Spinal cord endoplasmic reticulum stress associated with a microsomal accumulation of mutant superoxide dismutase-1 in an ALS model -- Kikuchi et al. 103 (15): 6025 Data Supplement - HTML Page - index.htslp -- Proceedings of the National Academy of Sciences

Kikuchi et al. 10.1073/pnas.0509227103.

Supporting Information

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Supporting Figure 6
Supporting Figure 7
Supporting Figure 8
Supporting Figure 9
Supporting Table 1
Supporting Figure 10
Supporting Figure 11
Supporting Text

Supporting Figure 6

Fig. 6. Western blot analysis of the cleaved p50 fragment of ATF6 in homogenates from spinal cords of transgenic SOD1^G93A, SOD1^G85R, and SOD1^WT mice and nontransgenic littermates. The p50 fragment was detected in end-stage SOD1^G93A and SOD1^G85R mice but not in the other groups.

Supporting Figure 7

Fig. 7. (A) Immunoblot analysis of procaspase-12 and its cleaved fragment in spinal cords of SOD1^G93A and SOD1^G85R mice as well as nontransgenic control mice. (B) Enzymatic activity of caspase-12 in spinal cords of end-stage transgenic SOD1^G93A mice and of nontransgenic littermates. Spinal cord caspase-12 activity expressed as relative fluorescence units (RFU) is significantly higher in transgenic SOD1^G93A mice. Values are means ± SEM (n = four to six per group). *P < 0.05; Student’s t test.

Supporting Figure 8

Fig. 8. Microsomal fractions of 5-month-old transgenic SOD1, SOD1^G93A, and nontransgenic mice were analyzed by Western blot by using antibodies directed against human and mouse SOD1, as well as synaptotagmin and cytochrome oxidase (COX) as markers for other membranous components to assess contamination. Microsomal fractions from transgenic SOD1^WT and SOD1^G93A mice show both human (hSOD1) and mouse SOD1 (mSOD1), whereas those from nontransgenic controls show only mSOD1. Synaptotagmin and COX are found in negligible amounts in the microsomal fractions, confirming their purity, but yield clear bands in the crude mitochondrial fraction used as positive control. Asterisks refer to high molecular weight SOD1 species detected in the transgenic SOD1^G93A microsomal fractions only.

Supporting Figure 9

Fig. 9. Indicated amounts of recombinant human SOD1^WT and SOD1^G93A protein were subjected to SDS/PAGE followed by Coomassie brilliant blue staining or immunoblotting with a rabbit anti-SOD1 antibody. Each band density was measured and analyzed by linear regression analysis. In both Coomassie brilliant blue staining and immunoblotting with anti-SOD1 antibody, neither the slopes nor intercepts differed (ANCOVA, P > 0.7) between human SOD1^WT and SOD1^G93A.

Supporting Figure 10

Fig. 10. (A) Immunoprecipitation with an anti-SOD1 antibody followed by immunoblot using anti-BiP and anti-SOD1 antibodies. (B) Immunoprecipitation with an anti-SOD1 antibody using spinal microsomal fractions of SOD1^G93A mice at different disease stages and nontransgenic mice.

Supporting Figure 11

Fig. 11. (A) Schematic representation of in vitro synthesized BiP deletion mutants. N44: 44-kDa N-terminal catalytic domain; PBD: peptide binding domain; CTT: C-terminal tail. (B) For analysis of the interaction domain of BiP with hSOD1, GST-full length BiP, GST-D N44, GST-D PBD, GST-D CTT, or GST control protein immobilized on glutathione-Sepharose was incubated with in vitro synthesized hSOD1^G93A. Western blotting shows that the PBD domain is responsible for the interaction with hSOD1^G93A.

Table 1. Quantification of immunogold labeling for SOD1 in anterior horn motor neurons and cerebellar Purkinje cells

Gold particles/μm ER
	Genotype	Cytosol	ER	ER/cytosol
Spinal cord
	NTG	2.09 ± 0.34	0.54 ± 0.34	0.29 ± 0.17
	WT	4.08 ± 0.28	1.21 ± 0.15	0.27 ± 0.09
	G93A	3.54 ± 0.38	2.47 ± 0.27	0.73 ± 0.14*
Cerebellum
	NTG	11.82 ± 1.69	5.08 ± 1.46	0.43 ± 0.13
	WT	13.68 ± 2.14	5.63 ± 1.96	0.41 ± 0.14
	G93A	6.41 ± 0.62	4.04 ± 0.65	0.63 ± 0.10

Gold particles per micrometer endoplasmic reticulum (ER) were counted in the cytosol (within 30 nm of either side of the ER) and over the ER lumen. Ratios between ER and cytosolic labeling were established for either genotype. Anterior horn motor neurons from transgenic SOD1^G93A had a significantly higher ER/cytosol ratio of SOD1 than the two other genotypes. In cerebellar Purkinje cells, no significant difference in the ER/cytosol ratio of SOD1 was found among the different genotypes. Values represent means ± SEM. (n = 23-30 ER profiles per group; *P<0.05, one-way ANOVA, Student-Newman-Keuls Method).

Supporting Text

Protein preparation and Western blot analysis.
For phosphorylated protein kinase/endoribonuclease-1 a (IRE1a ), the homogenization buffer contained 50 mM NaF and 1 mM Na₃VO₄. Phosphorylation of IRE1a was confirmed by treating samples with protein phosphatase 1 (Roche Diagnostics). The primary antibodies used in this study for the various immunoblots are as follows: mouse anti-ATF6 (1:50-1:100; Imgenex, San Diego, CA), rabbit anti-IRE1a (1:500: Santa Cruz Biotechnology), rabbit anti-ATF4 (1:500; Santa Cruz Biotechnology), rabbit anti-caspase-12 (1:200; Santa Cruz Biotechnology), mouse anti-b -actin antibody (1:10,000; Sigma), sheep anti-SOD1 (1:3,000; Calbiochem), rabbit anti-SOD1 (1:1,000, Biodesign International, Saco, ME), mouse anti-synaptotagmin (1:1,000; Stressgen Biotechnologies, San Diego), mouse anti-cytochrome oxidase (1:1,000; Molecular Probes), rabbit anti-calnexin (1:2,000, Stressgen), mouse anti-GAPDH (1:5,000; Chemicon), rat anti-immunoglobulin-binding protein (BiP) (1:50; Santa Cruz Biotechnology), and mouse anti-BiP (1:50; BD Transduction Laboratories). After incubation (overnight, 4°C) with the primary antibody the membranes were probed (1 h, 25°C) with the appropriate horseradish-peroxidase (HRP)-conjugated secondary antibody. The bands were revealed by using chemiluminescence (SuperSignal, Pierce) and quantified by using a FluorChem 8800 digital image system (Alpha Innotech, San Leandro, CA). RT-PCR and detection for XBP1 mRNA splicing form.
RNA was first reverse-transcribed by using an XBP1-specific antisense primer, mXBP1.4AS: 5'-GCA CCT TCT AGA AGC TAC ACT AGC A-3'. Nested PCR using the sense primer mXBP1.3S (5'-AAA CAG AGT AGC AGC GCA GAC TGC-3') and antisense primer mXBP1.2AS (5'-GGA TCT CTA AAA CTA GAG GCT TGG TG-3') amplified a 600-bp cDNA product encompassing the IRE1 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. After digestion by PstI, proper identities of PCR products were confirmed by automatic sequencing (ABI Prism-310 Genetic Analyzer; Perkin-Elmer). Pre-PstI digested XBP1 mRNA density was used as control. Enzymatic activity assay for caspase-12
. Caspase-12 enzyme activity of the spinal cord from end-stage transgenic superoxide dismutase-1 (SOD1)^G93A mice, and age-matched nontransgenic littermates was measured by the Caspase-12 Fluorometric Assay Kit according to the manufacturer’s instructions (BioVision, Mountain View, CA). Biotinylation procedure.
Aliquots of microsomal fractions were exposed to Sulfo-NHS-SS-Biotin (30 min, 4°C) in the absence or presence of 1% Triton X-100. The reaction was then quenched, and excess biotinylation reagent in the supernatant was removed by the use of desalt spin columns (Pierce Biotechnology). Lysis of membranes of the sample portion without detergent was performed by brief sonication in the kit lysis buffer. The lysates were then centrifuged (10,000 × g, 2 min) and supernatants were applied onto columns containing NeutrAvidin gel. After incubation (1 h, 25°C) the nonlabeled proteins were collected in the flow-through. After subsequent washes biotinylated proteins were eluted by adding SDS/PAGE sample buffer containing 50 mM DTT to the columns followed by incubation (1 h, 25°C). The eluates were collected by centrifugation (1,000 × g, 2 min). Immunofluorescence analysis.
The primary antibodies used for the immunohistochemical analyses were as follows: mouse anti-ATF6 (1:100; Imgenex, San Diego), chicken anti-200-kDa neurofilament heavy chain (1:100,000; Abcam, Inc., Cambridge, MA), mouse anti-SOD1 (1:1,000; Santa Cruz Biotechnology), rabbit anti-BiP (1:200; Stressgen), rat anti-BiP (1:50; Santa Cruz Biotechnology), or rabbit anti-Calnexin (1:2,000; Stressgen). Immunolabeling was visualized by the Alexa488-labeled donkey anti-mouse antibody (1:500; Molecular Probes), the rhodamine-labeled goat anti-chicken antibody (1:500, Abcam), the Alexa488-labeled donkey anti-rabbit antibody (1:500; Molecular Probes), the Texas red-labeled goat anti-rabbit antibody (1:500; Vector Laboratories, Burlingame, CA), or the Texas red-labeled goat anti-rat antibody (1:500; Vector Laboratories). Immunoelectron microscopy.
After fixation by intracardial perfusion with cold 0.5% glutaraldehyde and 4% paraformaldehyde in a 0.1 M sodium phosphate buffer, small blocks of tissue were postfixed by immersion in the same fixative (12 h, 4°C), followed by dehydration in alcohols and subsequent embedding in LR White resin (Electron Microscopy Sciences, Hatfield, PA). The spinal cord anterior horn was identified on semithick sections stained with toluidine blue. The ultrathin sections of silver-gold color were collected on nickel grids, preincubated in 10% normal donkey serum in PBS (30 min, 25°C), and subsequently incubated (overnight, 4°C) with a rabbit anti-SOD1 antibody (1:250; Biodesign). After washing with PBS, the grids were incubated (1.5 h, 25°C) with 12-nm immunogold-conjugated secondary antibody (1:40; Jackson ImmunoResearch, West Grove, PA). The grids were stained with uranyl acetate. Preparation of plasmids.
The cDNAs of hSOD1^G93A were amplified by PCR by using an upstream primer (BamSOD) including a BamHI site (5'-CTT GGA TCC GCG ACG AAG GCC GTG-3') and a downstream primer (SODXho) including an XhoI site (5'-ATT CTC GAG TTA TTG GGC GAT CCC-3'). The amplified PCR products were digested with BamHI and XhoI restriction enzymes, and ligated into pGEX-6P-1(Amersham Pharmacia Biosciences). For the cloning of full-length BiP, mRNA derived from spinal cord was amplified by RT-PCR using the following primers: upstream including a BamHI site (5'-CCG GCC GGA TCC GAG GAG GAC AAG AAG GAG GA-3') and downstream including an XhoI site (5'-GGC CGG CTC GAG CTA CAA CTC GAT CTT TTT CTG AT-3'). The amplified PCR product was digested with BamHI and XhoI restriction enzymes and ligated into pGEX-6P-1. For construction of the deletion mutant of BiP for N terminus (D N44), peptide-binding domain (D PBD), and C terminus (D CTT), the inverted PCR was performed. The recombinant plasmid DNAs were transformed into competent cells of Escherichia coli strain BL-21 Star (DE3) (Invitrogen), and the positive clones were amplified. The purified recombinant plasmid DNAs were identified with sequencing. Purification of recombinant proteins.
The amplified bacteria containing the recombinant plasmid with the target protein were induced with 0.1 mM isopropyl-b -D-thiogalactose. The target proteins were expressed as GST fusion proteins and purified using glutathione-Sepharose 4B (Amersham Pharmacia Biosciences) according to the manufacturer’s protocol. The purified fusion proteins were restriction-digested on the beads with PreScission protease (Amersham Pharmacia Biosciences) in a digestion buffer (50 mM Tris· HCl, pH 7.5/150 mM NaCl/1 mM EDTA/1 mM DTT) overnight at 4°C. SOD1 antibody characterization.
The concentration of recombinant human SOD1^WTand SOD1^G93A protein was determined by the BCA method (Pierce Biotechnology) and diluted as shown in Fig. 9. Proteins were subjected to SDS/PAGE and stained with Imperial Protein Stain (Pierce Biotechnology), and duplicated SDS/PAGE was transferred to a nitrocellulose membrane for Western blotting by using a rabbit anti-SOD1 antibody (1:1,000; Biodesign). The bands were quantified by using a FluorChem 8800 digital image system (Alpha Innotech) and then analyzed statistically by GRAPHPAD PRISM 3.0 software (GraphPad, San Diego). In vitro
binding assays. Human SOD1^G93A protein (0.025 nmol) in 200 m l of binding buffer (20 mM Hepes, pH 6.8/100 mM KCl/5 mM MgCl₂/0.5% Nonidet P-40/2% glycerol/1 mM DTT/1 mM EDTA/1 mM ATP/4% BSA) was incubated with glutathione-Sepharose 4B bead-attached GST, GST-BiP, or GST-deletion mutants of BiP (0.2 nmol each) at 4°C overnight. The beads were washed four times with 400 m l of binding buffer and one time with PBS. Pellet was loaded on a 12% SDS/PAGE, and here, blots were probed with a sheep anti-SOD1 antibody (1:3,000; Calbiochem) and a goat anti-GST antibody (1:5,000, Amersham Pharmacia Biosciences). Immunoprecipitation.
Protein concentration was determined by the BCA method (Pierce Biotechnology). A sheep anti-SOD1 antibody (5 m l; Calbiochem) or a rabbit anti-BiP antibody (5 m l; a kind gift from Dr. L. M. Hendershot, St. Jude Children’s Research Hospital, Memphis, TN) was then added to 150 m g of protein extract and incubated overnight at 4°C. The antibody-antigen complex was then precipitated with Protein G beads (3 h, 4°C, with rotation; Amersham Pharmacia Biosciences). Immunoprecipitates were washed three times with 1 ml of 2-[N-morpholino]ethanesulfonic acid (Mes)-buffered solution, 20 m l of sample loading buffer were added to the final pellet, and samples were boiled. The supernatants were analyzed by SDS/PAGE, membranes were probed with a mouse anti-BiP antibody (1:50; BD Transduction Laboratories), and a sheep anti-SOD1 antibody (1:3,000; Calbiochem) or a mouse anti-ATF6 antibody (1:50; Imgenex).