Figure 1.

Expanded polyQ triggers ER stress through proteasomal dysfunction. (A) Activation of endogenous IRE1 and PERK by Q79. PC12-Q79 and PC12-Q14 cells (5 × 10⁶ each) were lysed at each time point after treatment with 2 μM thapsigargin (Tg) or removal of Tet, and analyzed by immunoprecipitation (IP)-immunoblotting (WB) with anti-IRE1α and anti-PERK antisera. (P-IRE1) Autophosphorylated IRE1; (P-PERK) autophosphorylated PERK. (B) Induction of CHOP by Q79. PC12-Q79, and PC12-Q14 cells (1 × 10⁶ each) were lysed with RIPA buffer at each time point after treatment with 2 μM thapsigargin (Tg) or removal of Tet, and analyzed by WB with anti-CHOP antiserum. (C) Activation of IRE1 and PERK by Q79 and proteasome inhibitor in primary neurons. Primary neurons (1 × 10⁶) derived from day 14.5 mouse embryos were infected with the indicated m.o.i. of Ad-β-gal, Ad-Q35, or Ad-Q79 for 48 h, or treated with 0.1 μM MG132 for 48 h or with 2 μM thapsigargin for 1 h. Cell lysates were analyzed by IP–WB with anti-IRE1α and anti-PERK antisera. (D) Induction of BiP and CHOP mRNA by Q79 and proteasome inhibitor in primary neurons. Results of RT–PCR following infection with the indicated m.o.i. of Ad-Q35 and Ad-Q79 for 48 h or treatments with MG132 (0.1 μM for 48 h) and thapsigargin (2 μM for 1 h). Expression of G3PDH was examined as a quantity control (bottom). (E) Inhibition of proteasomal activity by Q79. Primary neurons were infected with the indicated m.o.i. of Ad-β-gal or Ad-Q79 for the indicated time periods, or treated with the indicated concentration of MG132 for 1 h. The proteasomal activity was measured as described in Materials and Methods and is shown as fold increase relative to that of Ad-β-gal-infected (for Ad-Q79) or untreated (for MG132) cells. Data are means (±SE) of three independent experiments derived from independent embryos. (*) P < 0.05 relative to control; significance calculated by Student's t-test.