Generation of neuronal intranuclear inclusions by polyglutamine-GFP: Analysis of inclusion clearance and toxicity as a function of polyglutamine length

Recent evidence suggests that, in huntingtin and many other proteins, polyg lutamine repeats are a toxic stimulus in neurodegenerative diseases. To inv estigate 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-G FP, aggregated within the nuclei of transfected cells in a length- and time -dependent manner. Although Q35-GFP expression led to the development of se veral 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-as partyl-(OMe)-fluoromethylketone (BAF) only served to delay, not prevent, to xicity. In addition, aggregation and toxicity were not affected by other mo dulators of neuronal cell death such as genetic deletion of the proapoptoti c bcl-2 family member bax or addition of the protein synthesis inhibitor cy cloheximide. Lastly, nuclear condensation did not occur as part of the toxi city. These data suggest that polyglutamine-GFP expression is toxic to prim ary neurons but that the death is distinct from classical apoptosis.