AutoPepVax, a Novel Machine-Learning-Based Program for Vaccine Design: Application to a Pan-Cancer Vaccine Targeting EGFR Missense Mutations.

The current epitope selection methods for peptide vaccines often rely on epitope binding affinity predictions, prompting the need for the development of more sophisticated in silico methods to determine immunologically relevant epitopes. Here, we developed AutoPepVax to expedite and improve the in silico epitope selection for peptide vaccine design. AutoPepVax is a novel program that automatically identifies non-toxic and non-allergenic epitopes capable of inducing tumor-infiltrating lymphocytes by considering various epitope characteristics. AutoPepVax employs random forest classification and linear regression machine-learning-based models, which are trained with datasets derived from tumor samples. AutoPepVax, along with documentation on how to run the program, is freely available on GitHub. We used AutoPepVax to design a pan-cancer peptide vaccine targeting epidermal growth factor receptor (EGFR) missense mutations commonly found in lung adenocarcinoma (LUAD), colorectal adenocarcinoma (CRAD), glioblastoma multiforme (GBM), and head and neck squamous cell carcinoma (HNSCC). These mutations have been previously targeted in clinical trials for EGFR-specific peptide vaccines in GBM and LUAD, and they show promise but lack demonstrated clinical efficacy. Using AutoPepVax, our analysis of 96 EGFR mutations identified 368 potential MHC-I-restricted epitope-HLA pairs from 49,113 candidates and 430 potential MHC-II-restricted pairs from 168,669 candidates. Notably, 19 mutations presented viable epitopes for MHC I and II restrictions. To evaluate the potential impact of a pan-cancer vaccine composed of these epitopes, we used our program, PCOptim, to curate a minimal list of epitopes with optimal population coverage. The world population coverage of our list ranged from 81.8% to 98.5% for MHC Class II and Class I epitopes, respectively. From our list of epitopes, we constructed 3D epitope-MHC models for six MHC-I-restricted and four MHC-II-restricted epitopes, demonstrating their epitope binding potential and interaction with T-cell receptors. AutoPepVax's comprehensive approach to in silico epitope selection addresses vaccine safety, efficacy, and broad applicability. Future studies aim to validate the AutoPepVax-designed vaccines with murine tumor models that harbor the studied mutations.

Sodium butyrate ameliorates high glucose-suppressed neuronal mitophagy by restoring PRKN expression via inhibiting the RELA-HDAC8 complex.

Damaged mitochondria accumulation in diabetes is one of the main features that contribute to increased incidence of cognitive impairment by inducing apoptosis. Butyrate is a major metabolite produced by microbiota that has neuroprotective effects by regulating mitochondrial function. However, detailed mechanisms underlying how butyrate can regulate neuronal mitophagy remain unclear. Here, we examined the regulatory effects of sodium butyrate (NaB) on high glucose-induced mitophagy dysregulation, neuronal apoptosis, and cognitive impairment and its underlying mechanisms in human-induced pluripotent stem cell-derived neurons, SH-SY5Ys, and streptozotocin (STZ)-induced diabetic mice. In our results, diabetic mice showed gut-microbiota dysbiosis, especially a decreased number of butyrate-producing bacteria and reduced NaB plasma concentration. NaB ameliorated high glucose-induced neuronal mitochondrial dysfunction by recovering PRKN/Parkin-mediated mitophagy. High glucose-induced reactive oxygen species (ROS) and -inhibited PRKAA/AMPKα stimulated the RELA/p65-HDAC8 complex, which downregulated PRKN protein expression by binding to the PRKN promoter region. NaB restored PRKN expression by blocking RELA nuclear translocation and directly inhibiting HDAC8 in the nucleus. In addition, HDAC8 overexpression inhibited the positive effect of NaB on high glucose-induced mitophagy dysfunction and neuronal apoptosis. Oral administration of NaB improved cognitive impairment in diabetic mice by restoring mitophagy in the hippocampus. Taken together, NaB ameliorates neuronal mitophagy through PRKN restoration by inhibiting RELA-HDAC8 complexes, suggesting that NaB is an important substance for protecting neuronal apoptosis in diabetes-associated cognitive impairment.

Transcription Factor EB-Mediated Lysosomal Function Regulation for Determining Stem Cell Fate under Metabolic Stress.

Stem cells require high amounts of energy to replicate their genome and organelles and differentiate into numerous cell types. Therefore, metabolic stress has a major impact on stem cell fate determination, including self-renewal, quiescence, and differentiation. Lysosomes are catabolic organelles that influence stem cell function and fate by regulating the degradation of intracellular components and maintaining cellular homeostasis in response to metabolic stress. Lysosomal functions altered by metabolic stress are tightly regulated by the transcription factor EB (TFEB) and TFE3, critical regulators of lysosomal gene expression. Therefore, understanding the regulatory mechanism of TFEB-mediated lysosomal function may provide some insight into stem cell fate determination under metabolic stress. In this review, we summarize the molecular mechanism of TFEB/TFE3 in modulating stem cell lysosomal function and then elucidate the role of TFEB/TFE3-mediated transcriptional activity in the determination of stem cell fate under metabolic stress.

TRIM16-mediated lysophagy suppresses high-glucose-accumulated neuronal Aß.

ABBREVIATIONS: Aß: amyloid ß; AD: Alzheimer disease; AMPK: 5' adenosine monophosphate-activated protein kinase; CTSB: cathepsin B; CTSD: cathepsin D; DM: diabetes mellitus; ESCRT: endosomal sorting complex required for transport; FBXO27: F-box protein 27; iPSC-NDs: induced pluripotent stem cell-derived neuronal differentiated cells; LAMP1: lysosomal-associated membrane protein 1; LMP: lysosomal membrane permeabilization; LRSAM1: leucine rich repeat and sterile alpha motif containing 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; p-MAPT/tau: phosphorylated microtubule associated protein tau; ROS: reactive oxygen species; STZ: streptozotocin; TFE3: transcription factor E3; TFEB: transcription factor EB; TRIM16: tripartite motif containing 16; UBE2QL1: ubiquitin conjugating enzyme E2 Q family like 1; VCP: valosin containing protein.

Melatonin-mediated FKBP4 downregulation protects against stress-induced neuronal mitochondria dysfunctions by blocking nuclear translocation of GR.

The physiological crosstalk between glucocorticoid and melatonin maintains neuronal homeostasis in regulating circadian rhythms. However, the stress-inducing level of glucocorticoid triggers mitochondrial dysfunction including defective mitophagy by increasing the activity of glucocorticoid receptors (GRs), leading to neuronal cell death. Melatonin then suppresses glucocorticoid-induced stress-responsive neurodegeneration; however, the regulatory mechanism of melatonin, i.e., associated proteins involved in GR activity, has not been elucidated. Therefore, we investigated how melatonin regulates chaperone proteins related to GR trafficking into the nucleus to suppress glucocorticoid action. In this study, the effects of glucocorticoid on suppressing NIX-mediated mitophagy, followed by mitochondrial dysfunction, neuronal cell apoptosis, and cognitive deficits were reversed by melatonin treatment by inhibiting the nuclear translocation of GRs in both SH-SY5Y cells and mouse hippocampal tissue. Moreover, melatonin selectively suppressed the expression of FKBP prolyl isomerase 4 (FKBP4), which is a co-chaperone protein that works with dynein, to reduce the nuclear translocation of GRs among the chaperone proteins and nuclear trafficking proteins. In both cells and hippocampal tissue, melatonin upregulated melatonin receptor 1 (MT1) bound to Gαq, which triggered the phosphorylation of ERK1. The activated ERK then enhanced DNA methyltransferase 1 (DNMT1)-mediated hypermethylation of FKBP52 promoter, reducing GR-mediated mitochondrial dysfunction and cell apoptosis, the effects of which were reversed by knocking down DNMT1. Taken together, melatonin has a protective effect against glucocorticoid-induced defective mitophagy and neurodegeneration by enhancing DNMT1-mediated FKBP4 downregulation that reduced the nuclear translocation of GRs.

Ethanol-induced ceramide production causes neuronal apoptosis by increasing MCL-1S-mediated ER-mitochondria contacts.

Heavy alcohol consumption causes neuronal cell death and cognitive impairment. Neuronal cell death induced by ethanol may result from increased production of the sphingolipid metabolite ceramide. However, the molecular mechanisms of neuronal cell death caused by ethanol-induced ceramide production have not been elucidated. Therefore, we investigated the mechanism through which ethanol-induced ceramide production causes neuronal cell apoptosis using human induced-pluripotent stem cell-derived neurons and SH-SY5Y cells and identified the effects of ceramide on memory deficits in C57BL/6 mice. First, we found that ethanol-induced ceramide production was decreased by inhibition of the de novo synthesis pathway, mediated by serine palmitoyltransferase (SPT). The associated alterations of the molecules related to the ceramide pathway suggest that the elevated level of ceramide activated protein phosphatase 1 (PP1), which inhibited the nuclear translocation of serine/arginine-rich splicing factor 1 (SRSF1). This led to aberrant splicing of myeloid cell leukemia 1 (MCL-1) pre-mRNA, which upregulated MCL-1S expression. Our results demonstrated that the interaction of MCL-1S with the inositol 1, 4, 5-trisphosphate receptor (IP3R) increases calcium release from the endoplasmic reticulum (ER) and then activated ER-bound inverted formin 2 (INF2). In addition, we discovered that F-actin polymerization through INF2 activation promoted ER-mitochondria contacts, which induced mitochondrial calcium influx and mitochondrial reactive oxygen species (mtROS) production. Markedly, MCL-1S silencing decreased mitochondria-associated ER membrane (MAM) formation and prevented mitochondrial calcium influx and mtROS accumulation, by inhibiting INF2-dependent actin polymerization interacting with mitochondria. Furthermore, the inhibition of ceramide production in ethanol-fed mice reduced MCL-1S expression, neuronal cell death, and cognitive impairment. In conclusion, we suggest that ethanol-induced ceramide production may lead to mitochondrial calcium overload through MCL-1S-mediated INF2 activation-dependent MAM formation, which promotes neuronal apoptosis.

Silencing SIRT5 induces the senescence of UCB-MSCs exposed to TNF-α by reduction of fatty acid ß-oxidation and anti-oxidation.

Tumor necrosis factor-α (TNF-α) is an inflammatory cytokine involved in cell survival, apoptosis, and homeostasis. However, the regulatory effect of TNF-α on mesenchymal stem cell (MSC) redox regulation remains unknown. The process of delaying the senescence of MSCs and maintaining antioxidation mechanism is important in transplantation therapy to treat inflammatory diseases that result from restricted immunomodulatory effects of senescent MSCs. Thus, we examined the role of TNF-α-mediated signaling and its regulatory mechanisms on the senescence of umbilical cord blood-derived MSCs (UCB-MSCs) and identified its therapeutic efficacy in a collagen-induced arthritis (CIA) mouse model. We found that TNF-α increased fatty acid synthesis and lipid droplet (LD) formation through NF-κB/SREBP1-mediated FASN, SCD1, and DGAT2 expression, which protects UCB-MSCs from oxidative stress against accumulated toxic lipids. Additionally, DGAT2-mediated LD formation was regulated by TNF-α-activated TNF receptor (TNFR)1 signaling. We also found that storage of unsaturated FAs in LDs is regulated by SIRT5-dependent ß-oxidation of FAs, which reduces mitochondrial ROS (mtROS) accumulation. Particularly, mtROS homeostasis was maintained by superoxide dismutase 2 (SOD2) upregulation through TNFR2-mediated SIRT5/Nrf2 signaling. In a CIA mouse model, UCB-MSCs transfected with SIRT5 siRNA exhibited reduced therapeutic effects compared with UCB-MSCs transfected with NT siRNA. Overall, the results indicated that SIRT5 plays a central role in protecting TNF-α-induced UCB-MSC senescence through FA ß-oxidation and SOD2-mediated antioxidation.

Prenatal glucocorticoid exposure selectively impairs neuroligin 1-dependent neurogenesis by suppressing astrocytic FGF2-neuronal FGFR1 axis.

Exposure to maternal stress irreversibly impairs neurogenesis of offspring by inducing life-long effects on interaction between neurons and glia under raging differentiation process, culminating in cognitive and neuropsychiatric abnormalities in adulthood. We identified that prenatal exposure to stress-responsive hormone glucocorticoid impaired neurogenesis and induced abnormal behaviors in ICR mice. Then, we used human induced pluripotent stem cell (iPSC)-derived neural stem cell (NSC) to investigate how neurogenesis deficits occur. Following glucocorticoid treatment, NSC-derived astrocytes were found to be A1-like neurotoxic astrocytes. Moreover, cortisol-treated astrocytic conditioned media (ACM) then specifically downregulated AMPA receptor-mediated glutamatergic synaptic formation and transmission in differentiating neurons, by inhibiting localization of ionotropic glutamate receptor (GluR)1/2 into synapses. We then revealed that downregulated astrocytic fibroblast growth factor 2 (FGF2) and nuclear fibroblast growth factor receptor 1 (FGFR1) of neurons are key pathogenic factors for reducing glutamatergic synaptogenesis. We further confirmed that cortisol-treated ACM specifically decreased the binding of neuronal FGFR1 to the synaptogenic NLGN1 promoter, but this was reversed by FGFR1 restoration. Upregulation of neuroligin 1, which is important in scaffolding GluR1/2 into the postsynaptic compartment, eventually normalized glutamatergic synaptogenesis and subsequent neurogenesis. Moreover, pretreatment of FGF2 elevated neuroligin 1 expression and trafficking of GluR1/2 into the postsynaptic compartment of mice exposed to prenatal corticosterone, improving spatial memory and depression/anxiety-like behaviors. In conclusion, we identified neuroligin 1 restoration by astrocytic FGF2 and its downstream neuronal nuclear FGFR1 as a critical target for preventing prenatal stress-induced dysfunction in glutamatergic synaptogenesis, which recovered both neurogenesis and hippocampal-related behaviors.

Cyanidin 3-O-arabinoside suppresses DHT-induced dermal papilla cell senescence by modulating p38-dependent ER-mitochondria contacts.

BACKGROUND: Androgenetic alopecia (AGA) is a genetic disorder caused by dihydrotestosterone (DHT), accompanied by the senescence of androgen-sensitive dermal papilla cells (DPCs) located in the base of hair follicles. DHT causes DPC senescence in AGA through mitochondrial dysfunction. However, the mechanism of this pathogenesis remains unknown. In this study, we investigated the protective role of cyanidins on DHT-induced mitochondrial dysfunction and DPC senescence and the regulatory mechanism involved. METHODS: DPCs were used to investigate the effect of DHT on mitochondrial dysfunction with MitoSOX and Rhod-2 staining. Senescence-associated ß-galactosidase activity assay was performed to examine the involvement of membrane AR-mediated signaling in DHT-induced DPC senescence. AGA mice model was used to study the cyanidins on DHT-induced hair growth deceleration. RESULTS: Cyanidin 3-O-arabinoside (C3A) effectively decreased DHT-induced mtROS accumulation in DPCs, and C3A reversed the DHT-induced DPC senescence. Excessive mitochondrial calcium accumulation was blocked by C3A. C3A inhibited p38-mediated voltage-dependent anion channel 1 (VDAC1) expression that contributes to mitochondria-associated ER membrane (MAM) formation and transfer of calcium via VDAC1-IP3R1 interactions. DHT-induced MAM formation resulted in increase of DPC senescence. In AGA mice models, C3A restored DHT-induced hair growth deceleration, which activated hair follicle stem cell proliferation. CONCLUSIONS: C3A is a promising natural compound for AGA treatments against DHT-induced DPC senescence through reduction of MAM formation and mitochondrial dysfunction.

High glucose-mediated VPS26a down-regulation dysregulates neuronal amyloid precursor protein processing and tau phosphorylation.

BACKGROUND AND PURPOSE: The relationship between hyperglycaemia-induced retromer dysfunction impairing intracellular trafficking and Alzheimer's disease (AD) remains unclear, although diabetes mellitus (DM) is considered a risk factor for AD. Here, we investigated the effects of high glucose on the retromer and defined the dysregulation of mechanisms of amyloid precursor protein (APP) processing and tau phosphorylation. EXPERIMENTAL APPROACH: We used human induced-pluripotent stem cell-derived neuronal differentiated cells and SH-SY5Ys exposed to high glucose to identify the underlying mechanisms. Streptozotocin-induced diabetic mice were used to elucidate whether the retromer contributes to the AD-like pathology. KEY RESULTS: We found that vacuolar protein sorting-associated protein 26a (VPS26a) was decreased in the hippocampus of diabetic mice and high glucose-treated human neuronal cells. High glucose down-regulated VPS26a through ROS/NF-κB/DNA methyltransferase1-mediated promoter hypermethylation. VPS26a recovery blocked retention of APP and cation-independent mannose-6-phosphate receptor in endosomes and promoted transport to the trans-Golgi, which decreased Aß levels, and improved cathepsin D activity, reducing p-tau levels, respectively. Retromer enhancement ameliorated synaptic deficits, astrocyte over-activation, and cognitive impairment in diabetic mice. CONCLUSION AND IMPLICATIONS: In conclusion, VPS26a is a promising candidate for the inhibition of DM-associated AD pathogenesis by modulating APP processing and tau phosphorylation.

Natural Products in the Prevention of Metabolic Diseases: Lessons Learned from the 20th KAST Frontier Scientists Workshop.

The incidence of metabolic and chronic diseases including cancer, obesity, inflammation-related diseases sharply increased in the 21st century. Major underlying causes for these diseases are inflammation and oxidative stress. Accordingly, natural products and their bioactive components are obvious therapeutic agents for these diseases, given their antioxidant and anti-inflammatory properties. Research in this area has been significantly expanded to include chemical identification of these compounds using advanced analytical techniques, determining their mechanism of action, food fortification and supplement development, and enhancing their bioavailability and bioactivity using nanotechnology. These timely topics were discussed at the 20th Frontier Scientists Workshop sponsored by the Korean Academy of Science and Technology, held at the University of Hawaii at Manoa on 23 November 2019. Scientists from South Korea and the U.S. shared their recent research under the overarching theme of Bioactive Compounds, Nanoparticles, and Disease Prevention. This review summarizes presentations at the workshop to provide current knowledge of the role of natural products in the prevention and treatment of metabolic diseases.

Melatonin activates ABCA1 via the BiP/NRF1 pathway to suppress high-cholesterol-induced apoptosis of mesenchymal stem cells.

BACKGROUND: Retarded wound healing in patients with obesity contributes to a risk of complications associated with vascular insufficiency and oxidative stress. The high cholesterol levels of patients with obesity are associated with apoptosis of engrafted umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs). Melatonin contributes to the prevention of cholesterol accumulation in patients with obesity via a mechanism that is poorly understood. We therefore investigated the regulatory mechanism of melatonin in cholesterol-induced apoptosis. METHODS: The protective effects of melatonin on cholesterol-induced apoptosis were investigated in UCB-MSCs. We used a mouse model of induced obesity to show that melatonin treatment restored the survival rate of transplanted UCB-MSCs and their wound-healing capacity. The mean values of the treatment groups were compared with those of the control group using Student's t test, and differences among three or more groups were analyzed using one-way analysis of variance with Dunnett's multiple comparison test. RESULTS: Melatonin treatment increased the expression of ATP-binding cassette subfamily A member 1 (ABCA1), which reduced cholesterol accumulation and cholesterol-induced apoptosis. The mouse skin wound healing model showed that melatonin treatment restored the survival rate of transplanted UCB-MSCs and the wound-healing capacity of obese mice. Melatonin inhibited the expression of binding immunoglobulin protein (BiP) through the regulation of MT2/Sp1-dependent microRNA-597-5p. Melatonin decreased the co-localization of BiP with nuclear factor erythroid 2-related factor 1 (NRF1), which resulted in increased ABCA1 expression. CONCLUSION: Melatonin induced the efflux of intracellular cholesterol through ABCA1 to decrease apoptosis of UCB-MSCs via an MT2-dependent BiP/NRF1 pathway.

BNIP3L/NIX-mediated mitophagy protects against glucocorticoid-induced synapse defects.

Stress-induced glucocorticoids disturb mitochondrial bioenergetics and dynamics; however, instead of being removed via mitophagy, the damaged mitochondria accumulate. Therefore, we investigate the role of glucocorticoids in mitophagy inhibition and subsequent synaptic defects in hippocampal neurons, SH-SY5Y cells, and ICR mice. First, we observe that glucocorticoids decrease both synaptic density and vesicle recycling due to suppressed mitophagy. Screening data reveal that glucocorticoids downregulate BNIP3-like (BNIP3L)/NIX, resulting in the reduced mitochondrial respiration function and synaptic density. Notably, we find that glucocorticoids direct the glucocorticoid receptor to bind directly to the PGC1α promoter, downregulating its expression and nuclear translocation. PGC1α downregulation selectively decreases NIX-dependent mitophagy. Consistent with these results, NIX enhancer pre-treatment of a corticosterone-exposed mouse elevates mitophagy and synaptic density in hippocampus, improving the outcome of a spatial memory task. In conclusion, glucocorticoids inhibit mitophagy via downregulating NIX and that NIX activation represents a potential target for restoring synapse function.

Urolithin A suppresses high glucose-induced neuronal amyloidogenesis by modulating TGM2-dependent ER-mitochondria contacts and calcium homeostasis.

Hyperglycemia in diabetes mellitus (DM) patients is a causative factor for amyloidogenesis and induces neuropathological changes, such as impaired neuronal integrity, neurodegeneration, and cognitive impairment. Regulation of mitochondrial calcium influx from the endoplasmic reticulum (ER) is considered a promising strategy for the prevention of mitochondrial ROS (mtROS) accumulation that occurs in the Alzheimer's disease (AD)-associated pathogenesis in DM patients. Among the metabolites of ellagitannins that are produced in the gut microbiome, urolithin A has received an increasing amount of attention as a novel candidate with anti-oxidative and neuroprotective effects in AD. Here, we investigated the effect of urolithin A on high glucose-induced amyloidogenesis caused by mitochondrial calcium dysregulation and mtROS accumulation resulting in neuronal degeneration. We also identified the mechanism related to mitochondria-associated ER membrane (MAM) formation. We found that urolithin A-lowered mitochondrial calcium influx significantly alleviated high glucose-induced mtROS accumulation and expression of amyloid beta (Aß)-producing enzymes, such as amyloid precursor protein (APP) and ß-secretase-1 (BACE1), as well as Aß production. Urolithin A injections in a streptozotocin (STZ)-induced diabetic mouse model alleviated APP and BACE1 expressions, Tau phosphorylation, Aß deposition, and cognitive impairment. In addition, high glucose stimulated MAM formation and transglutaminase type 2 (TGM2) expression. We first discovered that urolithin A significantly reduced high glucose-induced TGM2 expression. In addition, disruption of the AIP-AhR complex was involved in urolithin A-mediated suppression of high glucose-induced TGM2 expression. Markedly, TGM2 silencing inhibited inositol 1, 4, 5-trisphosphate receptor type 1 (IP3R1)-voltage-dependent anion-selective channel protein 1 (VDAC1) interactions and prevented high glucose-induced mitochondrial calcium influx and mtROS accumulation. We also found that urolithin A or TGM2 silencing prevented Aß-induced mitochondrial calcium influx, mtROS accumulation, Tau phosphorylation, and cell death in neuronal cells. In conclusion, we suggest that urolithin A is a promising candidate for the development of therapies to prevent DM-associated AD pathogenesis by reducing TGM2-dependent MAM formation and maintaining mitochondrial calcium and ROS homeostasis.

Ethanol-activated CaMKII signaling induces neuronal apoptosis through Drp1-mediated excessive mitochondrial fission and JNK1-dependent NLRP3 inflammasome activation.

BACKGROUND: Neurodegeneration is a representative phenotype of patients with chronic alcoholism. Ethanol-induced calcium overload causes NOD-like receptor protein 3 (NLRP3) inflammasome formation and an imbalance in mitochondrial dynamics, closely associated with the pathogenesis of neurodegeneration. However, how calcium regulates this process in neuronal cells is poorly understood. Therefore, the present study investigated the detailed mechanism of calcium-regulated mitochondrial dynamics and NLRP3 inflammasome formation in neuronal cells by ethanol. METHODS: In this study, we used the SK-N-MC human neuroblastoma cell line. To confirm the expression level of the mRNA and protein, real time quantitative PCR and western blot were performed. Co-immunoprecipitation and Immunofluorescence staining were conducted to confirm the complex formation or interaction of the proteins. Flow cytometry was used to analyze intracellular calcium, mitochondrial dysfunction and neuronal apoptosis. RESULTS: Ethanol increased cleaved caspase-3 levels and mitochondrial reactive oxygen species (ROS) generation associated with neuronal apoptosis. In addition, ethanol increased protein kinase A (PKA) activation and cAMP-response-element-binding protein (CREB) phosphorylation, which increased N-methyl-D-aspartate receptor (NMDAR) expression. Ethanol-increased NMDAR induced intracellular calcium overload and calmodulin-dependent protein kinase II (CaMKII) activation leading to phosphorylation of dynamin-related protein 1 (Drp1) and c-Jun N-terminal protein kinase 1 (JNK1). Drp1 phosphorylation promoted Drp1 translocation to the mitochondria, resulting in excessive mitochondrial fission, mitochondrial ROS accumulation, and loss of mitochondrial membrane potential, which was recovered by Drp1 inhibitor pretreatment. Ethanol-induced JNK1 phosphorylation activated the NLRP3 inflammasome that induced caspase-1 dependent mitophagy inhibition, thereby exacerbating ROS accumulation and causing cell death. Suppressing caspase-1 induced mitophagy and reversed the ethanol-induced apoptosis in neuronal cells. CONCLUSIONS: Our results demonstrated that ethanol upregulated NMDAR-dependent CaMKII phosphorylation which is essential for Drp1-mediated excessive mitochondrial fission and the JNK1-induced NLRP3 inflammasome activation resulting in neuronal apoptosis. Video abstract.

Sodium butyrate inhibits high cholesterol-induced neuronal amyloidogenesis by modulating NRF2 stabilization-mediated ROS levels: involvement of NOX2 and SOD1.

The gut-brain axis is currently being studied as a therapeutic strategy for neurological diseases, especially Alzheimer's disease (AD). Obesity results in the gut microbiota dysbiosis, which includes butyrate-producing bacteria are reduced. Although sodium butyrate (NaB) has emerged as the potential therapeutic substance in AD, there is a lack of detailed results into what signaling pathways affect amyloidogenesis in AD induced by obesity. Thus, we investigated the regulatory role of NaB on amyloidogenesis in neuronal cells under high cholesterol. In our results, we verified that increased amyloid ß peptide (Aß) accumulation in the brain of obese mice and a reduction in butyrate-producing bacteria due to the gut microbiota dysbiosis induced by obesity. We showed that NaB decreased the expression levels of beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) and Aß accumulation induced by high cholesterol in SK-N-MC cells. We demonstrated that NaB was absorbed in cells through sodium-coupled monocarboxylate transporter 1 (SMCT1) and then inhibited high cholesterol-induced Aß accumulation. Subsequently, we also observed that reactive oxygen species (ROS) were overproduced because of increased NADPH oxidase 2 (NOX2) expression under high cholesterol. Meanwhile, NaB decreased NOX2 levels through a reduction of NF-κB activity, which ultimately inhibited Aß accumulation caused by high cholesterol. We demonstrated that NaB increased the expression levels of p21 under high cholesterol, contributing to p21/NRF2 (Nuclear factor erythroid 2-related factor 2) colocalization, which leads to NRF2 stabilization. NRF2 stabilization causes NF-κB inactivation, followed by NOX2 suppression and superoxide dismutase 1 (SOD1) upregulation. Thus, NaB with SOD1 silencing under high cholesterol did not eliminate excessive ROS, and eventually resulted in Aß accumulation. In conclusion, we demonstrated that NaB prevents excessive ROS through NOX2 suppression and SOD1 upregulation by p21/NRF2 pathway, which is critical for inhibiting BACE1-dependent amyloidogenesis in neuronal cells exposed to high cholesterol environment.

High glucose-mediated PICALM and mTORC1 modulate processing of amyloid precursor protein via endosomal abnormalities.

BACKGROUND AND PURPOSE: Although diabetes mellitus (DM) is an important risk factor for Alzheimer's disease (AD), the detailed mechanism(s) by which DM regulates amyloid ß (Aß) processing is still unclear. The longer residence time of amyloid precursor protein (APP) in endosomes is critical for Aß production and DM is known to cause endosomal dysregulation. Here we have examined the effects of high glucose on APP-producing endosomes and related signaling pathways. EXPERIMENTAL APPROACH: To identify the underlying mechanisms, we investigated the effects of high glucose on abnormalities in early endosomes and related signalling pathways in human neuroblastoma cells. In vivo, diabetic mice treated with pharmacological inhibitors were used to examine endosomal dysfunction. KEY RESULTS: The hippocampus of diabetic animals presented endosomal abnormalities and Aß up-regulation. High glucose increased Aß production through early endosomal enlargement achieved by increased lipid raft-mediated APP endocytosis. High glucose induced ROS-stimulated Sp1 activation, up-regulating phosphatidylinositol binding clathrin assembly protein (PICALM), clathrin heavy chain, and adaptor-related protein complex 2 alpha 1. PICALM facilitated clathrin-mediated APP endocytosis resulting in early endosomal enlargement. Meanwhile, AMPK/mTORC1-mediated autophagy defect and ROS- and mTORC1-mediated lysosomal dysfunction aggravated early endosomal enlargement under high glucose. Moreover, the increased Aß production and cognitive deficits in diabetic mice were reversed by inhibition of early endosomal enlargement. CONCLUSION AND IMPLICATIONS: High glucose induces early endosomal abnormalities through PICALM-induced APP endocytosis and mTORC1-inhibited endosomal clearance, up-regulating Aß production. Thus, targeting PICALM and mTORC1 to prevent endosomal disorders is a promising strategy for managing diabetes-induced AD.

Melatonin restores Muc2 depletion induced by V. vulnificus VvpM via melatonin receptor 2 coupling with Gαq.

BACKGROUND: Melatonin (5-methoxy-N-acetyltryptamine), a hormone produced in the pineal gland, has a variety of biological functions as an antioxidant, but a functional role of melatonin in the regulation of intestinal mucin (Muc) production during bacterial infection has yet to be described in detail. In this study, we investigate the effects of melatonin during Muc2 repression elicited by the Gram-negative bacterium V. vulnificus. METHODS: Mucus-secreting human HT29-MTX cells were used to study the functional role of melatonin during Muc2 depletion induced by the recombinant protein (r) VvpM produced by V. vulnificus. The regulatory effects of melatonin coupling with melatonin receptor 2 (MT2) on the production of reactive oxygen species (ROS), the activation of PKCδ and ERK, and the hypermethylation of the Muc2 promoter as induced by rVvpM were examined. Experimental mouse models of V. vulnificus infection were used to study the role of melatonin and how it neutralizes the bacterial toxin activity related to Muc2 repression. RESULTS: Recombinant protein (r) VvpM significantly reduced the level of Muc2 in HT29-MTX cells. The repression of Muc2 induced by rVvpM was significantly restored upon a treatment with melatonin (1 µM), which had been inhibited by the knockdown of MT2 coupling with Gαq and the NADPH oxidase subunit p47 phox. Melatonin inhibited the ROS-mediated phosphorylation of PKCδ and ERK responsible for region-specific hypermethylation in the Muc2 promoter in rVvpM-treated HT29-MTX cells. In the mouse models of V. vulnificus infection, treatment with melatonin maintained the level of Muc2 expression in the intestine. In addition, the mutation of the VvpM gene from V. vulnificus exhibited an effect similar to that of melatonin. CONCLUSIONS: These results demonstrate that melatonin acting on MT2 inhibits the hypermethylation of the Muc2 promoter to restore the level of Muc2 production in intestinal epithelial cells infected with V. vulnificus.

Transcriptomic Identification and Biochemical Characterization of HmpA, a Nitric Oxide Dioxygenase, Essential for Pathogenesis of Vibrio vulnificus.

Nitric oxide (NO) and its derivatives are important effectors of host innate immunity, disrupting cellular function of infecting pathogens. Transcriptome analysis of Vibrio vulnificus, an opportunistic human pathogen, identified a set of genes induced upon exposure to NO. Among them, VvhmpA (V. vulnificus hmpA), encoding a multidomain NO dioxygenase, was the most greatly induced upon exposure to NO and was thus further characterized. Absorption spectra demonstrated that VvHmpA is a heme protein in which the heme iron can exist in either reduced, NO-bound, or oxidized state. Biochemical studies revealed that VvHmpA is a flavohemoglobin containing equimolar amounts of heme and FAD as cofactors. The K M and k cat values of VvHmpA for NO at 37°C, the temperature encountered by V. vulnificus in the host, were greater than those at 30°C, indicating that VvHmpA detoxifies high levels of NO effectively during infection. Compared with the wild type, the VvhmpA mutant exhibited a lower NO-decomposition activity and impaired growth in the presence of NO in vitro. Also, the cytotoxicity and survival of the VvhmpA mutant infecting the NO-producing murine macrophage cells were lower than those of the wild type. Furthermore, the mouse lethality of the VvhmpA mutant was reduced compared to that of the parental wild type. The combined results revealed that VvHmpA is a potent virulence factor that is induced upon exposure to NO and important for the survival and pathogenesis of V. vulnificus during infection.