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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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Cell death associated with mutant SOD1 aggregates. Differentiated PC12 cells were transiently transfected with constructs encoding WT-YFP, G85R-YFP, or G93A-YFP. (A) Cell death after SOD1 protein expression. Individual cells were followed for a 48-h period, from 3 to 5 d after transfection, using time-lapse fluorescence microscopy. Cell death was calculated as a percentage of dead cells at the end of the 48-h period, compared with the total number of cells. Two-tailed t test analysis (95% confidence) was used to compare the statistical difference between data sets: ***, P < 0.001; *, P < 0.05. (B) Intensity of G85R-YFP whole cell fluorescence. Whole cell fluorescence, from 3 to 5 d after transfection, was measured in PC12 cells transiently transfected with G85R-YFP. Fluorescence was measured for each time frame (every 3 h). Completed aggregate formation is set as the point of fluorescence saturation and then normalized as the 48-h time-point. n = 17 cells without aggregates and n = 21 cells with aggregates. (C) Life-time of cells after mutant SOD1 aggregate formation. Images of individual cells were taken at 2 to 3 h intervals, during the 48-h period. For cells forming visible aggregates, life-time was determined from the time when aggregates appeared (t = 0) to the time when cell death was observed. For cells that underwent cell death without the presence of visual aggregates, t = 0 was determined 3 d after transfection corresponding to a time when levels of mutant SOD1 RNA and protein levels were readily detected. Normalized life-time, t = 0 to cell death, were depicted against the percentage of dead cells at the indicated times.
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fig5: Cell death associated with mutant SOD1 aggregates. Differentiated PC12 cells were transiently transfected with constructs encoding WT-YFP, G85R-YFP, or G93A-YFP. (A) Cell death after SOD1 protein expression. Individual cells were followed for a 48-h period, from 3 to 5 d after transfection, using time-lapse fluorescence microscopy. Cell death was calculated as a percentage of dead cells at the end of the 48-h period, compared with the total number of cells. Two-tailed t test analysis (95% confidence) was used to compare the statistical difference between data sets: ***, P < 0.001; *, P < 0.05. (B) Intensity of G85R-YFP whole cell fluorescence. Whole cell fluorescence, from 3 to 5 d after transfection, was measured in PC12 cells transiently transfected with G85R-YFP. Fluorescence was measured for each time frame (every 3 h). Completed aggregate formation is set as the point of fluorescence saturation and then normalized as the 48-h time-point. n = 17 cells without aggregates and n = 21 cells with aggregates. (C) Life-time of cells after mutant SOD1 aggregate formation. Images of individual cells were taken at 2 to 3 h intervals, during the 48-h period. For cells forming visible aggregates, life-time was determined from the time when aggregates appeared (t = 0) to the time when cell death was observed. For cells that underwent cell death without the presence of visual aggregates, t = 0 was determined 3 d after transfection corresponding to a time when levels of mutant SOD1 RNA and protein levels were readily detected. Normalized life-time, t = 0 to cell death, were depicted against the percentage of dead cells at the indicated times.

Mentions: To test whether aggregate formation is directly associated with neuronal cell death, differentiated PC12 cells expressing YFP, WT-YFP, G85R-YFP, or G93A-YFP were followed using live-cell time-lapse fluorescence microscopy. Individual cells were monitored, during a 48-h period (days 3 to 5 after transfection) for protein accumulation, aggregate appearance, and cell death, based on morphological change or propidium iodide (PI) staining (Fig. 5 and Video S1 available at http://www.jcb.org/cgi/content/full/jcb.200504050/DC1). Whereas WT-YFP-transfected cells did not form aggregates, the percentage of cells expressing aggregated G85R-YFP or G93A-YFP after 3 d of expression was 30 and 15%, respectively. Within the ensuing 48-h period, 15.3 ± 3.6% of nontransfected PC12 cells and 14.5 ± 3.5% of WT-YFP–transfected cells underwent cell death, whereas ∼90% (88.5 ± 1.2% for G85R-YFP and 90.3 ± 4.1% for G93A-YFP) of cells expressing a mutant SOD1 aggregate subsequently died (Fig. 5 A).


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)

Cell death associated with mutant SOD1 aggregates. Differentiated PC12 cells were transiently transfected with constructs encoding WT-YFP, G85R-YFP, or G93A-YFP. (A) Cell death after SOD1 protein expression. Individual cells were followed for a 48-h period, from 3 to 5 d after transfection, using time-lapse fluorescence microscopy. Cell death was calculated as a percentage of dead cells at the end of the 48-h period, compared with the total number of cells. Two-tailed t test analysis (95% confidence) was used to compare the statistical difference between data sets: ***, P < 0.001; *, P < 0.05. (B) Intensity of G85R-YFP whole cell fluorescence. Whole cell fluorescence, from 3 to 5 d after transfection, was measured in PC12 cells transiently transfected with G85R-YFP. Fluorescence was measured for each time frame (every 3 h). Completed aggregate formation is set as the point of fluorescence saturation and then normalized as the 48-h time-point. n = 17 cells without aggregates and n = 21 cells with aggregates. (C) Life-time of cells after mutant SOD1 aggregate formation. Images of individual cells were taken at 2 to 3 h intervals, during the 48-h period. For cells forming visible aggregates, life-time was determined from the time when aggregates appeared (t = 0) to the time when cell death was observed. For cells that underwent cell death without the presence of visual aggregates, t = 0 was determined 3 d after transfection corresponding to a time when levels of mutant SOD1 RNA and protein levels were readily detected. Normalized life-time, t = 0 to cell death, were depicted against the percentage of dead cells at the indicated times.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171239&req=5

fig5: Cell death associated with mutant SOD1 aggregates. Differentiated PC12 cells were transiently transfected with constructs encoding WT-YFP, G85R-YFP, or G93A-YFP. (A) Cell death after SOD1 protein expression. Individual cells were followed for a 48-h period, from 3 to 5 d after transfection, using time-lapse fluorescence microscopy. Cell death was calculated as a percentage of dead cells at the end of the 48-h period, compared with the total number of cells. Two-tailed t test analysis (95% confidence) was used to compare the statistical difference between data sets: ***, P < 0.001; *, P < 0.05. (B) Intensity of G85R-YFP whole cell fluorescence. Whole cell fluorescence, from 3 to 5 d after transfection, was measured in PC12 cells transiently transfected with G85R-YFP. Fluorescence was measured for each time frame (every 3 h). Completed aggregate formation is set as the point of fluorescence saturation and then normalized as the 48-h time-point. n = 17 cells without aggregates and n = 21 cells with aggregates. (C) Life-time of cells after mutant SOD1 aggregate formation. Images of individual cells were taken at 2 to 3 h intervals, during the 48-h period. For cells forming visible aggregates, life-time was determined from the time when aggregates appeared (t = 0) to the time when cell death was observed. For cells that underwent cell death without the presence of visual aggregates, t = 0 was determined 3 d after transfection corresponding to a time when levels of mutant SOD1 RNA and protein levels were readily detected. Normalized life-time, t = 0 to cell death, were depicted against the percentage of dead cells at the indicated times.
Mentions: To test whether aggregate formation is directly associated with neuronal cell death, differentiated PC12 cells expressing YFP, WT-YFP, G85R-YFP, or G93A-YFP were followed using live-cell time-lapse fluorescence microscopy. Individual cells were monitored, during a 48-h period (days 3 to 5 after transfection) for protein accumulation, aggregate appearance, and cell death, based on morphological change or propidium iodide (PI) staining (Fig. 5 and Video S1 available at http://www.jcb.org/cgi/content/full/jcb.200504050/DC1). Whereas WT-YFP-transfected cells did not form aggregates, the percentage of cells expressing aggregated G85R-YFP or G93A-YFP after 3 d of expression was 30 and 15%, respectively. Within the ensuing 48-h period, 15.3 ± 3.6% of nontransfected PC12 cells and 14.5 ± 3.5% of WT-YFP–transfected cells underwent cell death, whereas ∼90% (88.5 ± 1.2% for G85R-YFP and 90.3 ± 4.1% for G93A-YFP) of cells expressing a mutant SOD1 aggregate subsequently died (Fig. 5 A).

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