Figure 3.
Human superoxide dismutase 1 (hSOD1) localization in the nucleus of motor neurons in hSOD1-wild type and hSOD1-G93A transgenic (tg) mouse spinal cord in situ. (A, B) In hSOD1-wild type tg mouse spinal cord, hSOD1 immunoreactivity localizes to the cytoplasm and nucleus. hSOD1-wild type immunoreactivity appears to be stronger in the cytoplasm compared to the nucleus. This result mimics the distribution of endogenous mouse SOD1 and is characteristic of motor neurons in the ventral horn, identified by choline acetyltransferase (ChAT) immunostaining (A) and sensory neurons in the dorsal horn (B) of spinal cord. Motor neurons expressing hSOD1-wild type show cytoplasmic aggregates of hSOD1 (A, hatched white arrows), consistent with previous observations (16). (C-F) Spinal cords of hSOD1-G93A tg mice. In pre-symptomatic mouse spinal cord viewed at low magnification (C, D) and high magnification (C’, D’), hSOD1 immunoreactivity shows robust presence in the nucleus of motor neurons (as seen by ChAT immunostaining, a motor neuron cytoplasmic marker) in the ventral horn (C, C’), but hSOD1 is not present in nuclei of neurons in the dorsal horn (D, D’, white arrows). Some cytoplasmic hSOD1 aggregates are seen in motor neurons (C’, hatched white arrow). In spinal cords of hSOD1-G93A tg mice at near end-stage disease, motor neurons viewed at low magnification (E, F) and high magnification (E’, F’) have lost ChAT immunoreactivity and the nuclei show nuclear structure abnormalities and a loss of hSOD1 immunoreactivity (E, E’; white arrows). This is in stark contrast to the nuclei of the neurons of the dorsal horn that show enhanced immunoreactivity for hSOD1 (F, F’; white arrows). Nonneuronal cells, in both the dorsal and ventral horn, do not show the presence of hSOD1 (pink arrows) in their nuclei. Cytoplasmic aggregates of hSOD1-G93A are prominent in motor neurons (E’, hatched white arrow) and dorsal horn cells (F’, hatched white arrow) at end-stage disease. G, H: hSOD1 localization in mouse spinal cord using immunoperoxidase staining and diaminobenzidine as chromogen. Non-tg mouse spinal cord sections show no immunostaining for hSOD1 (G); same batch-stained spinal cord sections from pre-symptomatic hSOD1-G93A tg mice show marked immunolabeling of motor neurons (H, hatched arrows and inset) with marked accumulation of hSOD-G93A1 in the nucleus (H, white asterisks). Pre-symptomatically, there is prominent aggregation of hSOD1-G93A in the nucleus (H, inset, hatched black arrows) and only slight to modest aggregation of hSOD1 in the cytoplasm (H, inset solid black arrows). Scale bar in G (same for H) = 8 μm. Data are representative of 5 mice per genotype, ≥ 6 sections per mouse, and ≥10 neurons analyzed per section by confocal microscopy.