SOD1^mut interacts with Derlin-1. (A) ³⁵S-labeled p97, Ufd1, Npl4, VIMP, or Derlin-1 proteins were incubated with recombinant His-SOD1^wt or -SOD1^G93A proteins and immobilized on Ni-NTA beads (top left). The bottom part of the SDS-PAGE gel was stained with Coomassie brilliant blue dye (CBB). Amounts of incubated ³⁵S-labeled proteins are shown (input). (B) Lysates from HEK293 cells, transfected at the indicated combinations, were analyzed by IP-IB. (C) NSC34 cells were lysed after infection with Ad-Flag-SOD1^wt, Ad-Flag-SOD1^A4V, Ad-Flag-SOD1^G85R, or Ad-Flag-SOD1^G93A for 48 h and analyzed by IP-IB. (D) Extracts from spinal cords of mice were immunoprecipitated with an antibody to SOD1 (IP: SOD1) or control nonimmune antibody (IP: C) and analyzed by IB with antibodies to Derlin-1 and SOD1. The presence of Derlin-1 in the same lysates is shown. (E) Extracts from tissues of SOD1^wt mice and SOD1^G93A mice were analyzed by IB with antibodies to Derlin-1, MAP2, or p38. Interaction between SOD1^mut and Derlin-1 in tissues of SOD1^G93A mice was analyzed by IP-IB. (F) Spinal cord of SOD1^G93A mouse was homogenized and the microsomes were isolated from the post-nuclear and post-mitochondria homogenate. For alkaline extraction, the microsomes were incubated in 100 mM Na₂CO₃ (pH 7.5 or pH 10.9). After the incubation, the microsomes were pelleted and analyzed by IB with antibodies to Derlin-1, SOD1, and p97. Derlin-1 and p97 were used as controls for ER membrane-anchored protein and peripheral protein, respectively. (G) Schematic representation of various mutant forms of Derlin-1. Transmembrane domains of Derlin-1 are shown by black boxes. Identical residues of C-terminal fragment of Derlin family proteins are shaded. (H) HEK293 cells were transfected with various mutant forms of Derlin-1 and stained with antibody to HA. (I–K) Lysates from HEK293 cells, transfected at the indicated combinations, were analyzed by IP-IB.

SOD1^mut activates the IRE1–TRAF2–ASK1 pathway. (A) NSC34 cells were infected with Ad-SOD1^wt, Ad-SOD1^A4V, Ad-SOD1^G85R, or Ad-SOD1^G93A for 48 h. ASK1 activity was measured by in vitro kinase assay (IVK). (GST-SEK1KN^P) Phosphorylated GST-SEK1KN. Interaction between TRAF2 and ASK1 was analyzed by IP with antibody to TRAF2 and IB with antibody to ASK1. The presence of ASK1, TRAF2, and SOD1 in the same lysates is shown. (Lane 1) Kinase activity relative to the amount of ASK1 protein is shown as fold increase compared with control cells. (B) NSC34 cells were infected with adenoviruses and lentivirus at the indicated combinations. SOD1^mut-dependent IRE1–TRAF2–ASK1 complex formation were analyzed by IP-IB. (C) NSC34 cells, transfected with siRNA against Derlin-1 (si1) or nonspecific sequence (Control), were infected with Ad-SOD1^wt or Ad-SOD1^G93A for 48 h or treatment with 10 μM Thapsigargin for 2 h. Activation of IRE1 and ASK1 was analyzed by IP-IB with antibodies to IRE1α and by IVK using GST-SEK1KN as a substrate, respectively. The presence of ASK1, Derlin-1, Derlin-2, SOD1, and p38 in the same lysates is shown. (Lanes 1,5) Kinase activity relative to the amount of ASK1 protein is shown as fold increase compared with control cells. (D,E) NSC34 cells, transfected with siRNA against Derlin-1 (si1), Derlin-1 (si2), or nonspecific sequence (Control), were infected with Ad-SOD1^wt, Ad-SOD1^A4V, Ad-SOD1^G85R, or Ad-SOD1^G93A for 48 h or treatment with 2 μM thapsigargin for 2 h. Xbp-1 mRNA splicing was determined by RT–PCR. Interaction between TRAF2 and ASK1 was analyzed by IP with antibody to TRAF2 and IB with antibody to ASK1. The presence of TRAF2, ASK1, Derlin-1, and Flag-SOD1 in the same lysates is shown.

Schematic representation of the mechanism of SOD1^mut-induced motor neuron death through ER stress. See the text for details.

SOD1^mut triggers ER stress and inhibition of ERAD. (A) NSC34 cells were lysed after infection with Ad-SOD1^wt, Ad-SOD1^A4V, Ad-SOD1^G85R, or Ad-SOD1^G93A for 48 h or treatment with 2.5 μg/mL tunicamycin (a potent inducer of ER stress) for 2 h and analyzed by immunoprecipitation-immunoblotting (IP-IB) with antibodies to IRE1α or PERK. (P-IRE1) Activated IRE1; (P-PERK) activated PERK. The presence of SOD1 in the same lysates is shown. (B) NSC34 cells were transfected with SOD1^wt or SOD1^mut for 48 h. Expression of BiP and CHOP was examined by RT–PCR. (C) NSC34 cells were transfected with NHK and SOD1^wt or SOD1^mut. Cells were pulse-labeled with [³⁵S] methionine and cysteine and chased for the indicated time periods. Cell lysates were immunoprecipitated with antibody to α1AT. (NHK + CHO) Glycosylated NHK; (NHK − CHO) deglycosylated NHK. The relative radioactivities in NHK at different times of chase were calculated and are shown as fold decreases relative to the intensity observed at 0 h chase. (D) NSC34 cells were transfected with Venus^U and SOD1^wt or SOD1^mut. Cells were pulse-labeled with [³⁵S] methionine and cysteine and chased for the indicated time periods. Cell lysates were immunoprecipitated with antibody to GFP. The relative radioactivities in Venus^U at different times of chase were calculated and are shown as fold decreases relative to the intensity observed at 0 h chase.

SOD1^mut attenuates the retro-translocation of ERAD substrate on the components of ERAD. (A) Lysates from HEK293 cells, transfected with Derlin-1-HA, Myc-VIMP, and Flag-SOD1 at the indicated combinations, were analyzed by IP-IB. The presence of Derlin-1-HA and Flag-SOD1 in the same lysates is shown. (B) Lysates from HEK293 cells, transfected at the indicated combinations, were analyzed by IP-IB. The presence of p97 and Myc-SOD1 in the same lysates is shown. (C) Lysates from HEK293 cells, transfected at the indicated combinations, were analyzed by IP-IB. The presence of Derlin-1-HA and Myc-SOD1 in the same lysates is shown. (D) Lysates from HEK293 cells, transfected with NHK, Derlin-1-HA, Myc-VIMP, and Flag-SOD1 at the indicated combinations, were analyzed by IP-IB. The presence of Derlin-1-HA, Myc-VIMP, and Flag-SOD1 in the same lysates is shown. (E) HEK293 cells were transfected with NHK-Flag, Derlin-1-HA, Myc-SOD1, and HA-Ub at the indicated combinations and incubated with 0.25 μM MG132 for 18 h. NHK was immunoprecipitated with antibody to Flag. After incubation with the denaturing buffer containing 1% SDS, NHK was reimmunoprecipitated with antibody to Flag. Samples were immunoblotted with antibodies to HA and Flag. The presence of Derlin-1-HA and Myc-SOD1 in the same lysates is shown. Asterisks denote nonspecific bands and IgG.

Impairment of SOD1^mut–Derlin-1 interaction and deletion of ASK1 mitigate SOD1^mut-induced motor neurotoxicity. (A) Lysates from HEK293 cells, transfected at the indicated combinations, were analyzed by IP-IB. (B) NSC34 cells were infected with Ad-SOD1^wt, Ad-SOD1^G93A, Len-Venus-Derlin-1(CT4)-HA, and Len-Venus-HA at the indicated combinations for 48 h. Activation of IRE1 and PERK was examined as described in Figure 1A. Activation of ASK1 was analyzed by IVK using GST-MKK6KN as substrate. (ASK1^P) Autophosphorylated ASK1. (GST-MKK6KN^P) Phosphorylated GST-MKK6KN. Asterisk denotes nonspecific bands. (C) Wild-type mice spinal cord cultures were infected with lentivirus at the indicated combinations for 72 h. Cultures were fixed and doubly stained with antibodies to nonphosphorylated neurofilament (SMI32) (green) and GFAP (red). (D) ASK1^−/− mice spinal cord cultures were infected as indicated and stained with SMI32 antibody. (E) The percentage of total cell count of SMI32 antibody-positive cells is shown compared with control culture (wild type; n = 3); (ASK1^−/−; n = 5). Values are means ± SE of independent experiments. (*) P < 0.05; (**) P < 0.01; significance calculated by Student’s t-test. (#) P < 0.05 significance calculated by ANOVA. (F) The percentage of total cell count of anti-GFAP antibody-positive cells derived from wild-type mice is shown compared with control culture. Values are means ± SE of three independent experiments. (G) The onset of disease was determined by motor function deficit seen in rota-rod performance in SOD1^G93A mice in the presence or absence (ASK1^−/−) of ASK1. The cumulative probability of onset of rota-rod deficit was not significantly changed in SOD1^G93A/ASK1^−/− mice (n = 10; solid line) compared with SOD1^G93A mice (n = 10; dotted line). Probabilities of survival reveal prolongation of life span of SOD1^G93A/ASK1^−/− mice (n = 20; solid line) compared with SOD1^G93A mice (n = 20; dotted line). The data were analyzed by the Kaplan-Meier life test and by long-rank test. (H) Cresyl violet (Nissl)-stained paraffin sections of ventral horn from lumbar (level L3) spinal cords at end stage (age 34 wk) are shown. (I) Stereological analysis of motor neuron death. Numbers of motor neurons were determined by counting the large Nissl-positive neurons in the ventral horn. Five mice of each group were used for analysis. Thirty-week-old and 34-wk-old mice were sacrificed, and sections of lumbar spinal cord at levels L3, L4, and L5 were counted for each mouse. Values are means ± SE. (*) P < 0.05; (**) P < 0.01; significance calculated by Student’s t-test.