Wild-type SOD1 overexpression accelerates disease onset of a G85R SOD1 mouse

Abstract

Approximately 10% of amyotrophic lateral sclerosis (ALS) cases are familial (FALS), and approximately 25% of FALS cases are caused by mutations in Cu/Zn superoxide dismutase type 1 (SOD1). Mutant (MT) SOD1 is thought to be pathogenic because it misfolds and aggregates. A number of transgenic mice have been generated that express different MTSOD1s as transgenes and exhibit an ALS-like disease. Although one study found that overexpression of human wild-type (WT) SOD1 did not affect disease in G85R transgenic mice, more recent reports claim that overexpression of WTSOD1 in other MTSOD1 transgenic mice hastened disease, raising a possibility that the effect of WTSOD1 overexpression in this FALS mouse model is mutant-specific. In order to clarify this issue, we studied the effect of WTSOD1 overexpression in a G85R transgenic mouse that we recently generated. We found that G85R/WTSOD1 double transgenic mice had an acceleration of disease onset and shortened survival compared with G85R single transgenic mice; in addition, there was an earlier appearance of pathological and immunohistochemical abnormalities. The spinal cord insoluble fraction from G85R/WTSOD1 mice had evidence of G85R-WTSOD1 heterodimers and WTSOD1 homodimers (in addition to G85R homodimers) with intermolecular disulfide bond cross-linking. These studies suggest that WTSOD1 can be recruited into disease-associated aggregates by redox processes, providing an explanation for the accelerated disease seen in G85R mice following WTSOD1 overexpression, and suggesting the importance of incorrect disulfide-linked protein as key to MTSOD1 toxicity.

Figures

Figure 1.

Characterization of G85R and G85R/WTSOD1 transgenic mice. (A) A G85R transgenic mouse at ∼11 months of age. (B) Western blot of a spinal cord homogenate from a 6-month-old G85R mouse (lane 1), muscle homogenate from an 11-month-old G85R mouse (lane 2), and muscle homogenate from an 11-month-old non-transgenic littermate (lane 3) using an anti-SOD1 antibody that is directed against human as well as mouse SOD1. (C) Western blot using a human-specific anti-SOD1 antibody of a spinal cord homogenate from a G85R transgenic mouse (lane 1), a G85R/WTSOD1 transgenic mouse (lane 2) and a G93A transgenic mouse (lane 3). The arrows show that the G85R MTSOD1 (lane 1, lane 2—lower band) has a more rapid electrophoretic mobility than WTSOD1 (lane 2—upper band), which runs at the same mobility as G93A MTSOD1 (lane 3). Lane 2 contained a different amount of total protein in order to show the two bands and the ratio of G85R to WTSOD1. (D) G85R MTSOD1 has no SOD1 enzymatic activity. A protein extract (30 μg) from the spinal cord of a non-transgenic mouse (lane 1), WTSOD1 transgenic mouse (lane 2), a presymptomatic (lane 3) and endstage (lane 4) G85R single transgenic mouse, and a presymptomatic (lane 5), early disease phase (lane 6) and endstage (lane 7) G85R/WTSOD1 double transgenic mouse were electrophoresed on an SOD1 activity gel. Human SOD1 enzymatic activity is seen in the case of the WTSOD1 and G85R/WTSOD1 transgenic mice, but not the non-transgenic and G85R single transgenic mice.

Figure 2.

Clinical disease in G85R and G85R/WTSOD1 transgenic mice. (A) The G85R/WTSOD1 mouse has clear hind limb weakness at ∼150 days compared with two littermates, one a WTSOD1 transgenic mouse and one a G85R single transgenic mouse. (B) The age of mice at the onset of disease in G85R versus G85R/WTSOD1 transgenic mice. A round mark shows the age of disease onset of individual mice at a particular age that is indicated in the x-axis. (C) Survival in G85R versus G85R/WTSOD1 transgenic mice.

Figure 3.

Neuropathological and immunohistochemical studies of the anterior horn of the spinal cord. The first three columns show WTSOD1, G85R and G85R/WTSOD1 mice at 150 days, whereas the fourth column shows G85R mice at 350 days with respect to Nissl staining (first row), CTB immunoreactivity (second row), GFAP immunoreactivity (third row) and SOD1 immunoreactivity (fourth row). The scale bar = 20 µm.

Figure 4.

SOD1 immunoreactivity on western blots of the insoluble fraction of the spinal cord. The insoluble fractions from the spinal cord of mice were electrophoresed on SDS–polyacrylamide gels in the absence (A and B) or the presence (C) of β-mercaptoethanol (2-ME), and then western blotted with an overlay of human-specific anti-SOD1 antibody. In (A and C): lane 1—non-transgenic, lane 2—WTSOD1 (150 days), lane 3—G85R (150 days), lane 4—G85R (350 days), lane 5—G85R/WTSOD1 (60 days), lane 6—G85R/WTSOD1 (150 days) and lane 7—G93A (endstage, ∼130 days) transgenic mice. (B): lane 1—G85R/WTSOD1 mouse (150 days), lane 2—G85R mouse (350 days). Note that the gel in (A) was run longer than (C) (and therefore the monomers were electrophoresed off the gel) in order to increase the separation of bands of the predicted size of the G85R–G85R SOD1 homodimer, G85R–WTSOD1 heterodimer and WT–WTSOD1 homodimer [see arrows in (A), lane 6]; these three bands are seen more clearly in (B, lane 1). The WT–WTSOD1 homodimer (A, lane 6) has the same mobility as the G93A–G93A SOD1 homodimer (A, lane 7). These dimers and multimers are not detected in (C). The monomers (dashed arrows) are seen in (C), with the WT and G93A protein species having a slower electrophoretic mobility than G85R.

Figure 5.

SOD1 immunoreactivity on western blots of the insoluble fraction from varied tissues. An equal amount of protein of the insoluble fraction from the spinal cord and other tissues from a G85R/WTSOD1 double transgenic mouse (lanes 1–6) and from the spinal cord from a G85R single transgenic mouse (lane 7) was electrophoresed on SDS–polyacrylamide gels in the absence (A) and the presence (B) of β-mercaptoethanol (2-ME), and then western blotted with an overlay of human-specific anti-SOD1 antibody. In (A and B): lane 1—cerebrum, lane 2—cerebellum, lane 3—spinal cord, lane 4—liver, lane 5—muscle, lane 6—kidney, lane 7—spinal cord, lane 8—50 ng of SOD1 as a standard. Dimers that are present in (A) are disrupted and no longer detectable after β-mercaptoethanol treatment in (B). The positions of WT and G85R monomers in (A and B) are shown with arrowheads. The G85R/WTSOD1 double transgenic mouse was sacrificed at ∼160 days, whereas the G85R single transgenic mouse was sacrificed at ∼350 days.

Figure 6.

SOD1 immunoreactivity on western blots of the spinal cords of G85R/WTSOD1 mice at different ages. An equal amount of protein of the homogenate (containing the soluble and insoluble fraction) (A and B) and insoluble fraction (C and D) of the spinal cords from two G85R/WTSOD1 mice were electrophoresed on SDS–polyacrylamide gels in the absence (A and C) and presence (B and D) of β-mercaptoethanol and then western blotted with an overlay of human-specific anti-SOD1 antibody (upper panels) or anti-actin antibody (lower panels of A and B). Lane 1—presymptomatic (pre), lane 2—onset of weakness (wkness), lane 3—complete paralysis (end). The location of the WT and G85R is designated by the upper and lower arrowheads, respectively.