XBP-1 deficiency leads to autophagy-mediated degradation of mutant SOD1. (A, right panel) shXBP-1 and shControl cells were transfected with an expression vector for SOD1^G85R and, after 48 h, cells were treated for 8 h with MG132 (10 and 1 μM) or for 16 h with 10 mM 3-MA or 10 μM wortmannin, and SOD1 aggregation was analyzed by Western blot. (Bottom panel) A shorter exposure of the same Western blot shows monomeric SOD1. As control, SOD1^WT was expressed in shControl cells. (Left panel) Examples of SOD1 intracellular inclusions are presented visualized by fluorescent microscopy in cells treated with 3-MA or not. Arrows indicate intracellular SOD1-EGFP inclusions. Bar, 50 μm. (B) NSC34 shControl or shXBP-1 cells were transiently transfected with expression vectors for SOD1^WT-EGFP and SOD1^G85R-EGFP and stained with lysotracker and Hoechst, and the colocalization with SOD1 intracellular inclusions was determined by confocal microscopy. Bar, 10 μm. (C) SOD1^WT and SOD1^G85R were transiently expressed in shXBP-1 cells and, after 48 h, lysosomal activity was inhibited by treatment with 200 nM bafilomycin A₁ (Baf.) alone or in combination with 10 μg/mL pepstatin (Peps.) and E64d. SOD1 oligomers were visualized by Western blot after 16 h of treatment. HSP90 levels served as loading control. (Left panel) Examples of mutant SOD1 inclusions after lysosome inhibition. Bar, 50 μm. (D) shXBP-1 cells were transiently transduced with a shRNA lentiviral constructs against ATG5 mRNA or control vector. After 7 d, cells were transfected with SOD1^G85R-EGFP, and the levels of mutant SOD1 aggregation were evaluated by Western blot in total cell extracts. (Bottom panel) A shorter exposure (low expo.) of the same Western blot shows monomeric SOD1. HSP90 levels were monitored as loading control. (Left panel) Knockdown efficiency was monitored by real-time PCR and normalized with the levels of actin. Mean and standard deviation are presented. Data are representative of four determinations.

Reduced ERAD in XBP-1 knockdown cells and its connection with autophagy. (A) ERAD activity was measured in shXBP-1 and control cells by determining the degradation rate of the substrate CD3-δ-YFP by Western blot after 3 h of cycloheximide (cyclohex.) treatment to allow for the clearance of CD3-δ-YFP through the proteasome. As control, cells were cotreated with the proteasome inhibitor MG132. Nontransfected (NT) cells are also shown. (B) Mutant SOD1 aggregation was detected by Western blot analysis in NSC34 cells transduced with EDEM1 shRNA. Two different EDEM1 shRNA constructs (shEDEM) were used that reduced mRNA levels. Results are representative of three independent experiments. (Left panel) Knockdown levels were determined by real-time PCR. (C) In parallel, cells transduced with a shRNA construct against EDEM1 mRNA (construct 2 from B) or control vector were transiently transfected with an LC3-EGFP expression vector and the levels of autophagosomes were visualized and quantified after 48 h with a confocal microscope. Average and standard deviation are presented. Arrows indicate LC3-labeled autophagosomes. Bar, 10 μm.

XBP-1 deficiency increases autophagy levels in neurons of SOD1^G86R transgenic mice. (A) Autophagosomes were directly observed in the spinal cord of control of mSOD1^G86R transgenic mice on an XBP-1^WT or XBP-1^Nes−/− background by inmunofluorescence using an anti-LC3 antibody (green). Neurons were costained with an anti-NeuN antibody (red) and with Hoechst (nucleus, blue). Images are representative of the analysis of five different animals per group of ∼125–130 d of age. (Right panel) Quantification of the percentage of NeuN-positive cells containing LC3-positive vacuoles. Values represent average and standard deviation. P-value was calculated using Student's t-test. Bar, 10 μm. (B) The colocalization between LC3-positive dots (red) with neurons (NeuN, blue) or astrocytes (GFAP, green) was analyzed in the spinal cord of a mSOD1^G86R transgenic mice using coimmunofluorescence. A merged picture is presented where white arrows indicate astrocytes and red arrows indicate neurons. A negative control of staining without primary antibodies is presented. Bar, 20 μm. (C) Lysosomes were visualized in the samples in A after LAMP-2 staining (red). Colocalization with motoneurons was evaluated after ChAT (green) staining (white arrow). Total cells were stained with Hoechst (nucleus, blue). Quantification of LAMP-2-positive motoneurons is indicated in the inset. Bar, 20 μm. (D) Beclin-1, LC3, and Hsp90 expression were determined in spinal cord protein extracts from symptomatic mSOD1^G86R transgenic or control mice by Western blot. LC3-II form is indicated. Two representative animals are shown per group.

XBP-1/IRE1α deficiency in motoneurons reduces abnormal mutant SOD1 aggregation and toxicity. (A) NSC34 motoneuron-like cells were stably transduced with lentiviral vectors expressing shRNA against XBP-1 or control luciferase mRNA (shXBP-1 and shControl, respectively), and expression of XBP-1s or ATF4 (negative control) were analyzed after treatment with tunicamycin (500 ng/mL, Tm) by Western blot. Levels of Hsp90 served as loading control. (B) In parallel, IRE1α mRNA was targeted with shRNA and analyzed as described in A. (Top panel) The levels of XBP-1 mRNA splicing were monitored by RT–PCR in the same samples. (C) Levels of XBP-1s-dependent (edem, sec61, pdi, and wfs1) and XBP-1s-independent (bip and chop) genes were evaluated by real-time PCR in cells treated or not with 500 ng/mL Tm for 8 h. Average and standard deviation is presented of results representative of three independent experiments performed in duplicate. (D) shXBP-1, shIRE1α, and shControl cells were transiently transfected with expression vectors for human SOD1^WT-EGFP, SOD1^G93A-EGFP, and SOD1^G85R-EGFP fusion proteins. After 72 h, SOD1 intracellular inclusions were quantified by fluorescent microscopy (arrows). (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 performed. Average and standard deviation are presented. Bar, 20 μm. (E) In parallel, detergent-insoluble SOD1 protein aggregates were measured in cell extracts prepared in NP-40 and centrifuged at high speed. NP40-insoluble and NP40-soluble SOD1 was analyzed by Western blot. Of note, high-molecular-weight SOD1 aggregates are observed. shRNA cells: (M) Mock; (X) XBP-1; (I) IRE1α. SOD1 monomers are indicated by an arrowhead. Results are representative of at least four independent experiments. (F) shXBP-1 or shControl cells were transiently transfected with expression vector for SOD1^G93A. After 72 h, cell viability was monitored using the MTT assay and normalized to control nontreated (NT) cells. Data represent average and standard deviation of three determinations. (G) NSC34 cells were cotransfected with mutant SOD1-EGFP and an XBP-1s expression vector or empty pCDNA.3 vector. After 48 h, intracellular inclusions were observed by fluorescent microscopy (white arrowheads). (Insets) The percentage of cells harboring protein aggregates is indicated. Data represent average and standard deviation of three determinations. Bar, 50 μm. (Right panel) In parallel, SOD1 aggregation was determined by Western blot in total protein extracts. Data are representative of three independent experiments.

Inhibition of XBP-1 expression increases the levels of basal autophagy. (A) Number of cells with autophagosomes was quantified by confocal microscopy in shControl or shXBP-1 NSC34 cells after expressing a LC3-EGFP expression vector. Cells were classified into two groups: those that contained low LC3-EGFP dots or those that contained more than four LC3-EGFP dots. As control, cells were treated for 16 h with 10 mM 3-MA. (Top panel) Representative images of LC3-EGFP-positive vacuoles (arrows). Bar, 10 μm. Average and standard deviation are presented of three determinations. (B) To monitor the proteolytic activity of lysosomes, shXBP-1 and shControl cells were loaded with DQ-BSA for 16 h and analyzed by FACS to monitor the dequenching of the dye associated with lysosomal degradation. (n.s.) Nonstained cells. (Top panel) Representative images of DQ-BSA staining are presented. Bar, 10 μm. (C) Basal levels of the endogenous lipidated LC3-II form were monitored by Western blot in shControl (M) or shXBP-1 (X) cells under resting conditions. To monitor the flux of LC3 through the autophagy pathway, experiments were performed in the presence or absence of a lysosome inhibitor cocktail (Lys. Inh.) containing 200 nM bafilomycin A₁, 10 μg/mL pepstatin, and 10 μg/mL E64d for the indicated time points. (Right panel) As control, in shXBP-1 NSC34 cells, ATG5 was also knocked down with shRNAs and LC3-II levels were monitored by Western blot. (D) shControl and shXBP-1 cells were transiently transfected with a tandem monomeric LC3-RFP-GFP construct to monitor the active flux of LC3 though the autophagy pathway. After 48 h, LC3-positive dots were visualized by fluorescent microscopy in the red and green channels and the ratio between the number of red dots (autophagolysosomes, acidic compartment) versus colocalized yellow dots (representing autophagosomes) per cell was determined. Mean and standard deviation are presented. Representative overlapped fluorescent images are presented. Bar, 10 μm. (E,F) shControl, shXBP-1, and shIRE1α cells were maintained in rich culture medium or incubated in EBSS buffer for 2 h, and mitochondrial metabolism was determined using the MTT assay (E) or cell viability was monitored by propidium iodide staining (F). Overlapping phase contrast and propidium iodide staining images are shown. Average and standard deviation are presented of three to five determinations.

XBP-1 deficiency in the nervous system prolongs life span of mutant SOD1 transgenic mice and decreases the levels of apoptosis. (A) The levels of XBP-1 mRNA splicing (XBP-1s) and nonspliced (XBP-1u/s) were determined in the spinal cord of three symptomatic SOD1^G86R transgenic mice by RT–PCR. (B) The mRNA level of the XBP-1 target gene edem1 was analyzed by real-time PCR in total cDNA obtained from the spinal cord of five SOD1^G86R or four littermate control mice. All samples were normalized to β-actin levels. Average and standard deviation are presented. P-value was calculated using Student's t-test. (C) XBP-1^Nes−/− (N = 7) and control wild-type (N = 9) mice were bred onto SOD1^G86R transgenic mice and survival was evaluated in female animals. P-value was calculated with Kaplan-Meier statistics. (D) TUNEL-positive cells in the right half of the ventral horn were quantified in a total of five animals per group in mice at the late stage of the disease. Average and standard deviation are presented. Indicated P-value was calculated using Student's t-test.

Decreased mutant SOD1 aggregation in XBP-1-deficient mice and enhanced autophagy in sALS and fALS patients. (A) Electron microscopy sections of SOD1^G86R/XBP-1^Nes−/− mice were analyzed by immunogold staining of SOD1. Autophagosomes were identified by the presence of double-membrane vesicles containing cytosol and organelles such as mitochondria (M). (Bottom panel) A higher magnification of the areas marked with red squares is shown to depict the double membrane of the vesicle and SOD1 immunogold staining (see arrows). Images are representative of three independent experiments. (B) SOD1 aggregation was determined by Western blot in XBP-1^Nes−/− and control SOD1^G86R presymptomatic female mice of the same litter (90 d of age). Two animals per group are presented. As loading control, Hsp90 levels were monitored. (C) Colocalization of SOD1 and LC3 was determined by double-immunogold staining. Gold spheres were 5 nm (SOD1) and 10 nm (LC3). (Panel i) Low-magnification picture to identify motoneurons by their size, morphology, and location in the spinal cord. (N) Nucleus. (Panel ii) Negative control for staining. (Panels iii,iv) Two magnifications showing the colocalization of LC3 and SOD1 inside a dense structure (white arrow). (Panels v,vi) Two magnifications showing the colocalization of LC3 and SOD1 in an autophagosomal structure. White arrows indicate double membranes. Arrows in panels iv and vi are SOD1 (yellow), 5-nm gold colloid; and LC3 (red), 10-nm gold colloid. Representative pictures of the analysis of three animals per group in two independent experiments are presented. (D) Increased levels of autophagy in post-mortem samples of sALS and fALS cases. The levels of BECLIN-1, ATG5–ATG12 complex, LC3, EDEM1, and polyubiquitinated proteins were investigated in total protein extracts derived from spinal cord post-mortem samples from sALS and fALS patients and three healthy control subjects. HSP90 levels were analyzed as loading control.