TY - JOUR
T1 - Generation of neuronal intranuclear inclusions by polyglutamine-GFP
T2 - Analysis of inclusion clearance and toxicity as a function of polyglutamine length
AU - Moulder, Krista L.
AU - Onodera, Osamu
AU - Burke, James R.
AU - Strittmatter, Warren J.
AU - Johnson, Eugene M.
PY - 1999/1/15
Y1 - 1999/1/15
N2 - 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.
AB - 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.
KW - Aggregation
KW - Apoptosis
KW - Caspase
KW - Cerebellar granule neurons
KW - Ubiquitin
KW - cAMP
UR - http://www.scopus.com/inward/record.url?scp=0033556160&partnerID=8YFLogxK
U2 - 10.1523/jneurosci.19-02-00705.1999
DO - 10.1523/jneurosci.19-02-00705.1999
M3 - Article
C2 - 9880591
AN - SCOPUS:0033556160
VL - 19
SP - 705
EP - 715
JO - Journal of Neuroscience
JF - Journal of Neuroscience
SN - 0270-6474
IS - 2
ER -