Autophagy In SH-SY5Y Cells Research Articles

Rotenone and Its Derivative, Rotenoisin A, Induce Neurodegeneration Differentially in SH-SY5Y Cells.

Rotenone (ROT), the most significant rotenoid, which has shown anticancer activity, has also been reported to be toxic to normal cells, inducing Parkinson's disease (PD)-like neuronal loss with aggregation of α-synuclein (α-syn). To reduce the adverse effects of ROT, its derivative, rotenoisin A (ROA), is obtained by directly irradiating a ROT solution in methanol using γ-rays, which has been reported for potential anticancer properties. However, its PD-inducing effects have not yet been researched or reported. This study sought to compare the activities of ROA and ROT on the aggregation of α-syn, apoptosis, and autophagy in SH-SY5Y cells. ROA decreased cell survival less when compared with ROT on SH-SY5Y cells at 48 h in a dose-dependent manner. ROT (0.5 and 1 μM) and ROA (4 and 5 μM) decreased the expression of tyrosine hydroxylase. Western blot analysis of the Triton X-100 insoluble fraction revealed that both ROT and ROA significantly increased the levels of oligomeric, dimeric, and monomeric phosphorylated Serine129 α-syn and total monomeric α-syn. Moreover, both compounds decreased the proportion of neuronal nuclei, the neurofilament-heavy chain, and β3-tubulin. The phosphorylation of ERK and SAPK were reduced, whereas ROA did not act on Akt. Additionally, the increased Bax/Bcl-2 ratio further activated the downstream caspases cascade. ROT promoted the LC3BII/I ratio and p62 levels; however, different ROA doses resulted in different effects on autophagy while inducing PD-like impairments in SH-SY5Y cells.

Quercetin attenuated ropivacaine-induced neurotoxicity in SH-SY5Y cells through upregulating Pim1 and enhancing autophagy

BackgroundRopivacaine (Rop) is a local anesthetic that is widely used but is also potentially harmful. Quercetin (Quer) is a flavonoid component found in many plants and traditional Chinese medicines. It possesses anti-oxidant, anti-inflammatory, antitumor, and neuroprotective properties as a pharmaceutical. In Rop-induced neurotoxicity, the functions and molecular basis of Quer remain unclear. MethodsCell viability and proliferation were assessed using CCK-8 and 5-ethynyl-2′-deoxyuridine (EdU) incorporation assays, respectively. Apoptosis and autophagy were defined by both morphological criteria and markers such as caspase3 cleavage and monodansylcadaverine (MDC) staining. Western blot and immunofluorescence staining were evaluated. The target proteins were then predicted using molecular docking and validated at the cellular and protein levels. ResultsQuer was shown to significantly reduce Rop-induced viability inhibition and apoptosis in SH-SY5Y cells in a dose-dependent manner. Moreover, Quer reduced Rop-induced cytotoxicity by stimulating autophagy in SH-SY5Y cells by targeting the Pim1 protein, which was accomplished via the AMPK/mTOR pathway. ConclusionQuer relieved Rop-induced neurotoxicity in SH-SY5Y cells through upregulating Pim1 and enhancing autophagy, indicating that Quer may have potential therapeutic applications in the treatment of Rop-induced neurotoxicities.

FABP5-binding lipids regulate autophagy in differentiated SH-SY5Y cells.

The motor features of Parkinson's disease result from loss of dopaminergic neurons in the substantia nigra with autophagy dysfunction being closely linked to this disease. While a large body of work focusing on protein effectors of autophagy has been reported, regulation of autophagy by lipids has garnered far less attention. Therefore, we sought to identify endogenous lipid molecules that act as signaling mediators of autophagy in differentiated SH-SY5Y cells, a commonly used dopaminergic neuron-like cell model. In order to accomplish this goal, we assessed the role of a fatty acid-binding protein (FABP) family member on autophagy due to its function as an intracellular lipid chaperone. We focused specifically upon FABP5 due to its heightened expression in dopaminergic neurons within the substantia nigra and SH-SY5Y cells. Here, we report that knockdown of FABP5 resulted in suppression of autophagy in differentiated SH-SY5Y cells suggesting the possibility of an autophagic role for an interacting lipid. A lipidomic screen of FABP5-interacting lipids uncovered hits that include 5-oxo-eicosatetraenoic acid (5OE) and its precursor metabolite, arachidonic acid (AA). Additionally, other long-chain fatty acids were found to bind FABP5, such as stearic acid (SA), hydroxystearic acid (HSA), and palmitic acid (PA). The addition of 5OE, SA, and HSA but not AA or PA, led to potent inhibition of autophagy in SH-SY5Y cells. To identify potential molecular mechanisms for autophagy inhibition by these lipids, RNA-Seq was performed which revealed both shared and divergent signaling pathways between the lipid-treated groups. These findings suggest a role for these lipids in modulating autophagy through diverse signaling pathways and could represent novel therapeutic targets for Parkinson's disease.

BDNF alleviates Parkinson's disease by promoting STAT3 phosphorylation and regulating neuronal autophagy.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the gradual death of dopaminergic neurons. Brain-derived neurotrophic factor (BDNF) and its receptors are widely distributed throughout the central nervous system, which can promote the survival and growth of neurons and protect neurons. This study revealed that BDNF promotes STAT3 phosphorylation and regulates autophagy in neurons. The PD mouse model was established by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Moreover, SH-SY5Y cells were treated with 1-methyl-4-phenyl-pyridinium (MPP+) to establish a PD cell model. The level of BDNF was low in PD model mice and SH-SY5Y cells treated with MPP+. BDNF enhanced the levels of p-TrkB, P-STAT3, PINK1, and DJ-1. BDNF promoted autophagy, inhibited the level of p-α-syn (Ser129) and enhanced cell proliferation. The autophagy inhibitor 3-Methyladenine (3-methyladenine, 3-MA) reversed the protective effects of BDNF on neurons. BiFC assay results showed that there was a direct physical interaction between BDNF and STAT3, and coimmunoprecipitation experiments indicated an interaction between STAT3 and PI3K. The PI3K agonist Recilisib activated the PI3K/AKT/mTOR pathway, promoted autophagy, and alleviated neuronal cell damage. BDNF alleviates PD pathology by promoting STAT3 phosphorylation and regulating neuronal autophagy in SH-SY5Y cells and cultured primary neurons. Finally, BDNF has neuroprotective effects on PD model mice.

Histone Deacetylase 4 Inhibition Reduces Rotenone-Induced Alpha-Synuclein Accumulation via Autophagy in SH-SY5Y Cells

(1) Background: Parkinson’s disease (PD) is the most common movement disorder. Imbalanced protein homeostasis and α-syn aggregation are involved in PD pathogenesis. Autophagy is related to the occurrence and development of PD and can be regulated by histone deacetylases (HDACs). Various inhibitors of HDACs exert neuroprotective effects within in vitro and in vivo models of PD. HDAC4, a class Ⅱ HDAC, colocalizes with α-synuclein and ubiquitin in Lewy bodies and also accumulates in the nuclei of dopaminergic neurons in PD models. (2) Methods: In the present study, the gene expression profile of HDACs from two previously reported datasets in the GEO database was analyzed, and the RNA levels of HDAC4 in brain tissues were compared between PD patients and healthy controls. In vitro, SH-SY5Y cells transfected with HDAC4 shRNA or pretreated with mc1568 were treated with 1 μM of rotenone for 24 h. Then, the levels of α-syn, LC3, and p62 were detected using Western blot analysis and immunofluorescent staining, and cell viabilities were detected using Cell Counting Kit-8 (CCK-8). (3) Results: HDAC4 was highly expressed in PD substantia nigra and locus coeruleus. Mc1568, an inhibitor of HDAC4, decreased α-synuclein levels in rotenone-treated SH-SY5Y cells in a concentration-dependent manner and activated autophagy, which was impaired by rotenone. The knockdown of HDAC4 reversed rotenone-induced α-syn accumulation in SH-SY5Y cells and protected the neurons by enhancing autophagy. (4) Conclusions: HDAC4 is a potential therapeutic target for PD. The inhibition of HDAC4 by mc1568 or a gene block can reduce α-syn levels by regulating the autophagy process in PD. Mc1568 is a promising therapeutic agent for PD and other disorders related to α-syn accumulation.

Phloretin enhances autophagy by impairing AKT activation and inducing JNK-Beclin-1 pathway activation

Phloretin is a type of dihydrochalcone that is primarily found in apples and has been reported to possess various potent biological activities, such as anticancer, antioxidant and anti-inflammatory effects. Our previous study has shown that phloretin induces apoptosis in human glioblastoma. In this study, we found that phloretin induced autophagy in SH-SY5Y cells by decreasing p-AKT and p-mTOR levels in the AKT/mTOR pathway and increasing the activation of JNK, the phosphorylation of c-Jun and the expression of Beclin-1. Moreover, the upregulation of Beclin-1 was decreased by SP600125 or a siRNA against c-Jun. Furthermore, SP600125 and siRNAs against c-Jun and Beclin-1 inhibited phloretin-induced autophagy. In addition, inhibition of phloretin-induced autophagy by cotreatment with phloretin and 3-MA decreased phloretin-induced cytotoxicity to SH-SY5Y cells. In conclusion, our results suggest that the AKT/mTOR pathway and JNK-mediated Beclin-1 expression are involved in phloretin-induced autophagy. Phloretin can be used to protect neurons during phloretin treatment of glioblastoma.

PS-NPs Induced Neurotoxic Effects in SHSY-5Y Cells via Autophagy Activation and Mitochondrial Dysfunction.

Polystyrene nanoparticles (PS-NPs) are organic pollutants that are widely detected in the environment and organisms, posing potential threats to both ecosystems and human health. PS-NPs have been proven to penetrate the blood–brain barrier and increase the incidence of neurodegenerative diseases. However, information relating to the pathogenic molecular mechanism is still unclear. This study investigated the neurotoxicity and regulatory mechanisms of PS-NPs in human neuroblastoma SHSY-5Y cells. The results show that PS-NPs caused obvious mitochondrial damages, as evidenced by inhibited cell proliferation, increased lactate dehydrogenase release, stimulated oxidative stress responses, elevated Ca2+ level and apoptosis, and reduced mitochondrial membrane potential and adenosine triphosphate levels. The increased release of cytochrome c and the overexpression of apoptosis-related proteins apoptotic protease activating factor-1 (Apaf-1), cysteinyl aspartate specific proteinase-3 (caspase-3), and caspase-9 indicate the activation of the mitochondrial apoptosis pathway. In addition, the upregulation of autophagy markers light chain 3-II (LC3-II), Beclin-1, and autophagy-related protein (Atg) 5/12/16L suggests that PS-NPs could promote autophagy in SHSY-5Y cells. The RNA interference of Beclin-1 confirms the regulatory role of autophagy in PS-NP-induced neurotoxicity. The administration of antioxidant N-acetylcysteine (NAC) significantly attenuated the cytotoxicity and autophagy activation induced by PS-NP exposure. Generally, PS-NPs could induce neurotoxicity in SHSY-5Y cells via autophagy activation and mitochondria dysfunction, which was modulated by mitochondrial oxidative stress. Mitochondrial damages caused by oxidative stress could potentially be involved in the pathological mechanisms for PS-NP-induced neurodegenerative diseases.

MicroRNA-17-5p Protects against Propofol Anesthesia-Induced Neurotoxicity and Autophagy Impairment via Targeting BCL2L11.

Background Propofol (PPF) has been shown in studies to cause cognitive impairment and neuronal cell death in developing animals. PPF has been demonstrated to decrease the expression of microRNA-17-5p (miR-17-5p) in a recent study. Nonetheless, the function of miR-17-5p in PPF-induced neurotoxicity and related mechanisms is uncharacterized. Methods After the induction of neurotoxicity by treating the SH-SY5Y cells with PPF, qRT-PCR was conducted to evaluate the level of miR-17-5p. Using MTT and flow cytometry, cell viability and apoptosis rate were assessed, respectively. Interaction between miR-17-5p and BCL2 like 11 was (BCL2L11) studied using a Luciferase reporter assay. With the help of western blot analysis, we determined the level of proteins of apoptosis-related genes and autophagy-related markers. Results In SH-SY5Y cells, PPF treatment induced neurotoxicity and downregulated miR-17-5p expression. In SH-SY5Y cells post-PPF exposure, overexpression of miR-17-5p increased cell viability and decreased apoptosis. Consistently, miR-17-5p mimics mitigated PPF-generated autophagy via inhibition of Atg5, Beclin1, and LC3II/I level and elevation of p62 protein expression. In addition, BCL2L11, which was highly expressed in PPF-treated SH-SY5Y cells, was directly targeted by miR-17-5p. Further, in PPF-treated SH-SY5Y cells, overexpressed BCL2L11 counteracted the suppressing behavior of miR-17-5p elevation on PPF-induced apoptosis. Conclusion Overexpressed miR-17-5p alleviates PPF exposure-induced neurotoxicity and autophagy in SH-SY5Y cells via binding to BCL2L11, suggesting the possibility that miR-17-5p can serve as a candidate in the treatment of neurotoxicity (caused by PPF).

LncRNA NEAT1 promotes autophagy of neurons in mice by impairing miR-107-5p

ABSTRACT This work focused on the exploration of NEAT1 in Parkinson’s disease (PD) and aimed to explore its effects on PD and related molecular mechanisms. Two experimental models were initially constructed, including MPTP-induced mice in vivo and the MPP+-induced SH-SY5Y cell line in vitro. Immunofluorescence assays were conducted to determine the TH+ positive cell rate. Pole tests and rotarod tests were also performed for the visualization of behavioral changes in mice. Cellular apoptosis was determined using MTT and flow cytometry assays. Changes in the number of autophagosomes were obtained under a transmission electron microscope. The content of dopamine was confirmed by high performance liquid chromatography. The targeted interrelationship between miR-107-5p and NEAT1 was clarified via dual-luciferase reporter gene assays. Meanwhile, mRNA and protein expressions were also detected using qRT-PCR and Western blot respectively. Furthermore, the level of NEAT1 was positively correlated with MPP+ concentration. Interfering with NEAT1 in the present study promoted cellular proliferation and mediated SH-SY5Y cell apoptosis and autophagy treated with MPP+. An increase was discovered in TH positive neurons and suppressive autophagy in PD mice. miR-107-5p was then considered as a NEAT1 putative target involving apoptosis and autophagy of SH-SY5Y cells. Interfering with NEAT1 efficiently facilitated the viability of SH-SY5Y cells and drastically suppressed autophagy and apoptosis of PD mice induced by MPTP- via elevating miR-107-5p level, which indicated that lncRNA NEAT1 acted as a latent therapeutic factor for PD treatment.

Ellagic Acid Prevents α-Synuclein Aggregation and Protects SH-SY5Y Cells from Aggregated α-Synuclein-Induced Toxicity via Suppression of Apoptosis and Activation of Autophagy.

Parkinson’s disease (PD) is a neurodegenerative disease characterized by the loss of dopamine neurons and the deposition of misfolded proteins known as Lewy bodies (LBs), which contain α-synuclein (α-syn). The causes and molecular mechanisms of PD are not clearly understood to date. However, misfolded proteins, oxidative stress, and impaired autophagy are believed to play important roles in the pathogenesis of PD. Importantly, α-syn is considered a key player in the development of PD. The present study aimed to assess the role of Ellagic acid (EA), a polyphenol found in many fruits, on α-syn aggregation and toxicity. Using thioflavin and seeding polymerization assays, in addition to electron microscopy, we found that EA could dramatically reduce α-syn aggregation. Moreover, EA significantly mitigated the aggregated α-syn-induced toxicity in SH-SY5Y cells and thus enhanced their viability. Mechanistically, these cytoprotective effects of EA are mediated by the suppression of apoptotic proteins BAX and p53 and a concomitant increase in the anti-apoptotic protein, BCL-2. Interestingly, EA was able to activate autophagy in SH-SY5Y cells, as evidenced by normalized/enhanced expression of LC3-II, p62, and pAKT. Together, our findings suggest that EA may attenuate α-syn toxicity by preventing aggregation and improving viability by restoring autophagy and suppressing apoptosis.

Attenuation of the cytoprotection induced by hypoxic preconditioning upon transfection with BNIP3-siRNA in human neuroblastoma SH-SY5Y cells

Purpose The aim of this study was to investigate the functional role of hypoxic preconditioning (HPC) in human neuroblastoma cells. Methods BNIP3 small-interfering RNA (BNIP3-siRNA) sequence was synthesized and used to transfect human neuroblastoma SH-SY5Y cell lines. Thereafter, BNIP3 expression at mRNA and protein levels and its effects on the cell proliferation were analyzed. The most effective pair of siRNA was selected to knockdown the expression level of BNIP3. Moreover, the effects of HPC on oxygen-glucose deprivation/reperfusion (OGD/R)-induced apoptosis and autophagy in SH-SY5Y cells were explored to further reveal the possible mechanisms underlying HPC. Results BNIP3-siRNA attenuated the protective effects of HPC by decreasing the cell viability, increasing the enzymatic activity of caspase-3 and 9, increasing the rate of apoptosis, and increasing the protein expression level of activated caspase-3. Additionally, BNIP3-siRNA had no significant influence on the expression level of HIF-1α induced by HPC, while it substantially inhibited HPC-induced BNIP3/Beclin1 and autophagy. Conclusions HPC promoted autophagy through regulating BNIP3 to reduce OGD/R.

Resveratrol prevents haloperidol-induced mitochondria dysfunction through the induction of autophagy in SH-SY5Y cells

BackgroundHaloperidol is a commonly used antipsychotic drug and may increase neuronal oxidative stress associated with the side effects, including tardive dyskinesia and neurite withdraw. Autophagy plays a protective role in response to the accumulated reactive oxygen species (ROS) induced mitochondria damage. Resveratrol is an antioxidant compound having neuroprotective effects; however, it is unknown if resveratrol may stimulate autophagy and decrease mitochondria damage induced by haloperidol. HypothesisWe hypothesis that resveratrol stimulates the autophagic process and protects mitochondria lesion induced by haloperidol. MethodsMitoSOX™ Red Mitochondrial Superoxide Indicator and MitoTracker™ Green FM staining were used to measure the amount of the mitochondria ROS production and mitochondria mass in human SH-SY5Y cells treated with haloperidol and/or resveratrol. Autophagic related dyes and Western blot were applied to study the autophagic process and related protein expression. Besides, tandem monomeric mRFP-GFP-LC3 was used to investigate the fusion of autophagosome and lysosome. Transmission electron microscopy was used to investigate the mitochondrial and autophagic ultrastructures with or without haloperidol and resveratrol treatment. ResultsHaloperidol administration significantly increased mitochondria ROS and mitochondrial mass, indicating the increase of mitochondria dysfunction. Although haloperidol increased the autophagosomes and lysosome formation, the autophagosome-lysosome fusion and degradation were impaired. This was because we found an increased p62 after haloperidol treatment, an indication of autophagy incompletion. Importantly, resveratrol promoted the degradation of p62, upregulated the formation of autophagolysosome, and reversed haloperidol-induced mitochondria damage. ConclusionThese results collectively suggest that resveratrol may be introduced as a protective compound against haloperidol-induced mitochondria impairment and aberrant autophagy.

Activated Phosphoinositide 3-Kinase/Akt/Mammalian Target of Rapamycin Signal and Suppressed Autophagy Participate in Protection Offered by Licochalcone A Against Amyloid-β Peptide Fragment 25–35–Induced Injury in SH-SY5Y Cells

To assess effect of licochalcone A (LicA) on amyloid-β (Aβ) peptide fragment 25-35-induced nerve injury and reveal the potential molecular mechanisms involved. Viability of SH-SY5Y cells was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay after treatment with Aβ25-35 and/or LicA, following which apoptosis was detected by flow cytometry and Hoechst staining. Then, reactive oxygen species, glutathione, and superoxide dismutase were measured with flow cytometry and spectrophotometry. The ultrastructure of mitochondria was examined by transmission electron microscopy, and the biomarker proteins of autophagy, apoptosis, and phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway were measured with Western blotting. LicA improved cell viability and decreased lactate dehydrogenase leakage remarkably in Aβ25-35-induced injury in SH-SY5Y cells. After treatment with LicA, reactive oxygen species, glutathione, and superoxide dismutase levels in cells all were significantly decreased, which indicated that LicA has an antioxidative effect on Aβ25-35-induced oxidative injury. LicA could also significantly reduce Aβ25-35-induced autophagy in SH-SY5Y cells. In the cells injured by Aβ25-35, LicA prevented the transformation from light chain protein 3-I to light chain protein 3-II and reduced the levels of proteins GRP78, GRP94, CHOP, and Bax, but increased the levels of antiapoptotic protein and phosphorylation of PI3K, Akt, and mTOR. These effects of LicA were restored or suppressed by mTOR inhibitor rapamycin or PI3K inhibitor LY294002. LicA protects SH-SY5Y cells against Aβ25-35-induced injury, wherein suppressed autophagy and activated PI3K/Akt/mTOR signaling pathway are involved, and mTOR-dependent autophagy at least plays some role.

Inhibitors of Lipoxygenase and Cyclooxygenase-2 Attenuate Trimethyltin-Induced Neurotoxicity through Regulating Oxidative Stress and Pro-Inflammatory Cytokines in Human Neuroblastoma SH-SY5Y Cells.

Trimethyltin (TMT) is an environmental neurotoxin that mediates dopaminergic neuronal injury in the brain. In this study, we characterized the toxic mechanism and possible protective compounds against TMT-induced neurotoxicity in human dopaminergic neuroblastoma SH-SY5Y cells. Antioxidants such as melatonin, N-acetylcysteine (NAC), α-tocopherol, and allopurinol alleviated TMT toxicity. Apoptosis induced by TMT was identified by altered expression of cleaved caspase-3, Bax, Bcl-2, and Bcl-xL through Western blot analysis. The iron chelator deferoxamine ameliorated the alteration of apoptosis-related proteins through TMT exposure. TMT also induced delayed ultrastructural necrotic features such as mitochondrial swelling and cytoplasmic membrane rupture; NAC reduced these necrotic injuries. Esculetin, meloxicam, celecoxib, and phenidone decreased TMT toxicity. Elevation of the pro-inflammatory cytokines IL-1β, TNF-α, and NF-ĸB and reduction of the antioxidant enzymes catalase and glutathione peroxidase-1 (GPx-1) were induced by TMT and ameliorated by inhibitors of LOX and COX-2 enzymes. Both NMDA and non-NMDA antagonists attenuated TMT toxicity. The free calcium ion modulators nimodipine and BAPTA/AM contributed to neuronal survival against TMT toxicity. Inhibitors of the phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin pathway, an autophagy regulator, decreased TMT toxicity. These results imply that TMT neurotoxicity is the chief participant in LOX- and COX-2-mediated apoptosis, partly via necrosis and autophagy in SH-SY5Y cells.

LncRNA AC136007.2 alleviates cerebral ischemic-reperfusion injury by suppressing autophagy.

Differential expression and diagnostic significance of the long noncoding RNA (lncRNA) AC136007.2 has been reported in patients with acute ischemic stroke (AIS). However, its role on disease progression and outcome remains unclear. Here, we employed an oxygen-glucose deprivation/reperfusion (OGD/R) model in neuronal SH-SY5Y cells and performed middle cerebral artery occlusion (MCAO) surgery in rats to investigate the function of AC136007.2 in ischemia-reperfusion (I/R) injury. AC136007.2 expression was determined by RT-qPCR and cell viability was examined using CCK-8, Edu, LDH, and apoptosis assays. Pro-inflammatory cytokine expression was assessed using ELISA. OGD/R downregulated AC136007.2 expression in SH-SY5Y cells, decreased viability by inducing apoptosis, and stimulated secretion of TNF-α, IL-6, and IL-1β. In turn, lentivirus-mediated AC136007.2 overexpression significantly reversed these phenomena. LC3 immunofluorescence and western blotting analyses of LC3-I/II and Beclin-1 expression and AMPK/mTOR phosphorylation status showed that AC136007.2 suppressed autophagy in SH-SY5Y cells via inactivation of AMPK/mTOR signaling. Notably, incubation with the AMPK activator AICAR abolished the pro-survival effect of AC136007.2 upon OGD/R treatment. Importantly, intraventricular injection of AC136007.2 significantly reduced cerebral infarction and brain edema in MCAO rats, as shown by TTC staining and water content measurements. We conclude that AC136007.2 alleviates cerebral I/R injury by suppressing AMPK/mTOR-dependent autophagy.

Pyrethrum extract induces oxidative DNA damage and AMPK/mTOR-mediated autophagy in SH-SY5Y cells

Pyrethrum extract is used to produce the most widely applied botanical pesticides in agriculture. Though it primarily targets voltage-gated sodium channels in pests, its toxic effects in non-target systems, particularly in humans, is unclear. In this study, we investigated potential cytotoxic effects and their underlying mechanisms on human nerve cells in vitro. We found that pyrethrum extract exposure markedly inhibited cell viability and triggered oxidative DNA damage in human SH-SY5Y cells. It also induced LC3-II formation, upregulated Beclin-1 protein production, downregulated p62 protein production, and facilitated the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) and mechanistic target of rapamycin (mTOR). These results indicate that cytotoxic exposure to pyrethrum extract could be associated with AMPK/mTOR-mediated autophagy in human nerve cells. Furthermore, the oxidative DNA damage suggests that pyrethrum extract exerts severe toxic effects on human nerve cells. In conclusion, pyrethrum extract carries a risk to human health by inducing cytotoxicity.

TRPC6-Mediated Ca2+ Entry Essential for the Regulation of Nano-ZnO Induced Autophagy in SH-SY5Y Cells.

Recently, possible applications of zinc oxide nanoparticles (nano-ZnO) have been extensively studied owing to their ease of synthesis. However, the effect of nano-ZnO on the nervous system remains unclear. This study investigates the action of nano-ZnO on SH-SY5Y neuroblastoma cells. We found that nano-ZnO (0-50 µg/mL) induced a significant decrease in cell survival rate in a dose-dependent manner, and increased LC3 puncta formation. However, the apoptosis was not affected by nano-ZnO, because the protein levels of cytochrome c, caspase-3, Bcl-xL, and BAX were not varied by the nano-ZnO treatment. Nano-ZnO increased Ca2+ entry and the expression of TRPC6.The results suggested that nano-ZnO increased [Ca2+] through the TRPC-dependent Ca2+ influx, since Ca2+ influx can be prevented by the TRPC inhibitor. Furthermore, cells on nano-ZnO-treatment groups displayed loss of F-actin in a dose dependent manner, which also could be prevented by TRPC inhibitor. Herein, we demonstrated that the nano-ZnO activated TRPC6 channel, thereby increasing the Ca2+ flux and resulting in increased autophagy. Nano-ZnO could have possible anticancer effects in neuroblastoma by inhibiting the proliferation of tumor cells. However, we should also pay attention toward the biosecurity of nano materials.

LncRNA BDNF-AS promotes autophagy and apoptosis in MPTP-induced Parkinson’s disease via ablating microRNA-125b-5p

BackgroundsRecently, extensive evidence has indicated that the biological role of long non-coding RNAs (lncRNAs) in neurodegenerative diseases is becoming increasingly evident. The lncRNA brain-derived neurotrophic factor anti-sense (BDNF-AS) has been found to be dysregulated in Huntington’s Disease. However, the function of BDNF-AS in Parkinson's disease (PD) remains unknown. The purpose of this present study was to explore the effect of BDNF-AS on PD and its underlying molecular mechanisms. MethodsThe MPTP-induced mouse model of PD and MPP+-induced SH-SY5Y cell model were established. Immunofluorescence was performed to determine the number of TH + positive cells. Mice behavioral changes were detected by pole and rota-rod test. SH-SY5Y cells viability, apoptosis was detected by MTT assay and flow cytometry. The number of autophagosome was measured by transmission electron microscopy. Dopamine content was tested by high performance liquid chromatography. Dual-luciferase reporter gene assay was utilized to verify the correlation between BDNF-AS and miR-125b-5p. qRT-PCR and western blot were used to detect gene expression levels. ResultsOur results showed that BDNF-AS was up-regulated in MPTP-induced PD model and dopamine neurons, and MPP + treated SH-SY5Y cells, while miR-125b-5p was down-regulated. The expression of BDNF-AS was positively related with the MPP + concentration. BDNF-AS knockdown could significantly promote cell proliferation, while inhibit apoptosis and autophagy in SH-SY5Y cells treated by MPP + . Silencing BDNF-AS could also increase TH positive neurons and significantly suppress the autophagy of PD mice. Additionally, miR-125b-5p, a putative target gene of BDNF-AS, was involved in the effects of BDNF-AS on SH-SY5Y cell apoptosis and autophagy. ConclusionsOur study demonstrated that knockdown of BDNF-AS could elevate SH-SY5Y cell viability, inhibit autophagy and apoptosis in MPTP-induced PD models through regulating miR-125b-5p, suggesting that BDNF-AS might act as a potential therapeutic target for PD.

Manganese activates autophagy to alleviate endoplasmic reticulum stress–induced apoptosis via PERK pathway

Overexposure to manganese (Mn) is neurotoxic. Our previous research has demonstrated that the interaction of endoplasmic reticulum (ER) stress and autophagy participates in the early stage of Mn‐mediated neurotoxicity in mouse. However, the mechanisms of ER stress signalling pathways in the initiation of autophagy remain confused. In the current study, we first validated that ER stress–mediated cell apoptosis is accompanied by autophagy in SH‐SY5Y cells. Then, we found that inhibiting ER stress with 4‐phenylbutyrate (4‐PBA) decreased ER stress–related protein expression and reduced cell apoptosis, whereas blocking autophagy with 3‐methyladenine (3‐MA) increased cell apoptosis. These data indicate that protective autophagy was activated to alleviate ER stress–mediated apoptosis. Knockdown of the protein kinase RNA‐like ER kinase (PERK) gene inhibited Mn‐induced autophagy and weakened the interaction between ATF4 and the LC3 promoter. Our results reveal a novel molecular mechanism in which ER stress may regulate autophagy via the PERK/eIF2α/ATF4 signalling pathway. Additionally, Mn may activate protective autophagy to alleviate ER stress–mediated apoptosis via the PERK/eIF2α/ATF4 signalling pathway in SH‐SY5Y cells.

TMP21 regulates autophagy by modulating ROS production and mTOR activation

Autophagy is a catabolic cellular response to stress that has been liked to various human diseases. However, the precise involvement of autophagy in health and disease remains unclear. To explore the molecular mechanisms of autophagy, we investigated the effect of TMP21. We found that the down-regulation of TMP21 induced autophagy in SH-SY5Y cells. In addition, the enhanced autophagy observed upon TMP21 depletion was almost completely blocked in ATG5 knockout (KO) or ATG7-KO HeLa cells. Silencing of TMP21 in SH-SY5Y cells also increased the production of cellular reactive oxygen species (ROS). Accordingly, treatment with the ROS scavenger NAC suppressed autophagy activation as well as ROS production in TMP21-depleted cells. In addition, the inhibition of mTOR by treatment with Torin1 was mitigated in TMP21 overexpressing cells compared with that in control cells. Taken together, these results indicated that TMP21 could regulate autophagy by modulating ROS production and mTOR activation.