Mouse Neuroblastoma Cell Line Research Articles

New insight into the molecular etiopathogenesis of konzo: Cyanate could be a plausible neurotoxin contributing to konzo, contrary to thiocyanate

IntroductionChronic cassava-derived cyanide poisoning is associated with the appearance of konzo, a tropical spastic paraparesis due to selective upper motor neuron damage. Whether the disease is caused by a direct action of cyanide or its metabolites is still an open question. This preliminary study assessed the neurotoxic effects of thiocyanate (SCN) and cyanate (OCN), two cyanide metabolites hypothesized to be plausible toxic agents in konzo. MethodsCultured mouse neuroblastoma (Neuro-2A) and human neuroblastoma (SH-SY5Y) cell lines were incubated (24, 48, and 72hours) in sodium OCN or sodium SCN in a disease-relevant concentration range. Cell viability, caspase (3, 8, and 9) activities, and reactive oxygen species (ROS) generation were evaluated using appropriate assay kits. Additionally, electrophysiological responses induced by OCN and SCN in primary spinal cord neurons (from Sprague Dawley rats) were assessed by whole-cell patch-clamp techniques. ResultsBoth OCN and SCN were toxic in a dose-dependent way, even if SCN toxicity appeared at very high concentrations (30mM, corresponding to more than 100-fold higher than normal plasmatic levels), contrary to OCN (0.3-3mM). OCN was markedly more toxic in a poor culture medium (MEM; IC50 = 3.2mM) compared to a glucose- and amino acid-rich medium (DMEM; IC50=7.6mM). OCN treatment increased the ROS generation by 8.9 folds, as well as the Caspase-3, Caspase-8, and Caspase-9 activities by 3.2, 2.5, and 2.6 folds, respectively. Finally, OCN (and SCN to a lesser extent) induced depolarizing currents in primary spinal cord neurons, through an activation of ionotropic glutamate receptors. ConclusionOur results suggest OCN as the most plausible neurotoxic agent involved in konzo, while SCN toxicity could be questioned at such high concentrations. Also, they support apoptosis, oxidative stress, and excitotoxicity as probable mechanisms of OCN neurotoxicity.

Neuroglobin protects dopaminergic neurons in a Parkinson’s cell model by interacting with mitochondrial complex NDUFA10

The study aimed to validate the protective effect of neuroglobin (Ngb) in a cell model of Parkinson’s disease (PD) and explore its therapeutic potential. Lentivirus-Ngb (LvNgb) and siRNA-Ngb (siNgb) were used to achieve Ngb overexpression and knockdown, respectively, in a sporadic PD cell model. Apoptosis was evaluated by flow cytometry-based Annexin V/propidium iodide assays. Activation of the pro-apoptotic factor, Caspase-9, was detected by immunoblotting, and Complex I activities were detected by using enzyme-linked immunosorbent assay (ELISA). Mitochondrial dysfunction was examined by measuring the mitochondrial membrane potential (MMP), NAD+/NADH ratios, and reactive oxygen species (ROS) levels. Additionally, coimmunoprecipitation (Co-IP) assays were conducted in mouse neuroblastoma cell line 9D (MN9D) cells to determine the interactions of Ngb with the Complex I subunit NDUFA10. The results showed that Ngb overexpression reduced the percentages of apoptotic cells, total caspase-9 levels and restored Complex I activities in the PD cell model. Conversely, knockdown of Ngb resulted in an increase in apoptotic cells, higher total caspase-9 levels, and decreased Complex I activities. Furthermore, Ngb overexpression restored MMP and NAD+/NADH ratios and alleviated ROS-mediated oxidative stress in MN9D cells. Finally, Co-IP confirmed the interaction between Ngb and NDUFA10 in MN9D cells. In conclusion, Ngb protects MN9D cells against apoptosis by interacting with Complex I subunit NDUFA10, rescuing its activity and inhibiting the mitochondrial pathway of apoptosis in the MPP+-mediated PD model.

Mapping the lipidome in mitochondria-associated membranes (MAMs) in an invitro model of Alzheimer's disease.

The disruption of mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) plays a relevant role in Alzheimer's disease (AD). MAMs have been implicated in neuronal dysfunction and death since it is associated with impairment of functions regulated in this subcellular domain, including lipid synthesis and trafficking, mitochondria dysfunction, ER stress-induced unfolded protein response (UPR), apoptosis, and inflammation. Since MAMs play an important role in lipid metabolism, in this study we characterized and investigated the lipidome alterations at MAMs in comparison with other subcellular fractions, namely microsomes and mitochondria, using an invitro model of AD, namely the mouse neuroblastoma cell line (N2A) over-expressing the APP familial Swedish mutation (APPswe) and the respective control (WT) cells. Phospholipids (PLs) and fatty acids (FAs) were isolated from the different subcellular fractions and analyzed by HILIC-LC-MS/MS and GC-MS, respectively. In this invitro AD model, we observed a down-regulation in relative abundance of some phosphatidylcholine (PC), lysophosphatidylcholine (LPC), and lysophosphatidylethanolamine (LPE) species with PUFA and few PC with saturated and long-chain FA. We also found an up-regulation of CL, and antioxidant alkyl acyl PL. Moreover, multivariate analysis indicated that each organelle has a specific lipid profile adaptation in N2A APPswe cells. In the FAs profile, we found an up-regulation of C16:0 in all subcellular fractions, a decrease of C18:0 levels in total fraction (TF) and microsomes fraction, and a down-regulation of 9-C18:1 was also found in mitochondria fraction in the AD model. Together, these results suggest that the over-expression of the familial APP Swedish mutation affects lipid homeostasis in MAMs and other subcellular fractions and supports the important role of lipids in AD physiopathology.

Notoginsenoside R1 attenuates bupivacaine induced neurotoxicity by activating Jak1/Stat3/Mcl1 pathway

Bupivacaine, a common amide local anesthetic, can provide effective analgesia or pain relief but can also cause neurotoxicity, which remains a mounting concern in clinic and animal care. However, the precise underlying mechanisms have not been fully elucidated. A natural compound, notoginsenoside R1 (NG-R1) has been reported to exhibit a neuroprotective role in stress conditions. In this study, we explored the function and mechanism of NG-R1 in alleviating bupivacaine-induced neurotoxicity in mouse hippocampal neuronal (HT-22) and mouse neuroblastoma (Neuro-2a) cell lines. Our results exhibited that NG-R1 treatment can significantly rescue the decline of cell survival induced by bupivacaine. Tunel staining and western blotting showed that NG-R1 could attenuate BPV‑induced cell apoptosis. Besides, we focused on Mcl1 as a potential target as it showed opposite expression tendency in response to NG-R1 and bupivacaine exposure. Mcl1 knockdown blocked the inhibitory effect of NG-R1 on cell apoptosis against bupivacaine treatment. Intriguingly, we found that NG-R1 can upregulate Mcl1 transcription by activating Stat3 and promote its nuclear translocation. In addition, NG-R1 can also promote Jak1 phosphorylation and docking analysis provide a predicted model for interaction between NG-R1 and phosphorylated Jak1. Taken together, our results demonstrated that NG-R1 can attenuate bupivacaine induced neurotoxicity by activating Jak1/Stat3/Mcl1 pathway

Neuroprotective effect of olive leaf extract in the mouse neuroblastoma cell line (NB2a): A good model to evaluate the neuroprotective effects of natural products

Chronic neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases, whose prevalence increases with aging, cause serious health problems with inadequate treatment options. Long-term exposure to low doses of pesticides like chlorpyrifos has been linked to an increased risk of developing neurodegenerative diseases. This study assessed the effects of olive leaf extract (OLE) on cell viability and neurite outgrowth inhibition induced by chlorpyrifos (25 µM) using the mouse NB2a neuroblastoma cell line. High concentrations of OLE as 100 to 3000 µg/ml reduced cell viability, likely due to its anticancer properties. On the other hand, lower concentrations of OLE as 3, 10, 30 and 100 µg/ml significantly reversed the inhibition of neurite outgrowth caused by chlorpyrifos exposure, demonstrating a neuroprotective effect. In conclusion, the regular consumption of OLE may effectively slow or prevent the advancement of neurodegenerative diseases linked to long-term exposure to low-dose organophosphate pesticides. Furthermore, the chlorpyrifos-induced moderate neurotoxicity model can be employed as a valuable screening method for evaluating the neuroprotective potential of natural products, including various plant extracts.

New melatonin derivatives with donepezil fragment in Alzheimer’s disease as neuroprotectors

AbstractBackgroundAlzheimer’s disease is characterized by progressive, irreversible degeneration of acetylcholine‐producing neurons, cognitive impairment, and the accumulation of neurofibrillary tangles and amyloid plaques. The natural decline in melatonin levels with aging has been associated with insomnia and depression as well as the development of neurodegenerative disorders such as AD. Furthermore, the complexity of AD pathology has encouraged the design of molecules that are able to interact with two or more complementary targets, with the expectation that these multitarget ligands (MTL) may represent important advantages in the treatment of the disease. This study aimed to find new ChE inhibitors with improved toxicity.Method In vitro toxicity screening of the newly compounds on human neuroblastoma SH‐SY5Y, mouse neuroblastoma cell line Neuro‐2a and mouse fibroblast cells CCL‐1. The viability of the cells was assessed by MTT assay. The neuroprotective properties of the compounds were determined in a model of H2O2‐induced oxidative damage in SH‐SY5Y cells. For comparison, effects donepezil, melatonin and galanthamine were determined.ResultMost of the compounds have IC50 values over 100 µM. Compound 3f possess statistically significant protective effects. It should be noted that the neuroprotective effects were more pronounced than those of donepezil and similar to melatonin and rasagiline.ConclusionThe new series of melatonin derivatives with donepezil fragment exhibit low toxicity and a good in vitro safety profile. Further, in vitro protection study proved efficient prevention against oxidative stress in H2O2‐induced injury in SH‐SY5Ycells, especially by compound 3f and was proven as the most perspective for further investigations.Acknowledgments: The financial support by the National Science Fund of Bulgaria (Grant KP‐06‐Н63/11 14.12.2022) is gratefully acknowledged.

Differentiated neuroblastoma cells remain epigenetically poised for de-differentiation to an immature state.

Neuroblastoma is the most common extracranial solid tumor of childhood and accounts for a significant share of childhood cancer deaths. Prior studies utilizing RNA sequencing of bulk tumor populations showed two predominant cell states characterized by high and low expression of neuronal genes. Although cells respond to treatment by altering their gene expression, it is unclear whether this reflects shifting balances of distinct subpopulations or plasticity of individual cells. Using mouse and human neuroblastoma cell lines lacking MYCN amplification, we show that the antigen CD49b (also known as ITGA2) distinguishes these subpopulations. CD49b expression marked proliferative cells with an immature gene expression program, whereas CD49b-negative cells expressed differentiated neuronal marker genes and were non-cycling. Sorted populations spontaneously switched between CD49b expression states in culture, and CD49b-negative cells could generate rapidly growing, CD49b-positive tumors in mice. Although treatment with the chemotherapy drug doxorubicin selectively killed CD49b-positive cells in culture, the CD49b-positive population recovered when treatment was withdrawn. We profiled histone 3 (H3) lysine 27 acetylation (H3K27ac) to identify enhancers and super enhancers that were specifically active in each population and found that CD49b-negative cells maintained the priming H3 lysine 4 methylation (H3K4me1) mark at elements that were active in cells with high expression of CD49b. Improper maintenance of primed enhancer elements might thus underlie cellular plasticity in neuroblastoma, representing potential therapeutic targets for this lethal tumor.

Abstract A153: ALK proximitome reveal SLC3A2, part of the polyamine transporter, as a membrane interaction partner with growth promoting ability

Abstract Solute Carrier Family 3, Member 2 (SLC3A2 or 4F2hc) was identified as a potential ALK interacting partner in a BioID-proximity labeling screen in neuroblastoma cells. SLC3A2 is a multifunctional glycoprotein that mediates integrin-dependent signaling, acts as a trafficking chaperone for amino acid transporters, and regulates polyamine transportation. However, the functional consequences of an interaction between SLC3A2 and ALK are unclear. In this work we confirm that endogenous SLC3A2 and ALK interact in NB cells. This SLC3A2:ALK interaction is abrogated upon treatment with the ALK inhibitor lorlatinib. In agreement, stimulation of ALK with ALKAL2 ligand, resulted in SLC3A2 protein levels, which was decreased upon lorlatinib treatment. We show here that loss of ALK activity leads to suppressed SLC3A2 expression and reduced SLC3A2 protein stability in a panel of neuroblastoma cells. Solute Carrier Family 7 Member 3 (SLC7A5, also known as LAT1) is an amino acid transporter that forms a stable complex with SLC3A2 for localization at the plasma membrane, where they function. This complex is lost upon knockdown of SLC3A2, with concomitant inhibition of NB cell growth. To investigate the therapeutic potential of SLC3A2/SLC7A5 complex targeting, we performed monotreatment of NB cells with AMXT-1501 (a polyamine transport inhibitor) only showed a moderate effect in NB cells. In contrast, a combined lorlatinib/AMXT-1501 treatment resulted in synergistic inhibition of cell growth in ALK-driven primary-cultured mouse neuroblastoma cell lines. Taken together, our results show that SLC3A2 protein stability and its interaction with ALK depend on ALK signaling in ALK-driven neuroblastoma. The synergistic effect of combined ALK and polyamine transport inhibitors suggests a potential therapeutic option for ALK-driven neuroblastoma. Citation Format: Bengt Hallberg, Wei-Yun Lai, Tzu-Po Chuang, Ruth Palmer. ALK proximitome reveal SLC3A2, part of the polyamine transporter, as a membrane interaction partner with growth promoting ability [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A153.

Bardoxolone methyl inhibits the infection of rabies virus via Nrf2 pathway activation in vitro

BackgroundRabies is a widespread, fatal, infectious disease. Several antivirals against rabies virus (RABV) infection have been reported, but no approved, RABV-specific antiviral drugs that inhibit RABV infection in the clinic after symptom onset are available. Therefore, more effective drugs to reduce rabies fatalities are urgently needed. Bardoxolone methyl (CDDO-Me), an FDA-approved compound that has long been known as an antioxidant inflammatory modulator and one of the most potent nuclear factor erythroid-derived 2-like 2 (Nrf2) activators, protects myelin, axons, and CNS neurons by Nrf2 activation. Therefore, we investigated the potency of its anti-RABV activity in vitro.MethodsThe mouse neuroblastoma cell line Neuro2a (N2a) and three RABV strains of different virulence were used; the cytotoxicity and anti-RABV activity of CDDO-Me in N2a cells were evaluated by CCK-8 assay and direct fluorescent antibody (DFA) assay. Pathway activation in N2a cells infected with the RABV strains SC16, CVS-11 or CTN upon CDDO-Me treatment was evaluated by western blotting (WB) and DFA assay.ResultsCDDO-Me significantly inhibited infection of the three RABV strains of differing virulence (SC16, CVS-11 and CTN) in N2a cells. We also examined whether CDDO-Me activates the Nrf2-associated pathway upon infection with RABV strains of differing virulence. Nrf2, phosphorylated sequestosome (SQSTM1), SQSTM1, hemoglobin oxygenase (HO-1) and NAD(P)H dehydrogenase quinone 1 (NQO1) expression in N2a cells increased to varying degrees with CDDO-Me treatment, accompanied by Kelch-like ECH-associated protein 1 (Keap1) dissociation, upon infection with SC16, CVS-11 or CTN. The activation of SQSTM1 phosphorylation was significantly associated with the degradation of Keap-1 in CDDO-Me-treated N2a cells upon RABV infection. Furthermore, N2a cells pretreated with the Nrf2-specific inhibitor ATRA showed a significant decrease in HO-1 and NQO1 expression and a decrease in the anti-RABV efficacy of CDDO-Me. These inhibitory effects were observed upon infection with three RABV strains of differing virulence.ConclusionCDDO-Me inhibited RABV infection via Nrf2 activation, promoting a cytoprotective defense response in N2a cells. Our study provides a therapeutic strategy for RABV inhibition and neuroprotection during viral infection.

Activating ryanodine receptor improves isoflurane-induced cognitive dysfunction

BackgroundPostoperative cognitive dysfunction (POCD) is characterized by impaired learning and memory. 6 h duration isoflurane anesthesia is an important factor to induce POCD, and the dysfunction of ryanodine receptor (RyR) in the hippocampus may be involved in this process. We investigated the expression of RyR3 in the hippocampus of mice after 6-h duration isoflurane anesthesia, as well as the improvement of RyR receptor agonist caffeine on POCD mice, while attempting to identify the underlying molecular mechanism. MaterialsWe constructed a POCD model using 8-week-old male C57BL/6J mice that were exposed to 6-h duration isoflurane. Prior to the three-day cognitive behavioral experiment, RyR agonist caffeine were injected. Fear conditioning and location memory tests were used in behavioral studies. We also exposed the mouse neuroblastoma cell line Neuro-2a (N2A) to 6-h duration isoflurane exposure to simulate the conditions of in vivo cognitive dysfunction. We administered ryanodine receptor agonist (caffeine) and inhibitor (ryanodine) to N2a cells. Following that, we performed a series of bioinformatics analysis to discover proteins that are involved in the development of cognitive dysfunction. Rt-PCR and Western blot were used to assess mRNA level and protein expression. Results6-h duration isoflurane anesthesia induced cognitive dysfunction and increased RyR3 mRNA levels in hippocampus. The mRNA levels of RyR3 in cultured N2a cells after anesthesia were comparable to those in vivo, and the RyR agonist caffeine corrected the expression of some cognitive-related phenotypic proteins that were disturbed after anesthesia. Intraperitoneal injection of RyR agonist caffeine can improve cognitive function after isoflurane anesthesia in mice, and bioinformatics analyses suggest that CaMKⅣ may be involved in the molecular mechanism. ConclusionRyanodine receptor agonist caffeine may improve cognitive dysfunction in mice after isoflurane anesthesia.

Functional Interaction between Adenosine A2A and mGlu5 Receptors Mediates STEP Phosphatase Activation and Promotes STEP/mGlu5R Binding in Mouse Hippocampus and Neuroblastoma Cell Line.

(1) Background: Recently, we found that adenosine A2A receptor (A2AR) stimulation results in an increase in STEP phosphatase activity. In order to delve into the mechanism through which A2AR stimulation induced STEP activation, we investigated the involvement of mGlu5R since it is well documented that A2AR and mGlu5R physically and functionally interact in several brain areas. (2) Methods: In a neuroblastoma cell line (SH-SY5Y) and in mouse hippocampal slices, we evaluated the enzymatic activity of STEP by using a para-nitrophenyl phosphate colorimetric assay. A co-immunoprecipitation assay and a Western blot analysis were used to evaluate STEP/mGlu5R binding. (3) Results: We found that the A2AR-dependent activation of STEP was mediated by the mGlu5R. Indeed, the A2AR agonist CGS 21680 significantly increased STEP activity, and this effect was prevented not only by the A2AR antagonist ZM 241385, as expected, but also by the mGlu5R antagonist MPEP. In addition, we found that mGlu5R agonist DHPG-induced STEP activation was reversed not only by the mGlu5R antagonist MPEP but also by ZM 241385. Finally, via co-immunoprecipitation experiments, we found that mGlu5R and STEP physically interact when both receptors are activated (4) Conclusions: These results demonstrated a close functional interaction between mGlu5 and A2A receptors in the modulation of STEP activity.

High-throughput sequencing-based Detection of Japanese encephalitis virus and its effect on micro ribonucleic acid

it was to explore the mechanism of Japanese encephalitis virus (JEV) and micro ribonucleic acid (miRNA) under high-throughput sequencing. 20 experimental mice, with good growth status and no disease infection, were selected. The cells used in the experiment included mouse microglial cell line (BV2), mouse neuroblastoma cell line (NA), and mouse brain endothelial cell line (bEnd.3). JEV titration was performed with JEV-infected cells, ribonucleic acid (RNA) in the cells was extracted, and finally the miRNA high-throughput sequencing data was analyzed. Agarose gel electrophoresis showed that the 28S and 18S electrophoresis bands were bright. Among the miRNAs detected in mouse brain tissues, 2986 were down-regulated and 1251 were up-regulated. Among miRNAs detected in NA cells, 4238 the decreasing expression and 2356 were expressed increasingly. In reducing miRNA expression, 1 multiplicity of infection (MOI) of P3 strain infection was more significant than 0.1 MOI. 10 miRNAs with significantly decreasing expression were miR-466d-3p, miR-381–3p, miR-540–3p, miR-466a-3p, miR-467a-3p, miR-574–5p, miR-199a-5p, miR-467a-5p, miR-674–5p, and miR-376b-3p. These were all obviously down-regulated in JEV-infected BV2, NA, and bEnd.3 neurons. High-throughput sequencing of JEV-infected mouse brain tissues and mouse neuronal cells found that JEV infection led to down-regulation of overall miRNA expression in host cells.

Lactate promotes neuronal differentiation of SH-SY5Y cells by lactate-responsive gene sets through NDRG3-dependent and -independent manners

Lactate serves as the major glucose alternative to an energy substrate in the brain. Lactate level is increased in the fetal brain from the middle stage of gestation, indicating the involvement of lactate in brain development and neuronal differentiation. Recent reports show that lactate functions as a signaling molecule to regulate gene expression and protein stability. However, the roles of lactate signaling in neuronal cells remain unknown. Here, we showed that lactate promotes the all stages of neuronal differentiation of SH-SY5Y and Neuro2A, human and mouse neuroblastoma cell lines, characterized by increased neuronal marker expression and the rates of neurites extension. Transcriptomics revealed many lactate-responsive genes sets such as SPARCL1 in SH-SY5Y, Neuro2A, and primary embryonic mouse neuronal cells. The effects of lactate on neuronal function were mainly mediated through monocarboxylate transporters 1 (MCT1). We found that NDRG family member 3 (NDRG3), a lactate-binding protein, was highly expressed and stabilized by lactate treatment during neuronal differentiation. Combinative RNA-seq of SH-SY5Y with lactate treatment and NDRG3 knockdown shows that the promotive effects of lactate on neural differentiation are regulated through NDRG3-dependent and independent manners. Moreover, we identified TEA domain family member 1 (TEAD1) and ETS-related transcription factor 4 (ELF4) are the specific transcription factors that are regulated by both lactate and NDRG3 in neuronal differentiation. TEAD1 and ELF4 differently affect the expression of neuronal marker genes in SH-SY5Y cells. These results highlight the biological roles of extracellular and intracellular lactate as a critical signaling molecule that modifies neuronal differentiation.

Anti-CHAC1 exosomes for nose-to-brain delivery of miR-760-3p in cerebral ischemia/reperfusion injury mice inhibiting neuron ferroptosis

Ferroptosis plays a critical role in ischemic stroke, and anti-ferroptosis strategies were regarded as potentially effective measures. Based on ferroptosis-related mechanisms, this study aims to design and prepare anti-ferroptosis exosomes from adipose-derived mesenchymal stem cells (ADSC-Exo) for treating ischemic brain injury via intranasal (IN) administration. According to the bioinformatic analysis, CHAC1 was a key gene in the progress of ferroptosis in ischemic stroke. miR-760-3p can inhibit the expression of CHAC1 and may be abundant in ADSC-Exo. Therefore, ADSC-Exo were successfully isolated and the immunofluorescence showed that they can be efficiently delivered to the brain via IN administration. Additionally, IN administration of ADSC-Exo can effectively improve the neurobehavior function of mice after I/R, and improve the ferroptosis-related outcomes. As the immunofluorescence showed the co-localization of NeuN with CHAC1 obviously, we further evaluated the systematic effect of ADSC-Exo in an oxygen–glucose deprivation (OGD) mouse neuroblastoma cell line N2a model. The results showed that miR-760-3p in ADSC‐Exo contributed to their function in inhibiting ferroptosis by targeting CHAC1 in neurons. Collectively, the present study successfully designed and prepared anti-CHAC1 ADSC-Exo and suggested a promising exosome-based strategy for anti-ferroptosis therapy in cerebral ischemia/reperfusion injury.Graphical abstract

Elucidation of OSW-1-Induced Stress Responses in Neuro2a Cells.

OSW-1, a steroidal saponin isolated from the bulbs of Ornithogalum saundersiae, is a promising compound for an anticancer drug; however, its cytotoxic mechanisms have not been fully elucidated. Therefore, we analyzed the stress responses triggered by OSW-1 in the mouse neuroblastoma cell line Neuro2a by comparing it with brefeldin A (BFA), a Golgi apparatus-disrupting reagent. Among the Golgi stress sensors TFE3/TFEB and CREB3, OSW-1 induced dephosphorylation of TFE3/TFEB but not cleavage of CREB3, and induction of the ER stress-inducible genes GADD153 and GADD34 was slight. On the other hand, the induction of LC3-II, an autophagy marker, was more pronounced than the BFA stimulation. To elucidate OSW-1-induced gene expression, we performed a comprehensive gene analysis using a microarray method and observed changes in numerous genes involved in lipid metabolism, such as cholesterol, and in the regulation of the ER-Golgi apparatus. Abnormalities in ER-Golgi transport were also evident in the examination of secretory activity using NanoLuc-tag genes. Finally, we established Neuro2a cells lacking oxysterol-binding protein (OSBP), which were severely reduced by OSW-1, but found OSBP deficiency had little effect on OSW-1-induced cell death and the LC3-II/LC3-I ratio in Neuro2a cells. Future work to elucidate the relationship between OSW-1-induced atypical Golgi stress responses and autophagy induction may lead to the development of new anticancer agents.

Cytotoxicity, uptake and accumulation of selenium nanoparticles and other selenium species in neuroblastoma cell lines related to Alzheimer's disease by using cytotoxicity assays, TEM and single cell-ICP-MS

Alzheimer's disease (AD) is the most prevalent neurodegenerative disease, representing 80% of the total dementia cases. The “amyloid cascade hypothesis” stablishes that the aggregation of the beta-amyloid protein (Aβ42) is the first event that subsequently triggers AD development. Selenium nanoparticles stabilized with chitosan (Ch-SeNPs) have demonstrated excellent anti-amyloidogenic properties in previous works, leading to an improvement of AD aetiology. Here, the in vitro effect of selenium species in AD model cell line has been study to obtain a better assessment of their effects in AD treatment. For this purpose, mouse neuroblastoma (Neuro-2a) and human neuroblastoma (SH-SY5Y) cell lines were used. Cytotoxicity of selenium species, such as selenomethionine (SeMet), Se-methylselenocysteine (MeSeCys) and Ch-SeNPs, has been determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry methods. Intracellular localisation of Ch-SeNPs, and their pathway through SH-SY5Y cell line, have been evaluated by transmission electron microscopy (TEM). The uptake and accumulation of selenium species by both neuroblastoma cell lines have been quantified at single cell level by single cell- Inductively Coupled Plasma with Mass Spectrometry detection (SC-ICP-MS), with a previous optimisation of transport efficiency using gold nanoparticles (AuNPs) ((69 ± 3) %) and 2.5 mm calibration beads ((92 ± 8) %). Results showed that Ch-SeNPs would be more readily accumulated by both cell lines than organic species being accumulation ranges between 1.2 and 89.5 fg Se cell−1 for Neuro-2a and 3.1–129.8 fg Se cell−1 for SH-SY5Y exposed to 250 μM Ch-SeNPs. Data obtained were statistically treated using chemometric tools. These results provide an important insight into the interaction of Ch-SeNPs with neuronal cells, which could support their potential use in AD treatment.

Transferrin Enhances Neuronal Differentiation.

Although transferrin (Tf) is a glycoprotein best known for its role in iron delivery, iron-independent functions have also been reported. Here, we assessed apoTf (aTf) treatment effects on Neuro-2a (N2a) cells, a mouse neuroblastoma cell line which, once differentiated, shares many properties with neurons, including process outgrowth, expression of selective neuronal markers, and electrical activity. We first examined the binding of Tf to its receptor (TfR) in our model and verified that, like neurons, N2a cells can internalize Tf from the culture medium. Next, studies on neuronal developmental parameters showed that Tf increases N2a survival through a decrease in apoptosis. Additionally, Tf accelerated the morphological development of N2a cells by promoting neurite outgrowth. These pro-differentiating effects were also observed in primary cultures of mouse cortical neurons treated with aTf, as neurons matured at a higher rate than controls and showed a decrease in the expression of early neuronal markers. Further experiments in iron-enriched and iron-deficient media showed that Tf preserved its pro-differentiation properties in N2a cells, with results hinting at a modulatory role for iron. Moreover, N2a-microglia co-cultures revealed an increase in IL-10 upon aTf treatment, which may be thought to favor N2a differentiation. Taken together, these findings suggest that Tf reduces cell death and favors the neuronal differentiation process, thus making Tf a promising candidate to be used in regenerative strategies for neurodegenerative diseases.

Tributyltin activates the Keap1-Nrf2 pathway via a macroautophagy-independent reduction in Keap1.

Tributyltin (TBT) is an environmental chemical, which was used as an antifouling agent for ships. Although its use has been banned, it is still persistently present in ocean sediments. Although TBT reportedly causes various toxicity in mammals, few studies on the mechanisms of biological response against TBT toxicity exist. The well-established Keap1-Nrf2 pathway is activated as a cytoprotective mechanism under stressful conditions. The relationship between TBT and the Keap1-Nrf2 pathway remains unclear. In the present study, we evaluated the effect of TBT on the Keap1-Nrf2 pathway. TBT reduced Keap1 protein expression in Neuro2a cells, a mouse neuroblastoma cell line, after 6 hr without altering mRNA expression levels. TBT also promoted the nuclear translocation of Nrf2, a transcription factor for antioxidant proteins, after 12 hr and augmented the expression of heme oxygenase 1, a downstream protein of Nrf2. Furthermore, TBT decreased Keap1 levels in mouse embryonic fibroblast (MEF) cells, with the knockout of Atg5, which is essential for macroautophagy, as well as in wild-type MEF cells. These results suggest that TBT activates the Keap1-Nrf2 pathway via the reduction in the Keap1 protein level in a macroautophagy-independent manner. The Keap1-Nrf2 pathway is activated by conformational changes in Keap1 induced by reactive oxygen species or electrophiles. Furthermore, any unutilized Keap1 protein is degraded by macroautophagy. Understanding the novel mechanism governing the macroautophagy-independent reduction in Keap1 by TBT may provide insights into the unresolved biological response mechanism against TBT toxicity and the activation mechanism of the Keap1-Nrf2 pathway.

Progranulin upregulation by antisense oligonucleotides targeting regRNAs for treatment of FTD‐GRN

AbstractBackgroundHeterozygous mutations in progranulin (GRN) gene leading to progranulin protein (PGRN) haploinsufficieny are the major genetic cause of GRN‐related frontotemporal dementia (FTD‐GRN)1, 2 and 3. Decreased levels of PGRN disrupt lysosome homeostasis, cause aberrant microglia activation and neuronal cell death resulting in progressive behavioral changes, language deficits and memory impairment4. We hypothesize that upregulation of PGRN in both neuronal cells and microglia would be effective for treating FTD‐GRN patients.MethodRegulatory RNAs (regRNAs) are a class of noncoding RNAs that modulate gene transcription. We have developed an RNA Actuating Platform (RAPTM) that identified regRNAs and enables tunable upregulation of genes by specific targeting of regRNAs with antisense oligonucleotides (ASOs).ResultOur platform identified shared GRN regRNAs in human iPSC‐derived neuron and microglia like cells (iMGLs). ASOs targeting shared regRNAs were screened in a human neuroblastoma cell line to identify lead ASOs that upregulate GRN at least 2‐fold. We showed that the lead ASOs upregulate GRN in both human iPSC‐derived neurons and iMGL cells. Moreover, this level of GRN upregulation was sufficient to rescue staurosporine‐induced toxicity in FTD‐GRN patient iPSC‐derived neurons.Furthermore, we identified mouse Grn regRNA‐targeting ASOs that upregulate Grn transcription 2.5‐fold in mouse neuroblastoma cell lines and primary neurons. We tested these ASOs for efficacy in an FTD‐GRN mouse model (Grn+/tm1.1Far )5 by intracerebroventricular injection. Grn upregulation is observed across disease‐relevant brain regions, including cortex in the Grn haploinsufficient mice.ConclusionHere, we describe the development of oligonucleotide drug candidates that target regRNAs to upregulate GRN expression as a therapeutic approach for FTD‐GRN. We believe that our strategy will restore secreted and lysosomal PGRN levels in both microglia and neurons thereby decreasing neuroinflammation and neurotoxicity. We are developing these oligonucleotides as a disease modifying treatment for patients with FTD‐GRN.

Imipramine prevents Porphyromonas gingivalis lipopolysaccharide-induced microglial neurotoxicity

Porphyromonas gingivalis (P. gingivalis) is a Gram-negative anaerobe involved in the pathogenesis of chronic periodontitis, including local inflammation of the oral cavity. However, periodontal disease has recently been identified as a significant factor in the pathogenesis of neural diseases, including Alzheimer's disease. A virulence factor, P. gingivalis-lipopolysaccharide (LPS-PG), is involved in pro-inflammatory responses, not only in peripheral tissues but also in the brain. In this study, we examined whether P. gingivalis-induced brain inflammation could be ameliorated by pharmacotherapy, using in vivo and in vitro studies. In an animal experiment, peripheral administration of LPS-PG induced inflammation in the hippocampus via microglial activation, which was inhibited by pre-treatment with the antidepressant imipramine. Similarly, LPS-PG-induced inflammation in MG-6 cells, a mouse microglial cell line, was inhibited by pre-treatment with imipramine, which caused imipramine-induced inhibition of NF-κB signaling. Culture media obtained from LPS-PG-treated MG-6 cells induced neuronal cell death in Neuro-2A cells, a mouse neuroblastoma cell line, which was prevented by pre-treatment of MG-6 cells with imipramine. These results indicate that imipramine inhibits LPS-PG-induced inflammatory responses in microglia and ameliorates periodontal disease-related neural damage.