The effect of mutant SOD1 dismutase activity on non-cell autonomous degeneration in familial amyotrophic lateral sclerosis

Abstract

Mutant superoxide dismutase type 1 (MTSOD1), the most common known cause of familial amyotrophic lateral sclerosis (FALS), is believed to cause FALS as a result of a toxicity of the protein. MTSOD1s with full dismutase enzymatic activity (e.g., G37R) and without any enzymatic activity (e.g., G85R) cause FALS, demonstrating that the ability of MTSOD1 to cause FALS is not dependent on the dismutase activity; however, it remains unclear whether MTSOD1 dismutase activity can influence disease phenotype. In the present study, we selectively knocked down G85R expression in particular cell types of G85R mice. Results following knockdown of G85R in motor neurons (MNs)/interneurons of G85R mice were similar to results from a published study involving knockdown of G37R in G37R mice; however, G85R knockdown in microglia/macrophages induced a prolonged early and late disease phase while G37R knockdown in the same cells only affected late phase. These results show that: (i) MN as well as non-MN expression of G85R, like G37R, has a significant effect on disease in transgenic mice - indicating the role of non-cell autonomous degeneration in both dismutase-active and inactive MTSOD1s. (ii) The effect of MTSOD1 expression in microglia/macrophages varies with different mutants, and may be influenced by the MTSOD1's dismutase activity.

Figure 1

Cre recombination of G85R^flox. A) Representative Western blot of chicken embryo spinal cord homogenates harvested one day after electroporation and immunostained with an anti-SOD1 antibody that reacts with both human and chick SOD1 (Stressgen). Co-electroporation of G85R^flox and CMV:Cre plasmids (left lane) led to a prominent decrease in G85R expression compared to electroporation of G85R^flox plasmid plus control vector alone (right lane). B) qPCR quantitation of human SOD1 mRNA. See Materials and Methods for details. G85R MTSOD1 mRNA levels in the spinal cord of G85R^flox/Lhx3:Cre mice (black) were decreased by ~25% compared to G85R^flox mice (gray shaded). The asterisk indicates P < 0.05.

Figure 2

Plots of disease onset (A), end of early disease (B), and survival (C), and bar diagrams of the duration (with standard deviation) of the early phase (D) and late phase (E) of disease in G85R^flox/Lhx3:Cre vs. G85R^flox mice. In the Y axis of A–C, 1 refers to 100% of the number of animals, which is specified in the upper right. The asterisk indicates P < 0.001.

Figure 3

Neuropathological and immunohistochemical studies of the anterior horn of the lumbar spinal cord. The first three columns show WTSOD1, G85R^flox, and G85R^flox/Lhx3:Cre mice at 345 days while the fourth column shows G85R^flox/Lhx3:Cre mice at 405 days with respect to Nissl staining (row A), GFAP immunoreactivity (row B), and SOD1 immunoreactivity (row C). The scale bar = 50μm

Figure 4

Plots of onset (A), end of early disease (B), and survival (C), and bar diagrams of the duration (with standard deviation) of the early phase (D) and late phase (E) of disease in G85R^flox/CD11b:Cre vs. G85R^flox mice. In the Y axis of A–C, 1 refers to 100% of the number of animals. Please note the number of mice used in the survival is greater than the numbers used to plot onset and end of early disease. The asterisk in (D) indicates P < 0.01 and in (E) indicates P < 0.05.

Figure 5

Immunohistochemical studies of the anterior horn of the lumbar spinal cord. (A) Iba1 immunostaining of G85Rflox and G85Rflox/CD11b:Cre mice at 200, 300 and 340 days, and also G85Rflox/CD11b:Cre mice at 380 days; a WTSOD1 transgenic mouse is shown as a control. (B) The three columns of the upper panel show WTSOD1, G85Rflox, and G85Rflox/CD11b:Cre mice at 200 days, while the three columns of the lower panel show G85Rflox and G85Rflox/CD11b:Cre mice at 340 days and G85Rflox/CD11b:Cre mice at 380 days with respect to GFAP (top row) and SOD1 immunoreactivity (bottom row). The scale bar =50 μm

Figure 5

Immunohistochemical studies of the anterior horn of the lumbar spinal cord. (A) Iba1 immunostaining of G85Rflox and G85Rflox/CD11b:Cre mice at 200, 300 and 340 days, and also G85Rflox/CD11b:Cre mice at 380 days; a WTSOD1 transgenic mouse is shown as a control. (B) The three columns of the upper panel show WTSOD1, G85Rflox, and G85Rflox/CD11b:Cre mice at 200 days, while the three columns of the lower panel show G85Rflox and G85Rflox/CD11b:Cre mice at 340 days and G85Rflox/CD11b:Cre mice at 380 days with respect to GFAP (top row) and SOD1 immunoreactivity (bottom row). The scale bar =50 μm