Search Results (113)

Age-related neurodegeneration is characterized by oxidative stress and iron-dependent cell death, yet the neuroprotective mechanisms of folic acid in modulating ferroptosis remain unclear. This study systematically investigated the role of folic acid in inhibiting ferroptosis and attenuating neuronal damage in aging, with a focus on the solute carrier family 7 member 11 (SLC7A11)-glutathione (GSH)-glutathione peroxidase 4 (GPX₄) antioxidant pathway, using aged rats supplemented with folic acid (<0.1, 2.0, and 4.0 mg/kg·diet) for 22 months, with young adult rats as controls. Brain iron accumulation and ferroptosis-related proteins (SLC7A11, GPX₄, Ferritin heavy chain 1 (FTH1)) were evaluated. In vitro, HT-22 hippocampal neuronal cells were pre-treated with folic acid (0, 10, 20 μmol/L) for 72 h before combining with Erastin (10 μmol/L)-induced ferroptosis for an additional 24 h. Intracellular Fe²⁺, lipid peroxidation (LPO), malondialdehyde (MDA), reactive oxygen species (ROS), along with cystine, GSH, and ferroptosis-related protein levels were quantified. Stable sh-SLC7A11 knockdown and control (sh-NC) cell lines were used to validate the dependency of folic acid’s protective effects on SLC7A11 expression. Folic acid supplementation in aged rats dose-dependently reduced aging-related brain iron accumulation and enhanced the expression of SLC7A11, GPX₄, and FTH1. In Erastin-induced HT-22 cells, folic acid significantly mitigated ferroptosis hallmarks. Mechanistically, folic acid increased extracellular cystine uptake and intracellular GSH synthesis, thereby activating the SLC7A11-GSH-GPX₄ antioxidant pathway. Notably, molecular docking technique suggested that compared to GPX₄, folic acid stabilized SLC7A11’s active conformation. sh-SLC7A11 knockdown completely abolished folic acid-mediated protection against ferroptosis, as evidenced by restored loss of cystine, GSH and GPX₄ production. This study innovatively emphasized the critical role of folic acid supplementation in inhibiting ferroptosis by up-regulating the SLC7A11-GSH-GPX₄ antioxidant pathway, primarily through enhancing cystine availability and SLC7A11 expression. These findings established folic acid as a potential dietary intervention for aging-related neurodegenerative diseases characterized by neuronal ferroptosis, providing preclinical evidence for folic acid based neuroprotection. Full article

This study evaluates the effects of hypergravity (HG) on a neurodegenerative model in vitro, looking at how HG influences Tau protein aggregation in Mouse Hippocampal Neuronal Cells (HT22) induced by neurofibrillary tangle seeds. Overall, 50× g significantly, synergistically, reduced the Tau aggregate Area when combined with ERK-inhibitor PD-0325901, correlating with decreased phosphorylation at critical residues pS262 and pS396. These findings suggest HG treatments may help mitigate cytoskeletal damage linked to Tau aggregation. Full article

Inorganic arsenic [As(III) and As(V)] is a pervasive environmental contaminant in groundwater systems, early-life exposure to which is associated with an impaired cognitive ability and an increased risk of neurobehavioral disorders. Although the effect of As(III) on the neurons is well studied, the involvement of the microglia remains unclear. In this study, the effects of sodium arsenite (NaAsO₂) on microglial activation and the underlying NLRP3 inflammasome mechanism were determined. Pregnant rats were gavaged with NaAsO₂ (0, 1, 4, and 10 mg/kg body weight), which dissociates in aqueous solutions into bioactive arsenite species [As(OH)₃], from gestational day 1 (GD1) to postnatal day 21 (PND21). The results showed that As(III) induces learning and memory impairments and microglial activation in the hippocampus of offspring rats (PND21). Increased expression of NLRP3, the activation of caspase-1, and the production of interleukin-1β were observed in both the hippocampus of As(III)-exposed offspring rats and As(III)-exposed microglial BV2 cells under culture conditions. Interestingly, blocking the NLRP3 inflammasome using MCC950 mitigated its activation. Furthermore, inhibition of NADPH oxidase 2 (NOX2) using apocynin or specific siRNA significantly reduced As(III)-induced microglial NLRP3 inflammasome activation. In addition, inactivation of the microglial NLRP3 inflammasome or NOX2 markedly rescued As(III)-induced neurotoxicity in the hippocampal HT22 cells. Taken together, this study reveals that NOX2/NLRP3-inflammasome-dependent microglial activation promotes As(III)-induced learning and memory impairments in developmental rats. Full article

Mitochondrial transplantation (MTx) has emerged as a potential therapeutic approach for diseases associated with mitochondrial dysfunction, yet its scalability and cross-species feasibility remain underexplored. This study aimed to evaluate the dose-dependent uptake and molecular effects of xenogeneic mitochondrial transplantation (xeno-MTx) using rat-derived mitochondria in mouse neuronal systems. HT-22 hippocampal neuronal cells and a murine model of cardiac arrest-induced global cerebral ischemia were used to assess mitochondrial uptake, gene expression, and mitochondrial DNA presence. Donor mitochondria were isolated from rat pectoralis muscle and labeled with MitoTracker dyes. Flow cytometry and confocal microscopy revealed a dose-dependent increase in donor mitochondrial uptake in vitro. Quantitative PCR demonstrated a corresponding increase in rat-specific mitochondrial DNA and upregulation of Mfn2 and Bak1, with no changes in other fusion, fission, or apoptotic genes. Inhibitor studies indicated that mitochondrial internalization may involve actin-dependent macropinocytosis and cholesterol-sensitive endocytic pathways. In vivo, rat mitochondrial DNA was detected in mouse brains post–xeno-MTx, confirming donor mitochondrial delivery to ischemic tissue. These findings support the feasibility of xeno-MTx and its dose-responsive biological effects in neuronal systems while underscoring the need for further research to determine long-term functional outcomes and clinical applicability. Full article

PDE11A is a little-studied phosphodiesterase sub-family that breaks down cAMP/cGMP, with the PDE11A4 isoform enriched in the memory-related hippocampal formation. Age-related increases in PDE11A expression occur in human and rodent hippocampus and cause age-related cognitive decline of social memories. Interestingly, age-related increases in PDE11A4 protein ectopically accumulate in spherical clusters that group together in the brain to form linear filamentous patterns termed “PDE11A4 ghost axons”. The biophysical/physiochemical mechanisms underlying this age-related clustering are not known. Here, we determine if age-related clustering of PDE11A4 reflects liquid–liquid phase separation (LLPS; biomolecular condensation), and if PDE11A inhibitors can reverse this LLPS. We show human and mouse PDE11A4 exhibit several LLPS-promoting sequence features, including intrinsically disordered regions, non-covalent pi–pi interactions, and prion-like domains that were particularly enriched in the N-terminal regulatory region. Further, multiple bioinformatic tools predict PDE11A4 undergoes LLPS. Consistent with these predictions, aging-like PDE11A4 clusters in HT22 hippocampal neuronal cells were membraneless spherical droplets that progressively fuse over time in a concentration-dependent manner. Deletion of the N-terminal intrinsically disordered region prevented PDE11A4 LLPS despite equal protein expression between WT and mutant constructs. 1,6-hexanediol, along with tadalafil and BC11-38 that inhibit PDE11A4, reversed PDE11A4 LLPS in HT22 hippocampal neuronal cells. Interestingly, PDE11A4 inhibitors reverse PDE11A4 LLPS independently of increasing cAMP/cGMP levels via catalytic inhibition. Importantly, orally dosed tadalafil reduced PDE11A4 ghost axons in old mouse ventral hippocampus by 50%. Thus, PDE11A4 exhibits the four defining criteria of LLPS, and PDE11A inhibitors reverse this age-related phenotype both in vitro and in vivo. Full article

Background/Objectives: Diabetes frequently leads to cognitive impairment, encompassing issues with memory and executive function, as well as depression and anxiety. This study examines the impact of high-intensity interval exercise (HIIE) alongside glucagon-like peptide-1 receptor agonist (GLP-1 RA) semaglutide on cognitive dysfunction associated with diabetes. Methods: Db/db mice were divided into a control group, semaglutide group, HIIE group, and semaglutide combined with HIIE group to study metabolic and neurobehavioral effects. Cognitive and behavioral tests, hippocampal morphology, and molecular analyses (APP, BDNF, Aβ, p-Tau, PKA, AMPK) were performed. HT22 cells under high glucose were treated with semaglutide, L-lactate, PKA inhibitor H89, and AMPK inhibitor Compound C to validate mechanisms. Results: Over 8 weeks, both HIIE and semaglutide improved neuronal morphology and cognitive performance while reducing depression in db/db mice. However, the current study observed no synergistic effects. Both therapies decreased Aβ and p-Tau protein levels and increased BDNF levels in the hippocampus, likely through the AMPK and PKA signaling pathways, respectively. In vitro, HT22 cells under high glucose conditions exhibited elevated APP and p-Tau expression and reduced BDNF levels, which could be altered by L-lactate and semaglutide. The AMPK inhibitor Compound C and the PKA inhibitor H89 attenuated the increase in BDNF levels induced by L-lactate and semaglutide, but their combination mitigated this inhibitory effect. This study suggests that while HIIE and semaglutide improve cognitive function and reduce depression via BDNF, their combined use did not show the anticipated synergistic benefits due to potential antagonism between the AMPK and PKA pathways. Conclusions: This has important implications for designing exercise prescriptions for cognitive impairment in diabetics. Full article

Macelignan, from Myristica fragrans (nutmeg), is a bioactive compound with various pharmacological properties, including anti-inflammatory and neuroprotective activities. The purpose of this work was to investigate the antioxidant and anti-apoptotic effects of macelignan in glutamate-treated HT22 mouse hippocampal neurons. Macelignan was extracted and identified in a methanol extract of M. fragrans seeds. The DPPH was used to assess the antioxidative activity of macelignan. Glutamate (5 mM) was used to induce neurotoxicity in the HT22 cells. Neuroprotective effects were measured using relevant biochemical and imaging assays, including cell viability, ROS production, nuclear staining, apoptotic cell death, and protein expression. Macelignan markedly and concentration-dependently enhanced DPPH radical scavenging activity. In the HT22 cell model, glutamate induced cell damage by decreasing cell viability, promoting ROS generation, and increasing apoptotic cell death according to cell morphological changes. However, macelignan treatment restored cell viability, inhibited ROS generation concentration-dependently, and reduced apoptosis. Moreover, glutamate significantly up-regulated the phosphorylation of MAPK-pathway-related proteins, which was reversed by macelignan treatment. In conclusion, macelignan shows notable neuroprotective effects on oxidative stress and apoptotic cell death in glutamate-induced cells, and this study provides useful information on its potential therapeutic implications in neurological disorders. Full article

SET and MYND Domain-Containing 2 (Smyd-2), a specific protein lysine methyltransferase (PKMT), influences both histones and non-histones. Its role in cerebral ischemia/reperfusion (CIR), particularly in ferroptosis—a regulated form of cell death driven by lipid peroxidation—remains poorly understood. This study identifies the expression of Smyd-2 in the brain and investigates its relationship with neuronal programmed cell death (PCD). We specifically investigated how Smyd-2 regulates ferroptosis in CIR through its interaction with the Nuclear Factor Erythroid-2-related Factor-2 (Nrf-2)/Kelch-like ECH-associated protein (Keap-1) pathway. Smyd-2 knockout protects HT-22 cells from Erastin-induced ferroptosis but not TNF-α + Smac-mimetic-induced apoptosis/necroptosis. This neuroprotective effect of Smyd-2 knockout in HT-22 cells after Oxygen–Glucose Deprivation/Reperfusion (OGD/R) was reversed by Erastin. Smyd-2 knockout in HT-22 cells shows neuroprotection primarily via the Nuclear Factor Erythroid-2-related Factor-2 (Nrf-2)/Kelch-like ECH-associated protein (Keap-1) pathway, despite the concurrent upregulation of Smyd-2 and Nrf-2 observed in both the middle cerebral artery occlusion (MCAO) and OGD/R models. Interestingly, vivo experiments demonstrated that Smyd-2 knockout significantly reduced ferroptosis and lipid peroxidation in hippocampal neurons following CIR. Moreover, the Nrf-2 inhibitor ML-385 abolished the neuroprotective effects of Smyd-2 knockout, confirming the pivotal role of Nrf-2 in ferroptosis regulation. Cycloheximide (CHX) fails to reduce Nrf-2 expression in Smyd-2 knockout HT-22 cells. Smyd-2 knockout suppresses Nrf-2 lysine methylation, thereby promoting the Nrf-2/Keap-1 pathway without affecting the PKC-δ/Nrf-2 pathway. Conversely, Smyd-2 overexpression disrupts Nrf-2 nuclear translocation, exacerbating ferroptosis and oxidative stress, highlighting its dual regulatory role. This study underscores Smyd-2’s potential for ischemic stroke treatment by disrupting the Smyd-2/Nrf-2-driven antioxidant capacity, leading to hippocampal neuronal ferroptosis. By clarifying the intricate interplay between ferroptosis and oxidative stress via the Nrf-2/Keap-1 pathway, our findings provide new insights into the molecular mechanisms of CIR and identify Smyd-2 as a promising therapeutic target. Full article

Dementia with Lewy bodies (DLB) is a progressive neurodegenerative disorder marked by the accumulation of α-synuclein (αSyn), often co-existing with amyloid β (Aβ) pathology. Current treatments are largely symptomatic, highlighting a critical need for disease-modifying therapies. Evidence suggests that αSyn aggregates contribute to neuronal death in DLB, particularly when exacerbated by Aβ. Given the role of autophagy in clearing misfolded proteins, exploring agents that promote this pathway is essential for developing effective treatments. Ambroxol (AMBX), a mucolytic drug, has demonstrated potential in activating glucocerebrosidase (GCase), an enzyme that enhances lysosomal function and facilitates the autophagic clearance of toxic protein aggregates, including αSyn. This study aims to evaluate AMBX’s neuroprotective effects in a cellular model of DLB, with the goal of identifying new therapeutic agents that target the underlying pathology of DLB. In this study, HT-22 hippocampal neuronal cells were exposed to αSyn and Aβ, followed by AMBX treatment. Our results showed that AMBX significantly improved cell viability and reduced apoptosis in cells co-treated with αSyn and Aβ. Additionally, AMBX restored GCase activity, promoted autophagy, and reduced oxidative stress, which in turn mitigated αSyn aggregation and phosphorylation. These findings suggest that by activating GCase and enhancing autophagy, AMBX may help alleviate DLB-associated neurodegeneration. This study underscores the potential of AMBX as a therapeutic agent for DLB and supports further investigation in animal models and clinical trials to validate its efficacy in neurodegenerative disease contexts. Full article

Depression is a complex and common mental illness affecting physical and psychological health. Panax ginseng C. A. Mey is a traditional Chinese medicine with abundant pharmacological activity and applications in regulating mood disorders. 20 (S)-Protopanaxadiol is the major intestinal metabolite of ginsenoside and one of the active components in ginseng. In this study, we aimed to investigate the therapeutic effects of 20 (S)-Protopanaxadiol on neuronal damage and depression, which may involve mitochondrial dynamics. However, the mechanism underlying the antidepressant effects of 20 (S)-Protopanaxadiol is unelucidated. In the present study, we investigated the potential mechanisms underlying the antidepressant activity of 20 (S)-Protopanaxadiol by employing a corticosterone-induced HT22 cellular model and a chronic unpredicted mild stress (CUMS)-induced animal model in combination with a network pharmacology approach. In vitro, the results showed that 20 (S)-Protopanaxadiol ameliorated the corticosterone (CORT)-induced decrease in HT22 cell viability, decrease in 5-hydroxytryptamine (5-HT) levels, and increase in nitric oxide (NO) and malondialdehyde (MDA) levels. Furthermore, 20 (S)-Protopanaxadiol exerted improvement effects on the CORT-induced increase in HT22 cell mitochondrial reactive oxygen species, loss of mitochondrial membrane potential, and apoptosis. In vivo, the results showed that 20 (S)-Protopanaxadiol ameliorated depressive symptoms and hippocampal neuronal damage in CUMS mice, and sirtuin1 (SIRT1) and peroxisome proliferator-activated receptor-1-Alpha (PGC-1α) activity were activated in the hippocampus of mice, thereby alleviating mitochondrial dysfunction and promoting the clearance of damaged mitochondria. In both in vivo and in vitro models, after inhibiting SIRT1 expression, the protective effect of 20 (S)-Protopanaxadiol on mitochondria was significantly weakened, and dynamin-related protein 1 (DRP1)-mediated mitochondrial division was significantly reduced. These findings suggest that 20 (S)-Protopanaxadiol may exert neuroprotective and antidepressant effects by attenuating DRP1-mediated mitochondrial dysfunction and apoptosis by modulating the SIRT1/PGC-1α signaling pathway. Full article

Background: Hippocampal Neuroinflammation (HNF) is a critical driver of cognitive impairment. The lipopolysaccharide (LPS) accumulate amyloid beta (Aβ) and lead to HNF. The Bifidobacterium lactis (BL) 99 have anti-inflammatory ability. However, whether BL99-derived microbiota-derived vesicles (MV) could alleviate LPS-induced HNF remains unclear. Methods: To investigate, we used ultrafiltration with ultracentrifuge to extract BL99-derived-MV (BL99-MV). We used hippocampal neuronal HT22 cells (HT22) to establish the LPS-induced HNF model, and explored whether BL99-MV alleviate LPS-induced HNF. Results: The confocal microscopy showed that BL99-MV were taken up by HT22 and reduced the oxidative stress (ROS) level. The PCR showed that BL99-MV up-regulate IL-10 level, and down-regulate TNF-α, IL-1β, and IL-6. Transcriptomic analysis revealed 4127 differentially expressed genes, with 2549 genes upregulated and 1578 genes downregulated in the BL99-MV group compared to the LPS group. Compared to the LPS group, BL99-MV decreased FoxO6, IL-33, P53, and NFκB expression, but increased FoxO1 and Bcl2 expression. The WB showed that BL99-MV modulated NFκB, FoxO6, P53, Caspase9, and Caspase3 protein expression by reducing IL-33 expression in HT22. The findings demonstrated IL-33 as a regulator for FoxO6/P53 signaling. Conclusions: Here, we hypothesized that BL99-MV alleviated LPS-induced HNF to promote HT22 survival and synaptic development by regulating FoxO6/P53 signaling by targeting IL-33. Full article

Background: The peptidyl-prolyl isomerase (PIN1) plays a vital role in cellular processes, including intracellular signaling and apoptosis. While oxidative stress is considered one of the primary mechanisms of pathogenesis in brain ischemic injury, the precise function of PIN1 in this disease remains to be elucidated. Objective: We constructed a cell-permeable PEP-1–PIN1 fusion protein and investigated PIN1’s function in HT-22 hippocampal cells as well as in a brain ischemic injury gerbil model. Methods: Transduction of PEP-1–PIN1 into HT-22 cells and signaling pathways were determined by Western blot analysis. Intracellular reactive oxygen species (ROS) production and DNA damage was confirmed by DCF-DA and TUNEL staining. Cell viability was determined by MTT assay. Protective effects of PEP-1-PIN1 against ischemic injury were examined using immunohistochemistry. Results: PEP-1–PIN1, when transduced into HT-22 hippocampal cells, inhibited cell death in H₂O₂-treated cells and markedly reduced DNA fragmentation and ROS production. This fusion protein also reduced phosphorylation of mitogen-activated protein kinase (MAPK) and modulated expression levels of apoptosis-signaling proteins in HT-22 cells. Furthermore, PEP-1–PIN1 was distributed in gerbil hippocampus neuronal cells after passing through the blood–brain barrier (BBB) and significantly protected against neuronal cell death and also decreased activation of microglia and astrocytes in an ischemic injury gerbil model. Conclusions: These results indicate that PEP-1–PIN1 can inhibit ischemic brain injury by reducing cellular ROS levels and regulating MAPK and apoptosis-signaling pathways, suggesting that PIN1 plays a protective role in H₂O₂-treated HT-22 cells and ischemic injury gerbil model. Full article

In recent years, alongside research on mammalian-derived exosomes, there has been increasing interest in the physiological activities of plant-derived exosome-like nanoparticles (PDEN). The biocompatibility, minimal side effects, and diverse bioactive ingredients contained in PDEN make them valuable as potential therapeutic agents for an extensive range of diseases. In this study, we cost-effectively isolated exosome-like nanoparticles from green onion (Allium fistulosum) using polyethylene glycol and examined their biological activity in HT-22 cells exposed to glutamate. The isolated green onion-derived exosome-like nanoparticle (GDEN) had an average diameter of 167.4 nm and a zeta potential of −16.06 mV. GDEN effectively inhibited glutamate-induced Ca²⁺ influx and lipid peroxidation, thereby preventing ferroptotic cell death in HT-22 mouse hippocampal cells. Additionally, GDEN reduced the intracellular iron accumulation by modulating the expression of proteins associated with iron metabolism, including transferrin receptor 1, ferroportin 1, divalent metal transporter 1, and ferritin. Notably, GDEN upregulated the expression of glutathione peroxidase 4, a potent antioxidant protein involved in ferroptosis, along with an increase in glutathione synthesis. These findings indicate that GDENs have the potential to serve as bioactives from natural sources against glutamate-induced neuronal cell death, like ferroptosis. This study advances the investigation into the potential medical applications of GDEN and may provide a new approach for the utilization of these bioactive components against neuronal disorders. Full article

Leptin is a hormone produced by the small intestines and adipose tissue that promotes feelings of satiety. Leptin receptors (LepRs) are highly expressed in the hypothalamus, enabling central neural control of hunger. Interestingly, LepRs are also expressed in several other regions of the body and brain, notably in the cerebral cortex and hippocampus. These brain regions mediate higher-order sensory, motor, cognitive, and memory functions, which can be profoundly altered during periods of hunger and satiety. However, LepR expression in these regions has not been fully characterized on a cell-type-specific basis, which is necessary to begin assessing their potential functional impact. Consequently, we examined LepR expression on neurons and glia in the forebrain using a LepR-Cre transgenic mouse model. LepR-positive cells were identified using a ‘floxed’ viral cell-filling approach and co-labeling immunohistochemically for cell-type-specific markers, i.e., NeuN, VGlut2, GAD67, parvalbumin, somatostatin, 5-HT3R, WFA, S100β, and GFAP. In the cortex, LepR-positive cells were localized to lower layers (primarily layer 6) and exhibited non-pyramidal cellular morphologies. The majority of cortical LepR-positive cells were neurons, while the remainder were identified primarily as astrocytes or other glial cells. The majority of cortical LepR-positive neurons co-expressed parvalbumin, while none expressed somatostatin or 5-HT3R. In contrast, all hippocampal LepR-positive cells were neuronal, with none co-expressing GFAP. These data suggest that leptin can potentially influence neural processing in forebrain regions associated with sensation and limbic-related functions. Full article

Pharmacotherapy is the therapeutic mainstay in epilepsy; however, in about 30% of patients, epileptic seizures are drug-resistant. A ketogenic diet (KD) is an alternative therapeutic option. The mechanisms underlying the anti-seizure effect of a KD are not fully understood. Epileptic seizures lead to an increased energy demand of neurons. An improvement in energy provisions may have a protective effect. C8 and C10 fatty acids have been previously shown to activate mitochondrial function in vitro. This could involve sirtuins (SIRTs) as regulatory elements of energy metabolism. The aim of the present study was to investigate whether ß-hydroxybutyrate (ßHB), C8 fatty acids, C10 fatty acids, or a combination of C8 and C10 (250/250 µM) fatty acids, which all increase under a KD, could up-regulate SIRT1, -3, -4, and -5 in HT22 hippocampal murine neurons in vitro. Cells were incubated for 1 week in the presence of these metabolites. The sirtuins were measured at the enzyme (fluorometrically), protein (Western blot), and gene expression (PCR) levels. In hippocampal cells, the C8, C10, and C8 and C10 incubations led to increases in the sirtuin levels, which were not inferior to a ßHB incubation as the ‘gold standard’. This may indicate that both C8 and C10 fatty acids are important for the antiepileptic effect of a KD. A KD may be replaced by nutritional supplements of C8 and C10 fatty acids, which could facilitate the diet. Full article