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.

Anandamide and WIN 55212-2 Afford Protection in Rat Brain Mitochondria in a Toxic Model Induced by 3-Nitropropionic Acid: an In Vitro Study.

Mitochondrial dysfunction plays a key role in the development of neurodegenerative disorders. In contrast, the regulation of the endocannabinoid system has been shown to promote neuroprotection in different neurotoxic paradigms. The existence of an active form of the cannabinoid receptor 1 (CB1R) in mitochondrial membranes (mitCB1R), which might exert its effects through the same signaling mechanisms as the cell membrane CB1R, has been shown to regulate mitochondrial activity. Although there is evidence suggesting that some cannabinoids may induce protective effects on isolated mitochondria, substantial evidence on the role of cannabinoids in mitochondria remains to be explored. In this work, we developed a toxic model of mitochondrial dysfunction induced by exposure of brain mitochondria to the succinate dehydrogenase inhibitor 3-nitropropionic acid (3-NP). Mitochondria were also pre-incubated with the endogenous agonist anandamide (AEA) and the synthetic CB1R agonist WIN 55212-2 to evaluate their protective effects. Mitochondrial reduction capacity, reactive oxygen species (ROS) formation, and mitochondrial swelling were assessed as toxic markers. While 3-NP decreased the mitochondrial reduction capacity and augmented mitochondrial ROS formation and swelling, both AEA and WIN 55212-2 ameliorated these toxic effects. To explore the possible involvement of mitCB1R activation on the protective effects of AEA and WIN 55212-2, mitochondria were also pre-incubated in the presence of the selective CB1R antagonist AM281, which completely reverted the protective effects of the cannabinoids to levels similar to those evoked by 3-NP. These results show partial protective effects of cannabinoids, suggesting that mitCB1R activation may be involved in the recovery of compromised mitochondrial activity, related to reduction of ROS formation and further prevention of mitochondrial swelling.

Neuroprotective and antioxidant effects of docosahexaenoic acid (DHA) in an experimental model of multiple sclerosis.

Multiple sclerosis (MS) is a chronic demyelinating disease, whose etiology is not yet fully understood, although there are several factors that can increase the chances of suffering from it. These factors include nutrition, which may be involved in the pathogenesis of the disease. In relation to nutrition, docosahexaenoic acid (DHA), an omega-3 polyunsaturated fatty acid (n-3 PUFA), has emerged as an important player in the regulation of neuroinflammation, being considered a pleiotropic molecule. This study aimed to evaluate the effect of DHA supplementation on clinical state and oxidative stress produced by experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Twenty-five Dark Agouti rats which were used divided into Control Group, Control+Vehicle Group, Control+DHA Group, EAE Group, and EAE+DHA Group. DHA was administered for 51 days by intraperitoneal (i.p.) injection at a dose of 40 mg/kg, once a day, 5 days a week. DHA supplementation produced a decrease in oxidative stress, as well as an improvement in the clinical score of the disease. DHA could exert a beneficial effect on the clinic of MS, through the activation of the antioxidant factor Nrf2.

Sodium chloride-induced changes in oxidative stress, inflammation, and dysbiosis in experimental multiple sclerosis.

Objectives: The high-salt diet (HSD) has been associated with cognitive dysfunction by attacking the cerebral microvasculature, through an adaptive response, initiated in the intestine and mediated by Th17 cells. In the animal model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE), it has been described that NaCl causes an increase in T cell infiltration in the central nervous system. NaCl also promotes macrophage response and Th17 cell differentiation, worsening the course of the disease. HSD may trigger an activation of the immune system and enhance inflammation. However, certain studies not only do not support this possibility, but support the opposite, as the effect of salt on immune cells may not necessarily be pathogenic. Therefore, this study aimed to evaluate the effect of an over intake of salt in rats with EAE, based on the clinical course, oxidative stress, markers of inflammation and the gut dysbiosis.Methods: 15 Dark Agouti rats were used, which were divided into control group, EAE group and EAE + NaCl group. Daily 0.027 g of NaCl dissolved in 300 µl of H2O was administered through a nasogastric tube for 51 days.Results: NaCl administration produced an improvement in clinical status and a decrease in biomarkers of oxidative stress, inflammation, and dysbiosis.Conclusion: The underlying mechanism by which NaCl causes these effects could involve the renin-angiotensin-aldosterone system (RAAS), which is blocked by high doses of salt.

Effect of the Combination of Different Therapies on Oxidative Stress in the Experimental Model of Multiple Sclerosis.

Oxidative stress is heavily involved in several pathological features of Multiple Sclerosis (MS), such as myelin destruction, axonal degeneration, and inflammation. Different therapies have been shown to reduce the oxidative stress that occurs in the animal model of MS, experimental autoimmune encephalomyelitis (EAE). Some of these therapies are transcranial magnetic stimulation (TMS), extra virgin olive oil (EVOO) and S-allyl cysteine (SAC). This study aims to test the antioxidant effect of these three therapies, to compare the efficacy of SAC versus TMS and EVOO, and to analyze the effect of combining SAC + TMS and SAC and EVOO. Seventy Dark Agouti rats were used, which were divided into Control group; Vehicle group; Mock group; SAC; EVOO; TMS; SAC + EVOO; SAC + TMS; EAE; EAE + SAC; EAE + EVOO; EAE + TMS; EAE + SAC + EVOO; EAE + SAC + TMS. The TMS consisted of an oscillatory magnetic field in the form of a sine wave with a frequency of 60 Hz and an amplitude of 0.7mT (EL-EMF) applied for two hours in the morning, once a day, five days a week. SAC was administered at a dose of 50 mg/kg body weight, orally daily, five days a week. EVOO represented 10% of their calorie intake in the total standard daily diet of rats AIN-93G. All treatments were maintained for 51 days. TMS, EVOO and SAC, alone or in combination, reduce oxidative stress, increasing antioxidant defenses and also lowering the clinical score. Combination therapies do not appear to be more potent than individual therapies against the oxidative stress of EAE or its clinical symptoms.

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.

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.

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.

The ultrastructure of muscle fibers and satellite cells in experimental autoimmune encephalomyelitis after treatment with transcranial magnetic stimulation.

In this study, we investigated the effect of transcranial magnetic stimulation (TMS) on the ultrastructure of muscle fibers and satellite cells in rats with experimental autoimmune encephalomyelitis (EAE). EAE-induced animals were treated with TMS (60 Hz at 0.7 mT) for 2 hours in the morning, once a day, 5 days a week, for 3 weeks, starting on day 15 post-immunization. The rats were sacrificed on day 36 post-immunization, and the soleus muscles were evaluated by light microscopy and transmission electron microscopy. Findings were compared with a non-treated EAE group. Electron microscopy analysis showed the presence of degenerated mitochondria, autophagic vacuoles, and altered myofibrils in non-treated EAE group. This correlates with the presence of acid phosphatase activity in muscle fibers and core-targetoid lesions with desmin immunohistochemistry. Most myonuclei in the EAE group showed apoptotic features. In contrast, EAE induced-TMS treated animals had less ultrastructural changes in the mitochondria and the myofibrils, together with less frequent apoptotic nuclear features. Peripheral desmin+ protrusions, as a marker of active satellite cells, were significantly increased in TMS-treated group. This correlates ultrastructurally with the presence of active features in satellite cells in the TMS group. In conclusion, the attenuation of ultrastructural alterations in muscle fibers and activation response of satellite cells caused by EAE indicated that skeletal muscle had a regenerative response to TMS.

SARS-CoV-2 infection in multiple sclerosis patients: interaction with treatments, adjuvant therapies, and vaccines against COVID-19.

The SARS-CoV-2 pandemic has raised particular concern for people with Multiple Sclerosis, as these people are believed to be at increased risk of infection, especially those being treated with disease-modifying therapies. Therefore, the objective of this review was to describe how COVID-19 affects people who suffer from Multiple Sclerosis, evaluating the risk they have of suffering an infection by this virus, according to the therapy to which they are subjected as well as the immune response of these patients both to infection and vaccines and the neurological consequences that the virus can have in the long term. The results regarding the increased risk of infection due to treatment are contradictory. B-cell depletion therapies may cause patients to have a lower probability of generating a detectable neutralizing antibody titer. However, more studies are needed to help understand how this virus works, paying special attention to long COVID and the neurological symptoms that it causes.

COVID-19 contagion forecasting framework based on curve decomposition and evolutionary artificial neural networks: A case study in Andalusia, Spain.

Many types of research have been carried out with the aim of combating the COVID-19 pandemic since the first outbreak was detected in Wuhan, China. Anticipating the evolution of an outbreak helps to devise suitable economic, social and health care strategies to mitigate the effects of the virus. For this reason, predicting the SARS-CoV-2 transmission rate has become one of the most important and challenging problems of the past months. In this paper, we apply a two-stage mid and long-term forecasting framework to the epidemic situation in eight districts of Andalusia, Spain. First, an analytical procedure is performed iteratively to fit polynomial curves to the cumulative curve of contagions. Then, the extracted information is used for estimating the parameters and structure of an evolutionary artificial neural network with hybrid architectures (i.e., with different basis functions for the hidden nodes) while considering single and simultaneous time horizon estimations. The results obtained demonstrate that including polynomial information extracted during the training stage significantly improves the mid- and long-term estimations in seven of the eight considered districts. The increase in average accuracy (for the joint mid- and long-term horizon forecasts) is 37.61% and 35.53% when considering the single and simultaneous forecast approaches, respectively.

Melatonin and multiple sclerosis: antioxidant, anti-inflammatory and immunomodulator mechanism of action.

BACKGROUND: Melatonin is an indole hormone secreted primarily by the pineal gland that showing anti-oxidant, anti-inflammatory and anti-apoptotic capacity. It can play an important role in the pathophysiological mechanisms of various diseases. In this regard, different studies have shown that there is a relationship between Melatonin and Multiple Sclerosis (MS). MS is a chronic immune-mediated disease of the Central Nervous System. AIM: The objective of this review was to evaluate the mechanisms of action of melatonin on oxidative stress, inflammation and intestinal dysbiosis caused by MS, as well as its interaction with different hormones and factors that can influence the pathophysiology of the disease. RESULTS: Melatonin causes a significant increase in the levels of catalase, superoxide dismutase, glutathione peroxidase, glutathione and can counteract and inhibit the effects of the NLRP3 inflammasome, which would also be beneficial during SARS-CoV-2 infection. In addition, melatonin increases antimicrobial peptides, especially Reg3ß, which could be useful in controlling the microbiota. CONCLUSION: Melatonin could exert a beneficial effect in people suffering from MS, running as a promising candidate for the treatment of this disease. However, more research in human is needed to help understand the possible interaction between melatonin and certain sex hormones, such as estrogens, to know the potential therapeutic efficacy in both men and women.

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.

EHO-85: A Multifunctional Amorphous Hydrogel for Wound Healing Containing Olea europaea Leaf Extract: Effects on Wound Microenvironment and Preclinical Evaluation.

The prevalence of chronic wounds is increasing due to the population aging and associated pathologies, such as diabetes. These ulcers have an important socio-economic impact. Thus, it is necessary to design new products for their treatment with an adequate cost/effectiveness ratio. Among these products are amorphous hydrogels. Their composition can be manipulated to provide a favorable environment for ulcer healing. The aim of this study was to evaluate a novel multifunctional amorphous hydrogel (EHO-85), containing Olea europaea leaf extract, designed to enhance the wound healing process. For this purpose, its moistening ability, antioxidant capacity, effect on pH in the wound bed of experimental rats, and the effect on wound healing in a murine model of impaired wound healing were assessed. EHO-85 proved to be a remarkable moisturizer and its application in a rat skin wound model showed a significant antioxidant effect, decreasing lipid peroxidation in the wound bed. EHO-85 also decreased the pH of the ulcer bed from day 1. In addition, in mice (BKS. Cg-m +/+ Leprdb) EHO-85 treatment showed superior wound healing rates compared to hydrocolloid dressing. In conclusion, EHO-85 can speed up the closure of hard-to-heal wounds due to its multifunctional properties that are able to modulate the wound microenvironment, mainly through its remarkable effect on reactive oxygen species, pH, and moistening regulation.

Effects of transcranial magnetic stimulation on neurobiological changes in Alzheimer's disease (Review).

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by cognitive decline and brain neuronal loss. A pioneering field of research in AD is brain stimulation via electromagnetic fields (EMFs), which may produce clinical benefits. Noninvasive brain stimulation techniques, such as transcranial magnetic stimulation (TMS), have been developed to treat neurological and psychiatric disorders. The purpose of the present review is to identify neurobiological changes, including inflammatory, neurodegenerative, apoptotic, neuroprotective and genetic changes, which are associated with repetitive TMS (rTMS) treatment in patients with AD. Furthermore, it aims to evaluate the effect of TMS treatment in patients with AD and to identify the associated mechanisms. The present review highlights the changes in inflammatory and apoptotic mechanisms, mitochondrial enzymatic activities, and modulation of gene expression (microRNA expression profiles) associated with rTMS or sham procedures. At the molecular level, it has been suggested that EMFs generated by TMS may affect the cell redox status and amyloidogenic processes. TMS may also modulate gene expression by acting on both transcriptional and post­transcriptional regulatory mechanisms. TMS may increase brain cortical excitability, induce specific potentiation phenomena, and promote synaptic plasticity and recovery of impaired functions; thus, it may re­establish cognitive performance in patients with AD.