Autophagy In SH-SY5Y Cells Research Articles

Activated cathepsin L is associated with the switch from autophagy to apoptotic death of SH-SY5Y cells exposed to 6-hydroxydopamine

Autophagy and apoptosis are common responses to pathological damage in the process of Parkinson's disease (PD), and lysosome dysfunction may contribute to the etiology of PD's neurodegenerative process. In this study, we demonstrated that the neurotoxin 6-hydroxydopamine (6-OHDA) increased autophagy in SH-SY5Y cells, as determined by detection of the lysosome marker lysosomal-associated membrane protein1, the autophagy protein light chain 3 (LC3)-II and the autophagy substrate P62 protein. Meanwhile, autophagy repression with 3-methyladenine accelerated the activation of caspase-3 and PARP and aggravated the cell apoptotic death induced by 6-OHDA. Furthermore, we found that 6-OHDA treatment resulted in a transient increase in the intracellular and nuclear expression of cathepsin L (CTSL). The CTSL inhibitor, Z-FY-CHO, could promote autophagy, decrease accumulation of P62, and block activation of caspase-3 and PARP. Taken together, these results suggest that activation of autophagy may primarily be a protective process in SH-SY5Y cell death induced by 6-OHDA, and the nuclear translocation of CTSL could enhance the cell apoptotic cascade via disturbing autophagy-apoptotic systems in SH-SY5Y cells. Our findings highlight the potential role of CTSL in the cross talk between autophagy and apoptosis, which might be considered a therapeutic strategy for treatment of pathologic conditions associated with neurodegeneration.

Autophagy potentially protects against 2,3,7,8-tetrachlorodibenzo-p-Dioxin induced apoptosis in SH-SY5Y cells.

The environmental toxicant TCDD may elicit cytotoxic effects by inducing reactive oxygen species (ROS) generation. Autophagy is one of the first lines of defense against oxidative stress damage. Herein, we investigated whether autophagy played a regulatory role in TCDD-induced neurotoxicity. Here, we showed that TCDD exposure caused marked autophagy in SH-SY5Y cells, whose dose range was close to that inducing apoptosis. Electron microscopic and Western blot analyses revealed that TCDD induced autophagy at a starting dose of approximate 100 nM. Interestingly, 100-200 nM TCDD exposure resulted in obviously decreased cell viability and evident apoptotic phenotype. Furthermore, the levels of pro-apoptotic molecules, Bax and cleaved-PARP, increased significantly, whereas Bcl2 declined after exposed to 100 nM TCDD. In addition, the apoptosis was verified using flow cytometrical analysis. These data strongly suggested that TCDD induced both autophagy and apoptosis at a similar dose range in SH-SY5Y cells. Interestingly, pretreatment with ROS scavenger, N-acetyl-cysteine (NAC), could effectively block both TCDD-induced apoptosis and autophagy. More surprisingly, inhibition of autophagy with 3-methyladenine (3MA), remarkably augmented TCDD-induced apoptosis. The findings implicated that the onset of autophagy might serve as a protective mechanism to ameliorate ROS-triggered cytotoxic effects in human SH-SY5Y neuronal cells under TCDD exposure. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1068-1079, 2016.

The LRRK2 inhibitor GSK2578215A induces protective autophagy in SH-SY5Y cells: involvement of Drp-1-mediated mitochondrial fission and mitochondrial-derived ROS signaling.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene have been associated with Parkinson's disease, and its inhibition opens potential new therapeutic options. Among the drug inhibitors of both wild-type and mutant LRRK2 forms is the 2-arylmethyloxy-5-subtitutent-N-arylbenzamide GSK257815A. Using the well-established dopaminergic cell culture model SH-SY5Y, we have investigated the effects of GSK2578215A on crucial neurodegenerative features such as mitochondrial dynamics and autophagy. GSK2578215A induces mitochondrial fragmentation of an early step preceding autophagy. This increase in autophagosome results from inhibition of fusion rather than increases in synthesis. The observed effects were shared with LRRK2-IN-1, a well-described, structurally distinct kinase inhibitor compound or when knocking down LRRK2 expression using siRNA. Studies using the drug mitochondrial division inhibitor 1 indicated that translocation of the dynamin-related protein-1 has a relevant role in this process. In addition, autophagic inhibitors revealed the participation of autophagy as a cytoprotective response by removing damaged mitochondria. GSK2578215A induced oxidative stress as evidenced by the accumulation of 4-hydroxy-2-nonenal in SH-SY5Y cells. The mitochondrial-targeted reactive oxygen species scavenger MitoQ positioned these species as second messengers between mitochondrial morphologic alterations and autophagy. Altogether, our results demonstrated the relevance of LRRK2 in mitochondrial-activated pathways mediating in autophagy and cell fate, crucial features in neurodegenerative diseases.

Autophagy occurs within an hour of adenosine triphosphate treatment after nerve cell damage: the neuroprotective effects of adenosine triphosphate against apoptosis.

After hypoxia, ischemia, or inflammatory injuries to the central nervous system, the damaged cells release a large amount of adenosine triphosphate, which may cause secondary neuronal death. Autophagy is a form of cell death that also has neuroprotective effects. Cell Counting Kit assay, monodansylcadaverine staining, flow cytometry, western blotting, and real-time PCR were used to determine the effects of exogenous adenosine triphosphate treatment at different concentrations (2, 4, 6, 8, 10 mmol/L) over time (1, 2, 3, and 6 hours) on the apoptosis and autophagy of SH-SY5Y cells. High concentrations of extracellular adenosine triphosphate induced autophagy and apoptosis of SH-SY5Y cells. The enhanced autophagy first appeared, and peaked at 1 hour after treatment with adenosine triphosphate. Cell apoptosis peaked at 3 hours, and persisted through 6 hours. With prolonged exposure to the adenosine triphosphate treatment, the fraction of apoptotic cells increased. These data suggest that the SH-SY5Y neural cells initiated autophagy against apoptosis within an hour of adenosine triphosphate treatment to protect themselves against injury.

Pharmacological Activation of Sirt1 Ameliorates Polyglutamine-Induced Toxicity through the Regulation of Autophagy

Intracellular accumulation of polyglutamine (polyQ)-expanded Huntingtin (Htt) protein is a hallmark of Huntington’s disease (HD). This study evaluated whether activation of Sirt1 by the anti-cancer agent, β-lapachone (β-lap), induces autophagy in human neuroblastoma SH-SY5Y cells, thereby reducing intracellular levels of polyQ aggregates and their concomitant cytotoxicity. Treatment of cells with β-lap markedly diminished the cytotoxicity induced by forced expression of Htt exon 1 containing a pathogenic polyQ stretch fused to green fluorescent protein (HttEx1(97Q)-GFP). β-lap increased autophagy in SH-SY5Y cells, as evidenced by the increased formation of LC3-II and autolysosomes. Furthermore, β-lap reduced HttEx1(97Q)-GFP aggregation, which was significantly prevented by co-incubation with 3-methyladenine, an inhibitor of autophagy. β-lap increased Sirt1 activity, as shown by the increased deacetylation of the Sirt1 substrates, PARP-1 and Atg5, and the nuclear translocation of FOXO1. Both the induction of autophagy and attenuation of HttEx1(97Q)-GFP aggregation by β-lap were significantly prevented by co-incubation with sirtinol, a general sirtuin inhibitor or by co-transfection with shRNA against Sirt1. The pro-autophagic actions of β-lap were further investigated in a transgenic Caenorhabditis elegans (C. elegans) line that expressed Q67 fused to cyanine fluorescent protein (Q67). Notably, β-lap reduced the number of Q67 puncta and restored Q67-induced defects in motility, which were largely prevented by pre-treatment with RNAi against sir-2.1, the C. elegans orthologue of Sirt1. Collectively, these data suggest that β-lap induces autophagy through activation of Sirt1, which in turn leads to a reduction in polyQ aggregation and cellular toxicity. Thus, β-lap provides a novel therapeutic opportunity for the treatment of HD.

Autophagy-dependent and -independent involvement of AMP-activated protein kinase in 6-hydroxydopamine toxicity to SH-SY5Y neuroblastoma cells

The role of the main intracellular energy sensor adenosine monophosphate (AMP)-activated protein kinase (AMPK) in the induction of autophagic response and cell death was investigated in SH-SY5Y human neuroblastoma cells exposed to the dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA). The induction of autophagy in SH-SY5Y cells was demonstrated by acridine orange staining of intracellular acidic vesicles, the presence of autophagosome- and autophagolysosome-like vesicles confirmed by transmission electron microscopy, as well as by microtubule‐associated protein 1 light‐chain 3 (LC3) conversion and p62 degradation detected by immunoblotting. 6-OHDA induced phosphorylation of AMPK and its target Raptor, followed by the dephosphorylation of the major autophagy inhibitor mammalian target of rapamycin (mTOR) and its substrate p70S6 kinase (S6K). 6-OHDA treatment failed to suppress mTOR/S6K phosphorylation and to increase LC3 conversion, p62 degradation and cytoplasmatic acidification in neuroblastoma cells in which AMPK expression was downregulated by RNA interference. Transfection of SH-SY5Y cells with AMPK or LC3β shRNA, as well as treatment with pharmacological autophagy inhibitors suppressed, while mTOR inhibitor rapamycin potentiated 6-OHDA-induced oxidative stress and apoptotic cell death. 6-OHDA induced phosphorylation of p38 mitogen-activated protein (MAP) kinase in an AMPK-dependent manner, and pharmacological inhibition of p38 MAP kinase reduced neurotoxicity, but not AMPK activation and autophagy triggered by 6-OHDA. Finally, the antioxidant N-acetyl cysteine antagonized 6-OHDA-induced activation of AMPK, p38 and autophagy. These data suggest that oxidative stress-mediated AMPK/mTOR-dependent autophagy and AMPK/p38-dependent apoptosis could be valid therapeutic targets for neuroprotection.

Combination of N-(4-hydroxyphenyl) retinamide and apigenin suppressed starvation-induced autophagy and promoted apoptosis in malignant neuroblastoma cells

Autophagy is a catabolic process for recycling of cellular contents in response to metabolic stress in malignant tumors. We explored efficacy of the synthetic retinoid N-(4-hydroxyphenyl) retinamide (4-HPR) and the isoflavonoid apigenin (APG) in the serum-starved human malignant neuroblastoma cells. Combination of 0.5 μM 4-HPR and 50 μM APG synergistically decreased cell viability in the serum-starved neuroblastoma SH-SY5Y, SK-N-BE2, and IMR-32 cells. Acridine orange (AO) staining and LC3 II upregulation showed that serum-starvation for 12 and 24 h progressively increased the formation of acidic vesicular organelles (AVO) and autophagy in SH-SY5Y cells. Further, AO staining and flow cytometry showed blockage of formation of AVO and accumulation of auophagic population, respectively, following the treatment of the serum-starved SH-SY5Y cells with combination of 0.5 μM 4-HPR and 50 μM APG. Combination therapy downregulated autophagy inducing proteins such as Beclin 1, LC3 II, TLR-4, and Myd88 while upregulated autophagy inhibitory p-Akt/mTOR singaling pathway. Consistent with the hypothesis that inhibition of autophagy could induce apoptosis, we noticed inhibition of autophagy and induction of apoptosis in the serum-starved SH-SY5Y cells with the suppression of the survival factor NF-κB, upregulation of pro-apoptotic Bax, downregulation of anti-apoptotic Bcl-2, activation of caspase-3, and degradation of poly(ADP-ribose) polymerase (PARP) after combination therapy. Collectively, combination of 4-HPR and APG worked synergistically to suppress autophagy and promote apoptosis in human malignant neuroblastoma cells.

Neuroprotection of deferoxamine on rotenone-induced injury via accumulation of HIF-1α and induction of autophagy in SH-SY5Y cells

Hypoxia-inducible factor-1α (HIF-1α) is a transcription factor that activates the transcription of genes and is responsible for progression of cell survival and proliferation. The synthesis of HIF-1α can be stimulated via oxygen (O 2)-independent mechanisms; whereas, the degradation of HIF-1α is regulated via Fe 2+ and/or O 2-dependent enzyme prolyl hydroxylase (PHD). Aberrant iron accumulation, mitochondrial dysfunction and impairment of protein degradation system, such as autophagy, have been implicated in the pathogenesis of Parkinson's disease, among which, iron and mitochondrial dysfunction may enhance the enzyme activity of prolyl hydroxylase and cause the decrease of HIF-1α. Recent reports have indicated that HIF-1α may induce autophagy under hypoxic condition. Considering the metabolic characteristics of HIF-1α under the pathogenesis of Parkinson's disease, we speculated that compounds that might stabilize HIF-1α could prevent neuronal injury caused by excessive iron or mitochondrial injury under normoxic condition. Deferoxamine is one of iron chelators that may accumulate HIF-1α due to the decreased degradation of HIF-1α via inhibition of prolyl hydroxylase activity. In this study, we showed that the protein level of HIF-1α was decreased in rotenone or MPP +-treated SH-SY5Y cell models of Parkinson's disease. We demonstrated that deferoxamine caused accumulation of HIF-1α accompanied by the enhancement of autophagy in SH-SY5Y cells. When HIF-1α gene was inhibited, deferoxamine-induced autophagy was suppressed accordingly, indicating that deferoxamine-induced autophagy was dependent on the expression of HIF-1α. Our results also showed that deferoxamine attenuated rotenone-induced apoptosis, which was blocked when HIF-1α or autophagy related gene Beclin 1 was suppressed. In summary, the present study indicated that the level of HIF-1α was decreased under the situation when mitochondrial complex I was inhibited, and the neuroprotective role of deferoxamine in rotenone-induced apoptosis could be partially explained by its effects on the accumulation of HIF-1α and HIF-1α-mediated induction of autophagy.

Loss of PINK1 Function Promotes Mitophagy through Effects on Oxidative Stress and Mitochondrial Fission

Mitochondrial dysregulation is strongly implicated in Parkinson disease. Mutations in PTEN-induced kinase 1 (PINK1) are associated with familial parkinsonism and neuropsychiatric disorders. Although overexpressed PINK1 is neuroprotective, less is known about neuronal responses to loss of PINK1 function. We found that stable knockdown of PINK1 induced mitochondrial fragmentation and autophagy in SH-SY5Y cells, which was reversed by the reintroduction of an RNA interference (RNAi)-resistant plasmid for PINK1. Moreover, stable or transient overexpression of wild-type PINK1 increased mitochondrial interconnectivity and suppressed toxin-induced autophagy/mitophagy. Mitochondrial oxidant production played an essential role in triggering mitochondrial fragmentation and autophagy in PINK1 shRNA lines. Autophagy/mitophagy served a protective role in limiting cell death, and overexpressing Parkin further enhanced this protective mitophagic response. The dominant negative Drp1 mutant inhibited both fission and mitophagy in PINK1-deficient cells. Interestingly, RNAi knockdown of autophagy proteins Atg7 and LC3/Atg8 also decreased mitochondrial fragmentation without affecting oxidative stress, suggesting active involvement of autophagy in morphologic remodeling of mitochondria for clearance. To summarize, loss of PINK1 function elicits oxidative stress and mitochondrial turnover coordinated by the autophagic and fission/fusion machineries. Furthermore, PINK1 and Parkin may cooperate through different mechanisms to maintain mitochondrial homeostasis.

Regulation of Autophagy by Extracellular Signal-Regulated Protein Kinases During 1-Methyl-4-Phenylpyridinium-Induced Cell Death

Increased autophagic vacuoles (AVs) occur in injured or degenerating neurons, under both developmental and pathological situations. Although regulation of starvation-induced autophagy has been extensively studied, less is known about autophagic responses to pathological damage. The neurotoxin 1-methyl-4-phenylpyridinium (MPP(+)) produces mitochondria-targeted injury, which contributes to parkinsonism induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine in mammals. Here, we demonstrate that MPP(+) elicited increased autophagy in SH-SY5Y cells, as assessed by electron microscopy, immunofluorescence for the autophagy protein LC3/Atg8, LC3 electrophoretic mobility shift, mitochondrial degradation, and monodansylcadaverine staining for late AVs/autolysosomes. During nutrient deprivation, class III phosphatidylinositol-3 kinase (PI3K) stimulates autophagy in concert with the autophagy-regulatory protein beclin 1/Atg6. Although PI3K inhibitors and RNA interference knockdown of beclin 1 effectively inhibited autophagy elicited by amino acid deprivation, neither reduced MPP+-induced autophagic stress. In contrast, inhibition of mitogen-activated protein kinase/extracellular signal-regulated protein kinase kinase reduced AV content, mitochondrial degradation, and cell death in MPP+-treated cells. RNA interference studies targeting core Atg proteins also reduced AV content and cell death. Likewise, in primary midbrain dopaminergic neurons, MPP+ elicited increased AV content, which was reversed by inhibition of mitogen-activated protein kinase/extracellular signal-regulated protein kinase kinase but not PI3K. These results implicate a role for extracellular signal-regulated protein kinase (ERK) signaling upstream of MPP+-elicited autophagic stress. Moreover, pathological stimulation of beclin 1-independent autophagy is associated with neuronal cell death.