An examination of alpha B-crystallin as a modifier of SOD1 aggregate pathology and toxicity in models of familial amyotrophic lateral sclerosis

Abstract

Amyotrophic lateral sclerosis is a progressively paralytic neurodegenerative disease that can be caused by mutations in Cu,Zn-superoxide dismutase 1 (SOD1). Transgenic mice that over-express mutant SOD1 develop paralysis and accumulate aggregates of mutant protein in the brainstem and spinal cord. The present study uses a cell culture model to demonstrate alpha B-crystallin is capable of reducing aggregation of mutant SOD1. To test the role of alpha B-crystallin in modulating SOD1 aggregation in vivo, alpha B-crystallin deficient mice were bred to mice expressing two different SOD1 mutants (G37R and L126Z). Although completely eliminating alpha B-crystallin reduced the interval to disease endstage by 20-30 days in mice expressing either mutant, there were no detectable changes in the levels of sedimentable, SOD1 aggregates in the spinal cord of symptomatic mice. Because alpha B-crystallin is most abundantly expressed in muscle, we expected that the loss of this chaperone would leave this tissue vulnerable to mutant SOD1 aggregation. However, there was no evidence of mutant SOD1 aggregation in the muscle of mice lacking alpha B-crystallin. Our findings indicate that a significant perturbation to the protein homeostasis network of muscle is not sufficient to induce the aggregation of misfolded mutant SOD1. These outcomes have implications regarding the role of chaperones in modulating the tissue specific accumulations of misfolded SOD1.

Figure 1

αB-crystallin reduces mutant SOD1 aggregation in cell culture. HEK293FT cells were transfected with mutant SOD1 (4 μg) alone, with mutant SOD1 (2 μg) and αB-crystallin (2 μg), or with mutant SOD1 (2 μg) and GFP (2 μg). Cell lysates were extracted in non-ionic detergent and analyzed in 18% Tris-Glycine gels as described in Supplemental Information. Immunoblots were probed with m/hSOD1 antiserum. A. Detergent-insoluble (20 μg). B. Detergent-soluble (5 μg). C. Quantification of aggregation propensity measured as a ratio of detergent insoluble to soluble SOD1 and graphed with error bars representing the SEM. *, significantly different from mutant SOD1 (p<0.05). °, significantly different from WT SOD1 (p<0.05). The image shown is representative of 3 repetitions of the experiment. Data from at least 3 experiments were used to quantify aggregation propensities in panel C. Note, cells expressing G37R, G37R/αB, and G37R/GFP were harvested 48 hours after transfection; all others were harvested 24 hours post-transfection.

Figure 2

Reduction or elimination of αB-crystallin in mutant SOD1 transgenic mice modestly alters the age at which mice reach end-stage criteria. Kaplan-Meier survival curves of mutant SOD1 transgenic mice with WT (black), heterozygous (red), or homozygous null (blue) αB-crystallin. A. Gn.G37R mice. B. Gn.L126Z mice. Note: PrP.G37R mice did not develop symptoms (n=10 per condition).

Figure 3

SOD1 aggregation is absent in muscle tissue. Muscle tissues from hindlimbs (gastrocnemius and soleus) were isolated from mice and extracted in non-ionic detergent and analyzed by SDS-PAGE in 18% Tris-Glycine gels. Immunoblots were probed with hSOD1 antiserum. A. Detergent-insoluble (20 μg). B. Detergent-soluble (5 μg). The image shown is representative of 2 repetitions of the experiment.

Figure 4

Reduction or elimination of αB-crystallin in SOD1 transgenic mice does not alter aggregation in spinal cord tissue. Spinal cords were extracted in non-ionic detergent and electrophoresed in 18% Tris-Glycine gels. Immunoblots were probed with hSOD1 antiserum. A. Detergent-insoluble (20 μg). B. Detergent-soluble (5 μg). The image shown is representative of 3 repetitions of the experiment.

Figure 5

Ubiquitin-positive structures do not change in location or abundance in the absence of αB-crystallin. Mice were perfused with 4% paraformaldehyde and spinal cord tissue was immersed in sucrose prior to cryostat sectioning. Sections were stained with ubiquitin (A, D, G, J) and GFAP (B, E, H, K), followed by staining with secondary fluorescent antibodies: anti-rabbit-AlexaFluor 568 (A, D, G, J) and anti-mouse-AlexaFluor 488 (B, E, H, K). Ventral horn of spinal cord is shown at an original magnification of 40x. The image shown is representative of at least 3 repetitions of the experiment. Scale bar – 40 microns.