Abstract
Proteasomal stress and the accumulation of polyubiquitinated proteins are key features of numerous neurodegenerative disorders. Previously we demonstrated that stabilization of p53 and activation of its target gene, puma (p53-upregulated mediator of apoptosis), mediated proteasome inhibitor-induced apoptosis in cancer cells. Here we demonstrated that Puma also contributed to proteasome inhibitor-induced apoptosis in mouse neocortical neurons. Although protection afforded by puma gene deletion was incomplete, we found little evidence indicating contributions from other proapoptotic BH3-only proteins. Attenuation of bax expression did not further reduce Puma-independent apoptosis, suggesting that pathways other than the mitochondrial apoptosis pathway were activated. Real-time imaging experiments in wild-type and puma-deficient neurons using a fluorescence resonance energy transfer (FRET)-based caspase sensor confirmed the involvement of a second cell death pathway characterized by caspase activation prior to mitochondrial permeabilization and, more prominently, a third, caspase-independent and Puma-independent pathway characterized by rapid cell shrinkage and nuclear condensation. This pathway involved lysosomal permeabilization in the absence of autophagy activation and was sensitive to cathepsin but not autophagy inhibition. Our data demonstrate that proteasomal stress activates distinct cell death pathways in neurons, leading to both caspase-dependent and caspase-independent apoptosis, and demonstrate independent roles for Puma and lysosomal permeabilization in this model.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Apoptosis / drug effects
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Apoptosis / genetics
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Apoptosis / physiology
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Apoptosis Regulatory Proteins / deficiency
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Apoptosis Regulatory Proteins / genetics
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Apoptosis Regulatory Proteins / metabolism*
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Autophagy
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BH3 Interacting Domain Death Agonist Protein / deficiency
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BH3 Interacting Domain Death Agonist Protein / genetics
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Base Sequence
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Bcl-2-Like Protein 11
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Caspase 3 / metabolism
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Cathepsins / metabolism
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Cytochromes c / metabolism
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DNA Primers / genetics
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Fluorescence Resonance Energy Transfer
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Gene Expression
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Lysosomes / drug effects
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Lysosomes / metabolism
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Membrane Potential, Mitochondrial
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Membrane Proteins / deficiency
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Membrane Proteins / genetics
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Mice
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Mice, Inbred C57BL
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Mice, Knockout
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Models, Neurological
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Nerve Degeneration / genetics
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Nerve Degeneration / metabolism
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Nerve Degeneration / pathology
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Neurons / cytology*
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Neurons / drug effects
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Neurons / metabolism*
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Protease Inhibitors / pharmacology
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Proteasome Inhibitors*
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Proto-Oncogene Proteins / deficiency
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Proto-Oncogene Proteins / genetics
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Proto-Oncogene Proteins c-bcl-2 / genetics
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RNA, Small Interfering / genetics
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Stress, Physiological
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Tumor Suppressor Proteins / deficiency
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Tumor Suppressor Proteins / genetics
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Tumor Suppressor Proteins / metabolism*
Substances
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Apoptosis Regulatory Proteins
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BH3 Interacting Domain Death Agonist Protein
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Bcl-2-Like Protein 11
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Bcl2l11 protein, mouse
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Bid protein, mouse
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DNA Primers
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Membrane Proteins
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PUMA protein, mouse
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Pmaip1 protein, mouse
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Protease Inhibitors
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Proteasome Inhibitors
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Proto-Oncogene Proteins
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Proto-Oncogene Proteins c-bcl-2
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RNA, Small Interfering
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Tumor Suppressor Proteins
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Cytochromes c
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Cathepsins
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Casp3 protein, mouse
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Caspase 3