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. 2012 May 15;21(10):2245-62.
doi: 10.1093/hmg/dds040. Epub 2012 Feb 14.

Targeting the UPR transcription factor XBP1 protects against Huntington's disease through the regulation of FoxO1 and autophagy

Affiliations

Targeting the UPR transcription factor XBP1 protects against Huntington's disease through the regulation of FoxO1 and autophagy

Rene L Vidal et al. Hum Mol Genet. .

Abstract

Mutations leading to expansion of a poly-glutamine track in Huntingtin (Htt) cause Huntington's disease (HD). Signs of endoplasmic reticulum (ER) stress have been recently reported in animal models of HD, associated with the activation of the unfolded protein response (UPR). Here we have investigated the functional contribution of ER stress to HD by targeting the expression of two main UPR transcription factors, XBP1 and ATF4 (activating transcription factor 4), in full-length mutant Huntingtin (mHtt) transgenic mice. XBP1-deficient mice were more resistant to developing disease features, associated with improved neuronal survival and motor performance, and a drastic decrease in mHtt levels. The protective effects of XBP1 deficiency were associated with enhanced macroautophagy in both cellular and animal models of HD. In contrast, ATF4 deficiency did not alter mHtt levels. Although, XBP1 mRNA splicing was observed in the striatum of HD transgenic brains, no changes in the levels of classical ER stress markers were detected in symptomatic animals. At the mechanistic level, we observed that XBP1 deficiency led to augmented expression of Forkhead box O1 (FoxO1), a key transcription factor regulating autophagy in neurons. In agreement with this finding, ectopic expression of FoxO1 enhanced autophagy and mHtt clearance in vitro. Our results provide strong evidence supporting an involvement of XBP1 in HD pathogenesis probably due to an ER stress-independent mechanism involving the control of FoxO1 and autophagy levels.

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Figures

Figure 1.
Figure 1.
XBP1 deficiency in the nervous system protects against experimental HD. (A) DARPP32 levels were analyzed in the striatum of 6-month-old mice by western blot analysis. Hsp90 was monitored as loading control. Lower panel: The levels of DARPP32 were quantified from XBP1WT (n= 3), XBP1WT-mHttQ128 (n= 5) and XBP1Nes−/−-mHttQ128 (n= 7) mice (left panel). Mean and SEM are presented. (B) Immunohistochemistry analysis of DARPP32 levels in the striatum of 6-month-old mice. Scale bar, 50 μm. Quantification of the number of neurons (C) and (D) apoptotic cells was performed in 10 different regions of the striatum; each region corresponds to 0.08 mm2. Four independent animals were analyzed for each group. (E) Motor performance was monitored with the rotarod assay in XBP1 wild-type or deficient animals bred onto the YAC128 HD mouse model (XBP1WT-mHttQ128 or XBP1Nes−/−-mHttQ128, respectively; 6–9 animals per group) in 4-month-old animals. Rotarod values were normalized to the performance of XBP1WT mice as a value of 1. Mean and SEM are presented. *P< 0.05 and **P< 0.01, calculated with Student's t-test. In all experiments, littermates were employed for comparison.
Figure 2.
Figure 2.
XBP1 deficiency in the nervous system decreases mHtt levels in the YAC128 HD model. (A) Levels of Huntingtin (Htt) were measured in the striatum of 6-month-old mice by western blot analysis using the anti-Htt clone mEM48 antibody. XBP1WT animals were analyzed as control. Hsp90 was monitored as loading control. Htt levels were quantified in striatum extracts from XBP1WT (n= 3), XBP1WT-mHttQ128 (n= 8) and XBP1Nes−/−-mHttQ128 (n= 13) mice (left panel). Mean and SEM are presented. (B) In parallel, Htt relative levels were determined using anti-Htt clone 1HU-4C8 antibody. Htt levels were quantified in the brain striatum of XBP1WT (n= 3), XBP1WT-mHttQ128 (n= 3) and XBP1Nes−/−-mHttQ128 (n= 3) mice (left panel). Mean and SEM are presented. *P< 0.05, calculated with Student's t-test. (C) The mRNA level of the human huntingtin gene was analyzed by real-time PCR in total cDNA obtained from the brain striatum of XBP1Nes−/−-mHttQ128 or littermate control mice. All samples were normalized to β-actin levels. Average and SEM of the analysis of three animals per group are shown. (D) mHtt levels were analyzed using anti-polyQ clone 3B5H10 antibody in the striatum of 12-month-old mice (upper panel). Right panel: mHtt levels were quantified in ATF4WT (n= 3), ATF4WT-mHttQ128 (n= 4) and ATF4−/−-mHttQ128 (n= 4) (bottom panel). In addition to Hsp90, γ-tubulin levels were analyzed as loading control. In all experiments, littermates were employed for comparison.
Figure 3.
Figure 3.
mHtt levels in an HD knock-in model on an XBP1-deficient background. (A) XBP1Nes−/− mice were bred with a knock-in heterozygous HD mouse model (HdhQ111/Q7). Levels of Huntingtin (Htt) and mHtt were measured in the striatum of 6-month-old mice by western blot analysis using the anti-Htt clone 1HU-4C8 antibody (left panel). XBP1WT animals were analyzed as control. Hsp90 was monitored as loading control. Total Htt levels were quantified in striatum extracts from XBP1WT (n= 6), XBP1WT-HdhQ111/Q7 (n= 7) and XBP1Nes−/−-HdhQ111/Q7 (n= 6) mice (right panel). (B) The band corresponding to mHtt levels was quantified from experiments presented in (A) to calculate the relative levels normalized with the expression of HdhQ111/Q7 control mice. (C) In parallel, mHtt relative levels were determined using anti-polyQ clone 3B5H10 antibody, and relative mHtt levels were quantified (left panel). Mean and SEM are presented. *P< 0.05, calculated with Student's t-test. (D) ATF4−/− mice were bred with a knock-in heterozygous HD mouse model (HdhQ111/Q7). Levels of Htt and mHtt were measured in the striatum of 6-month-old mice by western blot analysis using the anti-polyQ clone 3B5H10 antibody. Mean and SEM are presented of the analysis of ATF4WT (n= 3), ATF4WT-HdhQ111/Q7 (n= 6) and ATF4−/−-HdhQ111/Q7(n= 5). In all panels, statistical analysis was performed with Student's t-test. #P= 0.07, &P= 0.06.
Figure 4.
Figure 4.
XBP1/IRE1α deficiency in neuronal cells reduces abnormal mHtt aggregation. (A) NSC34 motoneuron-like cells were stably transduced with lentiviral vectors expressing shRNA against XBP1, IRE1α or control luciferase mRNA (shXBP1, shIRE1α and shCTR, respectively), followed by transient transfection with expression vectors for polyQ79-EGFP and polyQ11-EGFP as control. After 72 h, polyQ79-EGFP intracellular inclusions were quantified by fluorescent microscopy (right panel). Left panel: The number of cells displaying intracellular inclusions was quantified in a total of at least 300 cells per experiment. Results are representative of four independent experiments. Average and standard deviation are presented. Scale bar, 20 μm. (B) In parallel, detergent-insoluble polyQ79-EGFP aggregates were measured in cell extracts prepared in Triton-X100 buffer followed by western blot analysis in 4–12% polyacrylamide gradient gels. shRNA cells: Control (C), XBP1 (X) or IRE1α (I). Results are representative of at least four independent experiments. Two independent experiments per condition are presented. (C) NSC34 cells were co-transfected with an expression vector for XBP1s or empty vector (mock) in the presence of a polyQ79-EGFP expression vector. After 72 h, polyQ79-EGFP intracellular inclusions (right panel) were quantified by fluorescent microscopy (arrowheads). Left panel: The number of cells displaying intracellular inclusions was quantified in at least 300 cells per experiment. Results are representative of four independent experiments. Mean and SEM are presented. Scale bar, 20 μm. (D) Detergent-insoluble polyQ79-EGFP aggregates were measured in cell extracts prepared in Triton X-100 buffer from Neuro2A shXBP1 or shCTR cells. As control, polyQ11-EGFP was expressed in both cell lines. Levels of Hsp90 served as loading control. (E) Neuro2A shXBP1 or shCTR cells were transiently transfected with expression vectors for human mHtt (exon 1) GFP-mHttQ85. After 72 h, GFP-mHttQ85 aggregates were measured in cell extracts prepared in Triton X-100 and analyzed by western blot. Levels of Hsp90 served as loading control. Left panel: mHtt aggregates were quantified and the results are representative of four independent experiments. Mean and SEM are presented. (F) shCTR Neuro2a cells were transiently transfected with mHttQ138-mRFP or mHttQ138Δ5-13-mRFP constructs in the presence of cytochrome b5-EGFP (Cyt b5-EGFP) to label the ER. After 24 h, cells were visualized by confocal microscopy. Nucleus was stained with DAPI. Scale bar, 20 μm. (G) Neuro2A shXBP1 (black bar) or shCTR (white bar) cells were transiently transfected with mHttQ138-mRFP or mHttQ138Δ5-13-mRFP. After 48 h, mHtt intracellular inclusions were quantified in at least 200 cells per experiment. Mean and SEM are presented. *P< 0.05 and **P< 0.01, calculated with Student's t-test.
Figure 5.
Figure 5.
XBP1 deficiency leads to autophagy-mediated degradation of mHtt in cellular models of HD. (A) NSC34 shXBP1, shIRE1α or shCTR cells were transfected with an expression vector for polyQ79-EGFP, and after 48 h cells were treated with MG132 (10 and 1 μm) for 8 h, or with 10 mm 3-MA or 10 μm wortmannin for 16 h and polyQ79-EGFP aggregation analyzed by western blot in 4–12% polyacrylamide gradient gels. (B) Examples of polyQ79-EGFP intracellular inclusions in shXBP1 cells untreated or treated with 3-MA are presented, visualized by fluorescent microscopy. Scale bar, 50 μm. (C) PolyQ79-EGFP aggregates observed in (A) were quantified in each treatment and for comparison normalized to the value obtained in non-treated shCTR cells. (D) NSC34 cells were transiently transfected with expression vectors for polyQ11-EGFP and polyQ79-EGFP. After 24 h, cells were stained with lysotracker and DAPI, and the co-localization with polyQ79-EGFP intracellular inclusions was determined by confocal microscopy. Scale bar, 10 μm. (E) NSC34 shCTR or shXBP1 cells were transiently transfected with expression vectors for LC3-EGFP and stained with DAPI and visualized by confocal microscopy. After 48 h, cells were treated with 10 mm 3-MA for 16 h. Scale bar, 20 μm. (F) To monitor LC3 flux through the autophagy pathway in Neuro2A shCTR or shXBP1, cells were treated or not with a lysosome inhibitor cocktail (lys. inh.) containing 200 nm bafilomycin A1, 10 μg/ml pepstatin and 10 μg/ml E64d for indicated time points and endogenous LC3 levels monitored by western blot. Hsp90 levels served as loading control. (G) Kinetics of the accumulation of LC3-II protein showed in (F) was quantified and normalized to Hsp90 levels and then to non-treated shCTR cells.
Figure 6.
Figure 6.
Loss of XBP1 targets mHtt to the autophagy pathway. (A) The accumulation of APG-like structures was visualized by EM in neurons of the striatum of XBP1Nes−/−-mHttQ128 mice at 6 months of age. Scale bars, 230 nm (a1) and 600 nm (a2). Images represent the analysis of three independent animals. Neurons were identified by their morphology at low magnification. (B) ER structure was visualized by EM in neurons of the striatum of mice at 6 months of age. Scale bar, 300 nm. Left panel: ER dilatation was quantified from XBP1WT (n= 3), XBP1Nes−/− (n= 3), XBP1WT-mHttQ128 (n= 3) and XBP1Nes−/−-mHttQ128 (n= 3) mice as described in Materials and Methods. Mean and SEM are presented. As an example, the perimeter of the ER was marked with a black line with a gray area. *P<0.05 calculated with Student's t-test. (C) Brain extracts from indicated animals were subjected to subcellular fractionation to purify fractions enriched in different organelles including autophagosomes (APG), autophagolysosomes (APGL), ER and cytosol (cyt). (D) Quantification of Htt levels in the autophagic compartments relative to XBP1WT is shown (XBP1WT-mHttQ128, n= 3; XBP1Nes−/−-mHttQ128, n= 3) using the anti-Htt clone 1HU-4C8 antibody. Mean and SEM are presented. (E) Biochemical characterization of the fractions isolated in (C). Immunoblot for the indicated marker proteins to verify the purity of the fractions was performed for the homogenate (H), cytosol (cyt), autophagosomes (APG), autophagolysosomes (APGL) and endoplasmic reticulum (ER) isolated from cortex and midbrain. (F) Immunogold staining and EM analysis of the brain striatum, using an anti-expanded polyQ antibody (clone 3B5H10) was performed in tissue derived from XBP1Nes−/−-mHttQ128 mice. (f1) Co-localization with ER membrane. Scale bar, 500 nm. (f2) A double-membrane APG-containing positive immunogold staining in its lumen. Scale bar, 300 nm. (f3) An example of co-localization of immunogold staining and Golgi apparatus membrane is shown. Scale bar, 500 nm. Data represent the analysis of three independent animals. Arrows indicate immunogold staining. N, nucleus; GA, Golgi apparatus; ER, endoplasmic reticulum.
Figure 7.
Figure 7.
XBP1 negatively regulates FoxO1 expression. (A) The levels of XBP1 mRNA splicing were analyzed in the striatum from XBP1Nes−/−-mHttQ128 and littermate control mice at 3 months of age. (B) The mRNA levels of indicated UPR-target genes were measured by real-time PCR in total cDNA obtained from the striatum of four XBP1Nes−/−-mHttQ128 mice or littermate control mice at 6 months of age. All samples were normalized to β-actin levels. Average and SEM of the analysis of three animals per group are shown. (C) FoxO1 levels were analyzed in the striatum of 6-month-old mice by western blot. Hsp90 was used as loading control. Left panel: The relative levels of FoxO1 were quantified from XBP1WT (n= 3) and XBP1Nes−/− (n= 3) mice and normalized with Hsp90 levels. Mean and SEM are presented. (D) The distribution of FoxO1 (green) was analyzed in the striatum of XBP1WT−-mHttQ128, XBP1Nes−/−-mHttQ128, XBP1WT and XBP1Nes−/− animals at 6 months of age. Co-staining with NeuN (red) and DAPI (blue) stain nucleus was performed. Images were visualized with a confocal microscope and represent the analysis of three animals per group. Scale bar, 50 μm. (E) The mRNA level of foxO1 was analyzed by real-time PCR in total cDNA obtained from the brain striatum of XBP1Nes−/− or littermate control mice. All samples were normalized to β-actin levels. Average and SEM of the analysis of three animals per group are shown. (F) Neuro2A cells were stably transduced with lentiviral vectors expressing shRNA against XBP1 or control luciferase mRNA (shXBP1 and shCTR, respectively). FoxO1 and XBP1s levels were monitored after treatment with Tm (5 μg/ml, Tm) for 8 h, using western blot analysis. Levels of Hsp90 were used as loading control. Quantification is presented at the bottom of the gel as fold change. (G) HEK cells were transiently transfected with expression vectors for HA-FoxO1 and different concentrations of XBP1s or empty vector (pCDNA.3). FoxO1 and XBP1s levels were analyzed by western blot. The asterisk indicates unspecific band used as loading control. (H) Neuro2A cells were co-transfect with expression vector for mHttQ138-mRFP with HA-FoxO1 or empty vector, and after 48 h, the number of RFP-positive cells containing mHtt inclusions was quantified. Mean and SEM are presented. **P< 0.01, calculated with Student's t-test. (I) Neuro2A cells were transiently transfected with an HA-FoxO1 expression vector or empty vector, and then treated for the indicated times with a cocktail of lysosomal inhibitors (200 nm bafilomycin A1, 10 μg/ml pepstatin and 10 μg/ml E64d). LC3-II flux was then monitored by western blot analysis and normalized with the loading control and as a fold change to the untreated control cells (quantification at the bottom).
Figure 8.
Figure 8.
XBP1s, ER stress and autophagy markers in HD human post-mortem samples. (A) The levels of mHtt (anti-polyQ), XBP1s, ATF4, CHOP, BiP and LC3 were measured in total protein extracts derived from the striatum of post-mortem samples from HD patients at disease stage III and healthy control subjects. These identified samples were obtained from the Harvard Brain Tissue Resource Center. HSP90 levels were analyzed as loading control. As positive control for ER stress, MEFs treated with 1 µg/ml Tm is presented. (B) For comparison, relative levels of XBP1s and LC3-II were quantified and plotted after normalization with HSP90 levels shown as arbitrary units.

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