To study the role of metal binding in aggregation and disease, we created transgenic mice expressing SODMD from the 12 kb human genomic SOD1 construct. In order to produce this construct, mutations were introduced in all 5 coding exons (Supplementary Material, Fig. S1
). Mutations that target each of the histidine residues known to be involved in metal binding (H46, H48, H63, H71, H80, H120) were introduced by building from a previously described genomic construct in which histidines at positions 46, 48, 63, and 120 were mutated as follows H46R/H48Q/H63G/H120G (termed the SOD-Quad) (Wang et al. 2003
). The additional mutations added were H71R and H80R (charge conserving experimental mutation at H71 and FALS mutation at H80). Additionally H43, which is located adjacent to the normal Cu binding site, was mutated to H43R, a FALS mutation. Finally, we mutated the two free cysteine residues at positions 6 and 111. Cysteine 6 was mutated to Gly (G) to encode an FALS mutation. Cysteine 111 was mutated to Ser (S), which is the amino acid at this position in mouse SOD1 and most other mammals (Wang et al. 2006
). Collectively these mutations eliminate all known primary and secondary Cu and Zn binding sites.
A total of seven founder lines were initially produced, from which we selected the highest expressing line (U-69) to expand (Supplementary Material, Fig. S2
). To compare the expression of mutant SOD1 in the U-69 SODMD mice to previously established lines of mutant SOD1 mice, we determined the levels of transgene mRNA by Northern blotting (). For each line of mice we harvested 3 animals of similar ages. Mice expressing the mutations G93A [Line Gur 1 (Gurney et al. 1994
)] or double histidine (H46R/H48Q) mutants [Line 139 (Wang et al. 2002
)] showed the highest mRNA levels, followed by WT L76, mice expressing the G37R mutant [Line 29 (Wong et al. 1995
)] and two different lines of L126Z SOD1 mice [Lines 44 and L45 (Wang et al. 2005a
)]. A line of L126Z mice [L171 (Wang et al. 2005a
)] expresses mutant SOD1 at levels that were equivalent to SODMD Line U69 mice ().
Comparison of transgene mRNA levels in spinal cords of mutant SOD1 transgenic mouse lines
Previous studies have observed that the levels of transgene expression relate to disease development in mutant SOD1 mice (Wong et al. 1995
;Wang et al. 2002
;Wang et al. 2005b
). To relate expression levels to onset we compiled data on the age at which different lines of mice reach endstage –defined by paralysis of the hindlimbs. When we compared such data with transgene expression levels, we observed that mice reaching end-stage at earlier ages, present higher levels of transgene expression (). For example, G93A mice express the highest levels and develop paralysis at the earliest times, around 5 months old [159.1 ± 4.03 days; data from our colony (Supplementary Material, Fig. S3
)], while the lowest expressing mice (L126Z L171) did not develop paralysis until they were 13 months old (400.3 ± 22.56 days; ; Supplementary Material, Fig. S3
). Additionally, correlation analysis demonstrated that the age to paralysis in transgenic mice is dependent on the level of mutant SOD1 mRNA (Supplementary Material, Fig. S4
). Thus, although the levels of transgene mRNA in the U-69 SODMD mice was on the lower end of the spectrum, we concluded that SODMD mice from line U-69 expressed the mutant within the range to induce an ALS phenotype if the mutation is pathogenic.
In our experience the levels of transgene mRNA in mouse spinal cords do not necessarily correlate with the steady-state levels of mutant SOD1 protein present in spinal cords because some mutants are relatively unstable. For example, the L126Z mutant accumulates to lower levels than predicted by mRNA levels (Wang et al. 2005a
). Thus, to determine protein levels in the U-69 mice relative to other mutants, we evaluated total SOD1 protein levels by immunoblotting of spinal cord extracts from young animals. Compared to the L126Z Line 171 mice, the levels of SODMD protein were similar (). Note, however, that the levels are much lower than that of WT SOD1 mice (Line 76) or mice that express the H46R/H48Q mutant (Line 139). These results, together with our analysis in mRNA SOD1 levels, suggest that disease development in SODMD would be predicted to occur around the age of onset of L126Z L171 mice. However, unexpectedly, the SODMD mice remained free of ALS-like symptoms, such as obvious hind limb weakness, gait disturbances, or paralysis out to 2 years of age. As the mice approached 2 years of age, we began to observe death due to other morbidities, but no symptoms of weakness or paralysis were noted. To alleviate un-necessary stress to the aged animals, no animals were aged longer than 24 months and no SODMD mice developed ALS-like symptoms by this age.
Protein levels in the spinal cord of SODMD mice resemble those of L126Z SOD1 mice
As noted above, the levels of transgene expression are critical in transgenic animal models. For example, mice expressing the G37R mutation under the mouse prion promoter do not develop motor neuron disease unless levels of expression are raised through breeding to homozygosity (Wang et al. 2005b
). Similar examples occur for a line of mice that express the D90A mutant of SOD1 (Jonsson et al. 2006
) and the G93A mutant via a vector that utilizes a Thy1 promoter (Jaarsma et al. 2008
). Thus, we self-crossed SODMD mice with the intention to increase SOD1 protein levels through homozygosity, which should translate into a more rapid disease development. A total of 20 transgene positive mice were produced from homozygous matings with the expectation that approximately 33% of the transgene positive animals (25% overall) should be homozygous. The number of transgene positive animals from these matings was not suggestive of any loss of viability of homozygous animals. However, we still failed to observe disease symptoms in any of these mice out to 24 months of age.
Several previous studies have established that co-expressing high levels of WT SOD1 with mutant SOD1 can accelerate disease onset and, moreover, induce disease in animals that express mutant protein at levels that are below threshold to develop ALS-like symptoms (Deng et al. 2006
;Jaarsma et al. 2008
;Deng et al. 2008
;Prudencio et al. 2010
). In an attempt to induce disease development in SODMD mice, we mated these mice to the Gur WT strain of mice that express WT SOD1 at very high levels. Out of 59 animals that resulted from such mating, 11 harbored both WT and SODMD transgenes. Animals were aged out to 24 months with none developing paralysis. Mice from the Gur WT strain develop a gait disturbance late in life and we observed this phenotype, but none progressed to overt motor neuron disease and paralysis. For comparison, we also mated our low expressing L126Z (line 171) mice to Gur WT mice, generating 4 mice that harbored both transgenes. Compared to mice expressing only the L126Z Line 171 transgenes, mice harboring with the WT and L126Z transgenes developed motor neuron disease 20-25 weeks earlier (Supplementary Material, Fig. S5
). Thus, the low level of expression of the SODMD transgene cannot explain the lack of effect by the co-expression of WT SOD1. Therefore, we conclude that, although the MD and L126Z variants exhibit similar stability profiles (ratio of steady-state protein to transgene mRNA is similar), the SODMD mutant is much less pathogenic than the L126Z mutant.