HT22 Mouse Hippocampal Neuronal Cells Research Articles

Benzofuran and Benzo[b]thiophene-2-carboxamide Derivatives as Modulators of Amyloid Beta (Aβ42) Aggregation.

A group of N-phenylbenzofuran-2-carboxamide and N-phenylbenzo[b]thiophene-2-carboxamide derivatives were designed and synthesized as a novel class of Aβ42 aggregation modulators. In the thioflavin-T based fluorescence aggregation kinetics study, compounds 4a, 4b, 5a and 5b possessing a methoxyphenol pharmacophore were able to demonstrate concentration dependent inhibition of Aβ42 aggregation with maximum inhibition of 54% observed for compound 4b. In contrast, incorporation of a 4-methoxyphenyl ring in compounds 4d and 5d led to a significant increase in Aβ42 fibrillogenesis demonstrating their ability to accelerate Aβ42 aggregation. Compound 4d exhibited 2.7-fold increase in Aβ42 fibrillogenesis when tested at the maximum concentration of 25 µM. These results were further confirmed by electron microscopy studies which demonstrates the ability of compounds 4a, 4b, 4d, 5a, 5b and 5d to modulate Aβ42 fibrillogenesis. Compounds 5a and 5b provided significant neuroprotection to mouse hippocampal neuronal HT22 cells against Aβ42-induced cytotoxicity. Molecular docking studies suggest that the orientation of the bicyclic aromatic rings (either benzofuran or benzo[b]thiophene) plays a major role in moderating their ability to either inhibit or accelerate Aβ42 aggregation. Our findings support the application of these novel derivatives as pharmacological tools to study the mechanisms of Aβ42 aggregation.

MiR-122 overexpression alleviates oxygen-glucose deprivation-induced neuronal injury by targeting sPLA2-IIA.

Ischemic stroke (IS) is a neurological disease with significant disability and mortality. MicroRNAs were proven to be associated with cerebral ischemia. Previous studies have demonstrated miR-122 downregulation in both animal models of IS and the blood of IS patients. Nonetheless, the role and mechanism of miR-122-5p in IS remain unclear. We established primary human and mouse astrocytes, along with HT22 mouse hippocampal neuronal cells, through oxygen-glucose deprivation/reoxygenation (OGD/R) treatment. To assess the impact of miR-122, we employed CCK8 assays, flow cytometry, RT-qPCR, western blotting, and ELISA to evaluate cell viability, apoptosis, reactive oxygen species (ROS) generation, and cytokine expression. A dual-luciferase reporter gene assay was employed to investigate the interaction between miR-122 and sPLA2-IIA. Overexpression of miR-122 resulted in decreased apoptosis, reduced cleaved caspase-3 expression, and increased cell viability in astrocytes and HT22 cells subjected to OGD/R. RT-qPCR and ELISA analyses demonstrated a decrease in mRNA and cytokine levels of interleukin (IL)-6 and tumor necrosis factor (TNF)-α in both astrocytes and HT22 cells following miR-122 overexpression. Moreover, miR-122 overexpression reversed OGD/R-induced ROS levels and 8-OHdG formation in astrocytes. Additionally, miR-122 overexpression decreased the mRNA and protein expression of inducible nitric oxide synthase (iNOS). Furthermore, we found that miR-122 attaches to the 3'-UTR of sPLA2-IIA, thereby downregulate its expression. Our study demonstrates that miR-122-mediated inhibition of sPLA2-IIA attenuates OGD/R-induced neuronal injury by suppressing apoptosis, alleviating post-ischemic inflammation, and reducing ROS production. Thus, the miR-122/sPLA2-IIA axis may represent a promising target for IS treatment.

Circular RNA circEfnb2 promotes cell injury after cerebral infarction by sponging miR-202-5p and regulating TRAF3 expression

BackgroundExogenous neural cell transplantation may be therapeutic for stroke, cerebral ischemic injury. Among other mechanisms, increasing findings indicated circular RNAs (circRNAs) regulate the pathogenesis progression of cerebral ischemia. Mmu_circ_0015034 (circEfnb2) was upregulated in focal cortical infarction established by middle cerebral artery occlusion (MCAO) in mice. Our study was designed to probe the molecular mechanism of circEfnb2 in the oxygen-glucose deprivation/reperfusion (OGD/R)-induced neuronal damage in cerebral ischemia. MethodsWe established an in vitro OGD/R cell model. CircEfnb2 and microRNA-202-5p (miR-202-5p) levels were detected using real-time quantitative polymerase chain reaction (RT-qPCR). Lactate dehydrogenase (LDH), malondialdehyde (MDA), and reactive oxygen species (ROS) levels were assessed using specific kits. Tumor necrosis factor-α (TNF-α) and Interleukin-1β (IL-1β) levels were examined using an Enzyme-linked immunosorbent assay (ELISA). Flow cytometry analysis evaluated cell apoptosis. Protein levels of B-cell lymphoma-2 (Bcl-2), Bcl-2 related X protein (Bax), cleaved caspase 3, and Tumor necrosis factor receptor-associated factor 3 (TRAF3) were determined using Western blot assay. ResultsOverall, circEfnb2 was highly expressed whereas miR-202-5p was decreased in OGD/R-treated mouse hippocampal neuronal HT22 cells compared to normal controls (both p > 0.05). From an in vitro functional perspective, circEfnb2 knockdown attenuated an OGD/R-triggered neuronal injury compared to controls (p > 0.05). Mechanically, circEfnb2 acted as a sponge of miR-202-5p; downregulation of miR-202-5p annulled the inhibitory roles of circEfnb2 silencing in an OGD/R-caused neuronal injury model. Our analysis showed that miR-202-5p directly targeted TRAF3 as enhanced TRAF3 abolished the effects of miR-202-5p in the OGD/R-induced neuronal injury. In vivo, lentivirus with a short hairpin (sh)-circEfnb2 inhibited cerebral injury, when injected into cerebral cortex in MCAO mice (p > 0.05). ConclusionOur results suggest that circEfnb2 deficiency may decrease OGD/R-induced HT22 cell damage by modulating the miR-202-5p/TRAF3 axis. This explanation may provide a new direction for cerebral infarction potential therapeutic targets.

Chemical Constituents of Ajuga Lupulina and their Anti-Ferroptosis Activity.

Five new compounds (1, 2, 7, 12, and 16), along with fifteen known ones, were isolated from Ajuga lupulina Maxim. Their structures were revealed by analysing spectroscopic data (MS, NMR), and experimental and calculated ECD spectra was used to deduce the absolute configuration. Compound 16, with eight carbon atoms, was firstly isolated from the nature. All the isolates were evaluated for their inhibitory effect on RSL3-induced ferroptosis in HT22 mouse hippocampal neuronal cells. Among them, the abietane-type diterpenoids (7-11) significantly inhibited ferroptosis with EC50 values of 0.83 μM, 2.05 μM, 0.96 μM, 1.47 μM, and 1.19 μM, respectively.

Tanreqing injection inhibits dengue virus encephalitis by suppressing the activation of NLRP3 inflammasome

BackgroundEncephalitis caused by dengue virus (DENV) is considered a manifestation of severe dengue. Tanreqing injection (TRQ) is a well-known Chinese patented medicine, which has been used to treat brain-related disorders by inhibiting inflammation. Nevertheless, the effects of TRQ on DENV encephalitis have not been studied. The aim of this study was to evaluate the effects of TRQ on DENV encephalitis and to explore its potential mechanisms.MethodsThe cytotoxicity of TRQ was examined by MTT assay, and the anti-DENV activities of TRQ in BHK-21 baby hamster kidney fibroblast were evaluated through CCK-8 and plaque assays. The expression levels of NO, IL1B/IL-1β, TNFα and IL6 were measured by qRT‒PCR and ELISA in the BV2 murine microglial cell line. The inhibitory effects of TRQ on NLRP3 inflammasome activation in BV2 cells were examined by Western blotting, qRT‒PCR and ELISA. The effects of TRQ on HT22 mouse hippocampal neuronal cells were examined by CCK-8 assay, morphology observation and flow cytometry. Moreover, a DENV-infected ICR suckling mouse model was developed to investigate the protective role of TRQ in vivo.ResultsTRQ decreased the release of NO, IL6, TNFα and IL1B from BV2 cells and inhibited the activation of NLRP3. The presence of the NLRP3 agonist nigericin reversed the anti-inflammatory activities of TRQ. Furthermore, TRQ inhibited the death of HT22 cells by decreasing IL1B in DENV-infected BV2 cells. In addition, TRQ significantly attenuated weight loss, reduced clinical scores and extended the survival in DENV-infected ICR suckling mice. Critically, TRQ ameliorated pathological changes in ICR suckling mice brain by inhibiting microglia and NLRP3 activation and decreasing the production of inflammatory factors and the number of dead neurons.ConclusionTRQ exerts potent inhibitory effects on dengue encephalitis in vitro and in vivo by reducing DENV-2-induced microglial activation and subsequently decreasing the inflammatory response, thereby protecting neurons. These findings demonstrate the potential of TRQ in the treatment of dengue encephalitis.

Neuroprotective effect of Aster yomena ethanolic extract in HT-22 and SK-N-MC cells based on antioxidant activity

The antioxidant potentials of ethanolic extracts derived from Aster yomena (A. yomena) were evaluated by assessing their total phenolic and flavonoid contents and radical scavenging activities. Our findings revealed that the 60% ethanolic extract of A. yomena exhibited the most robust antioxidant properties among all extracts tested. Specifically, the IC50 values for the 2,2′-azino-bis (3-ethyl benzothiazoline-6-sulfonic acid) and 1,1-diphenyl-2-picrylhydrazyl radical scavenging activities of the 60% ethanolic extract from A. yomena were determined to be 1,640.30 μg/mL and 2,655.10 μg/mL, respectively. Moreover, the inhibitory effect on malondialdehyde increased with the 60% ethanolic extract from A. yomena. To assess the neuroprotective effects, we examined the impact of the 60% ethanolic extract from A. yomena against H2O2-induced cytotoxicity in HT-22 (mouse hippocampal neuronal cell line) and SK-N-MC (human neuroblastoma cell line) cells. The results demonstrated a significant improvement in cell viability and reduced intracellular oxidative stress. Furthermore, the major bioactive compounds present in the 60% ethanolic extract from A. yomena were identified as chlorogenic acid and rutin through high-performance liquid chromatography (HPLC) analysis.

Oxymatrine Improves Oxidative Stress-Induced Senescence in HT22 Cells and Mice via the Activation of AMP-Activated Protein Kinase.

The accumulation of oxidative stress is one of the important factors causing cellular senescence. Oxymatrine (OM) is a natural quinolizidine alkaloid compound known for its antioxidant effects. This study aimed to investigate the anti-senescence potential of OM through oxidative stress-induced in vitro and in vivo models. By treating 600 μM of H2O2 to the HT22 mouse hippocampal neuronal cell line and by administering 150 mg/kg D-galactose to mice, we generated oxidative stress-induced senescence models. After providing 1, 2, and 4 μg/mL of OM to the HT22 mouse cell line and by administering 50 mg/kg OM to mice, we evaluated the enhancing effects. We evaluated different senescence markers, AMPK activity, and autophagy, along with DCFH-DA detection reaction and behavioral tests. In HT22 cells, OM showed a protective effect. OM, by reducing ROS and increasing p-AMPK expression, could potentially reduce oxidative stress-induced senescence. In the D-Gal-induced senescence mouse model, both the brain and heart tissues recovered AMPK activity, resulting in reduced levels of senescence. In neural tissue, to assess neurological recovery, including anxiety symptoms and exploration, we used a behavioral test. We also found that OM decreased the expression level of receptors for advanced glycation end products (RAGE). In heart tissue, we could observe the restoration of AMPK activity, which also increased the activity of autophagy. The results of our study suggest that OM ameliorates oxidative stress-induced senescence through its antioxidant action by restoring AMPK activity.

Nrf2-mediated protective effect of alpha-lipoic acid on synaptic oxidative damage and inhibition of PKC/ERK/CREB pathway in bisphenol A-exposed HT-22 cells

The harmful effects of bisphenol A (BPA) on learning and memory may involve hippocampal oxidative damage; however, the underlying mechanism remains unclear. Antioxidants that antagonize BPA-induced neuronal oxidative damage lack research. This study aimed to develop an in vitro model using the HT-22 mouse hippocampal neuronal cell line to investigate the neurotoxic mechanism of BPA and the protective effect of alpha-lipoic acid (ALA) on nuclear factor erythroid 2-related factor 2 (Nrf2) inhibition. The results showed that ALA reduced BPA-induced reactive oxygen species and neuronal nitric oxide synthase (nNOS) levels; however, inhibiting Nrf2 weakened the protective effects of ALA. BPA reduced mitochondrial complex I/III activity and ATP levels, but ALA ameliorated this damage. ALA improved the BPA-induced downregulation of the kelch-like ECH-associated protein 1 (keap1)/Nrf2 system, synaptic-related proteins, and the protein kinase C (PKC)/extracellular signal-regulated kinase (ERK)/cAMP response element binding protein (CREB) pathway; however, the protective effects of ALA were weakened when Nrf2 was inhibited. Our results suggest that BPA causes oxidative damage to HT-22 cells by damaging mitochondrial function, nNOS, and the keap1/Nrf2 system, thereby impairing synaptic-related proteins and the PKC/ERK/CREB pathway. ALA counters BPA-induced damage via Nrf2, which may be a significant target for the protective action of ALA.

Neuron-derived exosomes mediate sevoflurane-induced neurotoxicity in neonatal mice via transferring lncRNA Gas5 and promoting M1 polarization of microglia.

Sevoflurane exposure during rapid brain development induces neuronal apoptosis and causes memory and cognitive deficits in neonatal mice. Exosomes that transfer genetic materials including long non-coding RNAs (lncRNAs) between cells play a critical role in intercellular communication. However, the lncRNAs found in exosomes derived from neurons treated with sevoflurane and their potential role in promoting neurotoxicity remain unknown. In this study, we investigated the role of cross-talk of newborn mouse neurons with microglial cells in sevoflurane-induced neurotoxicity. Mouse hippocampal neuronal HT22 cells were exposed to sevoflurane, and then co-cultured with BV2 microglial cells. We showed that sevoflurane treatment markedly increased the expression of the lncRNA growth arrest-specific 5 (Gas5) in neuron-derived extracellular vesicles, which inhibited neuronal proliferation and induced neuronal apoptosis by promoting M1 polarization of microglia and the release of inflammatory cytokines. We further revealed that the exosomal lncRNA Gas5 significantly upregulated Foxo3 as a competitive endogenous RNA of miR-212-3p in BV2 cells, and activated the NF-κB pathway to promote M1 microglial polarization and the secretion of inflammatory cytokines, thereby exacerbating neuronal damage. In neonatal mice, intracranial injection of the exosomes derived from sevoflurane-treated neurons into the bilateral hippocampi significantly increased the proportion of M1 microglia, inhibited neuronal proliferation and promoted apoptosis, ultimately leading to neurotoxicity. Similar results were observed in vitro in BV2 cells treated with the CM from HT22 cells after sevoflurane exposure. We conclude that sevoflurane induces the transfer of lncRNA Gas5-containing exosomes from neurons, which in turn regulates the M1 polarization of microglia and contributes to neurotoxicity. Thus, modulating the expression of lncRNA Gas5 or the secretion of exosomes could be a strategy for addressing sevoflurane-induced neurotoxicity.

Effects of mitragynine on viability, proliferation, and migration of C6 rat glioma, SH-SY5Y human neuroblastoma, and HT22 immortalized mouse hippocampal neuron cell lines

Mitragynine (MG) is an indole alkaloid found in the extract of Mitragyna speciosa Korth native to Southeast Asia. Although MG is known for its pain-relieving and psychoactive effects, reports have suggested that it has therapeutic potential against neoplasms and psychiatric disorders. However, no evidence currently exists to support the effect of MG on brain tumors. This study aimed to investigate the antitumor effects of MG in C6 rat glioma and SH-SY5Y human neuroblastoma tumor cell lines compared with those in the non-tumor HT22 mouse hippocampal neuronal cell line. MTT assay for cell viability, clonogenic and wound healing assays for cell migration, Hoechst 33342/propidium iodide staining for nuclear morphology, and cell cycle distribution using flow cytometry were performed. MG at 125.47 μM (50 μg/ml) significantly reduced the viability of all cell lines, and the clonogenicity of C6 glioma cells began decreasing at 75.28 μM (30 μg/ml) of MG. Cell migration was inhibited in C6 and HT22 cells treated with 75.28 μM (30 μg/ml) of MG. Apoptotic nuclear condensation and fragmentation were observed in all cell lines treated with 125.47 μM (50 μg/ml) MG, whereas late-phase apoptotic cells were predominant in the group treated with 250.94 μM (100 μg/ml) of MG. The cell cycle assay results suggest that MG arrested the S phase in the C6 cell line and the G2/M phase in the HT22 cell lines. This study showed that MG induces cell death and cell cycle arrest, disrupting cell migration and reducing the clonogenicity of brain tumor cells.

Cooperative effects of SIRT1 and SIRT2 on APP acetylation.

Alzheimer's disease (AD) is an age-related neurodegenerative disorder characterized by amyloid-β (Aβ) deposition and neurofibrillary tangles. Although the NAD+ -dependent deacetylases SIRT1 and SIRT2 play pivotal roles in age-related diseases, their cooperative effects in AD have not yet been elucidated. Here, we report that the SIRT2:SIRT1 ratio is elevated in the brains of aging mice and in the AD mouse models. In HT22 mouse hippocampal neuronal cells, Aβ challenge correlates with decreased SIRT1 expression, while SIRT2 expression is increased. Overexpression of SIRT1 prevents Aβ-induced neurotoxicity. We find that SIRT1 impedes SIRT2-mediated APP deacetylation by inhibiting the binding of SIRT2 to APP. Deletion of SIRT1 reduces APP recycling back to the cell surface and promotes APP transiting toward the endosome, thus contributing to the amyloidogenic processing of APP. Our findings define a mechanism for neuroprotection by SIRT1 through suppression of SIRT2 deacetylation, and provide a promising avenue for therapeutic intervention of AD.

Attenuation of amyloid-β-induced mitochondrial dysfunction by active components of anthocyanins in HT22 neuronal cells.

Alzheimer's disease (AD) is a common form of neurodegenerative disease in the elderly. Amyloid-β (Aβ)-associated neurotoxicity is an important component of the neurodegenerative change in AD. Recent studies have revealed a beneficial effect of anthocyanins in improving learning and memory in AD animal models. Using cultured HT22 mouse hippocampal neuronal cells as an in vitro model, we examined in this study the protective effect of ten pure components of anthocyanins against Aβ 42-induced cytotoxicity and also investigated the mechanism of their protective effects. We found that treatment of HT22 cells with the pure components of anthocyanins dose-dependently rescued Aβ 42-induced cytotoxicity, with slightly different potencies. Using petunidin as a representative compound, we found that it enhanced mitochondrial homeostasis and function in Aβ 42-treated HT22 cells. Mechanistically, petunidin facilitated β-catenin nuclear translocation and enhanced the interaction between β-catenin and TCF7, which subsequently upregulated mitochondrial homeostasis-related protein Mfn2, thereby promoting restoration of mitochondrial homeostasis and function in Aβ 42-treated HT22 cells. Together, these results reveal thatthe pure components of anthocyanins have a strong protective effect in HT22 cells against Aβ 42-induced cytotoxicity by ameliorating mitochondrial homeostasis and function in a β-catenin/TCF-dependent manner.

Transcriptomic analysis of glutamate-induced HT22 neurotoxicity as a model for screening anti-Alzheimer’s drugs

Glutamate-induced neurotoxicity in the HT22 mouse hippocampal neuronal cell line has been recognized as a valuable cell model for the study of neurotoxicity associated with neurodegenerative diseases including Alzheimer’s disease (AD). However, the relevance of this cell model for AD pathogenesis and preclinical drug screening remains to be more elucidated. While there is increasing use of this cell model in a number of studies, relatively little is known about its underlying molecular signatures in relation to AD. Here, our RNA sequencing study provides the first transcriptomic and network analyses of HT22 cells following glutamate exposure. Several differentially expressed genes (DEGs) and their relationships specific to AD were identified. Additionally, the usefulness of this cell model as a drug screening system was assessed by determining the expression of those AD-associated DEGs in response to two medicinal plant extracts, Acanthus ebracteatus and Streblus asper, that have been previously shown to be protective in this cell model. In summary, the present study reports newly identified AD-specific molecular signatures in glutamate-injured HT22 cells, suggesting that this cell can be a valuable model system for the screening and evaluation of new anti-AD agents, particularly from natural products.

Biochemical mechanism of erastin-induced ferroptotic cell death in neuronal cells.

Ferroptosis is a new form of nonapoptotic cell death closely associated with glutathione (GSH) peroxidase 4 inhibition and/or GSH depletion, resulting in the accumulation of cellular iron and lipid peroxides. The exact mechanism by which GSH depletion causes the accumulation of reactive oxygen species (ROS) and lipid-ROS and subsequent ferroptotic cell death in neuronal cells remains unclear. In the present study, using immortalized HT22 mouse hippocampal neuronal cells as a model, we show that nitric oxide (NO) accumulation via protein disulfide isomerase (PDI)-mediated neuronal nitric oxide synthase (nNOS) activation plays a critical role in chemically-induced ferroptosis. Mechanistically, we find that erastin-induced GSH depletion leads to activation of PDI, which then mediates ferroptosis by catalyzing nNOS dimerization, followed by accumulation of cellular NO, ROS and lipid ROS and ultimately ferroptotic cell death. Pharmacological inhibition of PDI enzymatic activity or selective PDI knockdown can effectively abrogate erastin-induced ferroptosis in HT22 cells. The results of this study reveal an important role of PDI in mediating chemically induced ferroptosis in a neuronal cell model, and PDI may serve as a potential drug target for protection against GSH depletion-associated ferroptotic neuronal cell death.

Chemical constituents of Ajuga forrestii and their anti-ferroptosis activity

Six new neoclerodane diterpenoids (1–6), along with ten known compounds (7–16), were isolated from Ajuga forrestii. Their structures were elucidated by HRESIMS, 1D and 2D NMR, ECD calculation, and single-crystal X-ray diffraction analysis. The structure of a known neoclerodane diterpene ajudecunoid C (6) was revised based on the reported NMR empirical rules. All the isolates were evaluated for their inhibitory effect on RSL3-induced ferroptosis in HT22 mouse hippocampal neuronal cells. Among them, compounds 8, 9, and 12 significantly inhibited RSL3-induced ferroptosis with EC50 values of 0.45 μM, 0.076 μM, and 0.14 μM.

Discovery and optimization of olanzapine derivatives as new ferroptosis inhibitors

Ferroptosis is a new type of cell death associated with many human diseases. It is a new strategy to discover ferroptosis inhibitors for the treatment of ferroptosis-related diseases. Here the FDA-approved drug library containing 1160 molecules was screened for ferroptosis inhibitors in RSL3-induced HT22 mouse hippocampal neuronal cells. As a result, olanzapine showed potent ferroptosis inhibitory activity (EC50 = 1.18 μM). Structural optimization and the structure–activity relationships (SARs) analysis led to the synthesis of 41 new derivatives (4–44) and one known compound 45. Comparing with olanzapine, its derivative 36 showed nearly sixteen-folds improved ferroptosis inhibition and low cytotoxicity (EC50 = 0.074 μM, CC50 = 18.8 μM). Further mechanistic studies revealed that compound 36 specifically inhibited ferroptosis by its antioxidative ability. This work demonstrates that olanzapine protected RSL3-induced ferroptosis in HT22 cell, and its derivative 36 having nanomolar ferroptosis inhibitory activity merit to be developed for drugs against ferroptosis-related neurological diseases.

Quantitative omics analyses of NCOA4 deficiency reveal an integral role of ferritinophagy in iron homeostasis of hippocampal neuronal HT22 cells.

Neurons require iron to support their metabolism, growth, and differentiation, but are also susceptible to iron-induced oxidative stress and cytotoxicity. Ferritin, a cytosolic iron storage unit, mediates cellular adaptation to fluctuations in iron delivery. NCOA4 has been characterized as a selective autophagic cargo receptor facilitating the mobilization of intracellular iron from ferritin. This process named ferritinophagy results in the degradation of ferritin and the consequent release of iron into the cytosol. Here we demonstrate that NCOA4 is important for the adaptation of the HT22 mouse hippocampal neuronal cell line to cellular iron restriction. Additionally, we determined the pathophysiological implications of impaired ferritinophagy via functional analysis of the omics profile of HT22 cells deficient in NCOA4. NCOA4 silencing impaired ferritin turnover and was cytotoxic when cells were restricted of iron. Quantitative proteomics identified IRP2 accumulation among the most prominent protein responses produced by NCOA4 depletion in HT22 cells, which is indicative of functional iron deficiency. Additionally, proteins of apoptotic signaling pathway were enriched by those responsive to NCOA4 deficiency. Transcriptome profiles of NCOA4 depletion revealed neuronal cell death, differentiation of neurons, and development of neurons as potential diseases and bio functions affected by impaired ferritinophagy, particularly, when iron was restricted. These findings identify an integral role of NCOA4-mediated ferritinophagy in the maintenance of iron homeostasis by HT22 cells, and its potential implications in controlling genetic pathways of neurodevelopment and neurodegenerative diseases.

β‐Lactolin Improves Mitochondrial Dysfunction in Aβ‐treated Mouse Hippocampal Neuronal Cells and a Human iPSC‐Derived Neuronal Cell Model of Alzheimer’s Disease

AbstractBackgroundMitochondrial dysfunctions are a key hallmark of Alzheimer’s disease (AD). β‐Lactolin, a whey‐derived glycine–threonine–tryptophan–tyrosine tetrapeptide, has been previously reported to prevent AD‐like pathologies in an 5×FAD mouse model via regulation of microglial functions. However, the direct effect of β‐lactolin on neuronal cells and neuronal mitochondrial functions remains unknown. Here, we investigated the effects of β‐lactolin on mitochondrial dysfunctions in amyloid β (Aβ)‐treated mouse hippocampal neuronal HT22 cells and human induced‐pluripotent cell (hiPSC)‐derived AD model neurons.MethodMouse hippocampal neuronal HT22 cell line, and hiPSC‐derived AD model neuron (PSEN1 P117L mutation) and its wild‐type control were used in this study. HT22 cells were treated with β‐lactolin (1–100 nM) for 1 hr, and Aβ42 (1–10 μM) was added for the next 23 hrs. Oxygen consumption rate was measured using extracellular flux analyzer. Intracellular ATP level was determined by luciferase activity. Cells were stained with MitoTracker, JC‐1, or MitoSOX fluorescent dye, and then mitochondrial morphologies, membrane potential, and oxidative stress, respectively, were examined by high content image analysis. Cell viability was measured by MTT assay. Gene expression levels were evaluated by RT‐PCR. hiPSC‐derived wild‐type and AD neurons were cultured for 14 days under β‐lactolin treatment, and mitochondrial morphologies and membrane potentials were analyzed.ResultHT22 cells treated with both β‐lactolin and Aβ exhibited increased oxygen consumption rate and cellular ATP concentrations compared to cells treated with Aβ alone, suggesting that β‐lactolin improves mitochondrial respiration and energy production. In high content image analysis, β‐lactolin attenuated Aβ‐induced mitochondrial fragmentation, membrane potential decrease, and excess oxidative stress, eventually preventing neuronal cell death. Treatments with β‐lactolin increased gene expression of mitofusin‐2, which contribute to mitochondrial fusion events. Finally, β‐lactolin attenuated impairment in both mitochondrial morphologies and membrane potentials in hiPSC‐derived AD model neurons, suggesting β‐lactolin shows a suppressing effect on human AD pathologies.Conclusionβ‐lactolin improved AD‐related neuronal mitochondrial dysfunctions and suppressed neuronal cell death in both mouse and human AD model neuronal cells. The dual function of β‐lactolin on both neuron and microglia marks an advantage in AD prevention.

Neuroprotective effects of phenolic glycosides from Populus tomentiglandulosa roots in HT22 mouse hippocampal neuronal cells

The present work isolated a total of three novel phenolic glycosides (1 − 3), and 12 known compounds (4 − 15) from methanol extract of the roots of Populus tomentiglandulosa. The chemical structures of these compounds were determined by spectroscopic analyses (1D and 2D NMR and mass spectrometry). The compounds (5, 7, and 11) having the 1‑hydroxy-6-oxo-2-cyclohexenecarboxylate (HCH) moiety showed strong neuroprotective effects in glutamate-induced HT22 hippocampal cells. However, among these compounds, tremulacin (compound 5; TRC) was evaluated for its ability to prevent glutamate-induced apoptosis. TRC reduced the glutamate-induced apoptosis by downregulating of cleaved poly (ADP-ribose) polymerase (PARP), caspase-3, Bax, and phosphorylated mitogen-activated protein kinase (MAPK) pathways while upregulating Bcl-2 and Bid. Also, TRC triggered the expression of hemeoxygenase-1 (HO-1) and nuclear erythroid-2 related factor 2 (Nrf2) in a dose-dependent manner. Furthermore, TRC treatment attenuated intracellular ROS generation and MMP loss. Molecular docking results also demonstrated that TRC interacted with Kelch-like ECH-associated protein 1 (Keap1). Overall, this study revealed that TRC might be a promising treatment option for neurological diseases such as Alzheimer's and Parkinson's.

Regulation of retinoid mediated StAR transcription and steroidogenesis in hippocampal neuronal cells: Implications for StAR in protecting Alzheimer's disease

Retinoids (vitamin A and its derivatives) play pivotal roles in diverse processes, ranging from homeostasis to neurodegeneration, which are also influenced by steroid hormones. The rate-limiting step in steroid biosynthesis is mediated by the steroidogenic acute regulatory (StAR) protein. In the present study, we demonstrate that retinoids enhanced StAR expression and pregnenolone biosynthesis, and these parameters were markedly augmented by activation of the PKA pathway in mouse hippocampal neuronal HT22 cells. Deletion and mutational analyses of the 5′-flanking regions of the StAR gene revealed the importance of a retinoic acid receptor (RAR)/retinoid X receptor (RXR)-liver X receptor (LXR) heterodimeric motif at −200/−185 bp region in retinoid responsiveness. The RAR/RXR-LXR sequence motif can bind RARα and RXRα, and retinoid regulated transcription of the StAR gene was found to be influenced by the LXR pathway, representing signaling cross-talk in hippocampal neurosteroid biosynthesis. Steroidogenesis decreases during senescence due to declines in the central nervous system and the endocrine system, and results in hormone deficiencies, inferring the need for hormonal balance for healthy aging. Loss of neuronal cells, involving accumulation of amyloid beta (Aβ) and/or phosphorylated Tau within the brain, is the pathological hallmark of Alzheimer's disease (AD). HT22 cells overexpressing either mutant APP (mAPP) or mutant Tau (mTau), conditions mimetic to AD, enhanced toxicities, and resulted in attenuation of both basal and retinoid-responsive StAR and pregnenolone levels. Co-expression of StAR with either mAPP or mTau diminished cytotoxicity, and concomitantly elevated neurosteroid biosynthesis, pointing to a protective role of StAR in AD. These findings provide insights into the molecular events by which retinoid signaling upregulates StAR and steroid levels in hippocampal neuronal cells, and StAR, by rescuing mAPP and/or mTau-induced toxicities, modulates neurosteroidogenesis and restores hormonal balance, which may have important implications in protecting AD and age-related complications and diseases.