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

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.

Rosiglitazone activation of PPARγ-dependent signaling is neuroprotective in mutant huntingtin expressing cells

“…Alterations in the function of the stress sensors (e.g., Bip, CHOP and ASK1) are implicated in the pathogenesis of HD [14,35]. Here, it was observed that the transcript and protein levels of Bip and CHOP in mHtt expressing cells were signficantly higher than in control Htt expressing N2A cells (Fig.…”

“…mHtt induces ER stress at an early stage of HD, and pathogenic ER stress from an aging or stressful environment is severe in the late stages of HD [13,14]. Abnormal expression of ER stress genes (e.g., Herpud1, Rrs1, and 3D3/lyric) occurs in HD [35,69]. Following ER stress, CHOP is translocated to the nucleus and can induce cell death proteins and decrease the expression of Bcl-2 [71].…”

Approaches to imaging unfolded secretory protein stress in living cells

“…For example, expression of Bip1-GFP in C. elegans [64] has been successfully used to quantitatively monitor UPR activation in these organisms. In mammalian cells, our group generated a fluorescent UPR reporter by fusing a portion of the BiP promoter containing ER stress response elements (-169 ERSE) [65] to the red FP tdTomato [66]. This reporter exhibits significant upregulation following treatment of cells with ER stressors such as tunicamycin (Tm) or DTT or upon expression of mutant proteins known to induce the UPR, such as exon1 of the Huntington's disease-associated mutant polyQ protein, huntingtin [66].…”

“…FRAP relies on the ability of fluorescently tagged BiP (such as BiP-GFP) to freely diffuse and sample the entire volume of the ER lumen. When a BiP-GFP molecule encounters and binds a misfolded protein, diffusional mobility of BiP-GFP decreases [66, 81]. Changes in BiP-GFP mobility can be quantitated by calculating the protein diffusion coefficient ( D ) using inhomogeneous simulations [82].…”

“…CD3δ with a yellow FP tag (YFP) accumulates in cells treated with ER stressors [90]. Using fluorescence microscopy with a similar CD3δ-sfGFP tool, we observed UPR activation and accumulation of CD3δ-sfGFP in cells expressing the mutant huntingtin protein, which is associated with Huntington's disease, [66]. …”