Toxicity In PC12 Cells Research Articles

A multifunctional CeO2@SiO2-PEG nanoparticle carrier for delivery of food derived proanthocyanidin and curcumin as effective antioxidant, neuroprotective and anticancer agent

The nanoparticle systems could effectively overcome the drug delivery challenges of food bioactive compounds. In this study, a novel and effective multifunctional PEG modified CeO2@SiO2 nanoparticle (CSP-NPs) system was successfully fabricated. Food derived proanthocyanidin (PAC) and curcumin (Cur) were loaded onto CSP-NPs and formed as PAC-NPs and Cur-NPs. Fourier transform Infrared spectra, X-ray diffraction, scanning electron microscopy, transmission electron microscopy, and dynamic light scattering were used to characterize the prepared NPs. CSP-NPs, PAC-NPs and Cur-NPs displayed spherical shape with about 35–45 nm size. The bioactivity analysis revealed that CSP-NPs system could effectively deliver PAC and Cur to exhibit strong antioxidant activity, potent neuroprotective effect against Aβ1-42-mediated toxicity in PC-12 cells (recovered cell viability from 57.5% to 81.0% at the dose of 25 μg/mL) and effective antiproliferative effects on HepG2 and Hela cells. Besides, all prepared nanoparticles (0–100 µg/ml) used in this study showed no significant toxicity on cell models of antioxidative and neuroprotective activities, excepting for cancer cells, suggesting that these nanoparticles had the potential of being utilized in drug delivery. Therefore, CSP-NPs might be a promising delivery system for hydrophilic molecule proanthocyanidin and hydrophobic molecule curcumin against the oxidative damage, neurodegenerative diseases and cancer, which could facilitate the application of food derived nutrients in functional foods industry.

Selenium modulates inorganic mercury induced cytotoxicity and intrinsic apoptosis in PC12 cells

Mercury (Hg) in its all forms, including inorganic Hg (iHg) is an environmental contaminant due to toxicity and diseases in human. However, a little is known about the underlying mechanisms responsible for iHg toxicity. Selenium (Se) is an essential trace element, recognized as an antioxidant and protective agent against metal toxicities. The purpose of this research was to investigate ameliorations of Se counter to iHg-mediated toxicity in PC12 cells. Cytotoxic assays have been shown that iHg (5 μM) caused oxidative stress and intrinsic apoptosis via ROS generation, oxidizing glutathione, damaging DNA, degrading cell membrane integrity, down-regulating mTOR, p-mTOR, akt and ERK1, and up-regulating cleaved caspase 3 and cytochrome c release in PC12 cells 48 h after incubation. Co-treatment of Se (5 μM) inhibited intrinsic apoptosis and oxidative stress induced by iHg (5 μM) via inhibiting ROS formation, boosting GPx contents, increasing reduced glutathione, limiting DNA degradation, improving cell membrane integrity, up-regulating mTOR, p-mTOR, akt, ERK1 and caspase 3, and down-regulating cleaved caspase 3 and cytochrome c leakage in PC12 cells. In conclusion, these results recommended that excessive ROS generation acts a critical role in iHg-influenced oxidative stress and co-treatment of Se attenuates iHg-cytotoxicity through its antioxidant properties.

Effects of curcumin, D-pinitol alone or in combination in cytotoxicity induced by arsenic in PC12 cells

Arsenic is a well-known potent toxicant affecting people by causing various human diseases. Long-term exposure to arsenic has strong adverse health effects on liver and kidney disorders, and various forms of cancer. Contrarily, curcumin and D-pinitol are bioactive dietary compounds that have antioxidant properties. Both are used to treat a broad variety of human diseases. Thus, we hypothesized that both may have synergistic effects against arsenic-induced toxicity in PC12 cells. Cells were pretreated with curcumin (1, 2.5, 5 and 10 μM), D-pinitol (1, 2.5, 5 and 10 μM) alone or in combination, then exposed to sodium arsenite (10 μM). The final concentration of curcumin 2.5 μM and D-pinitol 5 μM was selected for combination treatment based on their highest protection at lowest concentration against arsenic toxicity. Results demonstrated that pretreatment of curcumin and D-pinitol and their combined treatment with arsenic rescued PC12 cells. Western blot analysis results showed that pretreatment of curcumin and D-pinitol and their combined treatment with arsenic significantly inhibited arsenic-induced cell death through up-regulation of pro-survival proteins and down-regulation of cell death-related proteins, although these protein expressions were negatively regulated by arsenic. Furthermore, the effect of combined treatment with curcumin and D-pinitol was stronger than individual treatment.

Curcumin alleviates arsenic-induced toxicity in PC12cells via modulating autophagy/apoptosis.

Arsenic is a recognized highly toxic contaminant, responsible for numerous human diseases and affecting many millions of people in different parts of the world. Contrarily, curcumin is a natural dietary polyphenolic compound and the main active ingredient in turmeric. Recently it has drawn great attention due to its diverse biological activities, strong antioxidant properties and therapeutic potential against many human ailments. In this study, we aimed to explore the protective effects and the regulatory role of curcumin on arsenic-induced toxicity and gain insights into biomolecular mechanism/s. Arsenic (10μM) treatment in PC12cells for 24h induced cytotoxicity by decreasing cell viability and intracellular glutathione level and increasing lactate dehydrogenase activity and DNA fragmentation. In addition, arsenic caused apoptotic cell death in PC12cells, which were confirmed from flow cytometry results. Moreover, arsenic (10μM) treatment significantly down-regulated the inhibition factors of autophagy/apoptosis; mTOR, Akt, Nrf2, ERK1, Bcl-x, Xiap protein expressions, up-regulated the enhanced factors of autophagy/apoptosis; ULK, LC3, p53, Bax, cytochrome c, caspase 9, cleaved caspase 3 proteins and eventually caused autophagic and apoptotic cell death. However, curcumin (2.5μM) pretreatment with arsenic (10μM) effectively saves PC12cells against arsenic-induced cytotoxicity through increasing cell viability, intracellular GSH level and boosting the antioxidant defense system, and limiting the LDH activity and DNA damage. Furthermore, pretreatment of curcumin with arsenic expressively alleviated arsenic-induced toxicity and cell death by reversing the expressions of proteins; mTOR, Akt, Nrf2, ERK1, Bcl-x, Xiap, ULK, LC3, p53, Bax, cytochrome c, caspase 9 and cleaved caspase 3. Our findings indicated that curcumin showed antioxidant properties through the Nrf2 antioxidant signaling pathway and alleviates arsenic-triggered toxicity in PC12cells by regulating autophagy/apoptosis.

Ajmalicine and Reserpine: Indole Alkaloids as Multi-Target Directed Ligands Towards Factors Implicated in Alzheimer's Disease.

Alzheimer’s disease (AD) is a multifactorial disorder characterized by exponential loss of memory and cognitive deficit involving several disease modifying targets (amyloid beta, beta-secretase, monoaminoxidase-B, and cholinesterase). The present study explores multi-target directed ligand approach using secondary metabolite reserpine (RES) and ajmalicine (AJM) obtained from Rauwolfia serpentina roots. Novel LCMS and HPLC methods were developed for identification and quantification of reserpine and ajmalicine. In vitro enzyme inhibition assays were performed to evaluate anti-cholinesterase, β-site amyloid cleaving enzyme (BACE-1) inhibition and monoamine oxidase-B (MAO-B) inhibition, further analyzed with in silico analysis. Anti-amyloidogenic potential was studied using anti-aggregation studies along with TEM and circular dichroism (CD) analysis. In vitro neuroprotective potential against Aβ toxicity and anti-oxidative stress was demonstrated using PC12 cell cultures. Reserpine is a more potent dual cholinesterase inhibitor than ajmalicine (IC50 values of 1.7 μM (AChE) and 2.8 μM (BuChE)). The anti-aggregation activity of reserpine (68%) was more than ajmalicine (56%). Both compounds demonstrated neuroprotective activity against Aβ42 (92%) and H2O2 (93%) induced toxicity in PC12 cells against controls. Phytocompounds also inhibited MAO-B and BACE-1 enzymes in concentration dependent manner. Molecular docking studies indicated the strong binding of compounds to the catalytic site of targets. This novel study demonstrated that reserpine and ajmalicine as a multi-target directed ligand that have disease modifying potential for amelioration of AD.

Neuroprotective effects of olanzapine against rotenone-induced toxicity in PC12 cells.

Olanzapine is an antipsychotic drug used to treat patients with schizophrenia due to its lower incidence of extrapyramidal symptoms. Previous studies have shown that olanzapine activates AMP-activated protein kinase (AMPK), and induce autophagy in SH-SY5Y cell line. In this study, we investigated whether olanzapine protected against rotenone-induced neurotoxicity in PC12 cells. We showed that treatment with olanzapine increased the phosphorylation of AMPK in both dose- and time-dependent manners in PC12 cells. In addition, olanzapine activated autophagy and increased autophagic vacuoles. Furthermore, olanzapine pretreatment could protect PC12 cells from rotenone-induced apoptosis. Besides, olanzapine pretreatment could suppress the rotenone-induced depolarization of mitochondrial potential and thus protect the cells. Moreover, pretreatment with specific AMPK inhibitor compound C or with autophagy inhibitor 3-methyladenine impaired the protective effect of olanzapine on rotenone-treated PC12 cells. In summary, our results show for the first time that olanzapine ameliorates rotenone-induced injury by activating autophagy through AMPK pathway.

Structural characterization and neuroprotective effect of a polysaccharide from Corydalis yanhusuo

In the present study, we investigated the neuroprotective effects of a purified Corydalis yanhusuo polysaccharide (CYP) on Aβ (25–35)-induced neurotoxicity and explored its underlying molecular mechanisms in PC12 cells. The results showed pretreatment with CYP (25, 50, and 100 μg/ml) prior to Aβ (25–35) exposure significantly protected PC12 cells from Aβ (25–35)-induced cell death, lactate dehydrogenase (LDH) release, DNA fragmentation, mitochondrial dysfunction and mitochondrial cytochrome c release. Moreover, Aβ (25–35)-induced increase of ratio between Bax and Bcl-2 protein expression was dramatically reversed by CYP pretreatment. Furthermore, the addition of CYP led to a significant repressing effect on the elevated protein expression of cleaved caspase-8, caspase-9, and caspase-3 in Aβ (25–35)-treated PC12 cells. Taken together, these findings indicated that protective effect of CYP against Aβ (25–35)-induced toxicity in PC12 cells was probably mediated by inhibition of apoptosis via both mitochondrial apoptotic pathway and death receptor pathway.

Investigating the protective actions of D-pinitol against arsenic-induced toxicity in PC12 cells and the underlying mechanism

Arsenic is awfully toxic metalloid responsible for many human diseases all over the world. Contrastingly, D-pinitol is a naturally occurring bioactive dietary compound has antioxidant properties. The objective of this study is to elucidate the protective actions of D-pinitol on arsenic-induced cytotoxicity and explore its controlling role in biomolecular mechanisms in PC12 cells. Obtained results demonstrated that co-exposure of D-pinitol with arsenic increases cell viability, decreases DNA damage and protects PC12 cells from arsenic-induced cytotoxicity by increasing glutathione (GSH) level and glutathione reductase (GR). Protein expression of western blot analysis showed that co-exposure of D-pinitol and arsenic significantly inhibited arsenic-induced autophagy which further suppressed apoptosis through up-regulation of survival factors; mTOR, p-mTOR, Akt, p-Akt, NF-кB, Nrf2, ERK1, GR, Bcl-x and down-regulation of death factors; p53, Bax, cytochrome c, LC3, although arsenic regulated those factors negatively. These results of this study suggested that D-pinitol protects PC12 cells from arsenic-induced cytotoxicity.

The antitoxic effects of quercetin and quercetin-conjugated iron oxide nanoparticles (QNPs) against H2O2-induced toxicity in PC12 cells.

BackgroundWe recently showed that quercetin-conjugated iron oxide nanoparticles (QNPs) promoted the bioavailability of quercetin (Qu) in the brain of rats and improved the learning and memory of diabetic rats. In this study, we characterized the modifications in the antitoxic effects of Qu after conjugation.Materials and methodsWe conjugated Qu to dextran-coated iron oxide nanoparticles (DNPs) and characterized DNPs and QNPs using FTIR, XRD, DLS, Fe-SEM, and EDX analyzes. The antiradical properties of Qu, DNPs, and QNPs were compared by 2, 2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity assay. Catalase-like activities of DNPs and QNPs were estimated using catalase activity assay kit, and the antitoxic effects of Qu and QNPs were evaluated with spectrophotometry, MTT assay, flow cytometry, and real-time q-PCR.ResultsQu had a stronger anti-radical activity than DNPs and its activity decreased after being conjugated to DNPs. The catalase-like activity of DNPs remained intact after conjugation. DNPs had less toxicity on PC12 cells viabilities as compared to free Qu, and the conjugation of Qu with DNPs attenuated its cytotoxicity. Furthermore, MTT assay results indicated 24 h pretreatment with Qu had more protective effects than QNPs against H2O2-induced cytotoxicity, while Qu and QNPs had the same effects for 48 and 72 h incubation. Although the total antioxidant capacity of Qu was attenuated after conjugation, the results of flow cytometry and real-time q-PCR confirmed that 24 h pretreatment with the low concentrations of Qu and QNPs had the similar antioxidant, anti-inflammatory, and anti-apoptotic effects against the cytotoxicity of H2O2.ConclusionQu and QNPs showed the similar protective activities against H2O2-induced toxicity in PC12 cells. Given the fact that QNPs have magnetic properties, they may serve as suitable carriers to be used in neural research and treatment.

N6-(2-hydroxyethyl)-adenosine from Cordyceps cicadae attenuates hydrogen peroxide induced oxidative toxicity in PC12 cells.

N6-(2-hydroxyethyl)-adenosine (HEA), is one of the active molecule found in Cordyceps cicadae. The protective effect of HEA against H2O2 induced oxidative damage in PC12 cells and the mechanism of action was investigated. The cells were exposed to varying concentrations of HEA (5-40μM) for a period of 24h and further incubated with 100μM of H2O2 for an another 12h. Cell viability, LDH release, MMP collapse, Ca2+ overload, antioxidant parameters (reactive oxygen species generation (ROS), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), inflammatory mediators (interleukins 6 and 1β (IL-6 and IL-1β), tumor necrosis factor alpha (TNF-α) and NF-kB were evaluated. The results obtained showed that cells exposed to H2O2 toxicity showed reduced cell viability, increased LDH, ROS and Ca2+ overload. However, prior treatment of PC12 cells with HEA increased cell viability, reduced LDH release, MMP collapse, Ca2+ overload and ROS generation induced by H2O2 toxicity. Furthermore, HEA also increased the activities of antioxidant enzymes and inhibited lipid peroxidation as well as reduced IL-6, IL-1β, TNF-α and NF-kB. Thus, our results provided insight into the attenuative effect of HEA against H2O2 induced cell death through its antioxidant action by reducing ROS generation, oxidative stress and protecting mitochondrial function.

Flavanone glycosides inhibit β-site amyloid precursor protein cleaving enzyme 1 and cholinesterase and reduce Aβ aggregation in the amyloidogenic pathway

Alzheimer's disease (AD) is a slow but progressive neurodegenerative disease. One of the pathological hallmarks of AD is the progressive accumulation of β-amyloid (Aβ) in the form of senile plaques, and Aβ insult to neuronal cells has been identified as one of the major causes of AD onset. In the present study, we investigated the anti-AD potential of four flavonoids, naringenin, didymin, prunin, and poncirin, by evaluating their ability to inhibit acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and β-site amyloid precursor protein cleaving enzyme 1 (BACE1). All four flavonoids displayed promising inhibitory activity against AChE, BChE, and BACE1. Structure–activity relationships suggested that glycosylation of naringenin at sugar moieties, and at different positions of the glycosidic linkage, might be closely associated with anti-AD potential. Kinetic and docking studies showed the lowest binding energy and highest affinity for the mixed, competitive, and non-competitive type inhibitors didymin, prunin, and poncirin. Hydrophobic interactions and the number of hydrogen bonds determined the strength of the protein–inhibitor interaction. We also examined the neuroprotective mechanisms by which flavonoids act against Aβ25–35-induced toxicity in PC12 cells. Exposure of PC12 cells to 10 μM Aβ25–35 for 24 h resulted in a significant decrease in cell viability. In addition, pretreatment of PC12 cells with different concentrations of flavonoids for 1 h significantly reversed the effects of Aβ. Furthermore, treatment with the most active flavonoid, didymin, significantly reduced BACE1, APPsβ, and C99 expression levels in a dose-dependent manner, without affecting amyloid precursor protein (APP) levels in the amyloidogenic pathway. Together, our results indicate that flavonoids, and in particular didymin, exhibit inhibitory activity in vitro, and may be useful in the development of therapeutic modalities for the treatment of AD.

Characterization of Crocetin-Monoglucuronide as a Neuron-Protective Metabolite of Crocin-1.

Crocins are important apocarotenoids in the extract of saffron (Crocus sativus L.) and fruits of gardenia (Gardenia jasminoides E.). Crocins have shown versatile biological activities and thus they have been considered to be promising therapeutics for neurodegenerative and heart diseases. Metabolism studies report that crocetin-monoglucuronide (CM) is produced and detected in rat blood plasma after oral administration of crocins. However, due to the lack of standard compound of CM, its unambiguous identification, quantification, and bioactivity studies have been hindered. CM is synthetized and its existence in blood plasma in Sprague-Dawley (SD) rats is quantified. The pharmacokinetic studies show that CM is produced in blood and brain after oral administration of crocin-1. It is then discovered that CM possesses neuroprotective activity against beta-amyloid (Aβ) toxicity in PC-12 cells and drosophila models. The enzymatic assay shows that CM can effectively inhibit AChE and docking studies indicate that CM may bind in the gorge channel of AChE. The work discovered that CM is a neuron-protective metabolite of the orally administrated crocin-1. It is demonstrated that AChE can be one of the targets of CM in its neuron-protective activity.

Inhibiting c-Jun N-terminal kinase (JNK)-mediated apoptotic signaling pathway in PC12 cells by a polysaccharide (CCP) from Coptis chinensis against Amyloid-β (Aβ)-induced neurotoxicity

In this study, we investigated the protective effect and possible mechanism of a polysaccharide (CCP) from Coptis chinensis against Amyloid-β protein (Aβ)-induced toxicity in PC12 cells. The results showed pretreatment with CCP significantly protected PC12 cells from Aβ25–35 induced cell death, lactate dehydrogenase (LDH) release, nuclear fragmentation, mitochondrial dysfunction and cytochrome c release from mitochondria. Furthermore, CCP (100 μg/ml) significantly inhibited Aβ25–35 induced c-Jun N-terminal kinase (JNK) phosphorylation, but not influence signal-regulated kinase (ERK) and P38 mitogen-activated protein kinase (p38MAPK) pathway, and interestingly, the promoting effect of CCP on PC12 cell survival was only blocked by pre-treatment with a SP600125 (JNK inhibitor). In addition, Aβ25–35-induced increase of Bax and cleaved caspase-3, as well as decrease of Bcl-2 protein expression was markedly reversed by CCP or SP600125. Thus, our results indicate that the neuroprotective effect of CCP is associated with JNK-dependent apoptotic pathway.

A combination of curcumin, vorinostat and silibinin reverses Aβ-induced nerve cell toxicity via activation of AKT-MDM2-p53 pathway.

Alzheimer’s disease (AD) is a significant health issue for the elderly and becoming increasingly common as the global population ages. Although many efforts have been made to elucidate its pathology, there is still a lack of effective clinical anti-AD agents. Previous research has shown the neuroprotective properties of a combination of curcumin and vorinostat. In this study, nine other neuroprotective agents were investigated to examine whether a three-drug combination of curcumin, vorinostat, and a new drug is more advantageous than the previous two-drug combination in alleviating amyloid beta (Aβ)-induced nerve cell toxicity. Cell viability assay was performed to screen these agents, and further validation tests, including determination of cellular oxidative stress, apoptosis, and activity of the AKT/MDM2/p53 pathway, were performed. Among the nine candidate compounds, only silibinin at 1 µM reduced Aβ25–35-induced toxicity in PC12 cells. The neuroprotective effects of 1 µM silibinin in combination with 5 µM curcumin and 0.5 µM vorinostat (CVS) was shown in PC12 cells, in which it decreased apoptosis and oxidative stress marker levels that were increased by 20 µM Aβ25–35. Western blotting results showed that CVS pretreatment significantly increased the phosphorylation of AKT, BAD, and MDM2, which resulted in decreased intracellular expression of p53. Further, immunofluorescence results showed reduced p53 levels in the nuclei of PC12 cells following CVS pretreatment, indicating a reduction in the p53-mediated transcriptional activity associated with Aβ25–35 exposure. In conclusion, our findings suggested that pretreatment with CVS protected PC12 cells from Aβ25–35-induced toxicity through modulation of the AKT/MDM2/p53 pathway. Thus, CVS may present a new therapeutic option for treating AD.

Sulforaphane prevents PC12 cells from oxidative damage via the Nrf2 pathway

The aim of this study was to investigate the protective effect of sulforaphane (SFN) on 1-methyl-4-phenyl pyridine ion (MPP+)-induced cytotoxicity and to investigate its possible mechanisms. Methods: PC12 cell toxicity induced by MPP+ served as a cell model of Parkinson's diseases. The cell culture + experiments were divided into four groups based on the different treatments, namely, vehicle control, SFN, MPP+ and SFN pretreatment plus MPP+. Cell viability and apoptosis were examined by MTT assay and flow cytometry, respectively. Expressions of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase 1 (HO-1) and nicotinamide quinone oxidoreductase 1 (NQO1) were detected using western blotting. Results: MPP+ reduced the survival rate of PC12 cells in a dose- and time-dependent manner. After 24-h treatment with 500 µmol/l MPP+, the survival rate of PC12 cells decreased to 58.2±0.03% of that in the control groups. Under the same conditions MPP+ resulted in significant apoptosis of PC12 cells (apoptosis rate: 30.4±0.6%). However, SFN pretreatment significantly attenuated the cell damage induced by MPP+. Furthermore, it was demonstrated that SFN reversed the reduction of Nrf2, HO-1 and NQO1 expression induced by MPP+. Conclusion: SFN may protect PC12 cells from MPP+-induced damage via activating the Nrf2-ARE (antioxidant responsive element) pathway.

Combination of cell metabolomics and pharmacology: A novel strategy to investigate the neuroprotective effect of Zhi-zi-chi decoction

Ethnopharmacological relevanceZhi-zi-chi Decoction(ZZCD), a traditional Chinese medicine formula, has been reported its potential protective effect on psychological sub-health diseases. However, there still remains a lack of molecular mechanism interpretation. Aim of the studyThis study was aimed at investigating the mechanism of glutamate-induced toxicity in PC12 cells and the neuroprotective effect of ZZCD based on a novel strategy of the combination of cell metabolomics and pharmacology. Materials and methodsThe PC12 cells were treated with glutamate to simulate neurotoxic cell model. Gas chromatography coupled with mass spectrometry based on cell metabolomics approach was performed to comprehensively investigate the molecular mechanism of glutamate-induced toxicity The cell viability and cytotoxicity analysis, the determination of glutathione reductase(GR), superoxide dismutase(SOD) and reactive oxygen species(ROS), apoptosis analysis and western blot analysis were performed to evaluate the neuroprotection of ZZCD. ResultsForty metabolites were identified as potential biomarkers in model cells by principal components analysis(PCA) and orthogonal partial least squares-discriminant analysis(OPLS-DA). Glutamate decreased the GR and SOD activities, increased the level of intracellular ROS, activated the apoptotic pathway, and induced the changes of energy metabolism, amino acid metabolism and lipid metabolism. In addition, the extract of ZZCD could reverse the disturbed metabolic pathways by regulating those potential biomarkers and exerted anti-oxidation and anti-apoptosis. ConclusionZZCD has neuroprotective effect and the novel strategy can be applicable for other traditional Chinese medicine formulas.

Santacruzamate A Ameliorates AD-Like Pathology by Enhancing ER Stress Tolerance Through Regulating the Functions of KDELR and Mia40-ALR in vivo and in vitro.

Aggregated amyloid-β protein (Aβ) and Aβ-induced neuronal apoptosis have been implicated as critical factors in the pathophysiology of Alzheimer’s disease (AD). Certain preclinical results have indicated that the increased accumulation of protein aggregates in AD-affected neurons activates the unfolded protein response (UPR), a pathological phenomenon, which predominantly mediates the aberrant endoplasmic reticulum (ER) stress and apoptotic cascades in neuronal cells. In the present study, we confirmed that Santacruzamate A (STA, a natural product isolated from a Panamanian marine cyanobacterium) attenuates Aβ protein fragment 25–35 (Aβ25–35)-induced toxicity in PC12 cells and rescues cognitive deficits in APPswe/PS1dE9 mice by enhancing ER stress tolerance. We first demonstrated the anti-apoptotic effects of STA by evaluating caspase-3 activity, annexin V/propidium iodide (PI) staining, and terminal deoxynucleotidyl transferase dUTP nick end labeling staining. Behavioral testing of STA-treated APPswe/PS1dE9 mice showed that the pronounced memory impairments were ameliorated and that the consolidated memories were stably maintained over a 2-week period. The mechanistic studies provided evidence that STA inhibited Aβ25–35-induced UPR and ER stress by regulating the ER retention signal (KDEL) receptor, which reinforced the retention of resident chaperones in the ER lumen. Furthermore, STA regulated the expression of the mitochondrial intermembrane space assembly protein 40 (Mia40) and augmenter of liver regeneration (ALR), which ultimately attenuated the mitochondrial fission and apoptosis pathways. Together, our present findings suggest that the KDEL receptor and Mia40-ALR play a role in mitigating Aβ25–35-induced neurotoxicity, which might in turn positively regulate learning and memory. These observations support that STA may be a promising agent for reversing the progression of AD.

Sodium azide induces mitochondria‑mediated apoptosis in PC12 cells through Pgc‑1α‑associated signaling pathway.

Sodium azide (NaN3), an inhibitor of cytochrome oxidase, induces the release of excitotoxins via an energy impairment and this, in turn, results in neurodegeneration. The present study aimed to investigate the toxic effects NaN3 on apoptosis of PC12 cells and its mechanism of action in peroxisome proliferator-activated receptor γ co-activator 1-α (Pgc-1α)-associated signaling pathways. To induce apoptosis, PC12 cells were exposed to NaN3 (0, 5, 10, 20, 40 and 80 mM) for 12, 24, 48 and 72 h. Cell viability was determined by CCK-8 assay. DAPI staining was employed to additionally examine apoptotic cells and their nuclear changes. Production of reactive oxygen species (ROS), mitochondrial membrane potential (ΔΨm) and apoptotic rate were also assessed by flow cytometry. Cellular ATP content was estimated by firefly luciferase assay. In addition, the expression levels of B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), phosphorylated (p)-Ca2+/calmodulin-dependent protein kinase (CaMK), p-p38 mitogen-activated protein kinase (p38 MAPK), Pgc-1α, nuclear respiratory factor (Nrf)-1, mitochondrial transcription factor A (Tfam), p-extracellular signal-regulated kinase (Erk)1/2, Nrf-2 and complex IV (Cox IV) were determined by western blot analysis. The data suggested that NaN3 may induce PC12 cell injury and dose-dependently decrease the cell viability. The expression levels of pro-apoptotic proteins Bax and cytochrome c were upregulated, while the expression levels of anti-apoptotic proteins procaspase-3 and Bcl-2 were downregulated. In addition, the phosphorylation of MAPK and Ca2+/calmodulin-dependent protein kinase II (CaMKII) family members including pan-calcineurin A was increased, in particular the ratios of p-p38/p38 and p-CaMKII/CaMKII. However, the expression levels of Pgc-1α and its associated proteins, including Nrf-1/2, Tfam and p-Erk1/2 were decreased. In addition, mitochondria were the target organelles of NaN3-induced toxicity in PC12 cells, which moderated the dissipation of ΔΨm, preserved the cellular ATP content, promoted the production of ROS and increased the apoptotic rate. These results suggested that NaN3 induced cell death in PC12 cells via Pgc-1α-associated signaling pathways and provided a theoretical basis for additional investigation of the neurotoxic mechanism of NaN3, with applications in neurodegenerative disorders.

17 Oxo Sparteine and Lupanine, Obtained from Cytisus scoparius, Exert a Neuroprotection against Soluble Oligomers of Amyloid-β Toxicity by Nicotinic Acetylcholine Receptors.

Alzheimer's disease (AD) is a neurodegenerative pathology, which is characterized by progressive and irreversible cognitive impairment. Most of the neuronal perturbations described in AD can be associated with soluble amyloid- β oligomers (SO-Aβ). There is a large amount of evidence demonstrating the neuroprotective effect of Nicotine neurotransmission in AD, mainly through nicotinic acetylcholine receptor (nAChR) activation and antiapoptotic PI3K/Akt/Bcl-2 pathway signaling. Using HPLC and GC/MS, we isolated and characterized two alkaloids obtained from C. scoparius, Lupanine (Lup), and 17- oxo-sparteine (17- ox), and examined their neuroprotective properties in a cellular model of SO-Aβ toxicity. Our results showed that Lup and 17- ox (both at 0.03μM) prevented SO-Aβ-induced toxicity in PC12 cells (Lup: 64±7%; 17- ox: 57±6%). Similar results were seen in hippocampal neurons where these alkaloids prevented SO-Aβ neurotoxicity (Lup: 57±2%; 17- ox: 52±3%) and increased the frequency of spontaneous calcium transients (Lup: 60±4%; 17- Ox: 40±3%), suggesting an enhancing effect on neural network activity and synaptic activity potentiation. All of the neuroprotective effects elicited by both alkaloids were completely blocked by α-bungarotoxin. Additionally, we observed that the presence of both Lup and 17- ox increased Akt phosphorylation levels (52±4% and 35±7%, respectively) in cells treated with SO-Aβ (3 h). Taken together, our results suggest that the activation of nAChR by Lup and 17- ox induces neuroprotection in different cellular models, and appears to be an interesting target for the development of new pharmacological tools and strategies against AD.

Melatonin receptor stimulation by agomelatine prevents Aβ-induced tau phosphorylation and oxidative damage in PC12 cells.

PurposeAs a novel antidepressant drug, agomelatine has good therapeutic effect on the mood disorder and insomnia in Alzheimer’s disease (AD). Recent studies have shown the neuroprotective function of agomelatine, including anti-oxidative and anti-apoptosis effect. However, it remains unclear whether agomelatine exerts neuroprotection in AD. Thus, the neuroprotective effect of agomelatine against amyloid beta 25–35 (Aβ25–35)-induced toxicity in PC12 cells was evaluated in this study.MethodsThe concentration of malondialdehyde (MDA), LDH, and ROS was investigated to evaluate oxidative damage. The expression of P-tau, tau, PTEN, P-Akt, Akt, P-GSK3β, and GSK3β proteins was assessed by Western blotting. Our results demonstrated that Aβ25–35 significantly increased the content of MDA, LDH, and ROS. Meanwhile, Aβ25–35 upregulated the expression of P-tau and PTEN as well as downregulated P-Akt and P-GSK3β expression. These effects could be blocked by agomelatine pretreatment. Furthermore, luzindole, the melatonin receptor (MT) antagonist, could reverse the neuroprotective effect of agomelatine.ConclusionThe results demonstrated that antidepressant agomelatine might prevent the tau protein phosphorylation and oxidative damage induced by Aβ25–35 in PC12 cells by activating MT-PTEN/Akt/GSK3β signaling. This study provided a novel therapeutic target for AD in the future.