4-hydroxy-2-nonenal and 3-methyladenine

The production of reactive species contributes to the age-dependent accumulation of dysfunctional mitochondria and protein aggregates, all of which are associated with neurodegeneration. A putative mediator of these effects is the lipid peroxidation product 4-hydroxynonenal (4-HNE), which has been shown to inhibit mitochondrial function, and accumulate in the postmortem brains of patients with neurodegenerative diseases. This deterioration in mitochondrial quality could be due to direct effects on mitochondrial proteins, or through perturbation of the macroautophagy/autophagy pathway, which plays an essential role in removing damaged mitochondria. Here, we use a click chemistry-based approach to demonstrate that alkyne-4-HNE can adduct to specific mitochondrial and autophagy-related proteins. Furthermore, we found that at lower concentrations (5-10 μM), 4-HNE activates autophagy, whereas at higher concentrations (15 μM), autophagic flux is inhibited, correlating with the modification of key autophagy proteins at higher concentrations of alkyne-4-HNE. Increasing concentrations of 4-HNE also cause mitochondrial dysfunction by targeting complex V (the ATP synthase) in the electron transport chain, and induce significant changes in mitochondrial fission and fusion protein levels, which results in alterations to mitochondrial network length. Finally, inhibition of autophagy initiation using 3-methyladenine (3MA) also results in a significant decrease in mitochondrial function and network length. These data show that both the mitochondria and autophagy are critical targets of 4-HNE, and that the proteins targeted by 4-HNE may change based on its concentration, persistently driving cellular dysfunction.

Topics: Adenine; Aldehydes; Animals; Autophagy; Cells, Cultured; Energy Metabolism; Mitochondria; Mitochondrial Dynamics; Mitochondrial Proteins; Neurons; Oxidative Stress; Primary Cell Culture; Rats

Several pathologies are associated with elevated levels of serum ferritin, for which growth inhibitory properties have been reported; however, the underlying mechanisms are still poorly defined. Previously we have described cytotoxic properties of isoferritins released from primary hepatocytes in vitro, which induce apoptosis in an iron and oxidative stress-dependent mode. Here we show that this ferritin species stimulates endosome clustering and giant endosome formation in primary hepatocytes accompanied by enhanced lysosomal membrane permeability (LMP). In parallel, protein modification by lipid peroxidation-derived 4-hydroxynonenal (HNE) is strongly promoted by ferritin, the HNE-modified proteins (HNE-P) showing remarkable aggregation. Emphasizing the prooxidant context, GSH is rapidly depleted and the GSH/GSSG ratio is substantially declining in ferritin-treated cells. Furthermore, ferritin triggers a transient upregulation of macroautophagy which is abolished by iron chelation and apparently supports HNE-P clearance. Macroautophagy inhibition by 3-methyladenine strongly amplifies ferritin cytotoxicity in a time- and concentration-dependent mode, suggesting an important role of macroautophagy on cellular responses to ferritin endocytosis. Moreover, pointing at an involvement of lysosomal proteolysis, ferritin cytotoxicity and lysosome fragility are aggravated by the protease inhibitor leupeptin. In contrast, EGF which suppresses ferritin-induced cell death attenuates ferritin-mediated LMP. In conclusion, we propose that HNE-P accumulation, lysosome dysfunction, and macroautophagy stimulated by ferritin endocytosis provoke lysosomal "metastability" in primary hepatocytes which permits cell survival as long as in- and extrinsic determinants (e.g., antioxidant availability, damage repair, EGF signaling) keep the degree of lysosomal destabilization below cell death-inducing thresholds.

Topics: Adenine; Aldehydes; Animals; Apoptosis; Autophagy; Culture Media, Conditioned; Endocytosis; Epidermal Growth Factor; Female; Ferritins; Glutathione; Glutathione Disulfide; Hepatocytes; Intracellular Membranes; Iron Chelating Agents; Leupeptins; Liver; Lysosomes; Molecular Imaging; Permeability; Primary Cell Culture; Protease Inhibitors; Protein Aggregates; Rats; Rats, Inbred F344

Vascular injury and chronic arterial diseases result in exposure of VSMCs (vascular smooth muscle cells) to increased concentrations of growth factors. The mechanisms by which growth factors trigger VSMC phenotype transitions remain unclear. Because cellular reprogramming initiated by growth factors requires not only the induction of genes involved in cell proliferation, but also the removal of contractile proteins, we hypothesized that autophagy is an essential modulator of VSMC phenotype. Treatment of VSMCs with PDGF (platelet-derived growth factor)-BB resulted in decreased expression of the contractile phenotype markers calponin and α-smooth muscle actin and up-regulation of the synthetic phenotype markers osteopontin and vimentin. Autophagy, as assessed by LC3 (microtubule-associated protein light chain 3 α; also known as MAP1LC3A)-II abundance, LC3 puncta formation and electron microscopy, was activated by PDGF exposure. Inhibition of autophagy with 3-methyladenine, spautin-1 or bafilomycin stabilized the contractile phenotype. In particular, spautin-1 stabilized α-smooth muscle cell actin and calponin in PDGF-treated cells and prevented actin filament disorganization, diminished production of extracellular matrix, and abrogated VSMC hyperproliferation and migration. Treatment of cells with PDGF prevented protein damage and cell death caused by exposure to the lipid peroxidation product 4-hydroxynonenal. The results of the present study demonstrate a distinct form of autophagy induced by PDGF that is essential for attaining the synthetic phenotype and for survival under the conditions of high oxidative stress found to occur in vascular lesions.

Topics: Actins; Adenine; Aldehydes; Animals; Aorta; Autophagy; Biomarkers; Calcium-Binding Proteins; Calponins; Gene Expression Regulation; Macrolides; Male; Microfilament Proteins; Microtubule-Associated Proteins; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Osteopontin; Oxidative Stress; Phenotype; Platelet-Derived Growth Factor; Primary Cell Culture; Rats; Rats, Sprague-Dawley; Signal Transduction; Vimentin

Several lines of evidence suggest that progressive dysfunction of the retinal pigment epithelium (RPE) is central to the pathogenesis of age-related macular degeneration (AMD). We previously demonstrated that protein modifications with lipid peroxidation products, such as 4-hydroxynonenal (HNE) and malondialdehyde (MDA), induce lysosomal dysfunction in RPE cells in vitro. Here, we investigated whether phagocytosis of modified photoreceptor outer segments (POS) affects lipofuscinogenesis and autophagy, two interrelated processes directly connected to lysosomal function. Incubation of human RPE cells with HNE- and MDA-modified POS resulted in pronounced intracellular accumulation of granular material with lipofuscin-like autofluorescence. After daily treatment with modified POS for 7 days, cellular autofluorescence increased 8.2-fold as quantified by flow cytometry. In the presence of the lysosomal inhibitor ammonium chloride, unmodified POS likewise induced an 8.0-fold increase in autofluorescence. Spectral profiles of cellular autofluorescence after incubation with modified POS were unchanged compared to incubation with native POS. Autophagy activity, measured as turnover of metabolically radiolabeled endogenous proteins, was reduced by both HNE- and MDA-modified POS by 40%. Autophagy inhibition by 3-methyladenine and lysosomal inhibition by ammonium chloride induced lipofuscinogenesis even in the absence of POS. In summary, our results demonstrate that induction of lysosomal dysfunction by lipid peroxidation-derived protein modifications results in increased lipofuscinogenesis and reduced autophagy activity in RPE cells in vitro. These mechanisms may contribute to RPE cell dysfunction and degeneration in AMD.

Topics: Adenine; Aldehydes; Ammonium Chloride; Animals; Autophagy; Cell Line; Flow Cytometry; Humans; Lipid Peroxidation; Lipofuscin; Lysosomes; Malondialdehyde; Microscopy, Fluorescence; Oxidative Stress; Phagocytosis; Retinal Photoreceptor Cell Outer Segment; Retinal Pigment Epithelium; Swine