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Structural properties and neuronal toxicity of amyotrophic lateral sclerosis-associated Cu/Zn superoxide dismutase 1 aggregates.

Matsumoto G, Stojanovic A, Holmberg CI, Kim S, Morimoto RI - J. Cell Biol. (2005)

Bottom Line: In contrast, the proteasome is sequestered within the aggregate structure, an event associated with decreased degradation of a proteasomal substrate.Through the use of time-lapse microscopy of individual cells, we show that nearly all (90%) aggregate-containing cells express higher levels of mutant SOD1 and died within 48 h, whereas 70% of cells expressing a soluble mutant SOD1 survived.Our results demonstrate that SOD1 G85R and G93A mutants form a distinct class of aggregate structures in cells destined for neuronal cell death.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Molecular Biology and Cell Biology, Rice Institute for Biomedical Research, Northwestern University, Evanston, IL 60208, USA.

ABSTRACT
The appearance of protein aggregates is a characteristic of protein misfolding disorders including familial amyotrophic lateral sclerosis, a neurodegenerative disease caused by inherited mutations in Cu/Zn superoxide dismutase 1 (SOD1). Here, we use live cell imaging of neuronal and nonneuronal cells to show that SOD1 mutants (G85R and G93A) form an aggregate structure consisting of immobile scaffolds, through which noninteracting cellular proteins can diffuse. Hsp70 transiently interacts, in a chaperone activity-dependent manner, with these mutant SOD1 aggregate structures. In contrast, the proteasome is sequestered within the aggregate structure, an event associated with decreased degradation of a proteasomal substrate. Through the use of time-lapse microscopy of individual cells, we show that nearly all (90%) aggregate-containing cells express higher levels of mutant SOD1 and died within 48 h, whereas 70% of cells expressing a soluble mutant SOD1 survived. Our results demonstrate that SOD1 G85R and G93A mutants form a distinct class of aggregate structures in cells destined for neuronal cell death.

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Mutant SOD1 forms aggregates containing mobile and immobile fractions. (A–H) Differentiated PC12 cells were transiently transfected for 3 d with constructs encoding WT-CFP/YFP, G85R-CFP/YFP, or G93A-CFP/YFP, as indicated. (A) Western analysis of SOD1 proteins. Whole cells extracts were harvested and analyzed by Western analysis. CFP-tagged proteins were detected with an anti-GFP antibody and γ-tubulin was used as a loading control. G85R migrates slightly faster than WT and G93A, as previously described (Bruijn et al., 1998). The anti-GFP antibody detects no obvious free CFP molecules. (B) SOD1 activity assay. Whole cell extracts were subjected to native-PAGE and SOD1 activities were detected by NBT-negative staining. The activities of both transfected and endogenous SOD1 are detected, as indicated. (C) Visualization of SOD1 proteins. CFP fusion proteins (CFP, cyan) were visualized by confocal microscopy and phase contrast microscopy (DIC). White arrows indicate aggregates. Images have been cropped to enlarge the cell body; an image showing neurites, a marker for neuronal differentiation, is found in Fig. S1. (D) Filter trap assay of SOD1 proteins. Whole cell extracts were treated with 1% SDS and subjected to filter trap analysis on a cellulose acetate membrane. Proteins that are retained on the membrane were detected with an anti-GFP antibody. (E) FRAP analysis of SOD1 proteins. Single scan images were obtained before photobleaching (Pre) of a region of interest (ROI; white box) and at the indicated times after photobleaching. Arrows indicate the photobleached area. Note that the intensity of the images is scaled differently between the samples. (F) Quantitative FRAP analysis of SOD1 proteins. The relative fluorescence intensity (RFI) was determined at each time point and is represented as the mean ± SEM (n ≥ 10 cells). (G) FLIP analysis of SOD1 proteins. Single scan images of a diffuse (open arrow) and aggregated region (closed arrow) were obtained before (Pre) and at the indicated times during continuous photobleaching of a region (white box). (H) Quantitative FLIP analysis of SOD1 proteins. The RFI was determined at each time point and is represented as the mean ± SEM (n = 5–10 cells). Bars, 10 μm.
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fig1: Mutant SOD1 forms aggregates containing mobile and immobile fractions. (A–H) Differentiated PC12 cells were transiently transfected for 3 d with constructs encoding WT-CFP/YFP, G85R-CFP/YFP, or G93A-CFP/YFP, as indicated. (A) Western analysis of SOD1 proteins. Whole cells extracts were harvested and analyzed by Western analysis. CFP-tagged proteins were detected with an anti-GFP antibody and γ-tubulin was used as a loading control. G85R migrates slightly faster than WT and G93A, as previously described (Bruijn et al., 1998). The anti-GFP antibody detects no obvious free CFP molecules. (B) SOD1 activity assay. Whole cell extracts were subjected to native-PAGE and SOD1 activities were detected by NBT-negative staining. The activities of both transfected and endogenous SOD1 are detected, as indicated. (C) Visualization of SOD1 proteins. CFP fusion proteins (CFP, cyan) were visualized by confocal microscopy and phase contrast microscopy (DIC). White arrows indicate aggregates. Images have been cropped to enlarge the cell body; an image showing neurites, a marker for neuronal differentiation, is found in Fig. S1. (D) Filter trap assay of SOD1 proteins. Whole cell extracts were treated with 1% SDS and subjected to filter trap analysis on a cellulose acetate membrane. Proteins that are retained on the membrane were detected with an anti-GFP antibody. (E) FRAP analysis of SOD1 proteins. Single scan images were obtained before photobleaching (Pre) of a region of interest (ROI; white box) and at the indicated times after photobleaching. Arrows indicate the photobleached area. Note that the intensity of the images is scaled differently between the samples. (F) Quantitative FRAP analysis of SOD1 proteins. The relative fluorescence intensity (RFI) was determined at each time point and is represented as the mean ± SEM (n ≥ 10 cells). (G) FLIP analysis of SOD1 proteins. Single scan images of a diffuse (open arrow) and aggregated region (closed arrow) were obtained before (Pre) and at the indicated times during continuous photobleaching of a region (white box). (H) Quantitative FLIP analysis of SOD1 proteins. The RFI was determined at each time point and is represented as the mean ± SEM (n = 5–10 cells). Bars, 10 μm.

Mentions: We monitored the in vivo dynamics of wild-type (WT) and mutant SOD1 proteins, as well as their molecular interactions in differentiated PC12 neuronal cells and HeLa cells, by expressing CFP and YFP fusions with WT (WT-CFP and WT-YFP) and mutant (G85R-CFP, G85R-YFP, G93A-CFP, G93A-YFP) SOD1 proteins. WT, G85R, and G93A fused to CFP (Fig. 1 A) or YFP (unpublished data) accumulated to equivalent levels in transiently transfected PC12 cells and both mutant proteins were detected as full-length products of expected molecular sizes. A similar level of protein accumulation was also detected in HeLa cells (unpublished data). WT-CFP and G93A-CFP displayed SOD1 enzymatic activity and inhibited the reduction of nitroblue tetrazolium (NBT) by superoxide, whereas G85R-CFP did not exhibit detectable SOD1 activity (Pasinelli et al., 1998) in either PC12 (Fig. 1 B) or HeLa cells (unpublished data). The subcellular localization of WT-CFP, G85R-CFP, and G93A-CFP was visualized in differentiated PC12 (Fig. 1 C) and HeLa cells (Fig. S1 available at http://www.jcb.org/cgi/content/full/jcb.200504050/DC1). Neuronal processes, characteristic of differentiated PC12 cells, are not apparent in these images and are visualized at lower magnification (Fig. S1). WT-CFP exhibited a diffuse pattern of protein localization throughout the cell, whereas large perinuclear aggregates were detected in cells expressing G85R-CFP and G93A-CFP. As a complement to the visual identification of aggregates, we performed a cellulose acetate filter retardation assay (Wanker et al., 1999; Wang et al., 2002). Whereas WT-CFP was soluble, both mutant proteins were trapped as 1% SDS-insoluble species, in PC12 (Fig. 1 D) and HeLa cells (unpublished data), indicative of aggregate formation (Wanker et al., 1999; Wang et al., 2002). From these results, we conclude that both mutant SOD1-YFP/CFP proteins exhibit a similar aggregation phenotype, whether expressed in PC12 neuronal cells or HeLa cells.


Structural properties and neuronal toxicity of amyotrophic lateral sclerosis-associated Cu/Zn superoxide dismutase 1 aggregates.

Matsumoto G, Stojanovic A, Holmberg CI, Kim S, Morimoto RI - J. Cell Biol. (2005)

Mutant SOD1 forms aggregates containing mobile and immobile fractions. (A–H) Differentiated PC12 cells were transiently transfected for 3 d with constructs encoding WT-CFP/YFP, G85R-CFP/YFP, or G93A-CFP/YFP, as indicated. (A) Western analysis of SOD1 proteins. Whole cells extracts were harvested and analyzed by Western analysis. CFP-tagged proteins were detected with an anti-GFP antibody and γ-tubulin was used as a loading control. G85R migrates slightly faster than WT and G93A, as previously described (Bruijn et al., 1998). The anti-GFP antibody detects no obvious free CFP molecules. (B) SOD1 activity assay. Whole cell extracts were subjected to native-PAGE and SOD1 activities were detected by NBT-negative staining. The activities of both transfected and endogenous SOD1 are detected, as indicated. (C) Visualization of SOD1 proteins. CFP fusion proteins (CFP, cyan) were visualized by confocal microscopy and phase contrast microscopy (DIC). White arrows indicate aggregates. Images have been cropped to enlarge the cell body; an image showing neurites, a marker for neuronal differentiation, is found in Fig. S1. (D) Filter trap assay of SOD1 proteins. Whole cell extracts were treated with 1% SDS and subjected to filter trap analysis on a cellulose acetate membrane. Proteins that are retained on the membrane were detected with an anti-GFP antibody. (E) FRAP analysis of SOD1 proteins. Single scan images were obtained before photobleaching (Pre) of a region of interest (ROI; white box) and at the indicated times after photobleaching. Arrows indicate the photobleached area. Note that the intensity of the images is scaled differently between the samples. (F) Quantitative FRAP analysis of SOD1 proteins. The relative fluorescence intensity (RFI) was determined at each time point and is represented as the mean ± SEM (n ≥ 10 cells). (G) FLIP analysis of SOD1 proteins. Single scan images of a diffuse (open arrow) and aggregated region (closed arrow) were obtained before (Pre) and at the indicated times during continuous photobleaching of a region (white box). (H) Quantitative FLIP analysis of SOD1 proteins. The RFI was determined at each time point and is represented as the mean ± SEM (n = 5–10 cells). Bars, 10 μm.
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fig1: Mutant SOD1 forms aggregates containing mobile and immobile fractions. (A–H) Differentiated PC12 cells were transiently transfected for 3 d with constructs encoding WT-CFP/YFP, G85R-CFP/YFP, or G93A-CFP/YFP, as indicated. (A) Western analysis of SOD1 proteins. Whole cells extracts were harvested and analyzed by Western analysis. CFP-tagged proteins were detected with an anti-GFP antibody and γ-tubulin was used as a loading control. G85R migrates slightly faster than WT and G93A, as previously described (Bruijn et al., 1998). The anti-GFP antibody detects no obvious free CFP molecules. (B) SOD1 activity assay. Whole cell extracts were subjected to native-PAGE and SOD1 activities were detected by NBT-negative staining. The activities of both transfected and endogenous SOD1 are detected, as indicated. (C) Visualization of SOD1 proteins. CFP fusion proteins (CFP, cyan) were visualized by confocal microscopy and phase contrast microscopy (DIC). White arrows indicate aggregates. Images have been cropped to enlarge the cell body; an image showing neurites, a marker for neuronal differentiation, is found in Fig. S1. (D) Filter trap assay of SOD1 proteins. Whole cell extracts were treated with 1% SDS and subjected to filter trap analysis on a cellulose acetate membrane. Proteins that are retained on the membrane were detected with an anti-GFP antibody. (E) FRAP analysis of SOD1 proteins. Single scan images were obtained before photobleaching (Pre) of a region of interest (ROI; white box) and at the indicated times after photobleaching. Arrows indicate the photobleached area. Note that the intensity of the images is scaled differently between the samples. (F) Quantitative FRAP analysis of SOD1 proteins. The relative fluorescence intensity (RFI) was determined at each time point and is represented as the mean ± SEM (n ≥ 10 cells). (G) FLIP analysis of SOD1 proteins. Single scan images of a diffuse (open arrow) and aggregated region (closed arrow) were obtained before (Pre) and at the indicated times during continuous photobleaching of a region (white box). (H) Quantitative FLIP analysis of SOD1 proteins. The RFI was determined at each time point and is represented as the mean ± SEM (n = 5–10 cells). Bars, 10 μm.
Mentions: We monitored the in vivo dynamics of wild-type (WT) and mutant SOD1 proteins, as well as their molecular interactions in differentiated PC12 neuronal cells and HeLa cells, by expressing CFP and YFP fusions with WT (WT-CFP and WT-YFP) and mutant (G85R-CFP, G85R-YFP, G93A-CFP, G93A-YFP) SOD1 proteins. WT, G85R, and G93A fused to CFP (Fig. 1 A) or YFP (unpublished data) accumulated to equivalent levels in transiently transfected PC12 cells and both mutant proteins were detected as full-length products of expected molecular sizes. A similar level of protein accumulation was also detected in HeLa cells (unpublished data). WT-CFP and G93A-CFP displayed SOD1 enzymatic activity and inhibited the reduction of nitroblue tetrazolium (NBT) by superoxide, whereas G85R-CFP did not exhibit detectable SOD1 activity (Pasinelli et al., 1998) in either PC12 (Fig. 1 B) or HeLa cells (unpublished data). The subcellular localization of WT-CFP, G85R-CFP, and G93A-CFP was visualized in differentiated PC12 (Fig. 1 C) and HeLa cells (Fig. S1 available at http://www.jcb.org/cgi/content/full/jcb.200504050/DC1). Neuronal processes, characteristic of differentiated PC12 cells, are not apparent in these images and are visualized at lower magnification (Fig. S1). WT-CFP exhibited a diffuse pattern of protein localization throughout the cell, whereas large perinuclear aggregates were detected in cells expressing G85R-CFP and G93A-CFP. As a complement to the visual identification of aggregates, we performed a cellulose acetate filter retardation assay (Wanker et al., 1999; Wang et al., 2002). Whereas WT-CFP was soluble, both mutant proteins were trapped as 1% SDS-insoluble species, in PC12 (Fig. 1 D) and HeLa cells (unpublished data), indicative of aggregate formation (Wanker et al., 1999; Wang et al., 2002). From these results, we conclude that both mutant SOD1-YFP/CFP proteins exhibit a similar aggregation phenotype, whether expressed in PC12 neuronal cells or HeLa cells.

Bottom Line: In contrast, the proteasome is sequestered within the aggregate structure, an event associated with decreased degradation of a proteasomal substrate.Through the use of time-lapse microscopy of individual cells, we show that nearly all (90%) aggregate-containing cells express higher levels of mutant SOD1 and died within 48 h, whereas 70% of cells expressing a soluble mutant SOD1 survived.Our results demonstrate that SOD1 G85R and G93A mutants form a distinct class of aggregate structures in cells destined for neuronal cell death.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Molecular Biology and Cell Biology, Rice Institute for Biomedical Research, Northwestern University, Evanston, IL 60208, USA.

ABSTRACT
The appearance of protein aggregates is a characteristic of protein misfolding disorders including familial amyotrophic lateral sclerosis, a neurodegenerative disease caused by inherited mutations in Cu/Zn superoxide dismutase 1 (SOD1). Here, we use live cell imaging of neuronal and nonneuronal cells to show that SOD1 mutants (G85R and G93A) form an aggregate structure consisting of immobile scaffolds, through which noninteracting cellular proteins can diffuse. Hsp70 transiently interacts, in a chaperone activity-dependent manner, with these mutant SOD1 aggregate structures. In contrast, the proteasome is sequestered within the aggregate structure, an event associated with decreased degradation of a proteasomal substrate. Through the use of time-lapse microscopy of individual cells, we show that nearly all (90%) aggregate-containing cells express higher levels of mutant SOD1 and died within 48 h, whereas 70% of cells expressing a soluble mutant SOD1 survived. Our results demonstrate that SOD1 G85R and G93A mutants form a distinct class of aggregate structures in cells destined for neuronal cell death.

Show MeSH
Related in: MedlinePlus