Two superoxide dismutase prion strains transmit amyotrophic lateral sclerosis-like disease

Abstract

Amyotrophic lateral sclerosis (ALS) is an adult-onset degeneration of motor neurons that is commonly caused by mutations in the gene encoding superoxide dismutase 1 (SOD1). Both patients and Tg mice expressing mutant human SOD1 (hSOD1) develop aggregates of unknown importance. In Tg mice, 2 different strains of hSOD1 aggregates (denoted A and B) can arise; however, the role of these aggregates in disease pathogenesis has not been fully characterized. Here, minute amounts of strain A and B hSOD1 aggregate seeds that were prepared by centrifugation through a density cushion were inoculated into lumbar spinal cords of 100-day-old mice carrying a human SOD1 Tg. Mice seeded with A or B aggregates developed premature signs of ALS and became terminally ill after approximately 100 days, which is 200 days earlier than for mice that had not been inoculated or were given a control preparation. Concomitantly, exponentially growing strain A and B hSOD1 aggregations propagated rostrally throughout the spinal cord and brainstem. The phenotypes provoked by the A and B strains differed regarding progression rates, distribution, end-stage aggregate levels, and histopathology. Together, our data indicate that the aggregate strains are prions that transmit a templated, spreading aggregation of hSOD1, resulting in a fatal ALS-like disease.

Figure 1. Epitope-mapping patterns of seeds and aggregates formed in inoculated mice.

(A) Epitope-mapping patterns of the strain A seed (analyzed on 4 different occasions to demonstrate the method’s variability), 4 terminally ill strain A–inoculated mice, and 4 terminally ill noninoculated hSOD1^G85R-Tg mice. The staining intensities with the 8 Abs were normalized against staining with the aa 57–72 Ab (set to 100%) to facilitate the comparison of patterns. (B) Patterns of the strain B seed (analyzed on 5 different occasions) and of 11 terminally ill strain B–inoculated hSOD1^G85R-Tg mice. The staining intensities were normalized against staining with the aa 111–127 Ab (set to 100%). Typical strain B patterns are seen. (C) Strain A aggregate deposition in spinal cord of a hSOD1^G85R mouse sacrificed 24 hours after the inoculation. The section was stained with the aa 131–153 Ab. No such staining was induced by a syringe stick alone (Supplemental Figure 4). Arrow indicates the position of the seed in the left side of the lumbar spinal cord. cc, central channel. Scale bar: 50 μm. Dotted line delineates the ventral horn.

Figure 2. Inoculation of strain A and B aggregates initiated spreading, templated hSOD1 aggregation and premature motor neuron disease.

(A) Blue line indicates the survival of mice inoculated with the strain A seed (n = 32); red line indicates the survival of mice inoculated with the strain B seed (n = 16); green line indicates the survival of mice inoculated with the control seed (n = 10); gray line indicates the survival of noninoculated mice (n = 101). (B) Strain A aggregation in whole spinal cords of mice analyzed with the epitope-mapping assay using the aa 57–72 Ab. Blue triangles represent asymptomatic, blue Xs early symptomatic, and blue circles terminally ill strain A–inoculated mice. Green and gray triangles represent asymptomatic and green and gray circles represent terminally ill control-inoculated and noninoculated mice, respectively. (C) Strain B aggregation analyzed with the aa 111–127 Ab. Symbols as in B, except that strain B–inoculated mice are shown in red. Dashed line indicates regression lines adapted from Supplemental Figure 7. Shaded areas in B and C indicate blank reactions (9).

Figure 3. Propagation of hSOD1 aggregation along the neuraxis.

(A and B) Strain A–inoculated mice. (A) Results for presymptomatic mice aged 16–47 days (n = 8). Individual mice in different colors; open and closed triangles show the left (inoculation) and right sides, respectively. In some cases (white triangles and black triangles), only 1 of the sides was available. (B) Light blue and dark blue Xs indicate the left and right sides of 3 early-onset mice, respectively. Light blue circles and dark blue circles indicate the left and right sides of terminally ill mice. (C) Terminally ill noninoculated mice (gray circles) and control-inoculated mice (green circles). (D–F) Strain B–inoculated mice. (D) Presymptomatic mice aged 25–103 days (n = 8); symbols as in A. (E) Red Xs and brown Xs indicate the left and right sides of 2 early-symptomatic 87- and 114-day-old mice, respectively. (F) Red circles and brown circles indicate the left and right sides of terminally ill mice. Shaded areas in A, B, and D indicate blank reactions (9).