Cytotoxicity In Neuro2A Cells Research Articles

Degradation of neurodegenerative disease-associated TDP-43 aggregates and oligomers via a proteolysis-targeting chimera

BackgroundAmyotrophic lateral sclerosis (ALS) associated with TAR DNA-binding protein 43 (TDP-43) aggregation has been considered as a lethal and progressive motor neuron disease. Recent studies have shown that both C-terminal TDP-43 (C-TDP-43) aggregates and oligomers were neurotoxic and pathologic agents in ALS and frontotemporal lobar degeneration (FTLD). However, misfolding protein has long been considered as an undruggable target by applying conventional inhibitors, agonists, or antagonists. To provide this unmet medical need, we aim to degrade these misfolding proteins by designing a series of proteolysis targeting chimeras (PROTACs) against C-TDP-43.MethodsBy applying filter trap assay, western blotting, and microscopy imaging, the degradation efficiency of C-TDP-43 aggregates was studied in Neuro-2a cells overexpressing eGFP-C-TDP-43 or mCherry-C-TDP-43. The cell viability was characterized by alarmarBlue assay. The beneficial and disaggregating effects of TDP-43 PROTAC were examined with the YFP-C-TDP-43 transgenic C. elegans by motility assay and confocal microscopy. The impact of TDP-43 PROTAC on C-TDP-43 oligomeric intermediates was monitored by fluorescence lifetime imaging microscopy and size exclusion chromatography in the Neuro-2a cells co-expressing eGFP-C-TDP-43 and mCherry-C-TDP-43.ResultsFour PROTACs with different linker lengths were synthesized and characterized. Among these chimeras, PROTAC 2 decreased C-TDP-43 aggregates and relieved C-TDP-43-induced cytotoxicity in Neuro-2a cells without affecting endogenous TDP-43. We showed that PROTAC 2 bound to C-TDP-43 aggregates and E3 ligase to initiate ubiquitination and proteolytic degradation. By applying advanced microscopy, it was further shown that PROTAC 2 decreased the compactness and population of C-TDP-43 oligomers. In addition to cellular model, PROTAC 2 also improved the motility of transgenic C. elegans by reducing the C-TDP-43 aggregates in the nervous system.ConclusionsOur study demonstrated the dual-targeting capacity of the newly-designed PROTAC 2 against both C-TDP-43 aggregates and oligomers to reduce their neurotoxicity, which shed light on the potential drug development for ALS as well as other neurodegenerative diseases.

Brucine-Induced Neurotoxicity by Targeting Caspase 3: Involvement of PPARγ/NF-κB/Apoptosis Signaling Pathway.

Brucine, a weak alkaline indole alkaloid, is one of the main bioactive and toxic constituents of Strychnos nux-vomica L., which exerts multiple pharmacological activities, such as anti-tumor, anti-inflammatory, and analgesic effect. However, its potential toxic effects limited its clinical application, especially central nervous system toxicity. The present study was designed to investigate the neurotoxicity and mechanism of brucine. Our results showed that brucine significantly induced Neuro-2a cells and primary astrocyte death, as evidenced by MTT assay and LDH release. Moreover, transcriptome analysis indicated that PPAR/NF-κB and apoptosis signaling pathways were involved in the brucine-induced cytotoxicity in Neuro-2a cells. Subsequently, in fact, brucine evidently inhibited PPARγ and promoted phosphorylation of NF-κB. Furthermore, PPARγ inhibitor aggravated the neurotoxicity, while NF-κB inhibitor substantially reversed brucine-induced neurotoxicity. Moreover, brucine also significantly induced neuronal apoptosis and triggered increase in ratio of Bax/Bcl-2 and level of cleaved caspase 3, as well as its activity as evidenced by TUNEL staining and Western blot. Furthermore, molecular docking analysis predicted that brucine directly bound to caspase 3. Intriguingly, a caspase 3 inhibitor (Z-DEVE-FMK) largely abolished the neurotoxicity of brucine. Our results reveal that brucine-induced neurotoxicity via activation of PPARγ/NF-κB/caspase 3-dependent apoptosis pathway. These findings will provide a novel strategy against brucine-induced neurotoxicity.

Protective Evaluation of Compounds Extracted from Root of Rhodiola rosea L. against Methylglyoxal-Induced Toxicity in a Neuronal Cell Line.

Rhodiola rosea L. (R. rosea) is one of the most beneficial medicinal plants and it is studied as an adaptogen. This study aims to evaluate the neuroprotective activity of compounds extracted from the root of R. rosea against methylglyoxal (MG)-induced apoptosis in neuro-2A (N2A) cells. The root of R. rosea was extracted with ethanol and partitioned with water, ethyl acetate, and n-butanol fractions to evaluate acetylcholinesterase (AChE) inhibitory activity and neuroprotective activity. The ethyl acetate fraction exhibited the highest values of AChE inhibitory activity (49.2% ± 3%) and cell viability (50.7% ± 4.8%) for neuroprotection. The structure identification of the most potential fraction (ethyl acetate fraction) revealed 15 compounds, consisting of three tannins, five flavonoids, and seven phenolics by infrared spectroscopy, nuclear magnetic resonance, and mass spectroscopy. All compounds were evaluated for their neuroprotective activity. Salidroside had the most potential neuroprotective activity. Gallic acid and methyl gallate had potential cytotoxicity in N2A cells. This study showed that R. rosea might have potential neuroprotective activities.

Protective effects of butein on corticosterone-induced cytotoxicity in Neuro2A cells

A functional understanding of the relationship between glucocorticoids and neuronal apoptosis induced by the production of reactive oxygen species (ROS) may lead to a novel strategy for the treatment or prevention of depression. Previous reports suggest that butein, a type of flavonoids, may be a potent candidate against depression-related neuronal cell apoptosis caused by oxidative stress; however, the protective effects of butein on damaged corticosterone (CORT)-treated neuronal cells has not been elucidated. In the present study, we examined the protective effect of butein on CORT-induced cytotoxicity and neurite growth during cell differentiation of mouse neuroblastoma Neuro2A (N2A) cells. Moreover, the effect on cultured cells by high concentrations of butein was confirmed. Our results demonstrate that CORT treatment significantly decreases cell viability and induces cell death. CORT was suggested to induce apoptosis via mitochondrial dysfunction and caspase-3 activation; this apoptosis may be attributed to DNA damage by ROS generation, found in this study to be significantly inhibited by pretreatment with butein. We found that CORT produced significant growth suppression of retinoic acid-induced neurite outgrowth in N2A cells; however, butein significantly increased neurite length and induced dose-dependent apoptotic cytotoxicity in N2A cells. This study suggests that low concentration of butein can prevent CORT-induced cytotoxicity in N2A cells, and provides preliminary results supporting some of the beneficial roles of butein in neuroprotection.

Effects of Carbazole Derivatives on Neurite Outgrowth and Hydrogen Peroxide-Induced Cytotoxicity in Neuro2a Cells.

Many studies have demonstrated that oxidative stress plays an important role in several ailments including neurodegenerative diseases and cerebral ischemic injury. Previously we synthesized some carbazole compounds that have anti-oxidant ability in vitro. In this present study, we found that one of these 22 carbazole compounds, compound 13 (3-ethoxy-1-hydroxy-8- methoxy-2-methylcarbazole-5-carbaldehyde), had the ability to protect neuro2a cells from hydrogen peroxide-induced cell death. It is well known that neurite loss is one of the cardinal features of neuronal injury. Our present study revealed that compound 13 had the ability to induce neurite outgrowth through the PI3K/Akt signaling pathway in neuro2a cells. These findings suggest that compound 13 might exert a neurotrophic effect and thus be a useful therapy for the treatment of brain injury.

A phosphomimetic mutant TDP-43 (S409/410E) induces Drosha instability and cytotoxicity in Neuro 2A cells

Two DNA/RNA binding proteins, TDP-43 and FUS/TLSU, are involved in RNA processing, and their aberrant mutations induce inherited amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitinated inclusions. Wild type TDP-43 and FUS (wtTDP-43 and wtFUS) are mainly localized in the nucleus and biochemically interact with the microRNA processing enzyme Drosha. In this study, we investigated Drosha stability in Neuro 2A cells by gain and loss of function studies of wtTDP-43 and wtFUS and cycloheximide mediated protein degradation assay. We also generated three different phosphomimetic mutants of TDP-43 (S379E, S403/404E and S409/410E) by using a site-directed mutagenesis method and examined Drosha stability to elucidate a correlation between the phosphorylated TDP-43 mutants and Drosha stability. Overexpression of wtTDP-43 and/or wtFUS increased Drosha stability in Neuro 2A cells and double knockdown of wtTDP-43 and wtFUS reduced its stability. However, knockdown of wtTDP-43 or wtFUS did not affect Drosha stability in Neuro 2A cells. Interestingly, a phosphomimetic mutant TDP-43 (S409/410E) significantly reduced Drosha stability via prevention of protein–protein interactions between wtFUS and Drosha, and induced cytotoxicity in Neuro 2A cells.Our findings suggest that TDP-43 and FUS controls Drosha stability in Neuro 2A cells and that a phosphomimetic mutant TDP-43 (S409/410E) which is associated with Drosha instability can induce neuronal toxicity.

Genipin Suppresses A23187-Induced Cytotoxicity in Neuro2a Cells

Genipin is an iridoid compound and an aglucon of geniposide isolated from Gardenia fructus. We have previously reported that genipin induces neurite outgrowth in PC12h and Neuro2a cells and protects against cytotoxicity induced by several conditions such as beta-amyloid peptide, serum deprivation, and oxidative stress in rat primary hippocampal neurons and Neuro2a cells. In this paper, we examined the protective effect of genipin on A23187 (a calcium ionophore)-induced cytotoxicity in Neuro2a cells. A23187 induced cytotoxicity in concentration- and time-dependent manners as assayed by measurements of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetazolium bromide (MTT) reduction activity and lactate dehydrogenase (LDH) release. The cytotoxicity was significantly suppressed by genipin in a concentration-dependent manner. A23187 also significantly activated caspase3/7, which is known to be the critical mediator of apoptosis, after 1 h, and the cytotoxicity was clearly blocked by an inhibitor of caspase 3/7. Furthermore, A23187 induced the expression of immunoglobulin-binding protein/glucose-regulated protein of 78 kDa (BiP/GRP78) protein, which is an endoplasmic reticulum (ER) stress marker protein, and the expression was suppressed by genipin. These results suggest that genipin protects Neuro2a cells from A23187-induced cytotoxicity mediated by caspase 3/7 and ER stress. Therefore, genipin may be effective in preventing neurodegeneration observed in Alzheimer's disease and Parkinson's disease involving ER stress.

Neuroprotective Action of Genipin on Tunicamycin-Induced Cytotoxicity in Neuro2a Cells

Accumulation of unfolding or misfolded proteins within the lumen of the endoplasmic reticulum (ER) triggers ER stress, and sustained ER stress ultimately leads to cell death. Both of these events are involved in the activation of glucose-regulated protein of 78 kDa (GRP78, also known as Bip), CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP, also known as growth arrest and DNA damage-inducible gene 153 (GADD153)), and caspase-12. ER stress has been shown to be involved in neurodegenerative disorders, such as Alzheimer, Parkinson, and polyglutamine diseases. We previously showed that genipin, a natural iridoid compound, has a protective effect against amyloid-beta (Abeta)-induced cytotoxicity. Here, we studied the protective effects of genipin on cytotoxicity induced in Neuro2a cells by the specific ER stress inducer tunicamycin (TM). TM treatment significantly reduced cell viability in a dose-dependent manner. Genipin dramatically rescued the cells against TM-induced cell death. In addition, genipin suppressed ER stress-induced upregulation of CHOP and GRP78. These data suggest that genipin is effective at protecting against neurodegenerative disorders.

Neuro2a Cell Death Induced by 6-Hydroxydopamine is Attenuated by Genipin

We have previously reported that genipin, a natural iridoid compound, shows neuritogenic activity in cultured rat pheochromocytoma PC12h and mouse neuroblastoma Neuro2a cells. 6-Hydroxydopamine (6-OHDA) is a dopaminergic neurotoxin putatively involved in the pathogenesis of Parkinson's disease (PD). Here, we studied the protective effects of genipin on 6-OHDA-induced cytotoxicity in Neuro2a cells. 6-OHDA treatment markedly reduced Neuro2a cell viability in a concentration-dependent manner causing DNA condensation and fragmentation. Genipin significantly protected the cells against the 6-OHDA-induced cytotoxicity. Genipin also protected the cells against hydrogen peroxide (H2O2)-induced cytotoxicity. It is known that 6-OHDA is rapidly and non-enzymatically oxidized by molecular oxygen to form H2O2 and the corresponding p-quinone. These data suggest that genipin is effective at protecting against neurodegeneration that involves oxidative stress, such as PD.