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Soluble epoxide hydrolase inhibitors (sEHI) are a new class of non-opioid analgesics, with a representative compound, EC5026, currently in clinical trials for the management of neuropathic pain [1]. Our group has recently designed, synthesized and pharmacologically evaluated novel series of potent benzohomoadamantane-based sEHI [2]. Herein, we report further medicinal chemistry around the abovementioned polycyclic scaffold to improve the potency and, particularly, the DMPK properties of previous hits. After an extensive in vitro screening cascade, molecular modeling, and in vivo pharmacokinetics studies, three candidates were selected for in vivo studies. Two compounds evaluated in a murine model of capsaicin-induced allodynia displayed potent anti-allodynic effect in a dose-dependent manner. Next, the most potent compound was evaluated in the cyclophosphamide-induced murine model of cystitis, a well-established model of visceral pain, presenting robust analgesic efficacy [3]. Finally, considering that chemotherapy-induced neuropathic pain (CINP), a severe side effect of several anticancer agents, is a largely unmet medical need [4], our third candidate was evaluated in a murine model of paclitaxel-induced neuropathic pain. CINP was performed by a daily injection of paclitaxel via i.p. (2 mg/kg), for 5 consecutive days. Mice developed neuropathic mechanical allodynia, which peaked on day 10 after the first paclitaxel administration ¿time when the acute effects of sEHI were tested. Subcutaneous administration of this candidate (2.5-5 mg/kg) completely reversed in a dose dependent manner the sensory hypersensitivity. Additionally, administration of the sEHI (5 mg/kg, s.c.) 30 min before each paclitaxel injection completely prevented the development of neuropathic allodynia. Collectively, these results suggest interstitial cystitis/pain bladder syndrome and CINP as possible new indications for sEHI. Acknowledgements: This work was funded by the Grant PID2020-118127RB-I

The soluble epoxide hydrolase (sEH) has been suggested as a pharmacological target for the treatment of several diseases, including pain-related disorders. Herein, we report further medicinal chemistry around new benzohomoadamantane-based sEH inhibitors (sEHI) in order to improve the drug metabolism and pharmacokinetics properties of a previous hit. After an extensive in vitro screening cascade, molecular modeling, and in vivo pharmacokinetics studies, two candidates were evaluated in vivo in a murine model of capsaicin-induced allodynia. The two compounds showed an anti-allodynic effect in a dose-dependent manner. Moreover, the most potent compound presented robust analgesic efficacy in the cyclophosphamide-induced murine model of cystitis, a well-established model of visceral pain. Overall, these results suggest painful bladder syndrome as a new possible indication for sEHI, opening a new range of applications for them in the visceral pain field.

Modulation of the risk of Alzheimer¿s disease (AD) may begin early in life. During embryo development and maturation, the brain receives maternal physiological influences and establishes new epigenetic patterns that build its level of resilience to late diseases. We treated wild-type pregnant mice with the soluble epoxide hydrolase (sEH) inhibitor TPPU until their pups were weaned. The male progenitors were AD transgenic mice from the strain 5XFAD. At two months of age, male and female mice were blindly analyzed for learning and memory performance and their brain tissue was preserved for further analysis. Once genotyped, we found that 5XFAD mice worn from vehicle-treated mothers showed a poor response in cognitive tests of object recognition and spatial location. Notably, those 5XFAD mice from TPPU-treated mothers showed similar performance to their wild-type siblings. At the molecular level, tau pathology shown by increased hippocampal p-tau levels in 5XFAD mice was totally prevented in those whose mothers were treated with TPPU. Furthermore, TPPU treatment also modified the expression of epigenetic markers. Overall, we confirmed the potential of the enzyme sEH as a new target to fight AD and demonstrated that its inhibition in the developing brain produces long-term preventive effects against neurodegeneration.

Physically active lifestyle has huge benefits for the health and well-being of people of all ages. However, excessive training can lead to severe cardiovascular events such as heart fibrosis and arrhythmia. In addition, strenuous exercise may impair brain plasticity. Here we investigate the presence of any deleterious effects induced by chronic exercise at the level of high intensity, although not reaching exhaustion. We analyzed cardiovascular, cognitive and cerebral molecular changes in young adult male mice submitted to treadmill running for eight weeks at moderate or high intensity regimens compared to sedentary mice. Exercised mice showed decreased weight gain that was significant for the high intensity level group. Exercised mice showed cardiac hypertrophy, but no morphological changes in the aorta. High-intensity training induced decrease in heart rate and increase in motor skills. However, it did not impair recognition or spatial memory and, accordingly, the expression of hippocampal neuroplasticity markers was maintained. Interestingly, catalase expression and proteasome enzymatic activity were increased in the cerebral cortex of the high-intensity trained group; both first-line mechanisms contributing to maintain redox homeostasis and prevent the accumulation of damaged proteins. Therefore, physical exercise at an intensity that induces adaptive cardiovascular changes in parallel increases antioxidant defenses to prevent brain damage and build resilience against neurodegeneration.

Physically active lifestyle has huge implications for the health and well-being of people of all ages. However, excessive training can lead to severe cardiovascular events such as heart fibrosis and arrhythmia. In addition, strenuous exercise may impair brain plasticity. Here we investigate the presence of any deleterious effects induced by chronic high-intensity exercise, although not reaching exhaustion. We analyzed cardiovascular, cognitive, and cerebral molecular changes in young adult male mice submitted to treadmill running for eight weeks at moderate or high-intensity regimens compared to sedentary mice. Exercised mice showed decreased weight gain, which was significant for the high-intensity group. Exercised mice showed cardiac hypertrophy but with no signs of hemodynamic overload. No morphological changes in the descending aorta were observed, either. High-intensity training induced a decrease in heart rate and an increase in motor skills. However, it did not impair recognition or spatial memory, and, accordingly, the expression of hippocampal and cerebral cortical neuroplasticity markers was maintained. Interestingly, proteasome enzymatic activity increased in the cerebral cortex of all trained mice, and catalase expression was significantly increased in the high-intensity group; both first-line mechanisms contribute to maintaining redox homeostasis. Therefore, physical exercise at an intensity that induces adaptive cardiovascular changes parallels increases in antioxidant defenses to prevent brain damage.

Modulation of Alzheimer′s disease (AD) risk begins early in life. During embryo development and postnatal maturation, the brain receives maternal physiological influences and establishes epigenetic patterns that build its level of resilience to late-life diseases. The soluble epoxide hydrolase inhibitor N-[1-(1-oxopropyl)-4-piperidinyl]-N′-[4-(trifluoromethoxy)phenyl] urea (TPPU), reported as ant-inflammatory and neuroprotective against AD pathology in the adult 5XFAD mouse model of AD, was administered to wild-type (WT) female mice mated to heterozygous 5XFAD males during gestation and lactation. Two-month-old 5XFAD male and female offspring of vehicle-treated dams showed memory loss as expected. Remarkably, maternal treatment with TPPU fully prevented memory loss in 5XFAD. TPPU-induced brain epigenetic changes in both WT and 5XFAD mice, modulating global DNA methylation (5-mC) and hydroxymethylation (5-hmC) and reducing the gene expression of some histone deacetylase enzymes (Hdac1 and Hdac2), might be on the basis of the long-term neuroprotection against cognitive impairment and neurodegeneration. In the neuropathological analysis, both WT and 5XFAD offspring of TPPU-treated dams showed lower levels of AD biomarkers of tau hyperphosphorylation and microglia activation (Trem2) than the offspring of vehicle-treated dams. Regarding sex differences, males and females were similarly protected by maternal TPPU, but females showed higher levels of AD risk markers of gliosis and neurodegeneration. Taken together, our results reveal that maternal treatment with TPPU impacts in preventing or delaying memory loss and AD pathology by inducing long-term modifications in the epigenetic machinery and its marks.

increase exponentially. Lifestyle factors such as nutrition, exercise, and education, among others, influence ageing progression, throughout life. Notably, the Central Nervous System (CNS) can benefit from nutritional strategies and dietary interventions that prevent signs of senescence, such as cognitive decline or neurodegenerative diseases such as Alzheimer¿s disease and Parkinson¿s Disease. The dietary polyphenol Resveratrol (RV) possesses antioxidant and cytoprotective effects, producing neuroprotection in several organisms. The oxidative stress (OS) occurs because of Reactive oxygen species (ROS) accumulation that has been proposed to explain the cause of the ageing. One of the most harmful effects of ROS in the cell is DNA damage. Nevertheless, there is also evidence demonstrating that OS can produce other molecular changes such as mitochondrial dysfunction, inflammation, apoptosis, and epigenetic modifications, among others. Interestingly, the dietary polyphenol RV is a potent antioxidant and possesses pleiotropic actions, exerting its activity through various molecular pathways. In addition, recent evidence has shown that RV mediates epigenetic changes involved in ageing and the function of the CNS that persists across generations. Furthermore, it has been demonstrated that RV interacts with gut microbiota, showing modifications in bacterial composition associated with beneficial effects. In this review, we give a comprehensive overview of the main mechanisms of action of RV in different experimental models, including clinical trials and discuss how the interconnection of these molecular events could explain the neuroprotective effects induced by RV.

Monomeric C-reactive protein (mCRP), the activated isoform of CRP, induces tissue damage in a range of inflammatory pathologies. Its detection in infarcted human brain tissue and its experimentally proven ability to promote dementia with Alzheimer’s disease (AD) traits at 4 weeks after intrahippocampal injection in mice have suggested that it may contribute to the development of AD after cerebrovascular injury. Here, we showed that a single hippocampal administration of mCRP in mice induced memory loss, lasting at least 6 months, along with neurodegenerative changes detected by increased levels of hyperphosphorylated tau protein and a decrease of the neuroplasticity marker Egr1. Furthermore, co-treatment with the monoclonal antibody 8C10 specific for mCRP showed that long-term memory loss and tau pathology were entirely avoided by early blockade of mCRP. Notably, 8C10 mitigated Egr1 decrease in the mouse hippocampus. 8C10 also protected against mCRP-induced inflammatory pathways in a microglial cell line, as shown by the prevention of increased generation of nitric oxide. Additional in vivo and in vitro neuroprotective testing with the anti-inflammatory agent TPPU, an inhibitor of the soluble epoxide hydrolase enzyme, confirmed the predominant involvement of neuroinflammatory processes in the dementia induced by mCRP. Therefore, locally deposited mCRP in the infarcted brain may be a novel biomarker for AD prognosis, and its antibody blockade opens up therapeutic opportunities for reducing post-stroke AD risk.

Cumulative evidence indicates that excessive consumption of calories from saturated fat contributes to the development of Alzheimer's disease (AD). Here, we assess the triggering and progression of AD pathology induced by a high-fat diet (HFD), and the effects of resveratrol, a polyphenol found in common dietary sources with pleiotropic neuroprotective activities. Over 16 weeks, male wild type (WT) and AD transgenic 5XFAD mice were fed a control diet, HFD (60% kcal from fat), or HFD supplemented with 0.1% resveratrol. Resveratrol protected against HFD-induced memory loss in WT mice and prevented memory loss in 5XFAD mice. Resveratrol also reduced the amyloid burden aggravated by HFD in 5XFAD, and protected against HFD-induced tau pathology in both WT and 5XFAD strains. At the mechanistic level, resveratrol inhibited the HFD-increased amyloidogenic processing of the amyloid precursor protein in both strains; it also restored abnormal high levels in the proteolytic activity of the ubiquitin-proteasome system induced by HFD, suggesting the presence of a compensatory mechanism to counteract the accumulation of aberrant proteins. Thus, our data suggest that resveratrol can correct the harmful effects of HFD in the brain and may be a potential therapeutic agent against obesity-related disorders and AD pathology.

Neuroinflammation is a risk factor for Alzheimer’s disease (AD). We sought to study the glial derangement in AD using diverse experimental models and human brain tissue. Besides classical pro-inflammatory cytokines, we analyzed chitinase 3 like 1 (CHI3L1 or YKL40) and triggering receptor expressed on myeloid cells 2 (TREM2) that are increasingly being associated with astrogliosis and microgliosis in AD, respectively. The SAMP8 mouse model of accelerated aging and AD traits showed elevated pro-inflammatory cytokines and activated microglia phenotype. Furthermore, 6-month-old SAMP8 showed an exacerbated inflammatory response to peripheral lipopolysaccharide in the hippocampus and null responsiveness at the advanced age (for this strain) of 12 months.

Neuroinflammation is present in Alzheimer’s disease (AD) and plays an important role in its physiopathology. Epoxyeicosatrienoic acids (EETs) are signalling molecules, with anti-inflammatory properties, which are metabolized by the enzyme soluble Epoxide Hydrolase (sEH). Inhibition of sEH may be a new therapy to reduce neuroinflammation. The aim of this study was to examine, in BV-2 microglial and SH-SY5Y neuroblastoma cell cultures, the anti-inflammatory and neuroprotective effect of UB-EV-52 (EV52), a newly synthesized sEH inhibitor (sEHi); TPPU, a known sEHi; and Melatonin (MEL), a reference anti-inflammatory. For this purpose, we analyzed their effects against neuroinflammatory processes (iNOS activation, leading to NO release; and NFĸB activation) induced by Lipopolysaccharide (LPS), a common pro-inflammatory compound; and C-reactive protein (CRP) and monomeric C-reactive protein (mCRP), as it has been reported that they may contribute to neuroinflammation in AD. Moreover, we studied the agent cytotoxic effects. Our results showed that TPPU was protective against NO secretion induced by LPS and by mCRP. EV52 did not show as much protection as TPPU in NO release induced by LPS, although the obtained TPPU and EV52 IC50 values were in the same concentration range. TPPU and EV52 both inhibited the analyzed inflammatory molecular pathways in BV-2 cells. Only CRP showed neurotoxic properties in both BV-2 and SH-SY5Y. Our study established the cell culture models and the optimal LPS, mCRP and CRP concentrations for testing anti-inflammatory agents and demonstrated the anti-inflammatory potency of TPPU. Therefore, these studies may contribute to find a treatment against neuroinflammation in neurodegenerative diseases.

Alzheimer’s Disease (AD) represents a serious problem for the public health due to the increase in the life expectancy that rises the number of patients affected. After ischemia or stroke, a high percent of patients develops ischemia. Inflammation in AD is widely present and it is considered as a contributor to the cognitive loss and neurodegeneration. Monomeric C reactive protein (mCRP) has been recently described as a possible link between ischemia, inflammation and dementia. Epoxyeicosatrienoic acids (EETs) have anti-inflammatory properties which disappear when the soluble Epoxide Hydrolase (sEH) metabolizes them to sub-products. Thus, inhibiting the sEH (with TPPU) or mCRP could be a potential target to treat AD. The aim of this study was to evaluate both mCRP and sEH effects in C57BL6 mice and compare them to a well-established AD mouse model: 5xFAD mice. Mice underwent stereotaxic surgery to administrate mCRP at the hippocampus. Several behavioral tests, Western Blot and RT-PCR analysis were done one month after surgery. mCRP treated mice showed recognition memory and spatial memory loss. TPPU conferred neuroprotection against cognitive loss, as well as an increase in synaptic plasticity. The molecular pathways through which mCRP and TPPU act are still unclear and further studies are planned. 5xFAD mice showed cognitive loss, amyloid pathology and neuroinflammation, as expected. Keywords

AIM: Neuroinflammation derived from ischemic damage increases the risk of both vascular dementia and Alzheimer¿s disease (AD). The monomeric C- reactive protein (mCRP) formed in the dissociation of the pentraxin CRP within the extracellular matrix of ischemic tissue, may be a causative link between stroke-associated inflammation and memory loss. We aimed to investigate the mechanism underlying neuroinflammation and loss of neuroplasticity induced by mCRP. METHODS: We used our established in vivo model of dementia by bilateral hippocampus injection of mCRP in C57BL/6j mice. Transgenic AD mice 5XFAD were used for comparison. Cell lines of microglia BV2 and HMC3 and neuroblastoma SH-SY5Y were treated with mCRP or CRP. Microglial cells were also treated with lipopolysaccharide. We assayed hippocampus tissue and cell culture extracts for gene expression and protein levels in a search of changes underlying neuroinflammation, neuroplasticity loss and neurodegeneration. RESULTS: In vivo treatment with mCRP induced long-term loss of learning and memory, analyzed up to 6 months. First molecular results revealed lower activation of signaling pathways related to plasticity early genes Arc and Egr1 in mCRP mice. Main changes in 5XFAD were related to oxidative stress and gliosis markers. A main effect on protein processing induced by mCRP was hyperphosphorylation of tau in AD-associated pathogenic residues such as Ser202 and Ser396. We are analyzing the protective effects of anti-inflammatory agents. CONCLUSIONS: Characterizing the mechanisms of mCRP-induced dementia might contribute to fight against AD incidence in the elderly.

Physical exercise performed regularly is known to improve health and to reduce the risk of age-related diseases. Furthermore, there is some evidence of cognitive improvement in physically active middle-aged and older adults. We hypothesized that long-term physically active middle-aged men may have developed brain resilience that can be detected with the analysis of peripheral blood markers. We aimed to analyze the activation of pathways potentially modulated by physical activity in a cohort of healthy amateur rugby players (n = 24) and control subjects with low physical activity (n = 25) aged 45¿65 years. We had previously reported neuropsychological improvement in immediate memory responses in the player group compared to the controls. Here, we tested the expression of selected genes of longevity, inflammation, redox homeostasis, and trophic signaling in whole blood mRNA. Analyses were also performed on blood samples of young (aged 15¿25 years) control subjects with low physical activity (n = 21). Physical activity and other lifestyle factors were thoroughly recorded with standardized questionnaires. Interestingly, middle-aged control subjects showed lower levels of expression of SIRT1, SIRT3, CAT, and SOD1 than the young controls, although rugby players maintained the expression levels of these genes at a young-like level. Middle-aged players showed lower levels of IL1B than the non-physically active groups. However, there was a tendency towards a decrease in trophic and transduction factors in middle-aged groups as compared to the young controls. A statistical study of Spearman¿s correlations supported a positive effect of sporting activity on memory and executive functions, and on peripheral gene expression of SIRT1, SIRT3 and downstream genes, in the middle-aged rugby players. Our results indicate that the SIRT1-SIRT3 axis, and associated neuroprotective signaling, may contribute to the anti-aging resilience of the brain mediated by physical exercise.

AIM: Physical activity practiced regularly is known to improve the well-being and to reduce the risk of age-related diseases. We aimed to determine the impact of long-term physical activity on memory maintenance and search for peripheral blood markers related to cognitive function that may be detectable at middle-age METHODS: We performed neuropsychological analyses to veteran amateur rugby players (n = 24, age: 46–68 y) and middleaged sedentary controls (n = 25, age: 47–67 y). We analyzed the expression of selected genes in whole blood mRNA of player and control groups and young sedentary controls (n = 21, age: 17–25 y). A young trained group (n = 16, age: 18–25 y) was added to analyze age-related response in circulating neurotrophins induced by chronic exercise

Aim: Physical exercise performed regularly is known to improve health and to reduce the risk of age-related diseases. We had previously reported improved immediate memory responses in a cohort of veteran rugby players as compared to control subjects with low physical activity. We hypothesized that long-term physically active middle-aged men have developed brain resilience that can be detected with the analysis of peripheral blood markers. Methods: We tested the expression of selected genes of longevity, inflammation, redox homeostasis, and trophic signaling in whole blood mRNA. Analyses were performed on blood samples of veteran rugby players (n=24) and controls (n=25) aged 45-65 year, and also young controls (aged 15-25 years) with low physical activity (n=21). Physical activity and other lifestyles were thoroughly recorded with standardized questionnaires. Results: Middle-aged control subjects showed lower levels of expression of SIRT1, SIRT3 and CAT than the young controls, although rugby players maintained the expression levels of these genes at a young-like level. No differences were detected in the inflammatory genes, although there was a tendency towards a decrease in trophic and transduction factors in middle-aged groups as compared to the young controls. A statistical study of Spearman’s correlations supported a positive effect of sporting activity on memory and executive functions through SIRT1, SIRT3 and antioxidant gene expression levels in the veteran rugby players. Conclusions: Our results suggest the involvement of the SIRT1-SIRT3 axis, and the downstream antioxidant and neuroprotective signaling, in the anti-aging resilience of the brain mediated by physical exercise

Aging is accompanied by a decline in memory and other brain functions. Physical exercise may mitigate this decline through the modulation of factors participating in the crosstalk between skeletal muscle and the brain, such as neurotrophins and oxidative stress parameters. We aimed to determine whether long term exercise training (35 ± 15 years) promotes memory maintenance in middle-aged men, and to characterize the changes in neurotrophic factors and lipid oxidation markers in peripheral blood samples in both middle-aged and young men. The neuropsychological analysis showed significant improvements in memory through the Free and Cued Immediate Recall tests, in the middle-aged trained individuals when compared to the sedentary ones. We found a significant decrease in the resting serum BDNF and plasma Cathepsin B (CTSB) levels in the trained groups at both middle and young ages. BDNF and CTSB levels were inversely correlated with weekly hours of exercise. We also found a significant decrease in plasma malondialdehyde, an index of lipid peroxidation, in middle-aged and young trained subjects. The positive impact of long-term exercise training by delaying the onset of physiological memory loss and the associated neurotrophic and redox peripheral modulation, suggests the effectiveness of exercise as preventive strategy against age-related memory loss and neurodegeneration.