Internalization of α-synuclein oligomers into SH-SY5Y cells

Aggregates of misfolded α-synuclein are a distinctive feature of Parkinson’s disease. Small oligomers of α-synuclein are thought to be an important neurotoxic agent, and α-synuclein aggregates exhibit prion-like behavior, propagating misfolding between cells. α-Synuclein is internalized by both passive diffusion and active uptake mechanisms, but how uptake varies with the size of the oligomer is less clear. We explored how α-synuclein internalization into live SH-SY5Y cells varied with oligomer size by comparing the uptake of fluorescently labeled monomers to that of engineered tandem dimers and tetramers. We found that these α-synuclein constructs were internalized primarily through endocytosis. Oligomer size had little effect on their internalization pathway, whether they were added individually or together. Measurements of co-localization of the α-synuclein constructs with fluorescent markers for early endosomes and lysosomes showed that most of the α-synuclein entered endocytic compartments, in which they were probably degraded. Treatment of the cells with the Pitstop inhibitor suggested that most of the oligomers were internalized by the clathrin-mediated pathway.     

Cryptotanshinione Inhibits β-Amyloid Aggregation and Protects Damage from β-Amyloid in SH-SY5Y Cells

The deposition of amyloid β-protein (Aβ) fibrils into plaques within the brain parenchyma and along cerebral blood vessels is a hallmark of Alzheimer’s disease (AD). Aβ42 oligomers and fibrils cause the breakdown of neural circuits, neuronal death and eventually dementia. Drugs that inhibit Aβ42 aggregation may be a novel direction in AD drug discovery. Cryptotanshinone (CTS), an active component of the medicinal herb Salvia miltiorrhiza, has been shown to improve learning and memory in several pharmacological models of AD. However, the effects of CTS on the Aβ aggregation and toxicity are unclear. The current work shows the effectiveness of CTS on the inhibition of Aβ42 aggregation and toxicity to human neuroblastoma cells. In this study, we demonstrated that CTS can inhibit Aβ42 spontaneous aggregation using thioflavin T fluorescence assay and transmission electron microscopy. Furthermore, we investigated the effects of CTS on Aβ-induced oxidative cell death in cultured SH-SY5Y cells. MTT and lactate dehydrogenase assays showed that CTS reduced the cytotoxicity induced by Aβ42. CTS also dramatically reduced Aβ42-induced cellular apoptosis and increased level of reactive oxygen species in these cells. Our study suggests that CTS may be useful in the inhibition or prevention of AD development and progression.     

In vitro optimization of retinoic acid�Cinduced neuritogenesis and TH endogenous expression in human SH-SY5Y neuroblastoma cells by the antioxidant Trolox

Though, it is quite well-known how retinoic acid (RA) is able to induce neuritogenesis in different in vitro models, the putative role exerted by reactive oxygen species (ROS) during this process still need to be further studied. For such purpose, we used a neuronal-like cell line (SH-SY5Y cells) in order to investigate whether the antioxidant Trolox (a hydrophilic analog of alpha-tocopherol) could have any effect on the number of RA-induced neurites, and how significant changes in cellular redox homeostasis may affect the cellular endogenous expression of tyrosine hydroxylase (TH). Our results show a significant enhancement of RA (10 μM)-induced neuritogenesis and TH endogenous expression, when cells were co-treated with Trolox (100 μM) for 7 days. Moreover, this effect was associated with an improvement in cellular viability. The mechanism seems to mainly involve PI3 K/Akt rather than MEK signaling pathway. Therefore, our data demonstrate that concomitant decreases in basal reactive oxygen species (ROS) production could exert a positive effect on the neuritogenic process of RA-treated SH-SY5Y cells.     

eIF2 kinases mediate β-lapachone toxicity in yeast and human cancer cells

β-lapachone (β-lap) is a novel anticancer agent that selectively induces cell death in human cancer cells, by activation of the NQO1 NAD(P)H dehydrogenase and radical oxygen species (ROS) generation. We characterized the gene expression profile of budding yeast cells treated with β-lap using cDNA microarrays. Genes involved in tolerance to oxidative stress were differentially expressed in β-lap treated cells. β-lap treatment generated reactive oxygen species (ROS), which were efficiently blocked by dicoumarol, an inhibitor of NADH dehydrogenases. A yeast mutant in the mitocondrial NADH dehydrogenase Nde2p was found to be resistant to β-lap treatment, despite inducing ROS production in a WT manner. Most interestingly, DNA damage responses triggered by β-lap were abolished in the nde2Δ mutant. Amino acid biosynthesis genes were also induced in β-lap treated cells, suggesting that β-lap exposure somehow triggered the General Control of Nutrients (GCN) pathway. Accordingly, β-lap treatment increased phosphorylation of eIF2α subunit in a manner dependent on the Gcn2p kinase. eIF2α phosphorylation required Gcn1p, Gcn20p and Nde2p. Gcn2p was also required for cell survival upon exposure to β-lap and to elicit checkpoint responses. Remarkably, β-lap treatment increased phosphorylation of eIF2α in breast tumor cells, in a manner dependent on the Nde2p ortholog AIF, and the eIF2 kinase PERK. These findings uncover a new target pathway of β-lap in yeast and human cells and highlight a previously unknown functional connection between Nde2p, Gcn2p and DNA damage responses.     

Retinoic acid–induced survival effects in SH-SY5Y neuroblastoma cells

Neuroblastoma is a malignant childhood cancer arising from the embryonic sympathoadrenal lineage of the neural crest. Retinoic acid (RA) is included in the multimodal therapy of patients with high-risk neuroblastoma to eliminate minimal residual disease. However, the formation of RA-resistant cells substantially lowers 5-year overall survival rates. To examine mechanisms that lead to treatment failure, we chose human SH-SY5Y cells, which are known to tolerate incubation with RA by activating the survival kinases Akt and extracellular signal-regulated kinase 1/2. Characterization of downstream pathways showed that both kinases increased the phosphorylation of the ubiquitin ligase mouse double minute homolog 2 (Mdm2) and thereby enhanced p53 degradation. When p53 signaling was sustained by blocking complex formation with Mdm2 or enhancing c-Jun N-terminal kinase (JNK) activation, cell viability was significantly reduced. In addition, Akt-mediated phosphorylation of the cell-cycle regulator p21 stimulated complex formation with caspase-3, which also contributed to cell protection. Thus, treatment with RA augmented survival signaling and attenuated basal apoptotic pathways in SH-SY5Y cells, which increased cell viability.     

Dopamine facilitates α-synuclein oligomerization in human neuroblastoma SH-SY5Y cells

Parkinson’s disease is characterized by selective loss of dopaminergic neurons in the substantia nigra and by the appearance of Lewy bodies. Fibrillar α-synuclein is the main component of Lewy bodies. Previous studies have suggested that dopamine promotes α-synuclein oligomerization and that partially aggregated or oligomeric α-synuclein could be cytotoxic. To confirm this hypothesis using cell cultures, we performed size exclusion chromatography as a pretreatment method prior to Western blotting to more clearly detect a small amount of α-synuclein oligomers in wild-type α-synuclein-overexpressing SH-SY5Y cells. Using this method, we confirmed that stable overexpression of α-synuclein in SH-SY5Y cells indeed increased the amounts of α-synuclein oligomers in these cells and exposure of the cells to dopamine for 6 h facilitated α-synuclein oligomerization. These dopamine-induced α-synuclein oligomers continued to exist for the following 24 h. However, the dopamine-treated cells did not undergo cell death or apoptosis in spite of the presence of increased oligomeric α-synuclein. Our data may contribute to the understanding of the mechanisms underlying α-synuclein oligomer formation and its suspected cytotoxicity toward dopaminergic neurons.     

Human Chorionic Gonadotropin Increases β-Cleavage of Amyloid Precursor Protein in SH-SY5Y Cells

Elevated levels of amyloid-β (Aβ) peptides, the main component of amyloid plaques in Alzheimer’s disease, are the result of excessive β- and γ-cleavage of the amyloid precursor protein (APP) and/or impaired Aβ clearance in the brain. It has been suggested that high concentrations of luteinizing hormone (LH) in women contribute to increased Aβ generation after menopause, but the mechanism for this is incompletely understood. We investigated the effect of human chorionic gonadotropin (hCG), an LH receptor agonist, on APP β-cleavage in the SH-SY5Y neuroblastoma cell line. Treatment of these cells with hCG-induced elevated β-cleavage in a dose-dependent manner: administration of 30 mIU but not 10 mIU/ml of hCG significantly increased sAPPβ levels in the cell medium 1.7-fold as measured by ELISA. These results support the notion that LH contributes to elevated Aβ levels at least in part by increasing β-cleavage of APP by β-site APP cleaving enzyme.     

Isomerization of Asp7 increases the toxic effects of amyloid β and its phosphorylated form in SH-SY5Y neuroblastoma cells

The generation of amyloid β (Aβ) toxic oligomers during the formation of senile plaques and amyloid fibrils is thought to play a central role in the onset and progression of Alzheimer’s disease. Aβ production is a physiological process, but the factors that trigger a transition to pathogenic Aβ aggregation remain unknown. Posttranslational modifications of Aβ could potentially induce the transition. The effects of Aβ and its modified forms containing isomerized Asp7, phosphorylated Ser8, or both, were studied in SH-SY5Y human neuroblastoma cells. Asp7 isomerization of was shown to increase cytotoxicity of both the intact and phosphorylated Aβ. An increase in cytotoxicity was not associated with an increased internalization of the isomerized Asp7-containing Aβ or an influence on the function of mitochondria or reduced glutathione and reactive oxygen species levels. The nitric oxide (NO) level was identified as a determinant of the cytotoxic effect of isomerized Asp7-containing peptides, a decrease in NO level correlating with an increase in cytotoxicity.     

Differential effects of α-tocopherol and N-acetyl-cysteine on advanced glycation end product-induced oxidative damage and neurite degeneration in SH-SY5Y cells

Advanced glycation end products (AGEs) result from non-enzymatic glycation of proteins and cause cellular oxidative stress in a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-dependent manner. Due to these effects, AGEs are implicated as a causal factor in diabetic complications. Several antioxidants, including vitamin E, improve cell viability and diminish markers of oxidative damage in cells exposed to AGEs. However, vitamin E has been studied in cell culture systems with primary focus on apoptosis and lipid peroxidation, while its influences on AGE-induced protein and DNA oxidation, intracellular antioxidant status and cell morphology remain largely unknown. Here, we verify the suppression of AGE-induced cell death and lipid peroxidation by 200μM α-tocopherol in SH-SY5Y cells. We report the partial inhibition of DNA oxidation and a decrease in protein carbonyl formation by α-tocopherol with no effects on intracellular GSH concentrations. We observed that 2mM N-acetyl cysteine (NAC) also had a suppressive effect on DNA and protein oxidation, but unlike α-tocopherol, it caused a marked increase in intracellular GSH. Finally, we compared the ability of both antioxidants to maintain neurites in SH-SY5Y cells and found that α-tocopherol had no effect on neurite loss due to AGEs, while NAC fully maintained cell morphology. Thus, while α-tocopherol suppressed AGE-induced macromolecule damage, it was ineffective against neurite degeneration. These results may implicate thiol oxidation and maintenance as a major regulator of neurite degeneration in this model.     

Potential mechanism of phytochemical-induced apoptosis in human prostate adenocarcinoma cells: Therapeutic synergy in genistein and β-lapachone combination treatment

Background  

Prostate cancer is the second leading cause of male death in the United States. The incidence increases most rapidly with age, and multiple genetic and epigenetic factors have been implicated in the initiation, progression, and metastasis of the cancer. Nevertheless, scientific knowledge of the molecular mechanisms underlying the disease is still limited; and hence treatment has only been partially successful. The objective of the current studies was to examine the role of caspase 3 (CPP32) and NAD(P)H:quinone oxidoreductase (NQO1) in the signaling of genistein-and β-lapachone (bLap)-induced apoptosis in human prostate carcinoma cells PC3.     

eIF2 kinases mediate β-lapachone toxicity in yeast and human cancer cells

β-lapachone (β-lap) is a novel anticancer agent that selectively induces celldeath in human cancer cells, by activation of the NQO1 NAD(P)H dehydrogenase and radicaloxygen species (ROS) generation. We characterized the gene expression profile ofbudding yeast cells treated with β-lap using cDNA microarrays. Genes involved intolerance to oxidative stress were differentially expressed in β-lap treated cells.β-lap treatment generated reactive oxygen species (ROS), which were efficientlyblocked by dicoumarol, an inhibitor of NADH dehydrogenases. A yeast mutant in themitocondrial NADH dehydrogenase Nde2p was found to be resistant to β-lap treatment,despite inducing ROS production in a WT manner. Most interestingly, DNAdamage responses triggered by β-lap were abolished in thende2Δ mutant. Amino acid biosynthesis genes were also induced inβ-lap treated cells, suggesting that β-lap exposure somehow triggered theGeneral Control of Nutrients (GCN) pathway. Accordingly, β-lap treatment increasedphosphorylation of eIF2α subunit in a manner dependent on the Gcn2p kinase.eIF2α phosphorylation required Gcn1p, Gcn20p and Nde2p. Gcn2p was also required forcell survival upon exposure to β-lap and to elicit checkpointresponses. Remarkably, β-lap treatment increased phosphorylation of eIF2αin breast tumor cells, in a manner dependent on the Nde2p ortholog AIF, and the eIF2kinase PERK. These findings uncover a new target pathway of β-lap in yeast and humancells and highlight a previously unknown functional connection between Nde2p, Gcn2p andDNA damage responses.     

Differential effects of α-tocopherol and N-acetyl-cysteine on advanced glycation end product-induced oxidative damage and neurite degeneration in SH-SY5Y cells

Advanced glycation end products (AGEs) result from non-enzymatic glycation of proteins and cause cellular oxidative stress in a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-dependent manner. Due to these effects, AGEs are implicated as a causal factor in diabetic complications. Several antioxidants, including vitamin E, improve cell viability and diminish markers of oxidative damage in cells exposed to AGEs. However, vitamin E has been studied in cell culture systems with primary focus on apoptosis and lipid peroxidation, while its influences on AGE-induced protein and DNA oxidation, intracellular antioxidant status and cell morphology remain largely unknown. Here, we verify the suppression of AGE-induced cell death and lipid peroxidation by 200 μM α-tocopherol in SH-SY5Y cells. We report the partial inhibition of DNA oxidation and a decrease in protein carbonyl formation by α-tocopherol with no effects on intracellular GSH concentrations. We observed that 2 mM N-acetyl cysteine (NAC) also had a suppressive effect on DNA and protein oxidation, but unlike α-tocopherol, it caused a marked increase in intracellular GSH. Finally, we compared the ability of both antioxidants to maintain neurites in SH-SY5Y cells and found that α-tocopherol had no effect on neurite loss due to AGEs, while NAC fully maintained cell morphology. Thus, while α-tocopherol suppressed AGE-induced macromolecule damage, it was ineffective against neurite degeneration. These results may implicate thiol oxidation and maintenance as a major regulator of neurite degeneration in this model.     

Oxidized LDL lipids increase β-amyloid production by SH-SY5Y cells through glutathione depletion and lipid raft formation

Elevated total cholesterol in midlife has been associated with increased risk of dementia in later life. We have previously shown that low-density lipoprotein (LDL) is more oxidized in the plasma of dementia patients, although total cholesterol levels are not different from those of age-matched controls. β-Amyloid (Aβ) peptide, which accumulates in Alzheimer disease (AD), arises from the initial cleavage of amyloid precursor protein by β-secretase-1 (BACE1). BACE1 activity is regulated by membrane lipids and raft formation. Given the evidence for altered lipid metabolism in AD, we have investigated a mechanism for enhanced Aβ production by SH-SY5Y neuronal-like cells exposed to oxidized LDL (oxLDL). The viability of SH-SY5Y cells exposed to 4 μg oxLDL and 25 µM 27-hydroxycholesterol (27OH-C) was decreased significantly. Lipids, but not proteins, extracted from oxLDL were more cytotoxic than oxLDL. In parallel, the ratio of reduced glutathione (GSH) to oxidized glutathione was decreased at sublethal concentrations of lipids extracted from native and oxLDL. GSH loss was associated with an increase in acid sphingomyelinase (ASMase) activity and lipid raft formation, which could be inhibited by the ASMase inhibitor desipramine. 27OH-C and total lipids from LDL and oxLDL independently increased Aβ production by SH-SY5Y cells, and Aβ accumulation could be inhibited by desipramine and by N-acetylcysteine. These data suggest a mechanism whereby oxLDL lipids and 27OH-C can drive Aβ production by GSH depletion, ASMase-driven membrane remodeling, and BACE1 activation in neuronal cells.     

Cytoprotective effects of the antioxidant phytochemical indicaxanthin in β-thalassemia red blood cells

Antioxidant phytochemicals are investigated as novel treatments for supportive therapy in β-thalassemia. The dietary indicaxanthin was assessed for its protective effects on human β-thalassemic RBCs submitted in vitro to oxidative haemolysis by cumene hydroperoxide. Indicaxanthin at 1.0–10 μM enhanced the resistance to haemolysis dose-dependently. In addition, it prevented lipid and haemoglobin (Hb) oxidation, and retarded vitamin E and GSH depletion. After ex vivo spiking of blood from thalassemia patients with indicaxanthin, the phytochemical was recovered in the soluble cell compartment of the RBCs. A spectrophotometric study showed that indicaxanthin can reduce perferryl-Hb generated in solution from met-Hb and hydrogen peroxide (H₂O₂), more effectively than either Trolox or vitamin C.

Collectively our results demonstrate that indicaxanthin can be incorporated into the redox machinery of β-thalassemic RBC and defend the cell from oxidation, possibly interfering with perferryl-Hb, a reactive intermediate in the hydroperoxide-dependent Hb degradation. Opportunities of therapeutic interest for β-thalassemia may be considered.     

Inhibition of branching morphogenesis and alteration of glycosaminoglycan biosynthesis in salivary glands treated with β-d-xyloside

The role of glycosaminoglycans (GAGs) in the branching morphogenesis of embryonic mouse salivary glands was investigated by culturing the glands in the presence of xylose derivatives which stimulate synthesis of the xyloselinked classes of GAGs. Branching morphogenesis is inhibited severely, but reversibly, by 0.5–1.0 mM π-nitrophenyl-β-d-xylopyranoside and the inhibition correlates with a stimulation of incorporation of [³H]glucosamine (1.8-fold) and [³⁵S]sulfate (almost 3-fold) into GAGs. The effect of β-xyloside on accumulation of newly synthesized GAG also occurs in the presence of the protein synthesis inhibitor cycloheximide, suggesting that the production of free GAG chains rather than proteoglycan-associated GAGs is being stimulated. The xyloside effects apparently do not result from general cytotoxicity of the derivatives, since similar concentrations of the α-anomer do not alter salivary branching or GAG synthesis, the rudiments resume morphogenesis when returned to control medium, and the effect on GAG synthesis is stimulatory rather than inhibitory. The study suggests that GAG biosynthesis plays an important role in salivary development, and that xylosides provide useful probes for characterizing the molecular events controlling branching morphogenesis.     

Association of connexin36 with zonula occludens-1 in HeLa cells, βTC-3 cells, pancreas, and adrenal gland

The PDZ domain-containing protein zonula occludens-1 (ZO-1), a well-established component of tight junctions, has recently been shown to interact with various connexin proteins that form gap junctions. We investigated the association of connexin36 (Cx36) with ZO-1 in various cultured cells and tissues. Punctate immunofluorescence labeling for Cx36 was detected in Cx36-transfected HeLa cells, TC-3 cells, pancreatic islets, and adrenal medulla. Immunofluorescence for ZO-1 was also punctate in cells and tissues, and was colocalized with Cx36 at points of cell–cell contact. Immunoprecipitation of either Cx36 or ZO-1 from cell lysates and tissue homogenates resulted in immunoblot detection of ZO-1 or Cx36, respectively, in immunoprecipitates. A 14-amino acid peptide corresponding to the carboxy-terminus of Cx36 showed binding capacity to the PDZ1 domain of ZO-1, which was eliminated after removal of the last 4 carboxy-terminus amino acids. Low micromolar concentrations of the 14-amino acid peptide produced up to 85% inhibition of Cx36 interaction with the PDZ1 domain of ZO-1. These results provide evidence for molecular interaction between Cx36 and ZO-1 in vitro, and in vivo, and suggest that the interference with Cx36/ZO-1 interaction by short carboxy-terminus peptides of Cx36 may be of value for functional studies of this interaction.*co-1^st authors