Free radical-dependent nuclear localization of Bcl-2 in the central nervous system of aged rats is not associated with Bcl-2-mediated protection from apoptosis.
We have previously reported that Bcl-2 is up-regulated in the CNS of aged F344 rats as a consequence of oxidative stress. In addition to increased levels of expression, we now report that there is a subcellular redistribution of Bcl-2 in the CNS of aged F344 rats. Using western blotting, we found Bcl-2 predominantly located in the cytosol of young rats. However, in aged rats Bcl-2 was found primarily in the nucleus. This distribution, in the hippocampus and cerebellum, was reversed by treatment with the nitrone spin trap N-tert-butyl-alpha-phenylnitrone (PBN). Paradoxically, PBN treatment in young rats had the opposite effect, changing Bcl-2 from predominantly cytosolic to nuclear. We also detected an increase in Bax in aged hippocampal samples (both nuclear and cytosolic), which was reversed by treatment with PBN. The distribution of Bcl-2 and Bax in the cytosol of aged rats dramatically decreased the Bcl-2/Bax ratio, a probable indicator of neuronal vulnerability, which was restored upon treatment with PBN. In order to assess the effect of nuclear association of Bcl-2 we used PC12 cells stably transfected with a Bcl-2 construct to which we added the nuclear localization sequence of the SV40 large T antigen to the N-terminus which resulted in nuclear targeting of Bcl-2. Measurement of cell death using lactate dehydrogenase assays showed that, contrary to wild-type Bcl-2, Bcl-2 localized to the nucleus was not effective in protecting cells from treatment with 250 microm H2O2. These results suggest that nuclear localization of Bcl-2 observed in the aged CNS may not reflect a protective mechanism against oxidative stress, a major component of age-associated CNS impairments.
Kaufmann
Joel A
JA
Department of Anatomy and Neurosciences, University of Texas Medical Branch at Galveston, Galveston, Texas 77555-1043, USA.
Perez
Mike
M
Zhang
WenRu
W
Bickford
Paula C
PC
Holmes
Douglas B
DB
Taglialatela
Giulio
G
D000367:Age Factors; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis / Q000502:physiology*; D002467:Cell Nucleus / Q000378:metabolism*; D002490:Central Nervous System / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D002531:Cerebellum / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D003497:Cyclic N-Oxides; D003600:Cytosol / Q000378:metabolism; D016166:Free Radical Scavengers / Q000494:pharmacology; D005609:Free Radicals / Q000378:metabolism*; D006624:Hippocampus / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D018696:Neuroprotective Agents / Q000494:pharmacology; D009589:Nitrogen Oxides / Q000494:pharmacology; D016877:Oxidants / Q000494:pharmacology; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology; D016716:PC12 Cells; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011916:Rats, Inbred F344; D051028:bcl-2-Associated X Protein
D000975:Antioxidants; C494828:Bax protein, rat; D003497:Cyclic N-Oxides; D016166:Free Radical Scavengers; D005609:Free Radicals; D018696:Neuroprotective Agents; D009589:Nitrogen Oxides; D016877:Oxidants; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D051028:bcl-2-Associated X Protein; C029217:phenyl-N-tert-butylnitrone