) were more evident at exposures to higher Tl+ concentrations. For both parameters, the effects of Tl+ were more prominent in C6 cells compared to U373 cells. The same trend was observed for cell viability, with Tl+ affecting this parameter in C6 cells at low concentrations, whereas U373 cells showed greater resistance, with significant changes observed only at the higher concentrations. C6 and U373 cells treated with Tl+ also showed morphological characteristics corresponding to apoptosis. The cytotoxic effects of Tl+ were also assessed in neural and astrocytic primary cultures from the whole rat brain. Primary neural and astrocytic cultures were less sensitive than C6 and U373 cells, showing changes in cell viability at 50 and 100 µM concentrations, respectively. Cell cycle in both brain tumor cell lines was altered by Tl+ in G1/G2 and S phases. In addition, when combined with temozolamide (500 µM), Tl+ elicited cell cycle alterations, increasing SubG1 population. Combined, our novel results characterize and validate the cytotoxic and antiproliferative effects of Tl+ in glioblastoma cells.

Alpha-Mangostin Alleviates the Short-term 6-Hydroxydopamine-Induced Neurotoxicity and Oxidative Damage in Rat Cortical Slices and in Caenorhabditis elegans.

The development, at the experimental level, of therapeutic strategies based on natural products to attenuate neurological alterations in degenerative disorders has gained attention. Antioxidant molecules exhibit both anti-inflammatory and neuroprotective properties. Alpha-mangostin (α-Man) is a natural xanthonoid isolated from the mangosteen tree with demonstrated antioxidant and cytoprotective properties. In this study, we investigated the antioxidant and protective properties of α-Man, both ex vivo and in vivo. We assessed the mitochondrial reductant capacity and oxidative damage to lipids in rat cortical slices, and several endpoints characteristic of physiological stress in the nematode, Caenorhabditis elegans (C. elegans), upon exposure to the parkinsonian neurotoxin, 6-hydroxydopamine (6-OHDA). In rat cortical slices, α-Man (25 and 50 µM) reduced the 6-OHDA (100 µM)-induced oxidative damage to lipid levels, but failed to reverse loss in cell viability. In wild-type (N2) C. elegans, α-Man (5-100 µM) protected against 6-OHDA (25 mM)-induced decrease in survival when administered either as pre- or post-treatment. Protective effects of α-Man were also observed on survival in the VC1772 strain (skn-1 KO-) exposed to 6-OHDA, though the extent of the protection was lesser than in the wild-type N2 strain. However, α-Man (5-50 µM) failed to attenuate the 6-OHDA-induced motor alterations in the N2 strain. The loss of lifespan induced by 6-OHDA in the N2 strain was fully reversed by high concentrations of α-Man. In addition, while 6-OHDA decreased the expression of glutathione S-transferase in the CL2166 C. elegans strain, α-Man preserved and stimulated the expression of this protein. α-Man (25 µM) also prevented 6-OHDA-induced dopaminergic neurodegeneration in the BZ555 C. elegans strain. Altogether, our novel results suggest that α-Man affords partial protection against several, but not all, short-term toxic effects induced by 6-OHDA in cortical slices and in a skn-1-dependent manner in C. elegans.

Chemical structure of three basic Asp-49 phospholipases A2 isolated from Crotalus molossus nigrescens venom with cytotoxic activity against cancer cells.

Snake venoms are complex mixtures of molecules with several biological activities. Among these molecules, the enzymes with phospholipase A2 activity have been extensively studied in the venoms from snakes because of their importance in the envenomation process and symptoms. The Mexican rattlesnake Crotalus molossus nigrescens is widely distributed in the Mexican plateau. Unlike other crotalids, its venom components have been poorly studied. Here, we characterized the phospholipase activity of one fraction isolated from the venom of this snake and we determined the cytotoxic and neurotoxic effects on brain tumor cells and neuronal primary cultures, respectively. After reverse phase chromatography, we obtained a fraction which was analyzed by mass spectrometry showing higher activity than that from a PLA2 from bee venom used as control. This fraction was enriched with three basic Asp49 phospholipases with molecular masses of 12.5, 13.9 and 14.2 kDa. Their complete amino acid sequences were determined, and their predicted tertiary structures were generated using the model building softwares I-tasser and Chimera. Viability assays revealed that the fraction showed cytotoxic activity against brain tumor cells (C6, RG2 and Daoy) with IC50 values ranging between 10 and 100 ng/ml, whereas an IC50 > 100 ng/ml was exerted in rat primary astrocytes. These findings might be relevant in oncological medicine due to their potential as anticancer agents and low neurotoxic effects compared to conventional drugs.

Platinum nanoparticles Protect Against Lipopolysaccharide-Induced Inflammation in Microglial BV-2 Cells via Decreased Oxidative Damage and Increased Phagocytosis.

Neuroinflammation and oxidative stress cooperate to compromise the function of the central nervous system (CNS). Colloidal platinum nanoparticles (Pt NPs) are ideal candidates for reducing the deleterious effects of neuroinflammation since they act as free radical scavengers. Here we evaluated the effects of Pt NPs on several markers of lipopolysaccharide (LPS)-induced inflammation in cultured BV-2 microglial cells. BV-2 cells were treated with increased dilutions (1-100 ppm) of Colloidal Pt and/or LPS (1-10 µg/mL) at different exposure times. Three different protocols of exposure were used combining Pt NPs and LPS: (a) conditioning-protective effect (pre-post-treat), (b) therapeutic effect (co-treat) and (c) conditioning-therapeutic effect (pre-co-treat). After exposure to LPS for 24 h, cells were used for assessment of cell viability, reactive oxygen species (ROS) generation, lactate dehydrogenase (LDH) activity, apoptosis and caspase-3 levels, cell proliferation, mitochondrial membrane potential, inducible nitric oxide (iNOS) activity, pro-inflammatory cytokine (IL-1ß, TNF-α and IL-6) levels, and phagocytic activity. Low concentrations (below or equal to 10 ppm) of Colloidal Pt prevented or ameliorated the LPS-induced increase in ROS formation, loss of mitochondrial membrane potential, induction of apoptosis, increase in LDH release, increase in pro-inflammatory cytokines and iNOS, inhibition of phagocytosis linked to microglial persistence in the M1 phase phenotype, loss of cell adhesion, differentiation and/or proliferation, as well as loss of cell viability. These protective effects were evident when cells were preconditioned with Pt NPs prior to LPS treatment. Collectively, the findings demonstrate that at low concentrations, Pt NPs can regulate the function and phenotype of BV-2 cells, activating protective mechanisms to maintain the microglial homeostasis and reduce inflammatory events triggered by the inflammatory insults induced by LPS. These preventive/protective effects on the LPS pro-inflammatory model are linked to the antioxidant properties and phagocytic activity of these NPs.

Protective Effects of Novel Substituted Triazinoindole Inhibitors of Aldose Reductase and Epalrestat in Neuron-like PC12 Cells and BV2 Rodent Microglial Cells Exposed to Toxic Models of Oxidative Stress: Comparison with the Pyridoindole Antioxidant Stobadine.

Aldose reductase (AR) catalyzes the conversion of glucose to sorbitol in a NADPH-dependent reaction, thereby increasing the production of reactive oxygen species (ROS). Since AR activation is linked to redox dysregulation and cell damage in neurodegenerative diseases, AR inhibitors (ARIs) constitute promising therapeutic tools for the treatment of these disorders. Among these compounds, the novel substituted triazinoindole derivatives cemtirestat (CMTI) and COTI, as well as the clinically employed epalrestat (EPA) and the pyridoindole-antioxidant stobadine (STB), were tested in both PC12 cells and BV2 microglia exposed to four different neurotoxic models. These include (1) oxidative stress with hydrogen peroxide (H2O2), (2) mitochondrial complex IV inhibition with NaN3, (3) endoplasmic reticulum-stress and lipotoxicity induced by palmitic acid/bovine serum albumin (PAM/BSA), and (4) advanced carbonyl compound lipotoxicity by 4-hydroxynonenal (4-HNE). All toxic compounds decreased cell viability and increased ROS formation in both PC12 and BV2 cells in a concentration-dependent manner (1-1000 µM; NaN3 < H2O2≈PAM/BSA < 4-HNE). In PC12 cells, EPA increased cell viability in all toxic models only at 1 µM, whereas CMTI restored baseline viability in all toxic models. COTI afforded protection against lipotoxicity, while STB only prevented H2O2-induced toxicity. Except for the 4-HNE model, EPA prevented ROS generation in all other toxic models, whereas CMTI, COTI, and STB prevented ROS production in all toxic models. In BV2 cells, EPA and CMTI restored baseline cell viability in all toxic models tested, while COTI and STB did not prevent the loss of viability in the NaN3 model. All ARIs and STB efficiently prevented ROS formation in all toxic models in a concentration-independent manner. The differential protective effects evoked by the novel ARIs and STB on the toxic models tested herein provide novel and relevant comparative evidence for the design of specific therapeutic strategies against neurodegenerative events associated with neurological disorders.

Redox-active phytoconstituents ameliorate cell damage and inflammation in rat hippocampal neurons exposed to hyperglycemia+Aß1-42 peptide.

Alzheimer's disease (AD) is the most common dementia causing progressive loss of memory and compromised cognitive functions. Although the neurotoxic mechanisms underlying AD have yet to be fully elucidated, hyperglycemia seems to trigger oxidative and inflammatory responses in the brain of afflicted patients. Removal of free radicals reduces the neurotoxic effects of hyperglycemia in AD models. In this study we investigated the neuroprotective effects of the antioxidant phytoconstituents oleuropein (OLE), rutin (RUT), luteolin (LUT) and S-allylcysteine (SAC) in an experimental model combining the exposure to high glucose (HG, mimicking chronic hyperglycemia) plus amyloid-ß peptide 1-42 (Aß1-42, mimicking AD) in primary hippocampal neurons. Cells were pre-treated with OLE, RUT, LUT or SAC (10-1000 nM), and then co-treated with high glucose (GLU, 150 mM) for 24 h plus 500 nM oligomeric Aß1-42 for 24 h more. Cell viability and reactive oxygen species (ROS) formation were assessed as indices of survival/toxicity and oxidative stress, respectively. Activity/expression of antioxidant enzymes, toxic adducts, inflammatory molecules, mitochondrial membrane potential (ΔΨm) and the pattern of amyloid aggregation were also assessed. The GLU + Aß1-42 treatment significantly decreased cell viability, increased ROS formation, reduced superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase activities, augmented Advanced Glycation End Products- and 4-hydroxynonenal-adducts generation, increased 3-nitrotyrosine and inflammatory outcomes such as inducible nitric oxide synthase, interleukin 1ß and Tumor Necrosis Factor α, decreased MMP and augmented amyloid aggregation. All phytoconstituents reduced in a differential manner all toxic endpoints, with SAC showing the highest efficacy in preventing loss of cell viability and oxidative damage, whereas RUT was most efficacious in mitigating inflammatory endpoints. Combined, the results of this study suggest that protection afforded by these compounds against GLU + Aß1-42-induced cell damage in hippocampal neurons is attributable to their properties as redox modulators, which might act through a concerted mechanism oriented to reduce oxidative stress and neuroinflammation.

Oleamide Induces Cell Death in Glioblastoma RG2 Cells by a Cannabinoid Receptor-Independent Mechanism.

The endocannabinoid system has been associated with antiproliferative effects in several types of tumors through cannabinoid receptor-mediated cell death mechanisms. Oleamide (ODA) is a CB1/CB2 agonist associated with cell growth and migration by adhesion and/or ionic signals associated with Gap junctions. Antiproliferative mechanisms related to ODA remain unknown. In this work, we evaluated the effects of ODA on cell viability and morphological changes in a rat RG2 glioblastoma cell line and compared these effects with primary astrocyte cultures from 8-day postnatal rats. RG2 and primary astrocyte cultures were treated with ODA at increasing concentrations (25, 50, 100, and 200 µM) for different periods of time (12, 24, and 48 h). Changes in RG2 cell viability and morphology induced by ODA were assessed by viability/mitochondrial activity test and phase contrast microscopy, respectively. The ratios of necrotic and apoptotic cell death, and cell cycle alterations, were evaluated by flow cytometry. The roles of CB1 and CB2 receptors on ODA-induced changes were explored with specific receptor antagonists. ODA (100 µM) induced somatic damage, detachment of somatic bodies, cytoplasmic polarization, and somatic shrinkage in RG2 cells at 24 and 48 h. In contrast, primary astrocytes treated at the same ODA concentrations exhibited cell aggregation but not cell damage. ODA (100 µM) increased apoptotic cell death and cell arrest in the G1 phase at 24 h in the RG2 line. The effects induced by ODA on cell viability of RG2 cells were independent of CB1 and CB2 receptors or changes in intracellular calcium transient. Results of this novel study suggest that ODA exerts specific antiproliferative effects on RG2 glioblastoma cells through unconventional apoptotic mechanisms not involving canonical signals.

Cannabinoid-profiled agents improve cell survival via reduction of oxidative stress and inflammation, and Nrf2 activation in a toxic model combining hyperglycemia+Aß1-42 peptide in rat hippocampal neurons.

Alzheimer's disease (AD) is a multifactorial neurodegenerative disorder linked to various converging toxic mechanisms. Evidence suggests that hyperglycemia induces oxidative stress, mitochondrial dysfunction, inflammation and excitotoxicity, all of which play important roles in the onset and progression of AD pathogenesis. The endocannabinoid system (ECS) orchestrates major physiological responses, including neuronal plasticity, neuroprotection, and redox homeostasis, to name a few. The multi-targeted effectiveness of the ECS emerges as a potential approach to treat AD. Here we characterized the protective properties of the endocannabinoids arachidonylethanolamide (AEA) and 2-arachidonoylglycerol (2-AG), the synthetic cannabinoids CP 55-940 and WIN 55,212-2, and the fatty acid amide hydrolase (FAAH) inhibitor URB597, on a combined hyperglycemia + oligomeric amyloid ß peptide (Aß1-42) neurotoxic model in primary hippocampal neurons which exhibit several AD features. Cells were treated with cannabinoid agents at increased concentrations (1 nM-1 µM) for 6 h, and then co-treated with 150 mM glucose (GLU, 24 h), followed by incubation with 500 nM Aß1-42 (24 h). Cell viability/survival, reactive oxygen species (ROS) levels, antioxidant enzyme (SOD, CAT, GPx and GRx) activities, biological products of oxidative damage (AGE and HNE adducts) and nitrosative stress (3-NT), several endpoints of inflammation (iNOS, IL-1ß and TNF-α), amyloid quantification, mitochondrial membrane potential, and the involvement of the Nrf2 pathway, were all evaluated. The combined high glucose + amyloid beta 1-42 (GLU + Aß1-42) condition decreased cell viability and mitochondrial membrane potential, while augmenting oxidative damage and inflammation. All agents tested preserved cell viability and stimulated mitochondrial membrane potential, while reducing all the evaluated toxic endpoints in a differential manner, with URB597 showing the highest efficacy. The neuroprotective efficacy of all cannabinoid agents, except for URB597, led to partial recruitment of specific antioxidant activity and Nrf2 pathway regulation. Our results support the neuroprotective potential of these agents at low concentrations against the damaging effects of GLU + Aß1-42, affording new potential modalities for the design of AD therapies.

S-Allylcysteine Protects Against Excitotoxic Damage in Rat Cortical Slices Via Reduction of Oxidative Damage, Activation of Nrf2/ARE Binding, and BDNF Preservation.

Neuroprotective approaches comprising different mechanisms to counteract the noxious effects of excitotoxicity and oxidative stress need validation and detailed characterization. Although S-allylcysteine (SAC) is a natural compound exhibiting a broad spectrum of protective effects characterized by antioxidant, anti-inflammatory, and neuromodulatory actions, the mechanisms underlying its protective role on neuronal cell damage triggered by early excitotoxic insults remain elusive. In this study, we evaluated if the preconditioning or the post-treatment of isolated rat cortical slices with SAC (100 µM) can ameliorate the toxic effects induced by the excitotoxic metabolite quinolinic acid (QUIN, 100 µM), and whether this protective response involves the early display of specific antioxidant and neuroprotective signals. For this purpose, cell viability/mitochondrial reductive capacity, lipid peroxidation, levels of reduced and oxidized glutathione (GSH and GSSG, respectively), the rate of cell damage, the NF-E2-related factor 2/antioxidant response element (Nrf2/ARE) binding activity, heme oxygenase 1 (HO-1) regulation, extracellular signal-regulated kinase (ERK1/2) phosphorylation, and the levels of tumor necrosis factor-alpha (TNF-α) and the neurotrophin brain-derived neurotrophic factor (BDNF) were all estimated in tissue slices exposed to SAC and/or QUIN. The incubation of slices with QUIN augmented all toxic endpoints, whereas the addition of SAC prevented and/or recovered all toxic effects of QUIN, exhibiting better results when administered 60 min before the toxin and demonstrating protective and antioxidant properties. The early stimulation of Nrf2/ARE binding activity, the upregulation of HO-1, the ERK1/2 phosphorylation and the preservation of BDNF tissue levels by SAC demonstrate that this molecule displays a wide range of early protective signals by triggering orchestrated antioxidant responses and neuroprotective strategies. The relevance of the characterization of these mechanisms lies in the confirmation that the protective potential exerted by SAC begins at the early stages of excitotoxicity and neurodegeneration and supports the design of integral prophylactic/therapeutic strategies to reduce the deleterious effects observed in neurodegenerative disorders with inherent excitotoxic events.