Results: 5

1.
Fig. 5.

Fig. 5. From: An over-oxidized form of superoxide dismutase found in sporadic amyotrophic lateral sclerosis with bulbar onset shares a toxic mechanism with mutant SOD1.

H2O2-induced formation of the toxic iperOxSOD1/Bcl-2 complex triggers mitochondrial damage in patient cells. Control and iperOxSOD1-sALS cells treated for 2 h with 100 μM H2O2 underwent morphological evaluation by transmission electron microscopy. IperOxSOD1-sALS cells show derangement of mitochondria matrix and disappearance of morphological structure.

Stefania Guareschi, et al. Proc Natl Acad Sci U S A. 2012 March 27;109(13):5074-5079.
2.
Fig. 3.

Fig. 3. From: An over-oxidized form of superoxide dismutase found in sporadic amyotrophic lateral sclerosis with bulbar onset shares a toxic mechanism with mutant SOD1.

Like mutSOD1, iperOxSOD1 colocalizes and aggregates with Bcl-2 in patient cells. Colocalization of SOD1 and Bcl-2 was evaluated by immunofluorescence with rabbit anti-SOD1 (A–C) and mouse anti-Bcl-2 (D–F) antibodies. The merging signals show colocalization of the two proteins within the aggregates in iperOxSOD1-sALS and mutSOD1-fALS patients after H2O2 treatment (G and H, arrows). sALS lymphoblasts with no iperOxSOD1 and no SOD1/Bcl-2 high molecular weight aggregates did not demonstrate colocalization of the two proteins (C, F, and I).

Stefania Guareschi, et al. Proc Natl Acad Sci U S A. 2012 March 27;109(13):5074-5079.
3.
Fig. 1.

Fig. 1. From: An over-oxidized form of superoxide dismutase found in sporadic amyotrophic lateral sclerosis with bulbar onset shares a toxic mechanism with mutant SOD1.

Identification of an iper-oxidized SOD1 in a subset of sALS patients. (A) Representative immunoblot of SOD1 oxidation detected by a derivatization assay of carbonyl compounds. Proteins extracted from sALS, fALS, and healthy control lymphoblasts, before and after H2O2 treatment, were precipitated with rabbit anti-SOD1 antibody and analyzed by Western blot using anti-DNPH (Upper) and sheep anti-SOD1 (Lower) antibodies. (B) Ratio of DNPH and precipitated SOD1. For each patient's line, samples were analyzed in triplicate in three independent blots (with and without H2O2), and densitometric analysis was performed on each blot. The graph represents the mean ± SD SOD1 oxidation for each experimental group. ANOVA revealed significantly higher oxidized SOD1 in a group of sALS lymphoblasts compared with lymphoblasts from mutSOD1-fALS and healthy controls (*P < 0.05). (C) Representative blot of derivatization assay of carbonyl compounds on total protein content from fALS, sALS, and healthy control lymphoblasts before and after H2O2 treatment, showing no differences in total oxidation levels.

Stefania Guareschi, et al. Proc Natl Acad Sci U S A. 2012 March 27;109(13):5074-5079.
4.
Fig. 2.

Fig. 2. From: An over-oxidized form of superoxide dismutase found in sporadic amyotrophic lateral sclerosis with bulbar onset shares a toxic mechanism with mutant SOD1.

Like mutSOD1, iperOxSOD1 aggregates in patient cells. (A) SOD1 appearance in lymphoblasts of sALS, mutSOD1-fALS, and controls analyzed by immunofluorescence using a rabbit antiSOD1 antibody. Confocal experiments revealed SOD1-positive aggregates (arrows) in both mutSOD1-fALS and iperOxSOD1-sALS patients before (a and c) and after (b and d) H2O2 treatment (100 μM, 2 h). Control and sALS lymphoblasts exhibited SOD1 aggregation only after H2O2 treatment (f–h). (B) Percentage (± SD) of cells containing SOD1-positive aggregates before and after H2O2 treatment. Counting was done selecting seven different fields for each group. MutSOD1-fALS and iperOxSOD1-sALS patients showed a significantly higher percentage of cells containing aggregates compared with both remaining sALS and control patients (P < 0.0001). (C) Representative blots showing the aberrant bond of SOD1 and Bcl-2 in mutSOD1-fALS and iperOxSOD1-sALS patients (arrows). Proteins extracted from fALS, iperOxSOD1-sALS, and control lymphoblasts treated with 100 μM H2O2 were precipitated as above, and membranes were stained with mouse anti–Bcl-2 antibody (Upper). SOD1 precipitation was confirmed by restaining the blot with a sheep anti-SOD1 antibody (Lower). After H2O2 treatment, high molecular weight aggregates positive for both Bcl-2 (Upper) and SOD1 (Lower) were detected in mutSOD1-fALS and iperOxSOD1-sALS patients, but not in healthy controls (or the remaining sALS patients). The SOD1/Bcl-2 complex was recovered in the presence of the precipitating antibody, but not with the corresponding IgGs, indicating specificity of the binding. TL, total lysate.

Stefania Guareschi, et al. Proc Natl Acad Sci U S A. 2012 March 27;109(13):5074-5079.
5.
Fig. 4.

Fig. 4. From: An over-oxidized form of superoxide dismutase found in sporadic amyotrophic lateral sclerosis with bulbar onset shares a toxic mechanism with mutant SOD1.

oxSOD1-mediated toxicity requires binding with Bcl-2 and induces a toxic rearrangement of Bcl-2 structure. (A) In isolated mitochondria, recombinant oxSOD1 induces cytochrome C release only in the presence of Bcl-2. Bcl-2–negative or Bcl-2–positive mitochondria isolated from HEK293T cells were incubated with 5 μL of recombinant oxSOD1 for 30 min. Cytochrome C immunoreactivity was measured in supernatants by ELISA. OxSOD1, but not WT SOD1, caused an increase in cytochrome C release from damaged mitochondria. Data are mean ± SD of three independent experiments performed in duplicate (P < 0.05) with CaCl2 as a positive control for maximal cytochrome C release. (B) Representative blot showing that Bcl-2 is conformationally modified in lymphoblasts of iperOxSOD1-sALS and mutSOD1-fALS patients. In mutSOD1-fALS and iperOxSOD1-sALS lymphoblasts, there is increased exposure of the toxic BH3 domain paralleled by decreased exposure of the pocket region, indicating a conformational change in the Bcl-2 protein. (C) Densitometric analysis of the BH3/pocket ratio. Each patient's line was analyzed in three independent experiments. The graph represents BH3 and pocket exposure normalized on the total amount of Bcl-2 in all samples. The BH3/pocket ratio was significantly higher in mutSOD1-fALS and iperOxSOD1s-ALS lymphoblasts compared with control and other sALS lymphoblasts. *P < 0.05; **P < 0.0001. (D) Exposure of BH3 domain also was assessed by flow cytometry using the conformation-specific anti-BH3 antibody. The shift in fluorescence of mutSOD1-fALS and iperOxSOD1-sALS lymphoblasts compared with controls indicates increased exposure of the BH3 domain.

Stefania Guareschi, et al. Proc Natl Acad Sci U S A. 2012 March 27;109(13):5074-5079.

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