Material and methods



Supplemental Research Data

Supplemental Methods

Materials and plasmids. Tunicamycin (Tm), wortmannin, 3-methyladenine, Bafilomycin A1 and MG-132 were purchased from Calbiochem EMB Bioscience Inc. Cell media, EBSS media and antibiotics were obtained from Life Technologies (Maryland, USA). Puromycin, pepstatin and E64D were purchased from Sigma. Fetal calf serum was obtained from Atlanta Biologicals. Hoechst, lysotraker, and Acridine orange were purchased from Molecular Probes. All transfections were performed using the Effectene reagent (Quiagen). DNA was purified with Qiagen kits.

SOD1-EGFP expression vectors were described before (Hetz et al., 2007). Primers were designed to introduce a SalI site to allow subcloning SOD1 mutants into pEGFP-N1 (Clontech, Palo Alto, CA) and to remove the SOD1 translation stop codon. Mutants were generated via site directed mutagenesis of the SOD1WT template using the Quick Change kit (Stratagene, La Jolla, CA). For the LC3-EGFP expression vectors the rat LC3 cDNA was cloned into the BglII and EcoRI sites of the pEGFP-C1 vector (Clontech laboratories) as described previously (Kabeya et al., 2000). The XBP-1s expression vector was previously described (Lee et al., 2003), where the cDNA was obtained from NIH3T3 cells treated with tunicamycin and the XBP-1s cDNA cloned into the pCDNA.3 vector between the HindIII and ApaI sites.

XBP-1 mRNA splicing assays. Two assays were employed to analyze XBP-1 mRNA splicing (Lisbona et al., 2009). In brief, for cell lines, PCR primers 5'- ACACGCTTGGGAATGGACAC-3' and 5'- CCATGGGAAGATGTTCTGGG-3' encompassing the spliced sequences in xbp-1 mRNA were used for the PCR amplification with AmpliTaq Gold polymerase (Applied Biosystem, Foster City, CA). We separated the PCR products by electrophoresis on a 2.5% agarose gel (Agarose-1000 Invitrogen, Carlsbad, CA) and visualized them by ethidium bromide staining. In tissue, a more sensitive assay was used in spinal cord extracts to monitor XBP-1 mRNA splicing. PCR using the sense primer mXBP1.3S (5'-AAACAGAGTAGCAGCGCAGACTGC-3') and antisense primer mXBP1.2AS (5'-GGATCTCTAAAACTAGAGGCTTGGTG-3') amplified a 600-bp cDNA product encompassing the IRE1α cleavage sites. The fragment was further digested by PstI to reveal a restriction site that is lost after IRE1-mediated cleavage and splicing of the mRNA.

Knockdown of UPR and autophagy components in motoneurons. We generated stable motoneuron cell lines with reduced levels of XBP-1, IRE1α, Beclin-1 and EDEM using methods previously described (Hetz et al., 2007) by targeting the respective mRNA with shRNA using the lentiviral expression vector pLKO.1 and puromycin selection. As control empty vector or a shRNA against the luciferase gene were employed. Constructs were generated by The Broad Institute (Boston, USA) based on different criteria for shRNA design (see ). We screened a total of five different constructs for each gene and selected the most efficient one for further studies. Targeting sequences identified for the mouse XBP-1, IRE1α, Beclin-1, EDEM1 (two constructs) mRNA are 5'-CCATTAATGAACTCATTCGTT-3', 5'-GCTCGTGAATTGATAGAGAAA-3', 5'-GCGGGAGTATAGTGAGTTTAA-3' , 5'-CCATATCATATCTGTGGACAA-3' and 5'-GCCCTTAAAGAGCATCTACAT-3', respectively. For ATG5 mRNA knock-down we employed a combination of five different targeting sequences including 5'-GCCAAGTATCTGTCTATGATA-3', 5'-CCTTGGAACATCACAGTACAT-3', 5'-GCAGAACCATACTATTTGCTT-3', 5'-GCATCTGAGCTACCCAGATAA-3'and 5'-CCCTGAAATGGCATTATCCAA-3'.

The NSC34 cell model was selected for this study because it has several valuable characteristics of motor neurons (Cashman et al., 1992), which include the ability to induce contraction in co-cultured muscle cells; the expression of neurofilaments; the generation of action potentials and the synthesis, storage and release of acetylcholine (Cashman et al., 1992). In addition, NSC34 cells induce acetylcholine receptor clusters on co-cultured myotubules (Cashman et al., 1992), and they are sensitive to mutant SOD1 neurotoxicity (Hetz et al., 2007). Cells were grown in the presence of 3 μg/ml puromycin to maintain selective pressure.

Assays for mutant SOD1 aggregation and detection of intracellular inclusions. We developed assays using the transient expression of human SOD1WT and the mutants SOD1G93A and SOD1G85R as EGFP fusion proteins. These constructs were employed to visualize and quantify the formation of intracellular SOD1 inclusions in living cells by fluorescent confocal microscopy. SOD1 oligomers were visualized in total cell extracts prepared in RIPA buffer and sonication, and then analyzed by Western blot. Alternatively, nuclear cell lysates were prepared in 1% NP-40 in PBS containing protease inhibitors. After solubilization on ice for 30 min, cell nuclei were precipitated by centrifugation at 3000 rpm for 5 min and cell extracts were centrifuged at 10,000 g for 10 min to collect NP-40 soluble and insoluble material. Pellets were resuspended in Western blot sampler buffer containing SDS.

Quantification of autophagy and cell viability. Different assays and control experiments were employed to monitor autophagy-related processes following the recommendations and precautions described in (Klionsky et al., 2008). Lysosomes or acidic compartments were visualized after staining with different dyes. Living cells were stained with 200 nM lysotraker or 600 nM Acridine orange for 45 min at 37°C and 5% CO2. Cells were washed three times with cold PBS and then fixed for 30 min with 4% formaldehyde on ice, then maintained in PBS containing 0.4% formaldehyde for immediate visualization on a confocal microscope. Alternatively, cells were loaded with DQ-BSA to monitor lysosomal activity as previously described (Klionsky et al., 2008). After calibrating the concentration and timing of pre-incubation we found that 20 μg/ml of DQ-BSA for 16h was the optimal condition for high quality staining.

Autophagy was monitored by analyzing LC3-positive dots or the levels of LC3-II by Western blot and its flux through the autophagosomal/lysosomal pathway as described (Klionsky et al., 2008). Autophagosomes were visualized after the expression of LC3-EGFP after the transient transfection of the lowest amounts of DNA titered to obtain the best signal to noise ratio (1/3 amount of recommended concentrations by transfection kit). All quantifications were performed for a total of at least 150 cells in duplicate for each independent experiment. As control, the background levels of puncta were examined by fluorescence from untagged GFP, where no dots were observed. In control experiments, cells were treated with 10 mM 3-MA to inhibit autophagy.

LC3 is initially synthesized in an unprocessed form, proLC3, which is converted into a proteolytically processed form lacking amino acids from the C terminus, LC3-I, and is finally modified into the PE-conjugated form. To monitor LC3-II dynamics protein samples were processed in cold RIPA and immediately analyzed by Western blot using 15% SDS-polyacrilamide gels. As internal control LC3-II levels were compared with Hsp90 levels. To follow the flow of LC3I/II through the autophagy pathway, cells were treated with a mix of 200 nM bafilomycin A1, 10 μg/ml pepstatin and 10 μg/ml E64d. In addition, the assays were established in standard assays of autophagy using cells grown in serum free RPMI (not shown). Alternatively, to monitor flux we transiently expressed a tandem monomeric RFP-GFP-tagged LC3 (Klionsky et al., 2008). The GFP signal of this fusion protein is sensitive to the acidic and/or proteolytic conditions of the lysosome lumen, whereas mRFP is more stable. Therefore, co-localization of both GFP and RFP fluorescence indicates a compartment that has not fused with a lysosome. In contrast, an mRFP signal without GFP corresponds to an autophagolysosome. Thus, the LC3 flux then can be followed in living cells in the absence of drug treatment. As control to trigger autophagy in vivo, mice were starved for 24h and then liver extracts were prepared for western blot analysis to monitor LC3 levels as previously described (Mizushima et al., 2004).

Cell viability was monitored using the MTT assay or propidium iodide staining (Lisbona et al., 2009). Alternatively, cell cultures were stained with 1 mg/ml of PI and visualized with a fluorescent microscope. Proteasomal activity was monitored using the fluorescent substrate GFPu by FACS analysis. ERAD was monitored after expressing CD3−δ−YFP alone, because it is a well-established ERAD substrate when expressed in the absence of the other receptor subunits.

Animal experimentation. XBP-1flox/flox mice were crossed with mice expressing Cre recombinase under the control of the Nestin promoter to achieve deletion of XBP-1 in the nervous system (XBP-1Nes-/-) (Hetz et al., 2008). We employed as an ALS model the SOD1G86R transgenic strain (the equivalent of human SOD1G85R) which were generated in the FVB/N strain (strain FVB-Tg(Sod1-G86R)M1Jwg/J, The Jackson Laboratory). The expression of the SOD1 mutant gene is driven by the endogenous SOD1 promoter. As shown in Supplemental Fig. S3A, the overexpression levels of mutant SOD1 are very low compared with other transgenic mice such as the SOD1G93A line, decreasing the possible non-specific effects of overexpression. In addition, SOD1G86R encodes an enzyme with low SOD activity, and thus expression of the altered enzyme does not significantly affect overall SOD1 cellular activity when added to the genome in the presence of two wild-type parental genes. All animal experiments were performed according to procedures approved by the Institutional Review Board’s Animal Care and Use Committee of Harvard School of Public Health (approved animal protocol 04137) and the Faculty of Medicine of the University of Chile (approved protocol CBA # 0208 FMUCH). The forth to sixth generations of XBP-1Nes-/-/SOD1G86R mice were used to expand the colony and obtain experimental groups.

Disease onset analysis. Disease onset was determined by visual observation of the appearance of abnormal limb-clasping, slight tremor felt in one of the hind-limbs, wobbly gait and the first signs of paralysis in one hind-limb. End disease stage was determined as the time at which an animal can no longer right itself within 30 s after being placed on its back as described (Hetz et al., 2007;Hetz et al., 2008). Collection of onset data began at five weeks when mice were trained for rotarod as described in Hetz, et. al 2008.  Disease progression was monitored at least once per week until mice reached 90 days, from which point observations were made every 1-4 days.  Parameters included rotarod, weight, and qualitative assessment of hind limb grasping, back arch, grooming behavior, and paralysis.

Onset was determined separately for rotarod, and body weight where the last measurement before a precipitous and sustained decline in the readout parameter was noted as the start of disease.  Observational data defined duration of the disease from onset until the first day at which hind limb paralysis reached a minimum of 50%. 

Tissue analysis. To monitor SOD1 pathogenesis in vivo, female animals were euthanized and tissue collected for histology at different time points depending on the analysis required. Spinal cord tissue was processed for immunohistochemistry using standard procedures as described (Hetz et al., 2007). Apoptotic cells in the ventral horn were quantified by using the TUNEL assay (Promega) as previously described (Hetz et al., 2007). Motoneurons were also directly visualized with anti-ChAT 1:50 or anti-NeuN 1:100 (Chemicon) staining as previously described (Hetz et al., 2007). In addition, staining of LC3 and lysosomes were performed with anti-LC3 1:100 (Cell Signaling Technology) and anti-LAMP-2 1:200 (Developmental Studies Hybridoma Bank). Staining of astrocytes was performed with an anti-GFAP antibody 1:400 (Chemicon or Jackson). Confocal microscopy was used to acquire images and then analysis was performed using the IP lab v 4.04 software (Beckon and Dickenson).

Western blot analysis of spinal cord extracts. 1 cm lumbar spinal cord tissue was collected and homogenized in RIPA buffer (20mM Tris pH 8.0, 150mM NaCl, 0.1% SDS, 0.5% DOC, 0.5% triton X-100) containing a protease inhibitor cocktail (Roche, Basel, Switzerland) by sonication. Protein concentration was determined by micro-BCA assay (Pierce, Rockford, IL). The equivalent of 30–50μg of total protein was loaded onto 4-12, 7.5, 12 or 15% SDS-PAGE minigels (Cambrex Biosciences) depending on the analysis as described above. The following antibodies and dilutions were used: anti-Grp78/Bip, anti-Grp58, anti-PDI, 1:2,000 (StressGene, San Diego, CA), anti-XBP-1, 1:2,000 (Biolegend), anti-GFP 1:1000, anti-Ubiquitin 1:2000, anti-ATF4, anti-Hsp90, anti-CHOP 1:2,000 (Santa Cruz, CA); anti-SOD1 1:3000 (Calbiochem), anti-LC3 1:500, anti-Beclin-1 1:2000, anti-ATG5 1:2000 (Cell Signaling Technology).

RNA extraction and RT-PCR. Total RNA was prepared from spinal cord tissue homogenated in cold PBS using Trizol (Invitrogen, Carlsbad, CA) and cDNA was synthesized with SuperScript III (Invitrogen, Carlsbad, CA) using random primers p(dN)6 (Roche, Basel, Switzerland). Quantitative real-time PCR reactions employing SYBR green fluorescent reagent were performed in an ABI PRISM 7700 system (Applied Biosystems, Foster City, CA). The relative amounts of mRNAs were calculated from the values of comparative threshold cycle by using β-actin as control. Primer sequences were designed by Primer Express software (Applied Biosystems, Foster City, CA) or obtained from the Primer Bank (). Real time PCR was performed as previously described(Lee et al., 2005) using the following primers: grp78/bip 5’-TCATCGGACGCACTTGGAA-3’ and 5’-CAACCACCTTGAATGGCAAGA-3’; grp58 5’- GAGGCTTGCCCCTGAGTATG-3’ and 5’-GTTGGCAGTGCAATCCAC C-3’; Chop/gadd153 5’-GTCCCTAGCTTGGCTGACAGA-3’ and 5’-TGGAGAGC GAGGGCTTTG-3’; xbp-1 5’-CCTGAGCCCGGAGGAGAA-3’ and.5’-CTCG AGCAGTCTGCGCTG-3’; pdi 5’- CAAGATCAAGCCCCACCTGAT-3’ and AGTTCGCCCCAACCAGTACTT; erdj4 5’- CCCCAGTGTCAAACTGTACCAG-3’ and 5’- AGCGTTTCCAATTTTCCATAAATT-3’; edem 5’- AAGCCCTCTGGAACTTGCG-3’ and 5’- AACCCAATGGCCTGTCTGG-3’; sec61a 5’- CTATTTCCAGGGCTTCCGAGT-3’ and 5’- AGGTGTTGTACTGGCCTCGGT-3’; herp 5’- CATGTACCTGCACCACGTCG-3’ and 5’- GAGGACCACCATCATCCGG-3’; actin 5’- TACCACCATGTACCCAGGCA-3’ and 5-‘ CTCAGGAGGAGCAATGATCTTGAT-3’; wfs-1 5'-CCATCAACATGCTCC CGTTC-3' and 5'-GGGTAGGCCTCGCCAT-3'; grp94, 5'-TGTATGTACGCCGCGTATTCA-3' and 5'-TCGGAATCCACAACACCTTTG-3'; atg5 5’-TGTGCTTCGAGATGTGTGGTT-3’ and 5’-GTCAAATAGCTGACTCTTGGCAA-3’

EM studies and immunogold staining. Autophagosomes were also visualized by transmission electron microscopy as in (Klionsky et al., 2008) and morphology examined as described in (Eskelinen, 2008). In addition, EM studies were carried out by the Harvard EM Core Facility and the P. Catholic University of Chile EM facility. Cells were fixed with 1.25% formaldehyde, 2.5% glutaraldehyde, 0.03% picric acid in 100mM sodium cacodylate buffer. After washing with 100mM sodium cacodylate buffer, tissues were treated for 1 h with 1% osmium tetroxide and 1.5% potassium ferrocyanide, and then 30 min with 0.5% uranyl acetate in 50mM maleate buffer, pH 5.15. After dehydration in ethanol, cells were treated for 1 h in propylenoxide and then embedded in Epon/Araldite resin. Ultrathin sections were collected on electron microscope grids and observed by using a JEOL 1200EX transmission electron microscope at an operating voltage of 60 kV.

For immuno-gold EM staining the conditions for labeling were established.to detect specific signals as we previously described (Court FA et al., 2008). Spinal cords were immersion fixed in 4% paraformaldehyde and 0,1% glutaraldehyde, cryoprotected in sucrose and frozen in liquid nitrogen. After thawing, the tissue was dehydrated in graded alcohol; embedded in LR white resin, and polymerized in a 60oC oven for 24 h. Pale gold ultrathin sections were collected on 200 mesh nickel grids. Grids with sections were blocked with 1% BSA in 1X PBS for one hour at room temperature and incubated with rabbit anti-LC3 (1:25, Cell Signaling Technologies) and sheep anti-SOD-1 (1:75, Calbiochem), 0,1% BSA in 1X PBS overnight at 4oC. Grids were washed and incubated with goat anti-rabbit IgG conjugated to 10 nm gold colloid (1:20, Ted Pella) and donkey anti-sheep IgG conjugated to 5 nm gold colloid (1:20, Ted Pella), 0,1% BSA in 1X PBS for 3 hours at room temperature; after washing, grids were fixed with 1% glutaraldehyde in 0,1M cacodylate buffer and air dried. Sections were contrasted with 1% uranyl acetate and lead citrate. Grids were examined with a Philips Tecnai 12 electron microscope operated at 80 kV. Negative films were developed and scanned.

ALS human post-mortem spinal cord samples. Human post mortem tissue from ALS and control subjects was obtained as frozen tissue from the Massachusetts General Hospital (Boston, USA) and then processed for biochemical analysis by homogenization of an equivalent section in PBS containing protease and phosphatase inhibitors with further dilution in RIPA buffer (20mM Tris pH 8.0, 150mM NaCl, 0.1% SDS, 0.5% DOC, 0.5% triton X-100) containing a protease inhibitor cocktail (Roche, Basel, Switzerland) and sonication for 10 seconds. Protein concentration was determined by micro-BCA assay (Pierce, Rockford, IL) and the equivalent of 50 μg of total protein was analyzed by Western blot as described above. The gender, genotype, age of death and identity numbers are indicated in Table 1 of supplemental information.

Reference List

Cashman,N.R., H.D.Durham, J.K.Blusztajn, K.Oda, T.Tabira, I.T.Shaw, S.Dahrouge, and J.P.Antel. 1992. Neuroblastoma x spinal cord (NSC) hybrid cell lines resemble developing motor neurons. Dev. Dyn. 194: 209-221.

Court FA, W.T.Hendriks, H.D.Macgillavry, J.Alvarez, and M.J.van. 2008. Schwann cell to axon transfer of ribosomes: toward a novel understanding of the role of glia in the nervous system. J. Neurosci. 28: 11024-11029.

Eskelinen,E.L. 2008. Fine structure of the autophagosome. Methods Mol. Biol. 445: 11-28.

Hetz,C., A.H.Lee, D.Gonzalez-Romero, P.Thielen, J.Castilla, C.Soto, and L.H.Glimcher. 2008. Unfolded protein response transcription factor XBP-1 does not influence prion replication or pathogenesis. Proc. Natl. Acad. Sci. U. S. A 105: 757-762.

Hetz,C., P.Thielen, J.Fisher, P.Pasinelli, R.H.Brown, S.Korsmeyer, and L.Glimcher. 2007. The proapoptotic BCL-2 family member BIM mediates motoneuron loss in a model of amyotrophic lateral sclerosis. Cell Death. Differ.

Kabeya,Y., N.Mizushima, T.Ueno, A.Yamamoto, T.Kirisako, T.Noda, E.Kominami, Y.Ohsumi, and T.Yoshimori. 2000. LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J. 19: 5720-5728.

Klionsky,D.J., H.Abeliovich, P.Agostinis, D.K.Agrawal, G.Aliev, D.S.Askew, M.Baba, E.H.Baehrecke, B.A.Bahr, A.Ballabio, et al., 2008. Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy. 4: 151-175.

Lee,A.H., G.C.Chu, N.N.Iwakoshi, and L.H.Glimcher. 2005. XBP-1 is required for biogenesis of cellular secretory machinery of exocrine glands. EMBO J. 24: 4368-4380.

Lee,A.H., N.N.Iwakoshi, and L.H.Glimcher. 2003. XBP-1 regulates a subset of endoplasmic reticulum resident chaperone genes in the unfolded protein response. Mol. Cell Biol. 23: 7448-7459.

Lisbona,F., D.Rojas-Rivera, P.Thielen, S.Zamorano, D.Todd, F.Martinon, A.Glavic, C.Kress, J.H.Lin, P.Walter, J.C.Reed, L.H.Glimcher, and C.Hetz. 2009. BAX inhibitor-1 is a negative regulator of the ER stress sensor IRE1alpha. Mol. Cell 33: 679-691.

Mizushima,N., A.Yamamoto, M.Matsui, T.Yoshimori, and Y.Ohsumi. 2004. In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker. Mol. Biol. Cell 15: 1101-1111.

Supplemental Figure Legends

Supplemental Figure S1. Distinct roles of UPR transcription factors in the aggregation of mutant SOD1. (A) NSC34 stably expressing shRNA constructs against XBP-1, ATF4, ATF6, or control mRNAs were transiently transfected with expression vectors for human SOD1G85R–EGFP fusion proteins. After 72h, detergent insoluble SOD1 protein aggregates were analyzed by Western blot. Of note, high molecular weight SOD1 aggregates are observed. SOD1 monomers are indicated with an arrow head. Results are representative of three independent experiments. (B) The efficiency of ATF6 knockdown was evaluated by Western blot analysis. (C) ATF4 knockdown was evaluated in cells treated with 500 ng/ml Tm for 4h by Western blot analysis. (D) NSC34 cells stably expressing shRNA constructs against XBP-1 (X), IRE1α (I), or control mRNA (M) were transiently transfected with expression vectors for human SOD1WT, SOD1G93R, or SOD1G85R-EGFP fusion proteins. After 72h, detergent insoluble SOD1 protein aggregates were measured in cell extracts prepared in RIPA and analyzed by Western blot using anti-GFP antibody. (E) As control to monitor the efficiency of transfection in knockdown cells, NSC34 cells stably expressing shRNA constructs against XBP-1, IRE1α, or control mRNA were transiently transfected with expression vectors for human SOD1WT–EGFP and, EGFP fluorescence was determined by FACS after 48h.

Supplemental Figure 2. Increased autophagy and lysosomal content in XBP-1 knockdown NSC34 cells. (A) Proteasome activity was monitored in NSC34 shXBP-1 and shControl cells after the expression of the ubiquitin-dependent proteasome substrate GFPu (cytosolic reporter) and FACS analysis. Of note, the basal GFPu expression (pick fluorescence) was similar in shControl and shXBP-1 cells indicating similar proteasome activity. As positive control, shXBP-1 cells were treated with MG-132 for 6h, which lead to increased GFPu basal fluorescence and the appearance of an additional pick at higher fluorescence emission. Also, autofluorescence of non-transfected shXBP-1 cells is shown as control. (B) NSC34 cells were transfected with SOD1G85R-EFGP expression vectors (green) and after 72h stained with lysotracker and Hoechst. Representative confocal fluorescent images are presented. shXBP-1 cells (ii) were employed and compared with control cells (i). Of note, expression of SOD1 mutants increases the content of lysosomes. Scale bar, 10 μm. (C) NSC34 cells were transduced with control (shControl) or a shRNA lentiviral construct against Beclin-1 mRNA (shBeclin-1), and levels of Beclin-1 and mutant SOD1 aggregation were evaluated by Western blot. Data are representative of three independent experiments. (D) NSC34 cells were stably transduced with lentiviral vectors expressing shRNA against XBP-1 or luciferase mRNAs (shXBP-1 and shControl respectively), and content of lysosomes (white arrows) was visualized after lysotracker staining and confocal microscopy (red fluorescence). Nuclei were stained with Hoechst (blue). Images are representative of two experiments performed in duplicate where a minimum of 50 cells were analyzed. Scale bar, 10 μm. (D) Right panel: NSC34 cells carrying indicated shRNA constructs were stained with acridine orange and fluorescent emission quantified by FACS. As a positive control to drastically increase lysosomal content, cells were starved in EBSS buffer for 1h. Left panel: Representative confocal images of acridine orange staining are also shown. Scale bar, 10 μm. (E) Autophagosomes were visualized in shXBP-1 NSC34 cells by electron microscopy. Two representative pictures (a and b) depicting different autophagosomal structures are shown with two magnifications. Multimembrane autophagosomes are indicated with an arrow.

Supplemental Figure S3. ER stress in mutant SOD1 transgenic mice. (A) Left panel: The expression levels of the ER stress inducible genes PDI, BiP, and CHOP were analyzed in spinal cord protein extracts from symptomatic hSOD1G93A, mSOD1G86R transgenic or control mice by Western blot. All animals were between 100 to 120 days of age. As controls, the expression levels of Hsp90 (loading control) and SOD1 are shown (right panel). Of note, human SOD1 (hSOD1), and mouse SOD1 (mSOD1) proteins are resolved in the analysis. Each lane represents a different animal. (B) The mRNA levels of XBP-1 target genes wfs-1, erdj4, chop, grp78/bip, ero1, grp58, herp and sec61 were analyzed by real time PCR in total cDNA obtained from the spinal cord from SOD1G86R or littermate control mice. All samples were normalized to β -actin levels and represent the average of four control and five SOD1 transgenic mice. Average and standard deviation are presented.

Supplemental Figure S4. XBP-1 deficiency has a higher effect on the life span of SOD1G86R female mice. (A) XBP-1Nes-/- and control wild type mice were bred onto SOD1G86R transgenic mice and survival monitored. Median survival of control or XBP-1Nes-/- was 110 days to 120 days, respectively. p = 0.5 determined by t-student test. Total number of mice: N = 16, XBP-1WT-SOD1G86R mice; N= 15, XBP-1Nes-/--SOD1G86R mice. Total mice include female animals presented in Fig. 5B and male mice of panel B. (B) XBP-1Nes-/- and control male mice were bred onto SOD1G86R transgenic mice and animal survival was evaluated. p values for median survival were non-significant. Total number of mice: N = 7, XBP-1WT-SOD1G86R mice; N= 8, XBP-1Nes-/--SOD1G86R mice. (C) Duration of the disease was calculated in male and female SOD1G86R transgenic mice that were XBP-1WT or XBP-1Nes-/- based on the time of disease onset determined by a sustained decline of rotarod performance. Data represent mean and standard deviation of four groups of animals including male XBP-1Nes-/--SOD1G86R (N=11), female XBP-1Nes-/--SOD1G86R (N=3) and controls male XBP-1WT-SOD1G86R (N=14), and female XBP-1WT-SOD1G86R (N=2). p values were not significant (n.s.) as calculated using Student's t-test between indicated groups. (D) In parallel a similar analysis as in C was performed using body weight loss as a disease onset parameter in male XBP-1Nes-/--SOD1G86R (N=11) and female XBP-1Nes-/--SOD1G86R (N=3) mice.

Supplemental Figure S5. Symptomatic SOD1 transgenic mice have increased numbers of LC3-positive autophagosomes in motoneurons. (A) Higher magnifications of equivalent images from experiment presented in Figure 6A. Autophagosomes were identified in the spinal cord of female XBP-1Nes-/-,mSOD1G86R transgenic mice by immunofluorescence using an anti-LC3 antibody (green). Neurons were co-stained with an anti-NeuN antibody (red) and total cells stained with Hoechst (blue). Motoneurons were identified by their morphology and staining with NeuN. Three representative colocalized images for the three staining are presented (I, ii, iii). White arrows indicate motoneurons containing autophagosomes. Scale bar, 25 μm. (B) Mutant SOD1 accumulates in autophagosomes in vivo. The presence of autophagosomes was visualized by transmission EM in the spinal cord of female control XBP-1Nes-/- or SOD1G86R;XBP-1Nes-/- mice at 90 days of age (pre-symptomatic). Motoneurons were first identified with a lower magnification by morphology, size, and localization in the spinal cord and the presence of autophagosomal-related structures in cytosol (red arrows) was analyzed at different magnifications. The presence of double membrane vesicles filled with cytosolic components was used as criteria for identification. (i) Morphology of a motoneuron in XBP-1Nes-/- mice, (ii) autophagosome in XBP-1Nes-/-;SOD1G86R mice, (iii) autophagolysosome in XBP-1Nes-/-;SOD1G86R mice. Red arrows indicate autophagosome-related structures. N: nucleus, M: mitochondria, ER: endoplasmic reticulum.

Supplemental Figure S6 Astrocyte activation is not affected by XBP-1 deficiency in SOD1G86R mice. The spinal cord of female XBP-1WT, XBP-1Nes-/- control or SOD1G86R transgenic mice were stained with an anti-GFAP antibody and astrocytes visualized by confocal microscopy. Two magnifications are presented for the same section. For SOD1G86R transgenic-XBP-1WT or –XBP-1Nes-/- mice, three independent animals are presented (I, ii and iii).

Supplemental Figure S7. Controls to monitor autophagy levels and accumulation of SOD1 aggregates in male and female animals. (A) SOD1 aggregation was determined by Western blot in XBP-1Nes-/- and control SOD1G86R male mice. Symptomatic mice were employed. As control, non-transgenic litter mate mice are presented. Each well represents an independent mouse indicated with numbers. Hsp90 levels were monitored as loading control. (B) Autophagosomes were stained in the spinal cord of male (N = 5) and female (N= 8) mSOD1G86R transgenic mice on an XBP-1Nes-/- background by inmunofluorescence using an anti-LC3 antibody. Neurons were co-stained with an anti-NeuN antibody and with Hoechst as shown in Figure 6A. p value was calculated using Student's t-test. (C) Female [F] and male [M] mice were maintained with food in their cages or starved for 24h to activate autophagy. Then animals were sacrificed and the levels of LC3-I/LC3-II were monitored in muscle protein extracts by Western blot analysis. Two animals per group are presented. Hsp90 levels were monitored as loading control. (D) The level of mutant SOD1 aggregation was determined in symptomatic male [M] and female [F] SOD1G86R transgenic mice. As control non-transgenic mice are presented. All animals were littermate controls. Hsp90 levels were monitored as loading control. (E) The mRNA levels of SOD1 were determined by real time PCR from total cDNA. Values were normalized by beta actin levels. Average and standard deviation are presented of three independent SOD1 transgenic mice or two non transgenic mice as a reference. p value was calculated using Student's t-test and was shown to be not significant (n.s.).

Supplemental Figure S8. (A) UPR activation in spinal cord post mortem samples from patients affected with sALS. The levels of XBP-1s, ATF4, GRP58, HSP70, and HSP90 were analyzed by Western blot in post-mortem spinal cord samples from healthy control or sALS patients (B) The signal intensity (arbitrary units, AU) of the bands was quantified to calculate average and standard deviation. Lower panel: Values were normalized by the levels of HSP90 in the same sample and the ratio between the average value of sALS and control samples was analyzed to depict the average fold induction.

Supplemental Figure S9. Working model: Homeostatic balance between the UPR and autophagy in the nervous system. Impairment of the IRE1α/XBP-1 pathway increases basal levels of autophagy associated with increased content of autophagosomes and lysosomes, leading to a selective clearance of mutant SOD1 aggregates. XBP-1 deficiency may alter the basal function of cellular processes modulated by XBP-1s such as proteasome-degradation of misfolded proteins through the ERAD generated during the protein folding process at the ER lumen. Accumulation of ERAD derived-substrates may trigger autophagy to overcome XBP-1 deficiency, and then eliminate damaged ER and proteins. Increased autophagy may have therapeutic effects in pathological conditions such as ALS.

Table 1. Identity of human spinal cord post mortem samples. Samples are presented in the order analyzed.

RB# Tissue type Diagnosis Gender Age

1. 8583 thoracic cord control M 65

2. 8582 thoracic cord control M 70

3. 3728 thoracic cord control F 76

4. 1969 thoracic cord sALS M 65

5. 10173 thoracic cord sALS F 70

6. 6012 thoracic cord sALS M 55

7. 5518 thoracic cord sALS F 45

8. 10172 thoracic cord sALS M 45

9. 6948 thoracic cord fALS M 51

10. 5519 thoracic cord fALS M 75

11. 5525 thoracic cord fALS M 75

12. 2956 thoracic cord fALS M 58

................
................

In order to avoid copyright disputes, this page is only a partial summary.

Google Online Preview   Download