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. 2011 Oct 1;124(Pt 19):3332-43.
doi: 10.1242/jcs.087510. Epub 2011 Sep 6.

Changes in BiP availability reveal hypersensitivity to acute endoplasmic reticulum stress in cells expressing mutant huntingtin

Patrick Lajoie  1 , Erik L Snapp
Affiliations

Changes in BiP availability reveal hypersensitivity to acute endoplasmic reticulum stress in cells expressing mutant huntingtin

Patrick Lajoie et al. J Cell Sci. .

Erratum in

  • J Cell Sci. 2012 Feb 1;125(Pt 3):789

Abstract

Huntington's disease (HD) is caused by expanded glutamine repeats within the huntingtin (Htt) protein. Mutant Htt (mHtt) in the cytoplasm has been linked to induction of the luminal endoplasmic reticulum (ER) stress pathway, the unfolded protein response (UPR). How mHtt impacts the susceptibility of the ER lumen to stress remains poorly understood. To investigate molecular differences in the ER in cells expressing mHtt, we used live-cell imaging of a sensitive reporter of the misfolded secretory protein burden, GFP fused to the ER chaperone BiP (also known as GRP78), which decreases in mobility as it binds increasing amounts of misfolded proteins. Striatal neurons expressing full-length mHtt showed no differences in BiP-GFP mobility and no evidence of UPR activation compared with wild-type cells at steady state. However, mHtt-expressing cells were acutely sensitive to misfolded secretory proteins. Treatment with ER stressors, tunicamycin or DTT, rapidly decreased BiP-GFP mobility in mHtt striatal cells and accelerated UPR activation compared with wild-type cells. mHtt-expressing cells exhibited decreased misfolded protein flux as a result of ER associated degradation (ERAD) dysfunction. Furthermore, UPR-adapted mHtt cells succumbed to misfolded protein stresses that could be tolerated by adapted wild-type cells. Thus, mHtt expression impairs misfolded secretory protein turnover, decreases the ER stress threshold, and increases cell vulnerability to insults.

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Figures

Fig. 1.
Fig. 1.
Analysis of the ER stress marker BiP in Httex1–GFP-transfected cells. (A) Immunoblot of BiP levels in N2a cells transiently transfected with Httex1–GFP vectors containing 23, 73 or 145 polyQ repeats for 16 hours. Untransfected cells treated with 5 μg/ml Tm for 8 h or left untreated as a control. Samples were lysed and separated by SDS-PAGE and immunoblotted with anti-BiP or anti-GFP. Equal loading was confirmed by reprobing with anti-β-actin. (B) Representative fluorescence images of N2a cells transiently co-transfected with either empty GFP or Httex1–GFP and ERSE tdTomato ODC vectors. A positive control was included of cells expressing GFP and ERSE TdTomato ODC treated with 5 μg/ml Tm for 16 hours. Scale bars: 20 μm. (C) N2a cells were transiently cotransfected with either empty GFP or Httex1–GFP and ERSE tdTomato ODC vectors. A positive control (as for B) was included. Plots show fluorescence intensities of both GFP, or Httex1–GFP, and ERSE TdTomato ODC for individual cells. *P<0.01; AU, arbitrary units.
Fig. 2.
Fig. 2.
Analysis of ER stress markers BiP and CHOP in STHdhQ7/7 and STHdhQ111/111 cells. (A) Immunoblot of BiP levels in STHdhQ7/7 and STHdhQ111/111 cells. Equal loading was confirmed by reprobing with anti-α-tubulin. (B) Representative fluorescence images of STHdhQ7/7 and STHdhQ111/111 cells untreated or treated with 5 μg/ml Tm for 16 hours and immunofluorescently labeled with anti-BiP and anti-CHOP. Scale bars: 20 μm. (C) STHdhQ7/7 and STHdhQ111/111 cells were transiently co-transfected with empty GFP and ERSE tdTomato ODC vectors. A positive control was included of STHdhQ7/7 cells treated with 5 μg/ml Tm for 16 hours. Plots show fluorescence intensities of both GFP, or Httex1–GFP, and ERSE TdTomato ODC for individual cells. *P<0.01; AU, arbitrary units.
Fig. 3.
Fig. 3.
Differential effect of expression of Httex1 and full-length Htt on the ER misfolded protein burden. (A) Illustration of how BiP availability distinguishes between states of homeostasis and stress. Upon acute ER stress, BiP–GFP binds to misfolded protein resulting in larger molecular complexes. An increase in complex size should result in decreased diffusional mobility. (B) Representative FRAP series of N2a cells transfected with either BiP–GFP or ER–GFP. Scale bars: 20 μm. (C,D) D values of individual N2a cells transiently co-transfected with Httex1-mCherry constructs containing 23, 73 or 145 polyQ repeats and BiP–GFP (C) or ER–GFP (D) for 16 hours, and analyzed by FRAP. Control (Ctl) cells were transfected with BiP–GFP or ER–GFP alone and treated cells received 5 μg/ml Tm for 4 hours. (E,F) D values of individual STHdhQ7/7 and STHdhQ111/111 cells transfected with BiP–GFP or ER–GFP for 16 hours and treated with 5 μg/ml Tm for 4 hours or left untreated, and analyzed by FRAP. Thick horizontal lines in C–F indicate mean D values. *P<0.05 **P<0.001.
Fig. 4.
Fig. 4.
mHtt expression is associated with increased ER stressor-induced cell death in striatal cells expressing full-length Htt. (A) STHdhQ7/7 and STHdhQ111/111 cells untreated (control) or treated with 5 μg/ml Tm for 16 hours. Cells were fixed and labeled with cleaved caspase-3 antibody and phalloidin. Scale bars: 20 μm. (B) The percentage of cells with cleaved caspase-3 staining. *P<0.01. (C) STHdhQ7/7 and STHdhQ111/111 cells treated with 1 (1.0 Tm) and 5 μg/ml Tm (5.0 Tm) for 16 hours. STHdhQ7/7 cells were also treated with 5 μM staurosporine for 5 hours as positive control (S). Cells were processed for immunoblotting for cleaved caspase-3. Immunoblots were then reprobed with anti-α-tubulin as a loading control.
Fig. 5.
Fig. 5.
BiP availability reveals increased sensitivity to ER stress in STHdhQ111/111 cells. (A) D values of single STHdhQ7/7 and STHdhQ111/111 cells transfected with BiP–GFP for 16 hours, and either left untreated (Ctl) or treated with 5 μg/ml Tm for 30 minutes and analyzed by FRAP. (B) D values of single STHdhQ7/7 and STHdhQ111/111 cells transfected with BiP–GFP for 16 hours and treated with 5 μg/ml Tm for 30 minutes, 1.5 hours and 4 hours. (C) D values of single STHdhQ7/7 and STHdhQ111/111 cells transfected with BiP–GFP for 16 hours and treated with 0.5 μg/ml Tm for 30 minutes, 1 hour and 2 hours. (D) D values of single STHdhQ7/7 and STHdhQ111/111 cells transfected with BiP–GFP for 16 hours and treated with 2.5 mM DTT. D values are binned into 20 minutes intervals. (E) D values of single STHdhQ7/7 and STHdhQ111/111 cells transfected with inert ER–RFP for 16 hours and treated with either 2.5 mM DTT for 30 minutes or 5 μg/ml Tm for 4 hours and analyzed by FRAP. (F) Immunoblots of the UPR reporter phosphorylated eIF2α from STHdhQ7/7 and STHdhQ111/111 cells treated with 5 μg/ml Tm for the indicated times. Equal loading was confirmed by reprobing with anti-α-tubulin. Statistically significant differences between treated and untreated cells for the same cell line (unless otherwise specified) are shown in parentheses above the data sets.
Fig. 6.
Fig. 6.
Reversibility of misfolded protein stress on BiP–GFP mobility in striatal cell. D values of single STHdhQ7/7 and STHdhQ111/111 cells transfected with BiP–GFP for 16 hours and treated with 5 mM DTT for 30 minutes followed by a 1-hour washout of the drug in one well and analyzed by FRAP. *P<0.01.
Fig. 7.
Fig. 7.
Adaptation to ER stress in striatal cells expressing full-length Htt. (A) Representative fluorescence images of STHdhQ7/7 and STHdhQ111/111 cells either left untreated or treated with 5 mM DTT for 30 minutes and followed by 16 hours washout of the drug. Cells were fixed and immunofluorescently stained with anti-BiP. Scale bar: 20 μm. (B) D values of single STHdhQ7/7 and STHdhQ111/111 cells transfected with BiP–GFP for 16 hours, and either left untreated (Naive) or treated with 5 mM DTT for 30 minutes (Adapted) and followed by a 16-hour washout of the drug. Naive or Adapted cells were subsequently challenged with 5 μg/ml Tm for 4 hours (+Tm), and analyzed by FRAP. (C) STHdhQ7/7 and STHdhQ111/111 cells either left untreated (Naive) or treated with 5 mM DTT for 30 minutes (Adapted) and followed by a 16-hour washout of the drug. Naive or Adapted cells were subsequently challenged with 5 μg/ml Tm for 16 hours, fixed and labeled with anti-cleaved caspase-3 and phalloidin. The percentage of cells with cleaved caspase staining was then determined. n>82 cells per data set. *P<0.01, **P<0.0005.
Fig. 8.
Fig. 8.
Accumulation of ERAD substrate in striatal cells expressing mHTT. (A) Representative fluorescence images of STHdhQ7/7 and STHdhQ111/111 cells co-transfected with either CD3δ–SFGFP or P450–GFP (green) and ER–RFP (red). Merge images are shown in the bottom panel. Scale bar: 20 μm. (B) The ratios of CD3δ–SFGFP/ER–RFP and P450–GFP/ER–RFP fluorescence intensities in STHdhQ7/7 and STHdhQ111/111 cells. (C) Mean fluorescent intensities of CD3δ–SFGFP, P450–GFP and ER–RFP in STHdhQ7/7 and STHdhQ111/111 cells. *P<0.05; n>25 cells for each data set.
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