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The Journal of Neuroscience, January 15, 1999, 19(2):705-715
Generation of Neuronal Intranuclear Inclusions by Polyglutamine-GFP: Analysis of Inclusion Clearance and Toxicity as a Function of Polyglutamine Length
Krista L. Moulder1, Osamu Onodera2, 4, James R. Burke2, 4, Warren J. Strittmatter2, 3, 4, and Eugene M. Johnson Jr1 1 Departments of Neurology and Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110, and Departments of 2 Medicine (Neurology), 3 Neurobiology, and 4 Deane Laboratory, Duke University Medical Center, Durham, North Carolina 27710
Recent evidence suggests that, in huntingtin and many other proteins, polyglutamine repeats are a toxic stimulus in neurodegenerative diseases. To investigate the mechanism by which these repeats may be toxic, we transfected primary rat cerebellar granule neurons with polyglutamine-green fluorescent protein (GFP) fusion constructs containing 19 (Q19-GFP), 35 (Q35-GFP), 56 (Q56-GFP), or 80 (Q80-GFP) glutamine residues. All constructs, except Q19-GFP, aggregated within the nuclei of transfected cells in a length- and time-dependent manner. Although Q35-GFP expression led to the development of several small aggregates per cell, these aggregates were cleared or degraded, and the cells remained viable. In contrast, Q80-GFP expression resulted in one or two large aggregates and induced cell death. Caspase activation was observed after Q80-GFP aggregation, but inhibition of caspases with Boc-aspartyl(OMe)-fluoromethylketone (BAF) only served to delay, not prevent, toxicity. In addition, aggregation and toxicity were not affected by other modulators of neuronal cell death such as genetic deletion of the proapoptotic bcl-2 family member bax or addition of the protein synthesis inhibitor cycloheximide. Lastly, nuclear condensation did not occur as part of the toxicity. These data suggest that polyglutamine-GFP expression is toxic to primary neurons but that the death is distinct from classical apoptosis.
Key words: aggregation; cerebellar granule neurons; apoptosis; caspase; ubiquitin; cAMP
Copyright © 1999 Society for Neuroscience 0270-6474/99/192705-11$05.00/0
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