NMDA receptor antagonists reduce amyloid-β deposition by modulating calpain-1 signaling and autophagy, rescuing cognitive impairment in 5XFAD mice

Overstimulation of N-methyl-D-aspartate receptors (NMDARs) is the leading cause of brain excitotoxicity and often contributes to neurodegenerative diseases such as Alzheimer's Disease (AD), the most common form of dementia. This study aimed to evaluate a new NMDA receptor antagonist (UB-ALT-EV) and memantine in 6-month-old female 5XFAD mice that were exposed orally to a chronic low-dose treatment. Behavioural and cognitive tests confirmed better cognitive performance in both treated groups. Calcium-dependent protein calpain-1 reduction was found after UB-ALT-EV treatment but not after memantine. Changes in spectrin breakdown products (SBDP) and the p25/p35 ratio confirmed diminished calpain-1 activity. Amyloid β (Aβ) production and deposition was evaluated in 5XFAD mice and demonstrated a robust effect of NMDAR antagonists on reducing Aβ deposition and the number and size of Thioflavin-S positive plaques. Furthermore, glycogen synthase kinase 3β (GSK3β) active form and phosphorylated tau (AT8) levels were diminished after UB-ALT-EV treatment, revealing tau pathology improvement. Because calpain-1 is involved in autophagy activation, autophagic proteins were studied. Strikingly, results showed changes in the protein levels of unc-51-like kinase (ULK1), beclin-1, microtubule-associated protein 1A/1B-light chain 3(LC3B-II)/LC3B-I ratio and lysosomal-associated membrane protein 1 (LAMP1) after NMDAR antagonist treatments, suggesting an accumulation of autophagolysosomes in 5XFAD mice, reversed by UB-ALT-EV. Likewise, treatment with UB-ALT-EV recovered a WT mice profile in apoptosis markers Bcl-2, Bax, and caspase-3. In conclusion, our results revealed the potential neuroprotective effect of UB-ALT-EV by attenuating NMDA-mediated apoptosis and reducing Aβ deposition and deposition jointly with the autophagy rescue to finally reduce cognitive alterations in a mice model of familial AD

Inhibition of 11β-HSD1 Ameliorates Cognition and Molecular Detrimental Changes after Chronic Mild Stress in SAMP8 Mice

Impaired glucocorticoid (GC) signaling is a significant factor in aging, stress, and neurodegenerative diseases such as Alzheimer’s disease. Therefore, the study of GC-mediated stress responses to chronic moderately stressful situations, which occur in daily life, is of huge interest for the design of pharmacological strategies toward the prevention of neurodegeneration. To address this issue, SAMP8 mice were exposed to the chronic mild stress (CMS) paradigm for 4 weeks and treated with RL-118, an 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) inhibitor. The inhibition of this enzyme is linked with a reduction in GC levels and cognitive improvement, while CMS exposure has been associated with reduced cognitive performance. The aim of this project was to assess whether RL-118 treatment could reverse the deleterious effects of CMS on cognition and behavioral abilities and to evaluate the molecular mechanisms that compromise healthy aging in SAMP8 mice. First, we confirmed the target engagement between RL-118 and 11β-HSD1. Additionally, we showed that DNA methylation, hydroxymethylation, and histone phosphorylation were decreased by CMS induction, and increased by RL-118 treatment. In addition, CMS exposure caused the accumulation of reactive oxygen species (ROS)-induced damage and increased pro-oxidant enzymes—as well as pro-inflammatory mediators—through the NF-κB pathway and astrogliosis markers, such as GFAP. Of note, these modifications were reversed by 11β-HSD1 inhibition. Remarkably, although CMS altered mTORC1 signaling, autophagy was increased in the SAMP8 RL-118-treated mice. We also showed an increase in amyloidogenic processes and a decrease in synaptic plasticity and neuronal remodeling markers in mice under CMS, which were consequently modified by RL-118 treatment. In conclusion, 11β-HSD1 inhibition through RL-118 ameliorated the detrimental effects induced by CMS, including epigenetic and cognitive disturbances, indicating that GC-excess attenuation shows potential as a therapeutic strategy for age-related cognitive decline and AD

Cognitive Decline and BPSD Are Concomitant with Autophagic and Synaptic Deficits Associated with G9a Alterations in Aged SAMP8 Mice

Behavioural and psychological symptoms of dementia (BPSD) are presented in 95% of Alzheimer's Disease (AD) patients and are also associated with neurotrophin deficits. The molecular mechanisms leading to age-related diseases are still unclear; however, emerging evidence has suggested that epigenetic modulation is a key pathophysiological basis of ageing and neurodegeneration. In particular, it has been suggested that G9a methyltransferase and its repressive histone mark (H3K9me2) are important in shaping learning and memory by modulating autophagic activity and synaptic plasticity. This work deepens our understanding of the epigenetic mechanisms underlying the loss of cognitive function and BPSD in AD. For this purpose, several tasks were performed to evaluate the parameters of sociability (three-chamber test), aggressiveness (resident intruder), anxiety (elevated plus maze and open field) and memory (novel object recognition test) in mice, followed by the evaluation of epigenetic, autophagy and synaptic plasticity markers at the molecular level. The behavioural alterations presented by senescence-accelerated mice prone 8 (SAMP8) of 12 months of age compared with their senescence-accelerated mouse resistant mice (SAMR1), the healthy control strain was accompanied by age-related cognitive deficits and alterations in epigenetic markers. Increased levels of G9a are concomitant to the dysregulation of the JNK pathway in aged SAMP8, driving a failure in autophagosome formation. Furthermore, lower expression of the genes involved in the memory-consolidation process modulated by ERK was observed in the aged male SAMP8 model, suggesting the implication of G9a. In any case, two of the most important neurotrophins, namely brain-derived neurotrophic factor (Bdnf) and neurotrophin-3 (NT3), were found to be reduced, along with a decrease in the levels of dendritic branching and spine density presented by SAMP8 mice. Thus, the present study characterizes and provides information regarding the non-cognitive and cognitive states, as well as molecular alterations, in aged SAMP8, demonstrating the AD-like symptoms presented by this model. In any case, our results indicate that higher levels of G9a are associated with autophagic deficits and alterations in synaptic plasticity, which could further explain the BPSD and cognitive decline exhibited by the model

Inhibition of 11β-HSD1 Ameliorates Cognition and Molecular Detrimental Changes after Chronic Mild Stress in SAMP8 Mice

Impaired glucocorticoid (GC) signaling is a significant factor in aging, stress, and neurodegenerative diseases such as Alzheimer's disease. Therefore, the study of GC-mediated stress responses to chronic moderately stressful situations, which occur in daily life, is of huge interest for the design of pharmacological strategies toward the prevention of neurodegeneration. To address this issue, SAMP8 mice were exposed to the chronic mild stress (CMS) paradigm for 4 weeks and treated with RL-118, an 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) inhibitor. The inhibition of this enzyme is linked with a reduction in GC levels and cognitive improvement, while CMS exposure has been associated with reduced cognitive performance. The aim of this project was to assess whether RL-118 treatment could reverse the deleterious effects of CMS on cognition and behavioral abilities and to evaluate the molecular mechanisms that compromise healthy aging in SAMP8 mice. First, we confirmed the target engagement between RL-118 and 11β-HSD1. Additionally, we showed that DNA methylation, hydroxymethylation, and histone phosphorylation were decreased by CMS induction, and increased by RL-118 treatment. In addition, CMS exposure caused the accumulation of reactive oxygen species (ROS)-induced damage and increased pro-oxidant enzymes as well as pro-inflammatory mediators through the NF-κB pathway and astrogliosis markers, such as GFAP. Of note, these modifications were reversed by 11β-HSD1 inhibition. Remarkably, although CMS altered mTORC1 signaling, autophagy was increased in the SAMP8 RL-118-treated mice. We also showed an increase in amyloidogenic processes and a decrease in synaptic plasticity and neuronal remodeling markers in mice under CMS, which were consequently modified by RL-118 treatment. In conclusion, 11β-HSD1 inhibition through RL-118 ameliorated the detrimental effects induced by CMS, including epigenetic and cognitive disturbances, indicating that GC-excess attenuation shows potential as a therapeutic strategy for age-related cognitive decline and AD

Pharmacological inhibition of soluble epoxide hydrolase protects cognitive impairment in a Niemann-Pick mice model

Niemann-Pick type C (NPC) disease is a childhood autosomal recessive inherited rare neurodegenerative disease characterized by accumulation of cholesterol and glycosphingolipids and where the autophagy-lysosome system and inflammatory processes are implicated in the pathogenesis of the disease. We follow a novel approach to deal with NPC disease, by modulating key features of the disease such as inflammation and autophagy, through inhibition of soluble epoxide hydrolase (sEH)

Environmental Enrichment Modified Epigenetic Mechanisms in SAMP8 Mouse Hippocampus by Reducing Oxidative Stress and Inflammaging and Achieving Neuroprotection

With the increase in life expectancy, aging and age-related cognitive impairments arebecoming one of the most important issues for human health. At the same time, ithas been shown that epigenetic mechanisms are emerging as universally importantfactors in life expectancy. The Senescence Accelerated Mouse P8 (SAMP8) strainexhibits age-related deterioration evidenced in learning and memory abilities and is auseful model of neurodegenerative disease. In SAMP8, Environmental Enrichment (EE)increased DNA-methylation levels (5-mC) and reduced hydroxymethylation levels (5-hmC), as well as increased histone H3 and H4 acetylation levels. Likewise, we foundchanges in the hippocampal gene expression of some chromatin-modifying enzymegenes, such asDnmt3b,Hdac1,Hdac2,Sirt2, andSirt6.Subsequently, we assessedthe effects of EE on neuroprotection-related transcription factors, such as the Nuclearregulatory factor 2 (Nrf2)-Antioxidant Response Element pathway and Nuclear Factorkappa Beta (NF-κB), which play critical roles in inflammation. We found that EE producesan increased expression of antioxidant genes, such asHmox1,Aox1, andCox2, andreduced the expression of inflammatory genes such asIL-6andCxcl10, all of this withinthe epigenetic context modified by EE. In conclusion, EE prevents epigenetic changesthat promote or drive oxidative stress and inflammagin

A Novel NMDA Receptor Antagonist Protects against Cognitive Decline Presented by Senescent Mice

Alzheimer’s disease (AD) is the leading cause of dementia. Non-competitive N-Methyl-D-aspartate (NMDA) receptor antagonist memantine improved cognition and molecular alterations after preclinical treatment. Nevertheless, clinical results are discouraging. In vivo efficacy of the RL-208, a new NMDA receptor blocker described recently, with favourable pharmacokinetic properties was evaluated in Senescence accelerated mice prone 8 (SAMP8), a mice model of late-onset AD (LOAD). Oral administration of RL-208 improved cognitive performance assessed by using the three chamber test (TCT), novel object recognition test (NORT), and object location test (OLT). Consistent with behavioural results, RL-208 treated-mice groups significantly changed NMDAR2B phosphorylation state levels but not NMDAR2A. Calpain-1 and Caspase-3 activity was reduced, whereas B-cell lymphoma-2 (BCL-2) levels increased, indicating reduced apoptosis in RL-208 treated SAMP8. Superoxide Dismutase 1 (SOD1) and Glutathione Peroxidase 1 (GPX1), as well as a reduction of hydrogen peroxide (H2O2), was also determined in RL-208 mice. RL-208 treatment induced an increase in mature brain-derived neurotrophic factor (mBDNF), prevented Tropomyosin-related kinase B full-length (TrkB-FL) cleavage, increased protein levels of Synaptophysin (SYN) and Postsynaptic density protein 95 (PSD95). In whole, these results point out to an improvement in synaptic plasticity. Remarkably, RL-208 also decreased the protein levels of Cyclin-Dependent Kinase 5 (CDK5), as well as p25/p35 ratio, indicating a reduction in kinase activity of CDK5/p25 complex. Consequently, lower levels of hyperphosphorylated Tau (p-Tau) were found. In sum, these results demonstrate the neuroprotectant role of RL-208 through NMDAR blockadeThis research was funded by Ministerio de Economía, Industria y Competitividad (Agencia Estatal de Investigación, AEI) and Fondo Europeo de Desarrollo Regional (MINECO-FEDER) (Projects SAF2017-82771-R, SAF2016-77703, SAF2015-68749 and SAF2017-90913), Xunta de Galicia (ED431C 2018/21) and Generalitat de Catalunya (2017 SGR 106)S

A Novel NMDA Receptor Antagonist Protects against Cognitive Decline Presented by Senescent Mice

Alzheimer's disease (AD) is the leading cause of dementia. Non-competitive N-Methyl-D-aspartate (NMDA) receptor antagonist memantine improved cognition and molecular alterations after preclinical treatment. Nevertheless, clinical results are discouraging. In vivo e cacy of the RL-208, a new NMDA receptor blocker described recently, with favourable pharmacokinetic properties was evaluated in Senescence accelerated mice prone 8 (SAMP8), a mice model of late-onset AD (LOAD). Oral administration of RL-208 improved cognitive performance assessed by using the three chamber test (TCT), novel object recognition test (NORT), and object location test (OLT). Consistent with behavioural results, RL-208 treated-mice groups significantly changed NMDAR2B phosphorylation state levels but not NMDAR2A. Calpain-1 and Caspase-3 activity was reduced, whereas B-cell lymphoma-2 (BCL-2) levels increased, indicating reduced apoptosis in RL-208 treated SAMP8. Superoxide Dismutase 1 (SOD1) and Glutathione Peroxidase 1 (GPX1), as well as a reduction of hydrogen peroxide (H2O2), was also determined in RL-208 mice. RL-208 treatment induced an increase in mature brain-derived neurotrophic factor (mBDNF), prevented Tropomyosin-related kinase B full-length (TrkB-FL) cleavage, increased protein levels of Synaptophysin (SYN) and Postsynaptic density protein 95 (PSD95). In whole, these results point out to an improvement in synaptic plasticity. Remarkably, RL-208 also decreased the protein levels of Cyclin-Dependent Kinase 5 (CDK5), as well as p25/p35 ratio, indicating a reduction in kinase activity of CDK5/p25 complex. Consequently, lower levels of hyperphosphorylated Tau (p-Tau) were found. In sum, these results demonstrate the neuroprotectant role of RL-208 through NMDAR blockade

Structure-Based Virtual Screening and in vitro and in vivo Analyses Revealed Potent Methyltransferase G9a Inhibitors as Prospective Anti-Alzheimer's Agents

G9a is a lysine methyltransferase able to di-methylate lysine 9 of histone H3, promoting the repression of genes involved in learning and memory. Novel strategies based on synthesizing epigenetic drugs could regulate gene expression through histone post-translational modifications and effectively treat neurodegenerative diseases, like Alzheimer's disease (AD). Here, potential G9a inhibitors were identified using a structure-based virtual screening against G9a, followed by in vitro and in vivo screenings. First, screening methods with the AD transgenic Caenorhabditis elegans strain CL2006, showed that the toxicity/function range was safe and recovered age-dependent paralysis. Likewise, we demonstrated that the best candidates direct target G9a by reducing H3 K9me2 in the CL2006 strain. Further characterization of these compounds involved the assessment of the blood-brain barrier-permeability and impact on amyloid-β aggregation, showing promising results. Thus, we present a G9a inhibitor candidate, F, with a novel and potent structure, providing both leads in G9a inhibitor design and demonstrating their participation in reducing AD pathology. Keywords: Alzheimer's disease; G9a methyltransferase; amyloid-β; epigenetics; structure based virtual screening

Temporal Integrative Analysis of mRNA and microRNAs Expression Profiles and Epigenetic Alterations in Female SAMP8, a Model of Age-Related Cognitive Decline

A growing body of research shows that epigenetic mechanisms are critically involved in normal and pathological aging. The Senescence-Accelerated Mouse Prone 8 (SAMP8) can be considered a useful tool to better understand the dynamics of the global epigenetic landscape during the aging process since its phenotype is not fully explained by genetic factors. Here we investigated dysfunctional age-related transcriptional profiles and epigenetic programming enzymes in the hippocampus of 2- and 9-month-old SAMP8 female mice using the Senescent-Accelerated Resistant 1 (SAMR1) mouse strain as control. SAMP8 mice presented 1,062 genes dysregulated at 2 months of age, and 1,033 genes at 9 months, with 92 genes concurrently dysregulated at both ages compared to age-matched SAMR1. SAMP8 mice showed a significant decrease in global DNA methylation (5-mC) at 2 months while hydroxymethylation (5-hmC) levels were increased in SAMP8 mice at 2 and 9 months of age compared to SAMR1. These changes were accompanied by changes in the expression of several enzymes that regulate 5-mC and methylcytosine oxidation. Acetylated H3 and H4 histone levels were significantly diminished in SAMP8 mice at 2-month-old compared to SAMR1 and altered Histone DeACetylase (HDACs) profiles were detected in both young and old SAMP8 mice. We analyzed 84 different mouse miRNAs known to be altered in neurological diseases or involved in neuronal development. Compared with SAMR1, SAMP8 mice showed 28 and 17 miRNAs differentially expressed at 2 and 9 months of age, respectively; 6 of these miRNAs overlapped at both ages. We used several bioinformatic approaches to integrate our data in mRNA:miRNA regulatory networks and functional predictions for young and aged animals. In sum, our study reveals interplay between epigenetic mechanisms and gene networks that seems to be relevant for the progression toward a pathological aging and provides several potential markers and therapeutic candidates for Alzheimer's Disease (AD) and age-related cognitive impairment