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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 form a disordered honeycomb-like porous structure. (A–C) Differentiated PC12 cells were transiently transfected with constructs encoding YFP together with G85R-CFP, G93A-CFP, or httQ78-CFP, as indicated. (A) Colocalization of YFP with G85R-CFP, G93A-CFP, or httQ78-CFP aggregates. YFP (YFP, yellow) and CFP fusion protein (CFP, cyan) were visualized by confocal microscopy and phase contrast microscopy (DIC). Colocalization was illustrated by merging YFP and CFP channels (Merge). Arrows indicate protein aggregates. (B and C) Differentiated PC12 cells were transiently transfected with constructs encoding YFP together with G85R-CFP or G93A-CFP, as indicated. (B) FLIP analysis of 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 (black box). (C) Quantitative FLIP analysis of YFP. 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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fig2: Mutant SOD1 aggregates form a disordered honeycomb-like porous structure. (A–C) Differentiated PC12 cells were transiently transfected with constructs encoding YFP together with G85R-CFP, G93A-CFP, or httQ78-CFP, as indicated. (A) Colocalization of YFP with G85R-CFP, G93A-CFP, or httQ78-CFP aggregates. YFP (YFP, yellow) and CFP fusion protein (CFP, cyan) were visualized by confocal microscopy and phase contrast microscopy (DIC). Colocalization was illustrated by merging YFP and CFP channels (Merge). Arrows indicate protein aggregates. (B and C) Differentiated PC12 cells were transiently transfected with constructs encoding YFP together with G85R-CFP or G93A-CFP, as indicated. (B) FLIP analysis of 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 (black box). (C) Quantitative FLIP analysis of YFP. The RFI was determined at each time point and is represented as the mean ± SEM (n = 5–10 cells). Bars, 10 μm.

Mentions: FRAP and FLIP analyses suggested that mutant SOD1 proteins formed porous aggregate structures through which other soluble cellular proteins may diffuse. This is in contrast to aggregates formed by mutant huntingtin and polyglutamine-YFP proteins, which we and others have shown form a solid and immobile core structure (Chai et al., 2002; Kim et al., 2002; Holmberg et al., 2004). To determine whether noninteracting soluble proteins can indeed diffuse through the mutant SOD1 aggregate structures, PC12 cells were transiently cotransfected with a construct encoding an aggregation-prone protein (G85R-CFP, G93A-CFP, or httQ78-CFP) and a soluble YFP or WT-YFP. YFP (Fig. 2 A) and WT-YFP (Fig. S2 available at http://www.jcb.org/cgi/content/full/jcb.200504050/DC1) proteins displayed a diffuse and uniform pattern of localization, both within and outside the mutant SOD1 aggregates. In contrast, aggregates formed by expression of an expanded polyglutamine-containing mutant huntingtin protein (httQ78-CFP) exhibited a solid core that completely excluded YFP (Fig. 2 A). These results suggested that soluble proteins can colocalize with the aggregate structures formed by mutant SOD1.


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 form a disordered honeycomb-like porous structure. (A–C) Differentiated PC12 cells were transiently transfected with constructs encoding YFP together with G85R-CFP, G93A-CFP, or httQ78-CFP, as indicated. (A) Colocalization of YFP with G85R-CFP, G93A-CFP, or httQ78-CFP aggregates. YFP (YFP, yellow) and CFP fusion protein (CFP, cyan) were visualized by confocal microscopy and phase contrast microscopy (DIC). Colocalization was illustrated by merging YFP and CFP channels (Merge). Arrows indicate protein aggregates. (B and C) Differentiated PC12 cells were transiently transfected with constructs encoding YFP together with G85R-CFP or G93A-CFP, as indicated. (B) FLIP analysis of 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 (black box). (C) Quantitative FLIP analysis of YFP. 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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fig2: Mutant SOD1 aggregates form a disordered honeycomb-like porous structure. (A–C) Differentiated PC12 cells were transiently transfected with constructs encoding YFP together with G85R-CFP, G93A-CFP, or httQ78-CFP, as indicated. (A) Colocalization of YFP with G85R-CFP, G93A-CFP, or httQ78-CFP aggregates. YFP (YFP, yellow) and CFP fusion protein (CFP, cyan) were visualized by confocal microscopy and phase contrast microscopy (DIC). Colocalization was illustrated by merging YFP and CFP channels (Merge). Arrows indicate protein aggregates. (B and C) Differentiated PC12 cells were transiently transfected with constructs encoding YFP together with G85R-CFP or G93A-CFP, as indicated. (B) FLIP analysis of 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 (black box). (C) Quantitative FLIP analysis of YFP. The RFI was determined at each time point and is represented as the mean ± SEM (n = 5–10 cells). Bars, 10 μm.
Mentions: FRAP and FLIP analyses suggested that mutant SOD1 proteins formed porous aggregate structures through which other soluble cellular proteins may diffuse. This is in contrast to aggregates formed by mutant huntingtin and polyglutamine-YFP proteins, which we and others have shown form a solid and immobile core structure (Chai et al., 2002; Kim et al., 2002; Holmberg et al., 2004). To determine whether noninteracting soluble proteins can indeed diffuse through the mutant SOD1 aggregate structures, PC12 cells were transiently cotransfected with a construct encoding an aggregation-prone protein (G85R-CFP, G93A-CFP, or httQ78-CFP) and a soluble YFP or WT-YFP. YFP (Fig. 2 A) and WT-YFP (Fig. S2 available at http://www.jcb.org/cgi/content/full/jcb.200504050/DC1) proteins displayed a diffuse and uniform pattern of localization, both within and outside the mutant SOD1 aggregates. In contrast, aggregates formed by expression of an expanded polyglutamine-containing mutant huntingtin protein (httQ78-CFP) exhibited a solid core that completely excluded YFP (Fig. 2 A). These results suggested that soluble proteins can colocalize with the aggregate structures formed by mutant SOD1.

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