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The Journal of Neuroscience, December 31, 2008, 28(53):14401-14415; doi:10.1523/JNEUROSCI.4751-08.2008
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Neurobiology of Disease
Cytoplasmic Inclusions of Htt Exon1 Containing an Expanded Polyglutamine Tract Suppress Execution of Apoptosis in Sympathetic Neurons
Matthew A. King,1,3 Christoph G. Goemans,1 Farida Hafiz,1 Jochen H. M. Prehn,3 Andreas Wyttenbach,1,2 andAviva M. Tolkovsky1 1Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, United Kingdom, 2Southampton Neuroscience Group, University of Southampton, Bassett Crescent East, Southampton SO16 7PX, United Kingdom, and 3Royal College of Surgeons in Ireland, Dublin 2, Ireland
Correspondence should be addressed to Aviva M. Tolkovsky at her present address: Centre for Brain Repair, ED Adrian Building, The Forvie Site, Robinson Way, Cambridge CB2 0PY, UK. Email: amt{at}mole.bio.cam.ac.uk
Proteins containing extended polyglutamine repeats cause at least nine neurodegenerative disorders, but the mechanisms of disease-related neuronal death remain uncertain. We show that sympathetic neurons containing cytoplasmic inclusions formed by 97 glutamines expressed within human huntingtin exon1–enhanced green fluorescent protein (Q97) undergo a protracted form of nonapoptotic death that is insensitive to Bax deletion or caspase inhibition but is characterized by mitochondrial dysfunction. By treating the neurons with combined cytosine arabinoside and NGF withdrawal, we demonstrate that Q97 confers a powerful resistance to apoptosis at multiple levels: despite normal proapoptotic signaling (elevation of P-ser15–p53 and BimEL), there is no increase of Puma mRNA or Bax activation, both necessary for apoptosis. Even restoration of Bax translocation with overexpressed Puma does not activate apoptosis. We demonstrate that this robust inhibition of apoptosis is caused by Q97-mediated accumulation of Hsp70, which occurs through inhibition of proteasomal activity. Thus, apoptosis is reinstated by short hairpin RNA-mediated knockdown of Hsp70. These findings explain the rarity of apoptotic death in Q97-expressing neurons. Given the proteasomal blockade, we test whether enhancing lysosomal-mediated degradation with rapamycin reduces Q97 accumulation. Rapamycin reduces the amount of nonpathological Q25 by 70% over 3 d, but Q97 accumulation is unaffected. Interestingly, Q47 inclusions form more slowly as a result of constitutive lysosomal degradation, but faster-forming Q97 inclusions escape lysosomal control. Thus, cytoplasmic Q97 inclusions are refractory to clearance by proteasomal and lysosomal systems, leading to a toxicity that dominates over neuroprotective Hsp70. Our findings may explain the rarity of apoptosis but the inevitable cell death associated with polyQ inclusion diseases.
Key words: Bim; cell death; Hsp70; lysosome; proteasome; Puma
Received Oct. 2, 2008; revised Nov. 2, 2008; accepted Nov. 18, 2008.
Correspondence should be addressed to Aviva M. Tolkovsky at her present address: Centre for Brain Repair, ED Adrian Building, The Forvie Site, Robinson Way, Cambridge CB2 0PY, UK. Email: amt{at}mole.bio.cam.ac.uk
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