Abstract
Apoptotic and autophagic cell death have been implicated, on the basis of morphological and biochemical criteria, in neuronal loss occurring in neurodegenerative diseases and it has been shown that they may overlap. We have studied the relationship between apoptosis and autophagic cell death in cerebellar granule cells (CGCs) undergoing apoptosis following serum and potassium deprivation. We found that apoptosis is accompanied by an early and marked proliferation of autophagosomal-lysosomal compartments as detected by electron microscopy and immunofluorescence analysis. Autophagy is blocked by hrIGF-1 and forskolin, two well-known inhibitors of CGC apoptosis, as well as by adenovirus-mediated overexpression of Bcl-2. 3-Methyladenine (3-MA) an inhibitor of autophagy, not only arrests this event but it also blocks apoptosis. The neuroprotective effect of 3-MA is accompanied by block of cytochrome c (cyt c) release in the cytosol and by inhibition of caspase-3 activation which, in turn, appears to be mediated by cathepsin B, as CA074-Me, a selective inhibitor of this enzyme, fully blocks the processing of pro-caspase-3. Immunofluorescence analysis demonstrated that cathepsin B, normally confined inside the lysosomal-endosomal compartment, is released during apoptosis into the cytosol where this enzyme may act as an execution protease. Collectively, these observations indicate that autophagy precedes and is causally connected with the subsequent onset of programmed death.
Publication types
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Comparative Study
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Research Support, Non-U.S. Gov't
MeSH terms
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Adenine / analogs & derivatives*
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Adenine / pharmacology
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Amino Acid Chloromethyl Ketones / pharmacology
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Animals
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Animals, Newborn
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Antigens, CD / metabolism
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Apoptosis / drug effects*
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Autophagy / physiology*
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Caspases / metabolism
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Cathepsins / metabolism
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Cell Size / drug effects
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Cell Survival / drug effects
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Cells, Cultured
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Cerebellum / cytology*
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Colforsin / pharmacology
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Coumarins / pharmacology
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Cysteine Proteinase Inhibitors / pharmacology
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DNA-Binding Proteins / pharmacology
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Dose-Response Relationship, Drug
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Drug Interactions
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Erythroid-Specific DNA-Binding Factors
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Extracellular Signal-Regulated MAP Kinases / metabolism
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Fluorescent Antibody Technique / methods
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Gene Expression Regulation
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Glycoside Hydrolases / metabolism
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Green Fluorescent Proteins / metabolism
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Lysosomal Membrane Proteins
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Lysosomes / physiology*
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Lysosomes / ultrastructure
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Microscopy, Electron / methods
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Microtubule Proteins / metabolism
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Neurons / drug effects*
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Neurons / ultrastructure
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Oligopeptides / pharmacology
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Potassium / pharmacology*
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Proto-Oncogene Proteins c-bcl-2 / metabolism
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RNA, Messenger / biosynthesis
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Rats
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Rats, Wistar
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Reverse Transcriptase Polymerase Chain Reaction / methods
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Teprotide / pharmacology
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Time Factors
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Transcription Factors / pharmacology
Substances
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Ac-aspartyl-glutamyl-valyl-aspartyl-aminomethylcoumarin
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Amino Acid Chloromethyl Ketones
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Antigens, CD
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Coumarins
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Cysteine Proteinase Inhibitors
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DNA-Binding Proteins
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Erythroid-Specific DNA-Binding Factors
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Lysosomal Membrane Proteins
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Microtubule Proteins
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Oligopeptides
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Proto-Oncogene Proteins c-bcl-2
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RNA, Messenger
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Transcription Factors
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acetyl-tyrosyl-valyl-alanyl-aspartyl-7-amino-4-methylcoumarinamide
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benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone
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Green Fluorescent Proteins
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Colforsin
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3-methyladenine
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Teprotide
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Extracellular Signal-Regulated MAP Kinases
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Glycoside Hydrolases
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beta-galactanase
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Cathepsins
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Caspases
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Adenine
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Potassium