1,720,979 research outputs found
Inhibition of PI3k Class III-Dependent Autophagy Prevents Apoptosis and Necrosis by Oxidative Stress in Dopaminergic Neuroblastoma Cells
No full textHydrogen peroxide (H(2)O(2)) is an extremely reactive oxidoradical that is normally produced as a by-product of the mitochondrial activity and also under several metabolic stress conditions. Autophagy, a lysosomal degradation pathway, is triggered by oxidative stress as a defensive response. How autophagy and death pathways are coordinated in cells subjected to oxidative stress is still poorly understood. In human neuroblastoma SH-SY5Y cells, 200microM H(2)O(2) rapidly induced the formation of LC3-positive autophagic vacuoles and of beclin1-Vps34 double-positive macroaggregates. Vacuolar LC3 and beclin1 aggregates did not form when oxidative stress was performed in cells pretreated with 3-methyladenine (3MA), an inhibitor of Vps34, or infected with a recombinant adenovirus expressing a dominant-negative mutant of Vps34. H(2)O(2) provoked the permeabilization of lysosomes (at 30 min) and of mitochondria, the concomitant oligomerization of bax, and eventually (at 2 h), cell death in about 50% of the cell culture. Inactivation of Vps34-dependent autophagy in oxidative-stressed cells abrogated lysosome leakage, bax activation, and caspase-dependent apoptosis and conferred protection for as long as 16 h. Inhibition of caspase activity (by ZVAD-fmk) did not trigger an alternative cell death pathway but rather afforded complete protection from oxidative toxicity, despite the ongoing generation of oxidoradicals and the cellular accumulation of autophagic vacuoles and of leaking lysosomes. On long-term (16 h) exposure to H(2)O(2), signs of necrotic cell death became apparent in LC3-positive cells, which could be prevented by ZVAD-fmk. The present data highlight the pivotal role of autophagy in H(2)O(2)-induced cell death in dopaminergic neuroblastoma cells
The Role of Cathepsin D in the Pathogenesis of Human Neurodegenerative Disorders
Full text linkIn familial neurodegenerative disorders, protein aggregates form continuously because of genetic mutations that drive the synthesis of truncated or unfolded proteins. The oxidative stress imposed by neurotransmitters and environmental neurotoxins constitutes an additional threat to the folding of the proteins and the integrity of organelle membranes in neurons. Failure in degrading such altered materials compromises the function of neurons and eventually leads to neurodegeneration. The lysosomal proteolytic enzyme Cathepsin D is the only aspartic-type protease ubiquitously expressed in all the cells of the human body, and it is expressed at high level in the brain. In general, cathepsin D mediated proteolysis is essential to neuronal cell homeostasis through the degradation of unfolded or oxidized protein aggregates delivered to lysosomes via autophagy or endocytosis. More specifically, many altered neuronal proteins that hallmark neurodegenerative diseases (e.g., the amyloid precursor, α-synuclein, and huntingtin) are physiologic substrates of cathepsin D and would abnormally accumulate if not efficiently degraded by this enzyme. Furthermore, experimental evidence indicates that cathepsin D activity is linked to the metabolism of cholesterol and of glycosaminoglycans, which accounts for its involvement in neuronal plasticity. This review focuses on the unique role of cathepsin D mediated proteolysis in the pathogenesis of human neurodegenerative diseases
Suppression of autophagy precipitates neuronal cell death following low doses of methamphetamine
No full textMethamphetamine abuse is toxic to dopaminergic neurons, causing nigrostriatal denervation and striatal dopamine loss. Following methamphetamine exposure, the number of nigral cell bodies is generally preserved, but their cytoplasm features autophagic-like vacuolization and cytoplasmic accumulation of alpha-synuclein-, ubiquitin- and parkin-positive inclusion-like bodies. Whether autophagy is epiphenomenal or it plays a role in the mechanism of methamphetamine toxicity and, in the latter case, whether its role consists of counteracting or promoting the neurotoxic effect remains obscure. We investigated the signaling pathway and the significance (protective vs. toxic) of autophagy activation and the convergence of the autophagic and the ubiquitin-proteasome pathways at the level of the same intracellular bodies in a simple cell model of methamphetamine toxicity. We show that autophagy is rapidly up-regulated in response to methamphetamine. Confocal fluorescence microscopy and immuno-electron microscopy studies demonstrated the presence of alpha-synuclein aggregates in autophagy-lysosomal structures in cells exposed to methamphetamine, a condition compatible with cell survival. Inhibition of autophagy either by pharmacologic or genetic manipulation of the class III Phosphatidylinositol-3 kinase-mediated signaling prevented the removal of alpha-synuclein aggregates and precipitated a bax-mediated mitochondrial apoptosis pathway
Amino acid response by Halofuginone in Cancer cells triggers autophagy through proteasome degradation of mTOR
Full text linkpeer reviewedIn the event of amino acid starvation, the cell activates two main protective pathways: Amino Acid starvation Response (AAR), to inhibit global translation, and autophagy, to recover the essential substrates from degradation of redundant self-components. Whether and how AAR and autophagy (ATG) are cross-regulated and at which point the two regulatory pathways intersect remain unknown. Here, we provide experimental evidence that the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) specifically located at the lysosome level links the AAR with the autophagy pathway.
METHODS:
As an inducer of the AAR, we used halofuginone (HF), an alkaloid that binds to the prolyl-tRNA synthetase thus mimicking the unavailability of proline (PRO). Induction of AAR was determined assessing the phosphorylation of the eukaryotic translation initiation factor (eIF) 2α. Autophagy was monitored by assessing the processing and accumulation of microtubule-associated protein 1 light chain 3 isoform B (LC3B) and sequestosome-1 (p62/SQSTM1) levels. The activity of mTORC1 was monitored through assessment of the phosphorylation of mTOR, (rp)S6 and 4E-BP1. Global protein synthesis was determined by puromycin incorporation assay. mTORC1 presence on the membrane of the lysosomes was monitored by cell fractionation and mTOR expression was determined by immunoblotting.
RESULTS:
In three different types of human cancer cells (thyroid cancer WRO cells, ovarian cancer OAW-42 cells, and breast cancer MCF-7 cells), HF induced both the AAR and the autophagy pathways time-dependently. In WRO cells, which showed the strongest induction of autophagy and of AAR, global protein synthesis was little if any affected. Consistently, 4E-BP1 and (rp)S6 were phosphorylated. Concomitantly, mTOR expression and activation declined along with its detachment from the lysosomes and its degradation by the proteasome, and with the nuclear translocation of transcription factor EB (TFEB), a transcription factor of many ATG genes. The extra supplementation of proline rescued all these effects.
CONCLUSIONS:
We demonstrate that the AAR and autophagy are mechanistically linked at the level of mTORC1, and that the lysosome is the central hub of the cross-talk between these two metabolic stress responses
Resveratrol induces cell death in colorectal cancer cells by a novel pathway involving lysosomal cathepsin D
No full textIn human colorectal cancer cells, the polyphenol resveratrol (RV) activated the caspase-dependent intrinsic pathway of apoptosis. This effect was not mediated via estrogen receptors. Pepstatin A, an inhibitor of lysosomal cathepsin D (CD), not (2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester, an inhibitor of cathepsins B and L, prevented RV cytotoxicity. Similar protection was attained by small interference RNA-mediated knockdown of CD protein expression. RV promoted the accumulation of mature CD, induced lysosome leakage and increased cytosolic immunoreactivity of CD. Inhibition of CD or its post-transcriptional down-regulation precluded Bax oligomerization, permeabilization of mitochondrial membrane, cytosolic translocation of cytochrome c, caspase 3 activation and terminal deoxinucleotidyl transferase-mediated dUTP-biotin nick end labeling positivity occurring in RV-treated cells. The present study identifies the lysosome as a novel target of RV activity and demonstrates a hierarchy of the proteolytic pathways involved in its cytotoxic mechanism in which the lysosomal CD acts upstream of the cytosolic caspase activation. Our data indicate that metabolic, pharmacologic or genetic conditions affecting CD expression and/or activity could reflect on the sensitivity of cancer cells to RV
Resveratrol-induced apoptosis depends on the lipid kinase activity of Vps34 and on the formation of autophagolysosomes
No full textFolding, activity and targeting of mutated human cathepsin D that cannot be processed into the double-chain form
No full textThe precursor of human cathepsin D (CD) is converted into the single-chain and the double-chain active polypeptides by subsequent proteolysis reactions taking place in the endosomal-lysosomal compartment and involving specific aminoacid sequences. We have mutagenized the region of aminoacids (comprising the beta-hairpin loop) involved in the latter proteolytic maturation step and generated a mutant CD that cannot be converted into the mature double-chain form. This mutant CD expressed in rodent cells reaches the lysosome and is stable as single-chain polypeptide, bears high-mannose type sugars, binds to pepstatin A and is enzymatically active, indicating that it is correctly folded. The present work provides new insights on the aminoacid region involved in the terminal processing of human CD and on the function of the processing beta-hairpin loop
Cathepsin D-Bax death pathway in oxidative stressed neuroblastoma cells
No full textHydrogen peroxide, the major oxidoradical species in the central nervous system, has been involved in neuronal cell death and associated neurodegenerative diseases. In this study, we have investigated the involvement of the lysosomal pathway in the cytotoxic mechanism of hydrogen peroxide in human neuroblastoma cells. Alteration of lysosomal and mitochondrial membrane integrity was shown to be an early event in the lethal cascade triggered by oxidative stress. Desferrioxamine (DFO), an iron chelator that abolishes the formation of reactive oxygen species within lysosomes, prevented lysosome leakage, mitochondrial permeabilization and caspase-dependent apoptosis in hydrogen peroxide-treated cells. Inhibition of cathepsin D, not of cathepsin B, as well as small-interference RNA-mediated silencing of the cathepsin D gene prevented hydrogen peroxide-induced injury of mitochondria, caspase activation, and TUNEL-positive cell death. Cathepsin D activity was shown indispensable for translocation of Bax onto mitochondrial membrane associated with oxidative stress. DFO abolished both the cytosolic relocation of Cathepsin D and the mitochondrial relocation of Bax in hydrogen peroxide-treated cells. siRNA-mediated down-regulation of Bax expression protected the cells from oxidoradical injury. The present study identifies the lysosome as the primary target and the axis cathepsin D-Bax as the effective pathway of hydrogen peroxide lethal activity in neuroblastoma cells
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