Cytoplasmic inclusions of Htt exon1 containing an expanded polyglutamine tract suppress execution of apoptosis in sympathetic neurons.
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.
King
Matthew A
MA
Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, UK.
Goemans
Christoph G
CG
Hafiz
Farida
F
Prehn
Jochen H M
JH
Wyttenbach
Andreas
A
Tolkovsky
Aviva M
AM
D000704:Analysis of Variance; D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D016572:Cyclosporine / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005091:Exons / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism* / Q000473:pathology; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism; D020932:Nerve Growth Factor / Q000494:pharmacology; D009474:Neurons / Q000166:cytology / Q000502:physiology*; D010455:Peptides / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D020917:Receptor, trkA / Q000378:metabolism; D050486:Serotonin Plasma Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D017783:Superior Cervical Ganglion / Q000166:cytology*; D013997:Time Factors; D014162:Transfection; D051028:bcl-2-Associated X Protein / Q000378:metabolism
D004791:Enzyme Inhibitors; D018840:HSP70 Heat-Shock Proteins; D010455:Peptides; C493230:SLC6A4 protein, human; D050486:Serotonin Plasma Membrane Transport Proteins; D051028:bcl-2-Associated X Protein; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C097188:polyglutamine; D016572:Cyclosporine; D020932:Nerve Growth Factor; D020917:Receptor, trkA