Protective effects of Peroxiredoxin 6 overexpression on amyloid β-induced apoptosis in PC12 cells

Abstract

Oxidative stress triggered by amyloid beta (Aβ) accumulation contributes substantially to the pathogenesis of Alzheimer's disease (AD). In the present study, we examined the involvement of the antioxidant activity of peroxiredoxin 6 (Prdx 6) in protecting against Aβ_25–35-induced neurotoxicity in rat PC12 cells. Treatment of PC12 cells with Aβ_25-35 resulted in a dose- and time-dependent cytotoxicity that was associated with increased accumulation of intracellular reactive oxygen species (ROS) and mitochondria-mediated apoptotic cell death, including activation of Caspase 3 and 9, inactivation of poly ADP-ribosyl polymerse (PARP), and dysregulation of Bcl-2 and Bax. This apoptotic signaling machinery was markedly attenuated in PC12 cells that overexpress wild-type Prdx 6, but not in cells that overexpress the C47S catalytic mutant of Prdx 6. This indicates that the peroxidase activity of Prdx 6 protects PC12 cells from Aβ_25-35-induced neurotoxicity. The neuroprotective role of the antioxidant Prdx 6 suggests its therapeutic and/or prophylactic potential to slow the progression of AD and limit the extent of neuronal cell death caused by AD.     

Changes in mitochondrial dynamics during amyloid β-induced PC12 cell apoptosis

Changes in mitochondrial morphology and dynamics influence mitochondrial function and ultimately damage neurons in Alzheimer’s disease (AD). Amyloid β (Aβ) is a major factor in the pathogenesis of AD. Although it has been proved that Aβ can affect the dynamics of mitochondria, there is little known on the precise dynamic process. Thus, MTT, Hoechst 33342, and Annexin V/PI analysis were used to study Aβ_25–35 neurotoxity on PC12 cells, live cell station and image processing were applied to study the moving parameters and characters of mitochondria. We also studied changes of mitochondrial membrane potential and reactive oxygen species production. The results showed that long-term exposure of PC12 cells to Aβ_25–35 resulted in increase of mitochondrial number and decrease of mitochondrial length and size, which presented fluctuated during early time and dramatic changes occurred after 6 h. Low concentration exposure caused little mitochondrial changes before 24 h while short time exposure induced mitochondrial fragmentation that could be recovered to normal. Mitochondrial membrane potential dissipation and reactive oxygen species production were observed, as well as apparent cell apoptosis with significant morphological changes. These data suggest that mitochondrial fission can be reversed during Aβ_25–35-induced PC12 cell apoptosis, depending on the concentration and exposure time of Aβ_25–35, which may be helpful in AD prevention and therapy.     

Macroautophagy-generated increase of lysosomal amyloid β-protein mediates oxidant-induced apoptosis of cultured neuroblastoma cells

Increasing evidence suggests the toxicity of intracellular amyloid β-protein (Aβ) to neurons, as well as the involvement of oxidative stress in Alzheimer disease (AD). Here we show that normobaric hyperoxia (exposure of cells to 40% oxygen for five days), and consequent activation of macroautophagy and accumulation of Aβ within lysosomes, induced apoptosis in differentiated SH-SY5Y neuroblastoma cells. Cells under hyperoxia showed: (1) increased numbers of autophagic vacuoles that contained amyloid precursor protein (APP) as well as Aβ monomers and oligomers, (2) increased reactive oxygen species production, and (3) enhanced apoptosis. Oxidant-induced apoptosis positively correlated with cellular Aβ production, being the highest in cells that were stably transfected with APP Swedish KM670/671NL double mutation. Inhibition of γ-secretase, prior and/or in parallel to hyperoxia, suggested that the increase of lysosomal Aβ resulted mainly from its autophagic uptake, but also from APP processing within autophagic vacuoles. The oxidative stress-mediated effects were prevented by macroautophagy inhibition using 3-methyladenine or ATG5 downregulation. Our results suggest that upregulation of macroautophagy and resulting lysosomal Aβ accumulation are essential for oxidant-induced apoptosis in cultured neuroblastoma cells and provide additional support for the interactive role of oxidative stress and the lysosomal system in AD-related neurodegeneration.     

The roles of integrin β4 in vascular endothelial cells

Integrin heterodimers play diverse and important roles in physiological and pathological processes, such as cell adhesion, migration, proliferation, differentiation, angiogenesis, and tumor progression, via the outside-in and/or inside-out signaling pathways. Aberrant functions of integrins have been implicated in the causation and intervention of multiple diseases. Integrin β(4), a laminin-5 (LN5) receptor, mainly locates in the adhesion structure of hemidesmosome (HD). Most of the previous researches concentrated on the role of integrin β(4) in cancer and cancer therapy, and a few focused on the physiological roles of normal mammalian cells. Recently, accumulating data reveal that integrin β(4) participates in cell death, macroautophagy (hereafter autophagy), senescence, and differentiation regulations in various cell types including human umbilical vein endothelial cells (HUVECs), mesenchymal stem cells, and mouse neural cells, implying the key roles of integrin β(4) in the physiological alteration of mammalian cells. Thus, the elucidation of integrin β(4)-mediated signaling may undoubtedly contribute to novel therapeutic strategies for various human diseases, such as vascular and neural disorders. We have reviewed the roles of integrin β(4) in neural cells. In the present review we will discuss the recent research progress in the inherent functions and pharmacological modulation of integrin β(4) in vascular endothelial cells.     

Inhibition of eIF2α dephosphorylation enhances TRAIL-induced apoptosis in hepatoma cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an inducer of cancer cell death that holds promise in cancer therapy. Cancer cells are more susceptible than normal cells to the cell-death-inducing effects of TRAIL. However, a variety of cancer cells are resistant to TRAIL through complex mechanisms. Here, we investigate the effects of inhibition of eukaryotic initiation factor 2 subunit α (eIF2α) dephosphorylation on TRAIL-induced apoptosis in hepatoma cells. Treatment of hepatoma cells with salubrinal, an inhibitor of eIF2α dephosphorylation, enhances TRAIL-induced eIF2α phosphorylation, CCAAT/enhancer-binding protein homologous protein (CHOP) expression and caspase activation. Salubrinal enhances TRAIL-induced apoptosis, which could be abrogated by caspase inhibitor. Overexpression of phosphomimetic eIF2α (S51D) enhances TRAIL-induced CHOP expression, caspase 7 and PARP cleavage and apoptosis. By contrast, overexpression of phosphodeficient eIF2α (S51A) abrogates the stimulation of TRAIL-induced apoptosis by salubrinal. Moreover, knockdown of growth arrest and DNA damage-inducible protein 34 (GADD34), which recruits protein phosphatase 1 to dephosphorylate eIF2α, enhances TRAIL-induced eIF2α phosphorylation, CHOP expression, caspase activation and apoptosis. Furthermore, the sensitization of hepatoma cells to TRAIL by salubrinal is dependent on CHOP. Knockdown of CHOP abrogates the stimulation of TRAIL-induced caspase activation and apoptosis by salubrinal. Combination of salubrinal and TRAIL leads to increased expression of Bim, a CHOP-regulated proapoptotic protein. Bim knockdown blunts the stimulatory effect of salubrinal on TRAIL-induced apoptosis. Collectively, these findings suggest that inhibition of eIF2α dephosphorylation may lead to synthetic lethality in TRAIL-treated hepatoma cells.     

Inhibition of aggregation of amyloid β42 by arginine-containing small compounds

Aggregations of proteins are in many cases associated with neurodegenerative diseases such as Alzheimer's (AD). Small compounds capable of inhibiting protein aggregation are expected to be useful for not only in the treatment of disease but also in probing the structures of aggregated proteins. In previous studies using phage display, we found that arginine-rich short peptides consisting of four or seven amino acids bound to soluble 42-residue amyloid β (Aβ42) and inhibited globulomer (37/48 kDa oligomer) formation. In the present study, we searched for arginine-containing small molecules using the SciFinder searching service and tested their inhibitory activities against Aβ42 aggregation, by sodium dodecyl sulfate (SDS)-PAGE and thioflavine T binding assay. Commercially available Arg-Arg-7-amino-4-trifluoromethylcoumarin was found to exhibit remarkable inhibitory activities to the formation of the globulomer and the fibril of Aβ42. This chimera-type tri-peptide is expected to serve as the seed molecule of a potent inhibitor of the Aβ aggregation process.     

PKCα mediates acetylcholine-induced activation of TRPV4-dependent calcium influx in endothelial cells

Transient receptor potential vanilloid channel 4 (TRPV4) is a polymodally activated nonselective cationic channel implicated in the regulation of vasodilation and hypertension. We and others have recently shown that cyclic stretch and shear stress activate TRPV4-mediated calcium influx in endothelial cells (EC). In addition to the mechanical forces, acetylcholine (ACh) was shown to activate TRPV4-mediated calcium influx in endothelial cells, which is important for nitric oxide-dependent vasodilation. However, the molecular mechanism through which ACh activates TRPV4 is not known. Here, we show that ACh-induced calcium influx and endothelial nitric oxide synthase (eNOS) phosphorylation but not calcium release from intracellular stores is inhibited by a specific TRPV4 antagonist, AB-159908. Importantly, activation of store-operated calcium influx was not altered in the TRPV4 null EC, suggesting that TRPV4-dependent calcium influx is mediated through a receptor-operated pathway. Furthermore, we found that ACh treatment activated protein kinase C (PKC) α, and inhibition of PKCα activity by the specific inhibitor Go-6976, or expression of a kinase-dead mutant of PKCα but not PKCε or downregulation of PKCα expression by chronic 12-O-tetradecanoylphorbol-13-acetate treatment, completely abolished ACh-induced calcium influx. Finally, we found that ACh-induced vasodilation was inhibited by the PKCα inhibitor Go-6976 in small mesenteric arteries from wild-type mice, but not in TRPV4 null mice. Taken together, these findings demonstrate, for the first time, that a specific isoform of PKC, PKCα, mediates agonist-induced receptor-mediated TRPV4 activation in endothelial cells.     

Evidence for lysosomal processing of amyloid β-protein precursor in cultured cells

Amyloid -protein precursor (ABPP) of Alzheimer's disease (AD) represents a family of proteins which includes the parent protein which generates a small (4 kD) fragment that self-assembles to form amyloid fibrils in AD. Thus, the normal and abnormal proteolysis of ABPP may be directly relevant to AD pathogenesis. We have examined the accumulation of ABPP in cultured rodent and human neuronal cell lines in the presence and absence of a battery of protease inhibitors using immunohistochemistry and Western blot analysis. Here we present evidence for a lysosomal pathway for the turnover of ABPP and discuss the relevance of these results to plaque pathology and abnormal ABPP immunostaining in AD.Special issue dedicated to Dr. Paola S. Timiras     

Shear stress regulates expression of death-associated protein kinase in suppressing TNFα-induced endothelial apoptosis

Death associated protein kinase (DAPK) is a positive regulator in tumor necrosis factor α (TNFα)‐induced apoptotic pathway, and DAPK expression is lost in cancer cells. In the vasculature, misdirected apoptosis in endothelial cells leads to pathological conditions such as inflammation and physiological shear stress is protective against apoptosis. Using bovine aortic endothelial cells, we found that DAPK expression increased, while the auto‐inhibitory phosphorylation of serine 308 decreased with shear stress at 12 dynes/cm² for 6 h. Quantitative RT‐PCR revealed a corresponding increase in DAPK mRNA [P < 0.01]. We found that after 18‐h TNFα induction, shearing cells for another 6 h significantly reduced apoptosis based on TUNEL staining [P < 0.05], although cell necrosis was not affected. Under the same conditions, we observed significantly decreased overall DAPK, as well as phospho‐serine 308 DAPK [P < 0.05] compared to TNFα treatment alone. Caspase‐3 and ‐7 activities downstream of DAPK were also attenuated. Shearing cells alone resulted in enhanced apoptosis, likely due to increased DAPK activity. Our findings were further supported by DAPK siRNA, which yielded contrary results. We present conclusive evidence for the first time that shear stress of up to 6 h up‐regulates DAPK expression and activation. However, in the presence of apoptotic stimuli such as TNFα, shear stress caused decrease in DAPK activity. In fact, long‐term shear stress of 24 h significantly reduced overall DAPK expression. Our findings strongly support a novel role for DAPK in mediating effects of shear stress in suppressing cytokine‐activated apoptosis. J. Cell. Physiol. 227: 2398–2411, 2012. © 2011 Wiley Periodicals, Inc.     

Shear stress modulation of IL-1β-induced E-selectin expression in human endothelial cells

Background

Endothelial cells (ECs) are continuously exposed to hemodynamic forces imparted by blood flow. While it is known that endothelial behavior can be influenced by cytokine activation or fluid shear, the combined effects of these two independent agonists have yet to be fully elucidated.

Methodology

We investigated EC response to long-term inflammatory cues under physiologically relevant shear conditions via E-selectin expression where monolayers of human umbilical vein ECs were simultaneously exposed to laminar fluid shear and interleukin-1ß (shear-cytokine activation) in a parallel plate flow chamber.

Results and Conclusion

Naïve ECs exposed to shear-cytokine activation display significantly higher E-selectin expression for up to 24 hr relative to ECs activated in static (static-cytokine). Peak E-selectin expression occurred after 8–12 hr of continuous shear-cytokine activation contrary to the commonly observed 4–6 hr peak expression in ECs exposed to static-cytokine activation. Cells with some history of high shear conditioning exhibited either high or muted E-selectin expression depending on the durations of the shear pre-conditioning and the ensuing shear-cytokine activation. Overall, the presented data suggest that a high laminar shear enhances acute EC response to interleukin-1ß in naïve or shear-conditioned ECs as may be found in the pathological setting of ischemia/reperfusion injury while conferring rapid E-selectin downregulation to protect against chronic inflammation.     

Hsp90 regulates O-linked β-N-acetylglucosamine transferase: a novel mechanism of modulation of protein O-linked β-N-acetylglucosamine modification in endothelial cells

O-linked β-N-acetylglucosamine (O-GlcNAc) modification of proteins is involved in many important cellular processes. Increased O-GlcNAc has been implicated in major diseases, such as diabetes and its complications and cardiovascular and neurodegenerative diseases. Recently, we reported that O-GlcNAc modification occurs in the proteasome and serves to inhibit proteasome function by blocking the ATPase activity in the 19S regulatory cap, explaining, at least in part, the adverse effects of O-GlcNAc modification and suggesting that downregulating O-GlcNAc might be important in the treatment of human diseases. In this study, we report on a novel mechanism to modulate cellular O-GlcNAc modification, namely through heat shock protein 90 (Hsp90) inhibition. We observed that O-linked β-N-acetylglucosamine transferase (OGT) interacts with the tetratricopeptide repeat binding site of Hsp90. Inhibition of Hsp90 by its specific inhibitors, radicicol or 17-N-allylamino-17-demethoxygeldanamycin, destabilized OGT in primary endothelial cell cultures and enhanced its degradation by the proteasome. Furthermore, Hsp90 inhibition downregulated O-GlcNAc protein modifications and attenuated the high glucose-induced increase in O-GlcNAc protein modification, including high glucose-induced increase in endothelial or type 3 isoform of nitric oxide synthase (eNOS) O-GlcNAcylation. These results suggest that Hsp90 is involved in the regulation of OGT and O-GlcNAc modification and that Hsp90 inhibitors might be used to modulate O-GlcNAc modification and reverse its adverse effects in human diseases.     

Novel off-target effect of tamoxifen — Inhibition of acid ceramidase activity in cancer cells

Acid ceramidase (AC), EC 3.5.1.23, a lysosomal enzyme, catalyzes the hydrolysis of ceramide to constituent sphingoid base, sphingosine, and fatty acid. Because AC regulates the levels of pro-apoptotic ceramide and mitogenic sphingosine-1-phosphate, it is considered an apt target in cancer therapy. The present study reveals, for the first time, that the prominent antiestrogen, tamoxifen, is a pan-effective AC inhibitor in the low, single digit micromolar range, as demonstrated in a wide spectrum of cancer cell types, prostate, pancreatic, colorectal, and breast. Prostate cancer cells were chosen for the detailed investigations. Treatment of intact PC-3 cells with tamoxifen produced time- and dose-dependent inhibition of AC activity. Tamoxifen did not impact cell viability nor did it inhibit AC activity in cell-free assays. In pursuit of mechanism of action, we demonstrate that tamoxifen induced time-, as early as 5 min, and dose-dependent, as low as 5 μM, increases in lysosomal membrane permeability (LMP), and time- and dose-dependent downregulation of AC protein expression. Assessing various protease inhibitors revealed that a cathepsin B inhibitor blocked tamoxifen-elicited downregulation of AC protein; however, this action failed to restore AC activity unless assayed in a cell-free system at pH 4.5. In addition, pretreatment with tamoxifen inhibited PC-3 cell migration. Toremifene, an antiestrogen structurally similar to tamoxifen, was also a potent inhibitor of AC activity. This study reveals a new, off-target action of tamoxifen that may be of benefit to enhance anticancer therapies that either incorporate ceramide or target ceramide metabolism.     

The neuroprotective effect of agmatine against amyloid β-induced apoptosis in primary cultured hippocampal cells involving ERK,Akt/GSK-3β, and TNF-α

Molecular Biology Reports - β-Amyloid peptide (Aβ), the major element of senile plaques in Alzheimer’s disease (AD), has been found to accumulate in brain regions critical for...     

Novel indole-based melatonin analogues: Evaluation of antioxidant activity and protective effect against amyloid β-induced damage

Oxidative stress has been recognized as a contributing factor in ageing and various diseases including cancer and neuropathological disorders. Indole derivatives such as the neurohormone melatonin (MLT) constitute an important class of therapeutic agent in medicinal chemistry. MLT can scavenge different reactive oxygen species and can also stimulate the synthesis of antioxidant enzymes. As a part of our ongoing studies, a series of new indole-based hydrazide/hydrazone derivatives were synthesized as MLT analogues. Their antioxidant activity was investigated in human erythrocytes by evaluating their reducing effect against oxidation of a redox-sensitive fluorescent probe. Possible inherent cytotoxicity of the compounds was investigated in CHO-K1 cells by lactate dehydrogenase leakage test. Protection of neuronal PC12 cells against amyloid β-induced damage was examined by MTT assay and their ability in reduction of ROS generation induced by amyloid β was tested. MLT analogues having an o-halogenated aromatic moiety exhibited effective antioxidant properties without having any membrane-damaging effect. Moreover, derivatives having o-halogenated and dihalogenated aromatic side chain significantly protected neuronal cells at concentrations of 10 and 100 μM. In conclusion, MLT derivatives represent promising scaffolds for discovery of effective antioxidant and neuroprotective agents.     

Inhibition of protein kinase B activity induces cell cycle arrest and apoptosis during early G₁ phase in CHO cells

Inhibition of PKB (protein kinase B) activity using a highly selective PKB inhibitor resulted in inhibition of cell cycle progression only if cells were in early G1 phase at the time of addition of the inhibitor, as demonstrated by time-lapse cinematography. Addition of the inhibitor during mitosis up to 2 h after mitosis resulted in arrest of the cells in early G1 phase, as deduced from the expression of cyclins D and A and incorporation of thymidine. After 24 h of cell cycle arrest, cells expressed the cleaved caspase-3, a central mediator of apoptosis. These results demonstrate that PKB activity in early G1 phase is required to prevent the induction of apoptosis. Using antibodies, it was demonstrated that active PKB translocates to the nucleus during early G1 phase, while an even distribution of PKB was observed through cytoplasm and nucleus during the end of G1 phase.     

Appearance of cell-bound β-galactosidase activity in Kluyveromyces bulgaricus resting cells

Summary The conditions involved in the appearance of cell-bound -galactosidase activity in Kluyveromyces bulgaricus resting cells were determined in relation to phospholipid content. The cells displayed an optimum -galactosidase activity when they were anaerobically growing in whey medium and kept at 25°C for more than 14 days. The activity was stimulated by a preliminary 3–5 hours shaking period and an adequate protein concentration of whey medium. The expression of the cell-bound -galactosidase was correlated to a 60% decrease in yeast phospholipid content.     

Oxidative stress induces macroautophagy of amyloid β-protein and ensuing apoptosis

There is increasing evidence for the toxicity of intracellular amyloid β-protein (Aβ) to neurons and the involvement of lysosomes in this process in Alzheimer disease (AD). We have recently shown that oxidative stress, a recognized determinant of AD, enhances macroautophagy and leads to intralysosomal accumulation of Aβ in cultured neuroblastoma cells. We hypothesized that oxidative stress promotes AD by stimulating macroautophagy of Aβ that further may induce cell death by destabilizing lysosomal membranes. To investigate such possibility, we compared the effects of hyperoxia (40% ambient oxygen) in cultured HEK293 cells that were transfected with an empty vector (Vector), wild-type APP (APPwt), or Swedish mutant APP (APPswe). Exposure to hyperoxia for 5 days increased the number of cells with Aβ-containing lysosomes, as well as the number of apoptotic cells, compared to normoxic conditions. The rate of apoptosis in all three cell lines demonstrated dependence on intralysosomal Aβ content (Vector < APPwt < APPswe). Furthermore, the degree of apoptosis was positively correlated with lysosomal membrane permeabilization, whereas inhibitors of macroautophagy and lysosomal function decreased oxidant-induced apoptosis and diminished the differences in apoptotic response between different cell lines. These results suggest that oxidative stress can induce neuronal death through macroautophagy of Aβ and consequent lysosomal membrane permeabilization, which may help explain the mechanisms behind neuronal loss in AD.     

The role of β-adrenergic receptor signaling in the proliferation of hemangioma-derived endothelial cells

Background

Infantile hemangioma (IH) is a benign vascular neoplasm that arises from the abnormal proliferation of endothelial cells and enhanced angiogenesis. Recently, propranolol has been found to be effective in the management of IH, suggesting that β-adrenergic receptors (β-ARs) may play an important role in the pathogenesis of IH.

Results

In the present study, we investigated the β-adrenergic signaling that is associated with hemangioma-derived endothelial cell (HemEC) proliferation. The results showed that both β₁- and β₂-ARs were expressed in HemECs. Stimulation of the β-ARs by isoprenaline induced cell proliferation and elevation of second messenger cAMP levels. The proliferation-promoting action of isoprenaline was abolished by a β₁-selective antagonist and was more effectively abolished by a β₂-selective antagonist; the mechanism for the action of the antagonists was a G₀/G₁ phase cell cycle arrest which was associated with decreased cyclin D1, CDK-4, CDK-6 and phospho-Rb expression. Pre-treatment of the cells with VEGFR-2 or ERK inhibitors also prevented the isoprenaline-mediated proliferation of cells. In agreement with the involvement of β-ARs and VEGFR-2 in the HemEC response, β-AR antagonists and the VEGFR-2 inhibitor significantly attenuated isoprenaline-induced ERK phosphorylation. Moreover, treating the cells with isoprenaline markedly increased VEGF-A expression and VEGFR-2 activity in a β₂-AR-dependent manner.

Conclusions

We have demonstrated that the activation of the β-ARs in the ERK pathway may be important mechanisms in promoting HemEC growth. Furthermore, stimulation of the β-AR may transactivate VEGFR-2 signaling and further increase HemEC proliferation.