Effects of N-alkyl quaternary quinuclidines on oxidative stress biomarkers in SH-SY5Y cells

Both translation arrest and proteasome stress associated with accumulation of ubiquitin-conjugated protein aggregates were considered as a cause of delayed neuronal death after transient global brain ischemia; however, exact mechanisms as well as possible relationships are not fully understood. The aim of this study was to compare the effect of chemical ischemia and proteasome stress on cellular stress responses and viability of neuroblastoma SH-SY5Y and glioblastoma T98G cells. Chemical ischemia was induced by transient treatment of the cells with sodium azide in combination with 2-deoxyglucose. Proteasome stress was induced by treatment of the cells with bortezomib. Treatment of SH-SY5Y cells with sodium azide/2-deoxyglucose for 15 min was associated with cell death observed 24 h after treatment, while glioblastoma T98G cells were resistant to the same treatment. Treatment of both SH-SY5Y and T98G cells with bortezomib was associated with cell death, accumulation of ubiquitin-conjugated proteins, and increased expression of Hsp70. These typical cellular responses to proteasome stress, observed also after transient global brain ischemia, were not observed after chemical ischemia. Finally, chemical ischemia, but not proteasome stress, was in SH-SY5Y cells associated with increased phosphorylation of eIF2α, another typical cellular response triggered after transient global brain ischemia. Our results showed that short chemical ischemia of SH-SY5Y cells is not sufficient to induce both proteasome stress associated with accumulation of ubiquitin-conjugated proteins and stress response at the level of heat shock proteins despite induction of cell death and eIF2α phosphorylation.

Oxidative stress plays a significant role in the development of Parkinson’s disease (PD). Previous studies implicate nuclear receptor subfamily 4 group A member 1 (NR4A1) in oxidative stress associated with PD. However, the molecular mechanism underlying the regulation of NR4A1 expression remains incompletely understood. In the present study, a PD cell model was established by using 1-methyl-4-phenylpyridinium (MPP+) in SH-SY5Y cells. Cell viability and apoptosis were assessed by using CCK-8 assay and flow cytometry, respectively. The activities of LDH and SOD, and ROS generation were used as an indicators of oxidative stress. ChIP-PCR was performed to detect the interaction between Yin Yang 1 (YY1) and the NR4A1 promoter. MPP+ treatment inhibited SH-SY5Y cell viability in a dose- and time-dependent manner. NR4A1 and YY1 expression were decreased in MPP+-treated SH-SY5Y cells. Increasing NR4A1 or YY1 alleviated MPP+-induced apoptosis and oxidative stress in SH-SY5Y cells, whereas reduction of NR4A1 aggravated MPP+-induced cell injury. Transcription factor YY1 facilitated NR4A1 expression by binding with NR4A1 promoter. In addition, in MPP+-treated SH-SY5Y cells, the inhibition of NR4A1 to apoptosis and oxidative stress was further enhanced by overexpression of YY1. The reduction of NR4A1 led to an elevation of apoptosis and oxidative stress in MPP+-induced SH-SY5Y cells, and this effect was partially reversed by the overexpression of YY1. In conclusion, YY1 suppresses MPP+-induced apoptosis and oxidative stress in SH-SY5Y cells by binding with NR4A1 promoter and boosting NR4A1 expression. Our findings suggest that NR4A1 may be a candidate target for PD treatment. HIGHLIGHTS NR4A1 and YY1 are decreased in MPP+-treated SH-SY5Y cells. NR4A1 prevents oxidative stress and apoptosis in MPP+-treated SH-SY5Y cells. YY1 binds with NR4A1 promoter and increases NR4A1 expression. YY1 enhances the inhibition of NR4A1 to SH-SY5Y cell apoptosis and oxidative stress.

TiO2 P25 Nanoparticles Induce Mitochondrial damage and Increased Glutathione Synthesis in SH-SY5Y Neural Cells.

Appoptosin (SLC25A38) is a pro-apoptotic protein, which is upregulated in Alzheimer's disease (AD) brains and plays an important role in promoting the pathological progress of AD. The aim of this study was to investigate the effects of curcumin from the rhizome of Curcuma longa on appoptosin-induced apoptosis in SH-SY5Y cells. SH-SY5Y cells were pretreated with curcumin, then transfected with appoptosin or vector. The apoptotic cells were detected with Annexin V staining analysis by flow cytometry. The expression of cleaved caspase-3, appoptosin, heme oxygenase-1 (HO-1) was examined using Western blotting. Intracellular level of ROS was measured with DCFH-DA staining by flow cytometry analysis. Mitochondrial membrane potential (ΔΨm) was detected with JC-1 staining under a fluorescence microscope and quantified by fluorescence ratio detection.Overexpression of appoptosin in SH-SY5Y cells markedly increased cell apoptosis accompanied by reduced HO-1 expression, increased intracellular heme level, ROS overproduction and ΔΨm impairment. Treatment of SH-SY5Y cells with curcumin (2.5-20 μmol/L) for 24 h did not significantly affect their viability. However, pretreatment with curcumin (2.5-20 μmol/L) dose-dependently attenuated all above-mentioned pathological changes in appoptosin-transfected SH-SY5Y cells. Overexpression of appoptosin in SH-SY5Y cells markedly increased cell apoptosis accompanied by reduced HO-1 expression, increased intracellular heme level, ROS overproduction and ΔΨm impairment. Treatment of SH-SY5Y cells with curcumin (2.5-20 μmol/L) for 24 h did not significantly affect their viability. However, pretreatment with curcumin (2.5-20 μmol/L) dose-dependently attenuated all above-mentioned pathological changes in appoptosin-transfected SH-SY5Y cells. Curcumin inhibits appoptosin-induced apoptosis in SH-SY5Y cells by upregulating the expression of HO-1, reducing the production of intracellular heme and ROS, and preventing the ΔΨm loss.

Methamphetamine (METH) is an addictive psychostimulant showing neurotoxicity through neuronal apoptosis and the neuro-inflammatory pathway. Lupenone, a lupane triterpenoid, is an isolated compound exhibiting anti-oxidative, anti-inflammation, and anti-diabetic activities. However, whether lupenone plays a protective role against apoptosis induced by METH in SH-SY5y neuroblastoma cells remains unknown. In the present study, we elucidated that lupenone had no toxicity to SH-SY5y cells at different concentrations. On the other hand, we found that the treatment of SH-SY5y cells with an optimal concentration of lupenone could lead to protection against cell death induced by METH. AnnexinV/PI apoptosis analysis revealed a dramatically reduced level of the apoptotic cell population in lupenon and METH treated SH-SY5y cells. Moreover, diminished expression of anti-apoptotic proteins, including Bcl-2, Caspase3, Caspase7, and Caspase8 in METH-exposed SH-SY5y cells, was significantly recovered by treatment with lupenone. This protection in the expression of anti-apoptotic proteins was due to an increased phosphorylation level of PI3K/Akt in METH-treated SH-SY5y cells pre-incubated with lupenone. These findings suggest that lupenone can protect SH-SY5y cells against METH-induced neuronal apoptosis through the PI3K/Akt pathway.

BackgroundLong non-coding RNA small molecule RNA host gene 1 (SNHG1) was previously identified to be relevant with Parkinson’s disease (PD) pathogenesis. This work aims to further elucidate the regulatory networks of SNHG1 involved in PD.Methods1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-hydrochloride (MPTP)-induced mice and 1-methyl-4-phenylpyridinium (MPP+)-treated SH-SY5Y cells were respectively constructed as the in vivo and in vitro PD models. Expression levels of SNHG1 and miR-153-3p were detected by qRT-PCR. Protein expression levels of phosphate and tension homology deleted on chromosome ten (PTEN) were measured by western blotting assay. Cell viability and apoptosis were determined by MTT and flow cytometry assays. The interactions among SNHG1, miR-153-3p and PTEN were identified by luciferase reporter assay, RNA immunoprecipitation, and/or RNA pull-down analysis.ResultsIncreased SNHG1 expression was found in midbrain of MPTP-induced PD mice and MPP+-treated SH-SY5Y cells. Overexpression of SNHG1 lowered viability and enhanced apoptosis in MPP+-treated SH-SY5Y cells. Moreover, SNHG1 acted as a molecular sponge to inhibit the expression of miR-153-3p. Furthermore, miR-153-3p-mediated suppression of MPP+-induced cytotoxicity was abated following SNHG1 up-regulation. Additionally, PTEN was identified as a direct target of miR-153-3p, and SNHG1 could serve as a competing endogenous RNA (ceRNA) of miR-153-3p to improve the expression of PTEN. Besides, enforced expression of PTEN displayed the similar functions as SNHG1 overexpression in regulating the viability and apoptosis of MPP+-treated SH-SY5Y cells. Finally, SNHG1 was found to activate PTEN/AKT/mTOR signaling pathway in SH-SY5Y cells by targeting miR-153-3p.ConclusionSNHG1 aggravates MPP+-induced cellular toxicity in SH-SY5Y cells by regulating PTEN/AKT/mTOR signaling via sponging miR-153-3p, indicating the potential of SNHG1 as a promising therapeutic target for PD.

Aging of the brain is characterized by marked changes in the expression levels of the neurotrophin receptors, TrkA and p75(NTR). An expression pattern in which TrkA predominates in younger animals switches to one in which p75(NTR) predominates in older animals. This TrkA-to-p75(NTR) switch is accompanied by activation of the second messenger ceramide, stabilization of beta-site amyloid precursor protein-cleaving enzyme-1 (BACE1), and increased production of amyloid beta-peptide (Abeta). Here, we show that the insulin-like growth factor-1 receptor (IGF1-R), the common regulator of lifespan and age-related events in many different organisms, is responsible for the TrkA-to-p75(NTR) switch in both human neuroblastoma cell lines and primary neurons from mouse brain. The signaling pathway that controls the level of TrkA and p75(NTR) downstream of the IGF1-R requires IRS2, PIP3/Akt, and is under the control of PTEN and p44, the short isoform of p53. We also show that hyperactivation of IGF1-R signaling in p44 transgenic animals, which show an accelerated form of aging, is characterized by early TrkA-to-p75(NTR) switch and increased production of Abeta in the brain.

Methylglyoxal (MG) is thought to have harmful effects on Alzheimer's disease (AD). On the other hand, taurine demonstrates promising potential for treating AD. Therefore, we examined the neuroprotective properties of taurine against MG in the SH-SY5Y cells. We used the MTT assay to evaluate the effects of taurine (0.5, 1, and 1.5 mg/mL) and MG (1200 and 2400 μM) on SH-SY5Y cell viability. We measured the expression of interleukin (IL)-6, IL-17, and IL-1β in the target cell line after treatment with MG and taurine. Following the treatment of SH-SY5Y cells with MG and taurine, we evaluated the expression levels of microRNA (miRNA)-101a, miRNA-137, miRNA-222, and miRNA-29c genes using real-time PCR. Furthermore, the Neurogenesis Plus RT² Profiler PCR array was utilized to identify the expression of genes associated with neurogenesis. The survival results indicated that increasing taurine concentrations reduces the toxicity of MG in SH-SY5Y cells. Treatment of SH-SY5Y cells with taurine + MG decreased the expression levels of IL-6, IL-17, and IL-1β compared to those treated with MG (p < 0.05). Treatment with taurine led to increased expression of miRNA-101a, miRNA-137, miRNA-222, and miRNA-29c, with the highest levels observed at a concentration of 1 mg/mL (p < 0.001). The results showed that when SH-SY5Y cells are exposed to MG (2400 μM) + taurine (1.5 mg/mL), genes SHH, BMP2, ERBB2, NEUROG2, BDNF, POU3F3, PARD3, PAX3, NR2E3, NRP2, CXCL1, and EGF had a significant increase (p < 0.005). Taurine protects SH-SY5Y cells from MG-induced toxicity by enhancing cell viability, reducing inflammation, upregulating genes associated with neurogenesis, and upregulating specific miRNAs. This suggests taurine's potential as a therapeutic agent for conditions like AD. However, further in vivo studies and clinical trials are necessary to validate the therapeutic potential of taurine for AD.

Two different series of symmetrical and asymmetrical azobenzenes containing terminal cholesteryl/adamantyl derivatives (SAC/SAA and AAC) with varying spacer lengths (alkyl chains) have been developed. The gelation and aggregation of these derivatives were studied relative to structural motifs, spacer lengths, solvent affinity, temperatures and light conditions. Among these derivatives, the cholesteryl derivatives that have short alkyl chains (<3) act as efficient gelators in a variety of solvents. However, the cholesteryl derivatives with longer alkyl chains (11 spacer) and adamantyl derivatives did not possess this ability. Self-assembled fibrous structures were constructed by gelators with short alkyl chains (<3), while flower-like structures were constructed by gelators with moderately longer alkyl chains (3-6) at their respective critical gelation concentrations (CGCs) according to SEM (Scanning Electron Microscopy) and TEM (Transmission Electron Microscopy) analyses. In some cases, a partial/weak gel was observed in different solvents, which exhibited uniform spherical nanoparticles at CGCs. These nanoparticles were further entangled to form interconnected fibrous structures when the concentration was increased above the CGC (according to the SEM and TEM analyses). Secondary forces (van der Waals/H-bonding) and π-π stacking played important roles in the aggregation of both series in the solvents according to variable temperature (1)H-NMR analysis. The reversibility of sol-gel transitions by light was studied with respect to solvent affinity. This study revealed that reversible transitions were only observed in the non-polar solvents, as supported by the FTIR analysis of the gelators in the various solvents. The thermal and mesomorphic behaviors of the gelators by DSC (Differential Scanning Calorimetry) and POM (Polarized Optical Microscopy) analyses revealed that the chiral nematic (N*) and cholesteric mesophase (Ch*) were exhibited by only the short and longer alkyl chain cholesteryl derivatives, respectively. However, the cholesteryl derivative without a spacer (AAC0) did not exhibit any liquid crystalline phase but acted as an efficient gelator relative to the other gelators in this study.

Kynurenine pathway (KP) metabolites are emerging as important factors of aging-related pathologies. Kynurenine (KYN) and its metabolites increase with age and are known to affect multiple organ systems including CNS, musculoskeletal, and vascular systems. Our group has previously shown that KYN and sub-KYN metabolites below the enzyme Kynurenine monooxygenase (KMO) can act to inhibit osteogenesis of bone marrow MSCs via senescence induction. However, inhibition of KMO, blocks KYN induced senescence and restores osteogenic potentials of MSCs. We are now expanding this work to see if KYN metabolites act on cells of the aging CNS in a similar fashion potentially helping to drive cognitive decline and Alzheimer’s disease (AD) progression. We report for the first time that treatment of neuroblastoma-like SH-SY5Y cells with KYN, and sub-KMO metabolites 3-hydroxykynurenine (3HK), or quinolinic acid (QA) significantly increased senescence-associate beta-galactosidase activity, and expression of senescence associated secretory phenotype (SASP) markers, p21, PAI-1, TIMP2 as well as Histone 3 K9methylation. Treatment of SH-SY5Y cells with KMO inhibitors prevented elevated expression of SASP proteins and histone methylation. This makes KMO an attractive target to inhibit kynurenine pathway effects. Taken together, these data suggest that KYN and its metabolites may contribute to pathogenesis of AD via inducing senescence-like changes and SASP-mediated neuroinflammation in neuronal cells. Inhibition of KMO may allow rescue of brain microenvironment, limit cognitive decline and AD progression in older persons. Blocking KYN downstream metabolite formation, by inhibiting KMO, presents a novel therapeutic target for a potential novel AD mechanism.

Tissue transglutaminase (tTG) is a calcium-dependent enzyme that catalyzes the posttranslational modification of proteins by transamidation of specific polypeptide-bound glutamine residues. Previous in vitro studies have demonstrated that the transamidating activity of tTG requires calcium and is inhibited by GTP. To investigate the endogenous regulation of tTG, a quantitative in situ transglutaminase (TG) activity assay was developed. Treatment of human neuroblastoma SH-SY5Y cells with retinoic acid (RA) resulted in a significant increase in tTG levels and in vitro TG activity. In contrast, basal in situ TG activity did not increase concurrently with RA-induced increased tTG levels. However, stimulation of cells with the calcium-mobilizing drug maitotoxin (MTX) resulted in increases in in situ TG activity that correlated (r2 = 0.76) with increased tTG levels. To examine the effects of GTP on in situ TG activity, tiazofurin, a drug that selectively decreases GTP levels, was used. Depletion of GTP resulted in a significant increase in in situ TG activity; however, treatment of SH-SY5Y cells with a combination of MTX and tiazofurin resulted in significantly less in situ TG activity compared with treatment with MTX alone. This raised the possibility of calcium-dependent proteolysis due to the effects of tiazofurin, because in vitro GTP protects tTG against proteolysis by trypsin. Studies with a selective membrane permeable calpain inhibitor indicated that tTG is likely to be an endogenous substrate of calpain, and that depletion of GTP increases tTG degradation after elevation of intracellular calcium levels. TG activity was also increased in response to activation of muscarinic cholinergic receptors, which increases intracellular calcium through inositol 1,4,5-trisphosphate generation. The results of these experiments demonstrate that selective changes in calcium and GTP regulate the activity and levels of tTG in situ.

After the treatment of human neuroblastoma SH-SY5Y cells with retinoic acid for 24 h, the expression of c-Ret receptor tyrosine kinase was greatly elevated. Treatment of SH-SY5Y cells with glial cell line-derived neurotrophic factor under serum-free conditions after incubation of cells with retinoic acid resulted in the phosphorylation of c-Ret receptor tyrosine kinase, with subsequent morphological changes that included formation of neurites and rounding of cell bodies within 24-48 h. The number of neurite-bearing cells decreased with increasing concentrations of mitogen-activated protein kinase-specific and phosphatidylinositol 3-kinase inhibitors. These observations suggest that retinoic acid induces the expression of glial cell line-derived neurotrophic factor-responsive c-Ret receptor tyrosine kinase and that a glial cell line-derived neurotrophic factor-c-Ret receptor tyrosine kinase-induced signal transduction system that might be involved in neurite outgrowth via pathways that include phosphatidylinositol 3-kinase and mitogen-activated protein kinase.

Background Bromuconazole is a widely used triazole against various fungi disease. It’s employment provokes harmful effects on the environment and human health. In the present study, we explored bromuconazole toxic effects in both rat brain tissue and SH-SY5Y cell line. Methods Male Wistar rats were administrated orally with Bromuconazole (NOEL/4, NOEL o and NOEL ×2) daily for consecutive 28 days. In addition, neuronal SH-SY5Y cell line was used. The rat brains and SH-SY5Y cells were collected and analysed for AChE activity, oxidative stress biomarkers, genotoxicity and histopathological alterations. Results Our results showed that rat exposure to bromuconazole at doses corresponding to NOEL/4, NOEL and NOEL ×2 caused brain histopathological alteration and decrease in acetylcholine esterase (AChE) activity. In SH-SY5Y cell line, bromuconazole strongly induced cell mortality with an IC50 about 250 µM. Bromuconazole induced also DNA damage as assessed by comet assay in both rat brain tissue and SH-SY5Y cell. Moreover, bromuconazole increased ROS production, malondialdehyde (MDA) and protein carbonyl (PC) levels and enhanced the enzymatic activities of catalase (CAT), superoxide dismutase (SOD), Glutathione-S-transferase (GST) and peroxidase (GPx) in the two studied systems. Conclusion Therefore, we can deduce that bromuconazole-caused neurotoxicity may be related to oxidative statue disturbance. HIGHLIGHTS Bromuconzole causes oxidative stress in the brain tissue of male Wistar rats. Bromuconazole enhances MDA, PC levels and induces DNA damage in rat brain. Bromuconazole provokes disturbance of the neuronal antioxidant system. Bromuconazole induces histopathological alterations in rat brain. Bromuconazole exposure induced cytotoxic effects and DNA damage in SH-SY5Y cells. Bromuconazole exposure induced oxidative stress in SH-SY5Ycells.

Collapsin response mediator protein 2 (CRMP2) binds to microtubules and regulates axon outgrowth in neurons. This action is regulated by sequential phosphorylation by the kinases cyclin-dependent kinase 5 (Cdk5) and glycogen synthase kinase 3 (GSK3) at sites that are hyperphosphorylated in Alzheimer disease. The increased phosphorylation in Alzheimer disease could be due to increases in Cdk5 and/or GSK3 activity or, alternatively, through decreased activity of a CRMP phosphatase. Here we establish that dephosphorylation of CRMP2 at the residues targeted by GSK3 (Ser-518/Thr-514/Thr-509) is carried out by a protein phosphatase 1 family member in vitro, in neuroblastoma cells, and primary cortical neurons. Inhibition of GSK3 activity using insulin-like growth factor-1 or the highly selective inhibitor CT99021 causes rapid dephosphorylation of CRMP2 at these sites. In contrast, pharmacological inhibition of Cdk5 using purvalanol results in only a gradual and incomplete dephosphorylation of CRMP2 at the site targeted by Cdk5 (Ser-522), suggesting a distinct phosphatase targets this residue. A direct comparison of dephosphorylation at the Cdk5 versus GSK3 sites in vitro shows that the Cdk5 site is comparatively resistant to phosphatase treatment. The presence of the peptidyl-prolyl isomerase enzyme, Pin1, does not affect dephosphorylation of Ser-522 in vitro, in cells, or in Pin1 transgenic mice. Instead, the relatively high resistance of this site to phosphatase treatment is at least in part due to the presence of basic residues located nearby. Similar sequences in Tau are also highly resistant to phosphatase treatment. We propose that relative resistance to phosphatases might be a common feature of Cdk5 substrates and could contribute to the hyperphosphorylation of CRMP2 and Tau observed in Alzheimer disease.

Parkinson’s disease (PD) is the second-most common neurodegenerative disease worldwide. Several studies have investigated PD for decades; however, the exact mechanism of disease development remains unknown. To study PD, SH-SY5Y cells are often treated with 6-hydroxydopamine (6-OHDA) or 1-methyl-4-phenylpyridinium (MPP+) to induce PD. To understand the mechanism of PD pathogenesis, we confirmed protein changes between 6-OHDA- and MPP+-treated SH-SY5Y cells via proteomics analysis using liquid chromatography coupled with tandem mass spectrometry. 6-OHDA-treated SH-SY5Y cells showed increased expression of electron transporter-related proteins compared to that in the control group, along with decreased expression in MPP+-treated SH-SY5Y cells. However, both down- and upregulation of electron transporter-related proteins increased mitochondrial dysfunction and apoptosis. These proteins were confirmed via protein–protein interaction network analysis using IPA and STRING to induce mitochondrial dysfunction and apoptosis. Cell-based experiments using flow cytometry verified that apoptosis and mitochondrial membrane potential were increased in both 6-OHDA- and MPP+-treated SH-SY5Y cells. Our results provide new insights into PD pathogenesis, thereby contributing to the understanding of the mechanisms of PD development.