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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 aggregates sequester the proteasome and impair its activity. (A–D) Differentiated PC12 cells were transiently transfected with constructs encoding LMP2-YFP and G85R-CFP. (A) FRAP analysis of LMP2-YFP. Single scan images were obtained before photobleaching (Pre) of a ROI (white or black box) and at the indicated times after photobleaching. Arrows indicate the photobleached area. (B) Quantitative FRAP analysis of LMP2-YFP. The RFI was determined at each time point and is represented as the mean ± SEM (n ≥ 10 cells). (C) FLIP analysis of LMP2-YFP. 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). (D) Quantitative FLIP analysis of LMP2-YFP. The RFI was determined at each time point and is represented as the mean ± SEM (n = 5–10 cells). Note: 62.3 ± 3.7% of total LMP2-YFP fluorescence intensity is associated with the G85R-CFP aggregate (n = 7 cells; see Materials and methods). (E and F) Stable differentiated Ubi-YFP/PC12 cells were transiently transfected with constructs encoding WT-CFP, G85R-CFP, or G93A-CFP. (E) Accumulation of a proteasomal substrate. Ubi-YFP (YFP, yellow) and CFP fusion proteins (CFP, cyan) were visualized by fluorescence microscopy. Mutant SOD1 protein aggregates (closed arrows) and diffuse SOD1 (open arrows) are indicated. (F) Quantification of Ubi-YFP fluorescence. The fold increase of Ubi-YFP intensity compared with the mean intensity of nontransfected cells is represented as the mean ± SEM (n = 28–56 cells). Bars, 10 μm. Two-tailed t test analysis (95% confidence) was used to compare the statistical difference between data sets: ***, P < 0.001; *, P < 0.05; ns, P > 0.05.
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fig4: Mutant SOD1 aggregates sequester the proteasome and impair its activity. (A–D) Differentiated PC12 cells were transiently transfected with constructs encoding LMP2-YFP and G85R-CFP. (A) FRAP analysis of LMP2-YFP. Single scan images were obtained before photobleaching (Pre) of a ROI (white or black box) and at the indicated times after photobleaching. Arrows indicate the photobleached area. (B) Quantitative FRAP analysis of LMP2-YFP. The RFI was determined at each time point and is represented as the mean ± SEM (n ≥ 10 cells). (C) FLIP analysis of LMP2-YFP. 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). (D) Quantitative FLIP analysis of LMP2-YFP. The RFI was determined at each time point and is represented as the mean ± SEM (n = 5–10 cells). Note: 62.3 ± 3.7% of total LMP2-YFP fluorescence intensity is associated with the G85R-CFP aggregate (n = 7 cells; see Materials and methods). (E and F) Stable differentiated Ubi-YFP/PC12 cells were transiently transfected with constructs encoding WT-CFP, G85R-CFP, or G93A-CFP. (E) Accumulation of a proteasomal substrate. Ubi-YFP (YFP, yellow) and CFP fusion proteins (CFP, cyan) were visualized by fluorescence microscopy. Mutant SOD1 protein aggregates (closed arrows) and diffuse SOD1 (open arrows) are indicated. (F) Quantification of Ubi-YFP fluorescence. The fold increase of Ubi-YFP intensity compared with the mean intensity of nontransfected cells is represented as the mean ± SEM (n = 28–56 cells). Bars, 10 μm. Two-tailed t test analysis (95% confidence) was used to compare the statistical difference between data sets: ***, P < 0.001; *, P < 0.05; ns, P > 0.05.

Mentions: FRAP data from Figs. 1F, 3B, and 4B were analyzed with GraphPad Prism software, as described in Supplementary Information, Methods online. Data are displayed as mean ± SEM. Two-tailed t test analysis (95% confidence) was used to compare the statistical difference between data sets: ***, P < 0.001; ns, P > 0.05.


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 aggregates sequester the proteasome and impair its activity. (A–D) Differentiated PC12 cells were transiently transfected with constructs encoding LMP2-YFP and G85R-CFP. (A) FRAP analysis of LMP2-YFP. Single scan images were obtained before photobleaching (Pre) of a ROI (white or black box) and at the indicated times after photobleaching. Arrows indicate the photobleached area. (B) Quantitative FRAP analysis of LMP2-YFP. The RFI was determined at each time point and is represented as the mean ± SEM (n ≥ 10 cells). (C) FLIP analysis of LMP2-YFP. 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). (D) Quantitative FLIP analysis of LMP2-YFP. The RFI was determined at each time point and is represented as the mean ± SEM (n = 5–10 cells). Note: 62.3 ± 3.7% of total LMP2-YFP fluorescence intensity is associated with the G85R-CFP aggregate (n = 7 cells; see Materials and methods). (E and F) Stable differentiated Ubi-YFP/PC12 cells were transiently transfected with constructs encoding WT-CFP, G85R-CFP, or G93A-CFP. (E) Accumulation of a proteasomal substrate. Ubi-YFP (YFP, yellow) and CFP fusion proteins (CFP, cyan) were visualized by fluorescence microscopy. Mutant SOD1 protein aggregates (closed arrows) and diffuse SOD1 (open arrows) are indicated. (F) Quantification of Ubi-YFP fluorescence. The fold increase of Ubi-YFP intensity compared with the mean intensity of nontransfected cells is represented as the mean ± SEM (n = 28–56 cells). Bars, 10 μm. Two-tailed t test analysis (95% confidence) was used to compare the statistical difference between data sets: ***, P < 0.001; *, P < 0.05; ns, P > 0.05.
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Related In: Results  -  Collection

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fig4: Mutant SOD1 aggregates sequester the proteasome and impair its activity. (A–D) Differentiated PC12 cells were transiently transfected with constructs encoding LMP2-YFP and G85R-CFP. (A) FRAP analysis of LMP2-YFP. Single scan images were obtained before photobleaching (Pre) of a ROI (white or black box) and at the indicated times after photobleaching. Arrows indicate the photobleached area. (B) Quantitative FRAP analysis of LMP2-YFP. The RFI was determined at each time point and is represented as the mean ± SEM (n ≥ 10 cells). (C) FLIP analysis of LMP2-YFP. 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). (D) Quantitative FLIP analysis of LMP2-YFP. The RFI was determined at each time point and is represented as the mean ± SEM (n = 5–10 cells). Note: 62.3 ± 3.7% of total LMP2-YFP fluorescence intensity is associated with the G85R-CFP aggregate (n = 7 cells; see Materials and methods). (E and F) Stable differentiated Ubi-YFP/PC12 cells were transiently transfected with constructs encoding WT-CFP, G85R-CFP, or G93A-CFP. (E) Accumulation of a proteasomal substrate. Ubi-YFP (YFP, yellow) and CFP fusion proteins (CFP, cyan) were visualized by fluorescence microscopy. Mutant SOD1 protein aggregates (closed arrows) and diffuse SOD1 (open arrows) are indicated. (F) Quantification of Ubi-YFP fluorescence. The fold increase of Ubi-YFP intensity compared with the mean intensity of nontransfected cells is represented as the mean ± SEM (n = 28–56 cells). Bars, 10 μm. Two-tailed t test analysis (95% confidence) was used to compare the statistical difference between data sets: ***, P < 0.001; *, P < 0.05; ns, P > 0.05.
Mentions: FRAP data from Figs. 1F, 3B, and 4B were analyzed with GraphPad Prism software, as described in Supplementary Information, Methods online. Data are displayed as mean ± SEM. Two-tailed t test analysis (95% confidence) was used to compare the statistical difference between data sets: ***, P < 0.001; ns, P > 0.05.

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