Dysfunctional mitochondria in age-related neurodegeneration: Utility of melatonin as an antioxidant treatment.

Mitochondria functionally degrade as neurons age. Degenerative changes cause inefficient oxidative phosphorylation (OXPHOS) and elevated electron leakage from the electron transport chain (ETC) promoting increased intramitochondrial generation of damaging reactive oxygen and reactive nitrogen species (ROS and RNS). The associated progressive accumulation of molecular damage causes an increasingly rapid decline in mitochondrial physiology contributing to aging. Melatonin, a multifunctional free radical scavenger and indirect antioxidant, is synthesized in the mitochondrial matrix of neurons. Melatonin reduces electron leakage from the ETC and elevates ATP production; it also detoxifies ROS/RNS and via the SIRT3/FOXO pathway it upregulates activities of superoxide dismutase 2 and glutathione peroxidase. Melatonin also influences glucose processing by neurons. In neurogenerative diseases, neurons often adopt Warburg-type metabolism which excludes pyruvate from the mitochondria causing reduced intramitochondrial acetyl coenzyme A production. Acetyl coenzyme A supports the citric acid cycle and OXPHOS. Additionally, acetyl coenzyme A is a required co-substrate for arylalkylamine-N-acetyl transferase, which rate limits melatonin synthesis; therefore, melatonin production is diminished in cells that experience Warburg-type metabolism making mitochondria more vulnerable to oxidative stress. Moreover, endogenously produced melatonin diminishes during aging, further increasing oxidative damage to mitochondrial components. More normal mitochondrial physiology is preserved in aging neurons with melatonin supplementation.

Melatonin Augments the Expression of Core Transcription Factors in Aged and Alzheimer's Patient Skin Fibroblasts.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder. Altered neurogenesis and the appearance of AD pathological hallmarks are fundamental to this disease. SRY-Box transcription factor 2 (Sox2), octamer-binding transcription factor 4 (Oct4), and Nanog are a set of core transcription factors that play a very decisive role in the preservation of pluripotency and the self-renewal capacity of embryonic and adult stem cells. These factors are critically involved in AD pathogenesis, senescence, and aging. Skin fibroblasts are emblematic of cellular damage in patients. We, therefore, in the present study, analyzed the basal expression of these factors in young, aged, and AD fibroblasts. AD fibroblasts displayed an altered expression of these factors, differing from aged and young fibroblasts. Since melatonin is well acknowledged for its anti-aging, anti-senescence and anti-AD therapeutic benefits, we further investigated the effects of melatonin treatment on the expression of these factors in fibroblasts, along with precise validation of the observed data in human neuroblastoma SH-SY5Y cells. Our findings reveal that melatonin administration augmented the expression levels of Sox2, Oct4, and Nanog significantly in both cells. Altogether, our study presents the neuroprotective potential and efficacy of melatonin, which might have significant therapeutic benefits for aging and AD patients.

A Complex Interplay Between Melatonin and RORß: RORß is Unlikely a Putative Receptor for Melatonin as Revealed by Biophysical Assays.

A nuclear retinoic acid receptor (RAR)-related orphan receptor ß (RORß) is strictly expressed in the brain, particularly in the pineal gland where melatonin is primarily synthesized and concentrated. The controversial issues regarding the direct interaction of melatonin toward ROR receptors have prompted us to investigate the potential melatonin binding sites on different ROR isoforms. We adopted computational and biophysical approaches to investigate the potential of melatonin as the ligand for RORs, in particular RORß. Herein, possible melatonin binding sites were predicted by molecular docking on human RORs. The results showed that melatonin might be able to bind within the ligand-binding domain (LBD) of all RORs, despite their difference in sequence homology. The predicted melatonin binding scores were comparable to binding energies with respect to those of melatonin interaction to the well-characterized membrane receptors, MT1 and MT2. Although the computational analyses suggested the binding potential of melatonin to the LBD of RORß, biophysical validation failed to confirm the binding. Melatonin was unable to alter the stability of human RORß as shown by the unaltered melting temperatures upon melatonin administration in differential scanning fluorometry (DSF). A thermodynamic isothermal titration calorimetry (ITC) profile showed that melatonin did not interact with human RORß in solutions, even in the presence of SRC-1 co-activator peptide. Although the direct interaction between the LBD of RORß could not be established, RORα and RORß gene expressions were increased upon 24 h treatment with µM-range melatonin. Our data, thus, support the studies that the nuclear effects of melatonin may not be directly mediated via its interaction with the RORß. These findings warrant further investigation on how melatonin interacts with ROR signaling and urge the melatonin research community for a paradigm shift in the direct interaction of melatonin toward RORs. The quest to identify nuclear receptors for melatonin in neuronal cells remains valid for the community to achieve.

Huperzine A Regulates the Physiological Homeostasis of Amyloid Precursor Protein Proteolysis and Tau Protein Conformation-A Computational and Experimental Investigation.

The beneficial actions of the natural compound Huperzine A (Hup A) against age-associated learning and memory deficits promote this compound as a nootropic agent. Alzheimer's disease (AD) pathophysiology is characterized by the accumulation of amyloid beta (Aß). Toxic Aß oligomers account for the cognitive dysfunctions much before the pathological lesions are manifested in the brain. In the present study, we investigated the effects of Hup A on amyloid precursor protein (APP) proteolysis in SH-SY5Y neuroblastoma cells. Hup A downregulated the expression of ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) and presenilin 1 (PS1) levels but augmented the levels of A disintegrin and metalloproteinase 10 (ADAM10) with significant decrement in the Aß levels. We herein report for the first time an in silico molecular docking analysis that revealed that Hup A binds to the functionally active site of BACE1. We further analyzed the effect of Hup A on glycogen synthase kinase-3 ß (GSK3ß) and phosphorylation status of tau. In this scenario, based on the current observations, we propose that Hup A is a potent regulator of APP processing and capable of modulating tau homeostasis under physiological conditions holding immense potential in preventing and treating AD like disorders.

Melatonin Inhibits Hypoxia-Induced Alzheimer's Disease Pathogenesis by Regulating the Amyloidogenic Pathway in Human Neuroblastoma Cells.

Stroke and Alzheimer's disease (AD) are prevalent age-related diseases; however, the relationship between these two diseases remains unclear. In this study, we aimed to investigate the ability of melatonin, a hormone produced by the pineal gland, to alleviate the effects of ischemic stroke leading to AD by observing the pathogenesis of AD hallmarks. We utilized SH-SY5Y cells under the conditions of oxygen-glucose deprivation (OGD) and oxygen-glucose deprivation and reoxygenation (OGD/R) to establish ischemic stroke conditions. We detected that hypoxia-inducible factor-1α (HIF-1α), an indicator of ischemic stroke, was highly upregulated at both the protein and mRNA levels under OGD conditions. Melatonin significantly downregulated both HIF-1α mRNA and protein expression under OGD/R conditions. We detected the upregulation of ß-site APP-cleaving enzyme 1 (BACE1) mRNA and protein expression under both OGD and OGD/R conditions, while 10 µM of melatonin attenuated these effects and inhibited beta amyloid (Aß) production. Furthermore, we demonstrated that OGD/R conditions were able to activate the BACE1 promoter, while melatonin inhibited this effect. The present results indicate that melatonin has a significant impact on preventing the aberrant development of ischemic stroke, which can lead to the development of AD, providing new insight into the prevention of AD and potential stroke treatments.

Cytotoxic stress caused by azalamellarin D (AzaD) interferes with cellular protein translation by targeting the nutrient-sensing kinase mTOR.

Analogs of pyrrole alkaloid lamellarins exhibit anticancer activity by modulating multiple cellular events. Lethal doses of several lamellarins were found to enhance autophagy flux in HeLa cells, suggesting that lamellarins may modulate protein homeostasis through the interference of proteins or kinases controlling energy and nutrient metabolism. To further delineate molecular mechanisms and their targets, our results herein show that azalamellarin D (AzaD) cytotoxicity could cause translational attenuation, as indicated by a change in eIF2α phosphorylation. Intriguingly, acute AzaD treatment promoted the phosphorylation of GCN2, a kinase that transduces the integrated stress response (ISR), and prolonged exposure to AzaD could increase the levels of the phosphorylated forms of eIF2α and the other ISR kinase protein kinase R (PKR). However, the effects of AzaD on ISR signalling were marginally abrogated in cells with genetic deletion of GCN2 and PKR, and evaluation of protein target engagement by cellular thermal shift assay (CETSA) revealed no significant interaction between AzaD and ISR kinases. Further investigation revealed that acute AzaD treatment negatively affected mechanistic target of rapamycin (mTOR) phosphorylation and signalling. The analyses by CETSA and computational modelling indicated that mTOR may be a possible protein target for AzaD. These findings indicate the potential for developing lamellarins as novel agents for cancer treatment.

Molecular Mechanisms Associated with Neurodegeneration of Neurotropic Viral Infection.

Viral infections of the central nervous system (CNS) cause variable outcomes from acute to severe neurological sequelae with increased morbidity and mortality. Viral neuroinvasion directly or indirectly induces encephalitis via dysregulation of the immune response and contributes to the alteration of neuronal function and the degeneration of neuronal cells. This review provides an overview of the cellular and molecular mechanisms of virus-induced neurodegeneration. Neurotropic viral infections influence many aspects of neuronal dysfunction, including promoting chronic inflammation, inducing cellular oxidative stress, impairing mitophagy, encountering mitochondrial dynamics, enhancing metabolic rewiring, altering neurotransmitter systems, and inducing misfolded and aggregated pathological proteins associated with neurodegenerative diseases. These pathogenetic mechanisms create a multidimensional injury of the brain that leads to specific neuronal and brain dysfunction. The understanding of the molecular mechanisms underlying the neurophathogenesis associated with neurodegeneration of viral infection may emphasize the strategies for prevention, protection, and treatment of virus infection of the CNS.

Novel functions of the ER-located Hsp40s DNAJB12 and DNAJB14 on proteins at the outer mitochondrial membrane under stress mediated by CCCP.

The endoplasmic reticulum (ER) membrane provides infrastructure for intracellular signaling, protein degradation, and communication among the ER lumen, cytosol, and nucleus via transmembrane and membrane-associated proteins. Failure to maintain homeostasis at the ER leads to deleterious conditions in humans, such as protein misfolding-related diseases and neurodegeneration. The ER transmembrane heat shock protein 40 (Hsp40) proteins, including DNAJB12 (JB12) and DNAJB14 (JB14), have been studied for their importance in multiple aspects of cellular events, including degradation of misfolded membrane proteins, proteasome-mediated control of proapoptotic Bcl-2 members, and assembly of multimeric ion channels. This study elucidates a novel facet of JB12 and JB14 in that their expression could be regulated in response to stress caused by the presence of ER stressors and the mitochondrial potential uncoupler CCCP. Furthermore, JB14 overexpression could affect the level of PTEN-induced kinase 1 (PINK1) expression under CCCP-mediated stress. Cells with genetic knockout (KO) of DNAJB12 and DNAJB14 exhibited an altered kinetic of phosphorylated Drp1 in response to the stress caused by CCCP treatment. Surprisingly, JB14-KO cells exhibited a prolonged stabilization of PINK1 during chronic exposure to CCCP. Cells depleted with JB12 or JB14 also revealed an increase in the mitochondrial count and branching. Hence, this study indicates the possible novel functions of JB12 and JB14 involving mitochondria in nonstress conditions and under stress caused by CCCP.

The role of melatonin in amyloid beta-induced inflammation mediated by inflammasome signaling in neuronal cell lines.

Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder. In addition to amyloid beta (Aß) and tau, neuroinflammation is a crucial element in the etiology of this disease. However, the relevance of inflammasome-induced pyroptosis to AD is unknown. We aimed to clarify whether the anti-inflammatory effects of melatonin could prevent Aß-mediated activation of the inflammasome. We demonstrated that Aß upregulated NOD-like receptor family pyrin domain-containing 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD, and cysteinyl aspartate-specific proteinase caspase (caspase 1) expression in SH-SY5Y neuroblastoma cells, resulting in the release of proinflammatory cytokines, including interleukin-1ß (IL-1ß), interleukin-18 (IL-18) and tumor necrosis factor (TNF-α). Melatonin prevented inflammasome signaling and excessive cytokine release caused by Aß. We found that ethyl 2[(2-chlorophenyl)(hydroxy) methyl]acrylate (INF-4E, NLRP3 and caspase 1 inhibitor) significantly abolished Aß-induced proinflammatory cytokine expression. The increase in cleaved-caspase 1, pro-IL18, and cleaved-IL18 caused by Aß suggested the occurrence of pyroptosis, which was further confirmed by the increased expression of N-terminal gasdermin D (N-GSDMD). Melatonin plays a protective role against Aß-induced inflammation via an inflammasome-associated mechanism that is essential in inducing the active forms of cytokines and pyroptosis. The ability of melatonin to inhibit inflammasome may represent a turning point in the treatment of AD progression.

Melatonin modulates the aggravation of pyroptosis, necroptosis, and neuroinflammation following cerebral ischemia and reperfusion injury in obese rats.

Obesity is well-established as a common comorbidity in ischemic stroke. The increasing evidence has revealed that it also associates with the exacerbation of brain pathologies, resulting in increasingly severe neurological outcomes following cerebral ischemia and reperfusion (I/R) damage. Mechanistically, pyroptosis and necroptosis are novel forms of regulated death that relate to the propagation of inflammatory signals in case of cerebral I/R. Previous studies noted that pyroptotic and necroptotic signaling were exacerbated in I/R brain of obese animals and led to the promotion of brain tissue injury. This study aimed to investigate the roles of melatonin on pyroptosis, necroptosis, and pro-inflammatory pathways occurring in the I/R brain of obese rats. Male Wistar rats were given a high-fat diet for 16 weeks to induce the obese condition, and then were divided into 4 groups: Sham-operated, I/R treated with vehicle, I/R treated with melatonin (10 mg/kg), and I/R treated with glycyrrhizic acid (10 mg/kg). All drugs were administered via intraperitoneal injection at the onset of reperfusion. The development of neurological deficits, cerebral infarction, histological changes, neuronal death, and glial cell hyperactivation were investigated. This study revealed that melatonin effectively improved these detrimental parameters. Furthermore, the processes of pyroptosis, necroptosis, and inflammation were all diminished by melatonin treatment. A summary of the findings is that melatonin effectively reduces ischemic brain pathology and thereby improves post-stroke outcomes in obese rats by modulating pyroptosis, necroptosis, and inflammation.

Antiviral effect of melatonin on Japanese encephalitis virus infection involves inhibition of neuronal apoptosis and neuroinflammation in SH-SY5Y cells.

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, causes high mortality rates in humans and it is the most clinically important and common cause of viral encephalitis in Asia. To date, there is no specific treatment for JEV infection. Melatonin, a neurotropic hormone, is reported to be effective in combating various bacterial and viral infections. However, the effects of melatonin on JEV infection have not yet been studied. The investigation tested the antiviral effects of melatonin against JEV infection and elucidated the possible molecular mechanisms of inhibition. Melatonin inhibited the viral production in JEV-infected SH-SY5Y cells in a time- and dose-dependent manner. Time-of-addition assays demonstrated a potent inhibitory effect of melatonin at the post-entry stage of viral replication. Molecular docking analysis revealed that melatonin negatively affected viral replication by interfering with physiological function and/or enzymatic activity of both JEV nonstructural 3 (NS3) and NS5 protein, suggesting a possible underlying mechanism of JEV replication inhibition. Moreover, treatment with melatonin reduced neuronal apoptosis and inhibited neuroinflammation induced by JEV infection. The present findings reveal a new property of melatonin as a potential molecule for the further development of anti-JEV agents and treatment of JEV infection.

Melatonin suppresses inflammation and blood‒brain barrier disruption in rats with vascular dementia possibly by activating the SIRT1/PGC-1α/PPARγ signaling pathway.

Chronic cerebral hypoxia (CCH) is caused by a reduction in cerebral blood flow, and cognitive impairment has been the predominant feature that occurs after CCH. Recent reports have revealed that melatonin is proficient in neurodegenerative diseases. However, the molecular mechanism by which melatonin affects CCH remains uncertain. In this study, we aimed to explore the role and underlying mechanism of melatonin in inflammation and blood‒brain barrier conditions in rats with CCH. Male Wistar rats were subjected to permanent bilateral common carotid artery occlusion (BCCAO) to establish the VAD model. Rats were randomly divided into four groups: Sham, BCCAO, BCCAO treated with melatonin (10 mg/kg), and BCCAO treated with resveratrol (20 mg/kg). All drugs were administered once daily for 4 weeks. Our results showed that melatonin attenuated cognitive impairment, as demonstrated by the Morris water maze tests. Furthermore, melatonin reduced the activation of inflammation by attenuating the phosphorylated nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor alpha (pIκBα), causing the suppression of proteins related to inflammation and inflammasome formation. Moreover, immunohistochemistry revealed that melatonin reduced glial cell activation and proliferation, which were accompanied by Western blotting results. Additionally, melatonin also promoted the expression of sirtuin-1 (SIRT1), peroxisome proliferator-activated receptor-gamma coactivator 1-alpha (PGC-1α), and peroxisome proliferator-activated receptor-gamma (PPARγ), causing attenuated blood‒brain barrier (BBB) disruption by increasing tight junction proteins. Taken together, our results prove that melatonin treatment modulated inflammation and BBB disruption and improved cognitive function in VaD rats, partly by activating the SIRT1/PGC-1α/PPARγ signaling pathway.

Cognitive impairment and changes of red blood cell components and serum levels of IL-6, IL-18, and L-tryptophan in methamphetamine abusers.

The deficit in cognitive function is more concerning in methamphetamine (MA) users. The cognitive deficit was suspected to be the consequence of neuroinflammation-induced neurological dysregulation. In addition, activating the key enzyme in the tryptophan metabolic pathway by pro-inflammatory cytokines results in metabolite toxicity, further generating cognitive impairments. However, the evidence for the role of neuroinflammation and tryptophan metabolites involved in MA-induced cognitive deficit needs more conclusive study. OBJECTIVES: This retrospective study aimed to determine blood-inflammatory markers, tryptophan metabolite-related molecules, and cognitive function in MA abusers compared to healthy control (HC) participants. METHODS: The cognitive functions were evaluated using Stroop, Go/No-Go, One Back Task (OBT), and Wisconsin Card Sorting Test-64 (WCST-64). Blood samples were analyzed for complete blood count (CBC) analysis, serum inflammatory cytokines interleukin (IL)-6 and IL-18 and tryptophan metabolites. RESULTS: MA group exhibited poor cognitive performance in selective attention, inhibition, working memory, cognitive flexibility, concept formation and processing speed compared to HC. Reduction in red blood cell (RBC) components but induction in white blood cells (WBCs) and IL-6 were observed in MA abusers, which might indicate anemia of (systemic chronic low-grade) inflammation. In addition, the depletion of precursor in the tryptophan metabolic pathway, L-tryptophan was also observed in MA users, which might represent induction in tryptophan metabolites. CONCLUSION: These findings emphasize that blood biomarkers might be a surrogate marker to predict the role of neuroinflammation and abnormal tryptophan metabolite in MA-induced cognitive impairments.

Characterization of endotoxin free protein production of brain-derived neurotrophic factor (BDNF) for the study of Parkinson model in SH-SY5Y differentiated cells.

Human neuronal cells are a more appropriate cell model for neurological disease studies such as Alzheimer and Parkinson's disease. SH-SY5Y neuroblastoma cells have been widely used for differentiation into a mature neuronal cell phenotype. The cellular differentiation process begins with retinoic acid incubation, followed by incubation with brain-derived neurotrophic factor (BDNF), a recombinant protein produced in E. coli cells. Endotoxin or lipopolysaccharide (LPS) is the major component of the outer membrane of bacterial cells that triggers the activation of pro-inflammatory cytokines and ultimately cell death. Consequently, any endotoxin contamination of the recombinant BDNF used for cell culture experiments would impact on data interpretation. Therefore, in this study, we expressed the BDNF recombinant protein in bacterial endotoxin-free cells that were engineered to modify the oligosaccharide chain of LPS rendering the LPS unable to trigger the immune response of human cells. The expression of DCX and MAP-2 in differentiated cells indicate that in-house and commercial BDNF are equally effective in inducing differentiation. This suggests that our in-house BDNF protein can be used to differentiate SH-SY5Y neuroblastoma cells without the need for an endotoxin removal step.

Melatonin Attenuates Methamphetamine-Induced Alteration of Amyloid ß Precursor Protein Cleaving Enzyme Expressions via Melatonin Receptor in Human Neuroblastoma Cells.

Alzheimer's disease (AD) is the most prominent neurodegenerative disease represented by the loss of memory and cognitive impairment symptoms and is one of the major health imperilments among the elderly. Amyloid (Aß) deposit inside the neuron is one of the characteristic pathological hallmarks of this disease, leading to neuronal cell death. In the amyloidogenic processing, the amyloid precursor protein (APP) is cleaved by beta-secretase and γ-secretase to generate Aß. Methamphetamine (METH) is a psychostimulant drug that causes neurodegeneration and detrimental cognitive deficits. The analogy between the neurotoxic and neurodegenerative profile of METH and AD pathology necessitates an exploration of the underlying molecular mechanisms. In the present study, we found that METH ineluctably affects APP processing, which might contribute to the marked production of Aß in human neuroblastoma cells. Melatonin, an indolamine produced and released by the pineal gland as well as other extrapineal, has been protective against METH-induced neurodegenerative processes, thus rescuing neuronal cell death. However, the precise action of melatonin on METH has yet to be determined. We further propose to investigate the protective properties of melatonin on METH-induced APP-cleaving secretases. Pretreatment with melatonin significantly reversed METH-induced APP-cleaving secretases and Aß production. In addition, pretreatment with luzindole, a melatonin receptor antagonist, significantly prevented the protective effect of melatonin, suggesting that the attenuation of the toxic effect on METH-induced APP processing by melatonin was mediated via melatonin receptor. The present results suggested that melatonin has a beneficial role in preventing Aß generation in a cellular model of METH-induced AD.

A Synopsis of Multitarget Potential Therapeutic Effects of Huperzine A in Diverse Pathologies-Emphasis on Alzheimer's Disease Pathogenesis.

Numerous challenges are confronted when it comes to the recognition of therapeutic agents for treating complex neurodegenerative diseases like Alzheimer's disease (AD). The perplexing pathogenicity of AD embodies cholinergic dysfunction, amyloid beta (Aß) aggregation, neurofibrillary tangle formation, neuroinflammation, mitochondrial disruption along with vicious production of reactive oxygen species (ROS) generating oxidative stress. In this frame of reference, drugs with multi target components could prove more advantageous to counter complex pathological mechanisms that are responsible for AD progression. For as much as, medicinal plant based pharmaco-therapies are emerging as potential candidates for AD treatment keeping the efficacy and safety parameters in terms of toxicity and side effects into consideration. Huperzine A (Hup A) is a purified alkaloid compound extracted from a club moss called Huperzia serrata. Several studies have reported both cholinergic and non-cholinergic effects of this compound on AD with significant neuroprotective properties. The present review convenes cumulative demonstrations of neuroprotection provided by Hup A in in vitro, in vivo, and human studies in various pathologies. The underlying molecular mechanisms of its actions have also been discussed. However, more profound evidence would certainly promote the therapeutic implementation of this drug thus furnishing decisive insights into AD therapeutics and various other pathologies along with preventive and curative management.

Melatonin improves cognitive function by suppressing endoplasmic reticulum stress and promoting synaptic plasticity during chronic cerebral hypoperfusion in rats.

Chronic cerebral hypoperfusion (CCH) is the most common cause of cognitive impairment, which is commonly found in Alzheimer's disease (AD) and vascular dementia (VaD). Recently, studies have demonstrated that melatonin is an effective treatment in various neurodegenerative diseases. In this study, we aimed to investigate the effects of melatonin on CCH-induced AD pathology, endoplasmic reticulum (ER) stress, and synaptic plasticity, all of which are correlated with the activation of oxidative stress, apoptosis, and cognitive impairment. CCH was induced in male Wistar rats by bilateral common carotid artery occlusion (2VO). After surgery, rats were treated with melatonin (10 mg/kg) or piracetam (600 mg/kg) by oral gavage once a day for 4 weeks. At the end of the experiment, all rats were assessed for memory impairment by using the Morris water maze test. Subsequently, rats were sacrificed, and brains were removed to determine the levels of beta-amyloid (Aß), malondialdehyde (MDA); the acetylcholinesterase (AChE) activity; subjected to terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL); and subjected to western blotting of proteins related to memory, AD pathology, oxidative stress, ER stress, and apoptosis. Melatonin alleviated brain injury during 2VO induction, as revealed by decreased the expression of AD markers, attenuated oxidative stress, suppressed the expression of proteins related to ER stress, apoptosis, and stimulated the expression of the synaptic markers resulting in promoted cognitive function. Therefore, our data demonstrated that melatonin ameliorated cognitive impairment in the 2VO model, and these beneficial effects were associated with reduction in oxidative stress, ER stress, and apoptosis.

Agomelatine Exerts an Anti-inflammatory Effect by Inhibiting Microglial Activation Through TLR4/NLRP3 Pathway in pMCAO Rats.

Cerebral ischemic stroke is one of the main causes of death and long-term disability worldwide. However, the mechanism is unclear, and treatments are limited. In this study, we aimed to investigate the anti-inflammatory effect of agomelatine in a permanent middle cerebral artery occlusion (pMCAO) model. Forty-eight male Wistar rats were randomly divided into four groups: sham, pMCAO + vehicle, pMCAO + agomelatine (40 mg/kg, i.p.), and pMCAO + melatonin (10 mg/kg, i.p.) groups. On day 1 after permanent cerebral ischemia, the animals were sacrificed, and brain tissues were collected for western blot analysis, and immunohistochemistry. Agomelatine treatment ameliorated inflammatory responses by decreasing the protein levels of trigger Toll-like receptor (TLR4)/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway components together with nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3) inflammasome components. In addition, agomelatine suppressed microglial activation and pyroptotic cell death after cerebral ischemic injury. These results suggest that agomelatine exerts an anti-inflammatory effect and attenuates brain damage by inhibiting microglial activation through the TLR4/NLRP3 signaling pathway.

The effects of agomelatine on endoplasmic reticulum stress related to mitochondrial dysfunction in hippocampus of aging rat model.

BACKGROUND: Agomelatine, a novel antidepressant, is a melatonin MT receptor agonist and serotonin 5HT2C receptor antagonist. In this study, agomelatine was used to investigate the molecular mechanisms of hippocampal aging associated with endoplasmic reticulum (ER) stress, mitochondrial dysfunction, and apoptosis, all of which led to short-term memory impairment. METHOD: Hippocampal aging was induced in male Wistar rats by d-galactose (D-gal) intraperitoneal injection (100 mg/kg) for 14 weeks. During the last 4 weeks of D-gal treatment, rats were treated with agomelatine (40 mg/kg) or melatonin (10 mg/kg). At the end of the experiment, all rats were assessed for short-term memory by using the Morris water maze test. Subsequently, rats were sacrified and the hippocampus was removed from each rat for determination of reactive oxygen species (ROS), malondialdehyde (MDA), and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays; and immunohistochemistry related to ER stress, mitochondrial dysfunction, and apoptosis. RESULTS: Agomelatine suppressed the expression of the aging-related proteins P16 and receptor for advanced glycation endproducts (RAGE), the expression of NADPH oxidase (NOX) 2 and 4, and ROS production. This treatment also shifted the morphology of astrocytes and microglia toward homeostasis. Furthermore, agomelatine decreased inositol-requiring enzyme 1 (pIRE1), protein kinase R-like endoplasmic reticulum kinase (pPERK), and chaperone binding immunoglobulin protein (BiP), leading to suppression of ER stress markers C/EBP homologous protein (CHOP) and caspase-12. Agomelatine reduced Ca2+ from the ER and stabilized the mitochondrial membrane stability, which was denoted by the BCL2 Associated X (Bax)/B-cell lymphoma 2 (Bcl2) balance. Agomelatine decreased cleaved caspase-3 production and the Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL)-positive area, and glutamate excitotoxicity was prevented via suppression of N-methyl-d-aspartate (NMDA) receptor subunit expression. Agomelatine exhibited effects that were similar to melatonin. CONCLUSION: Agomelatine improved neurodegeneration in a rat model of hippocampal aging by attenuating ROS production, ER stress, mitochondrial dysfunction, excitotoxicity, and apoptosis.