Glial nuclear aggregates of superoxide dismutase-1 are regularly present in patients with amyotrophic lateral sclerosis

Abstract

The most common cause of amyotrophic lateral sclerosis (ALS) is mutations in superoxide dismutase-1 (SOD1). Since there is evidence for the involvement of non-neuronal cells in ALS, we searched for signs of SOD1 abnormalities focusing on glia. Spinal cords from nine ALS patients carrying SOD1 mutations, 51 patients with sporadic or familial ALS who lacked such mutations, and 46 controls were examined by immunohistochemistry. A set of anti-peptide antibodies with specificity for misfolded SOD1 species was used. Misfolded SOD1 in the form of granular aggregates was regularly detected in the nuclei of ventral horn astrocytes, microglia, and oligodendrocytes in ALS patients carrying or lacking SOD1 mutations. There was negligible staining in neurodegenerative and non-neurological controls. Misfolded SOD1 appeared occasionally also in nuclei of motoneurons of ALS patients. The results suggest that misfolded SOD1 present in glial and motoneuron nuclei may generally be involved in ALS pathogenesis.

Fig. 1

Misfolded superoxide dismutase-1 (misSOD1) is present in spinal cord ventral horn of ALS patients. Immunohistochemistry was performed with the Ra 131–153 ab in all cases. a Presence of misSOD1 in glial cell nuclei of a FALS patient homozygous for the D90A SOD1 mutation. b MisSOD1 in glial cell nuclei of a FALS patient not linked to SOD1 mutations. c MisSOD1 in glial cell nuclei of a SALS patient. A motoneuron also shows granular somal inclusions. d Higher magnification of a motoneuron from a patient homozygous for the D90A mutation, showing the presence of misSOD1 in the nucleus (arrow) and widely spread in the soma. Strongly stained glial nuclei are also seen. Motoneurons from a SALS patient showing intranuclear staining (e) and somal staining (f) of misSOD1. Strongly stained glial nuclei are also present in the section. g MisSOD1 in glial cell nuclei (arrows) and in motor neuron nuclei (arrowhead) from a patient with FALS not linked to SOD1 mutations. h Four of 46 control patients showed weak misSOD1-staining in spinal cord glial cell nuclei. As an example, the figure shows a non-neurological control patient. i Material from a control patient with Alzheimer’s disease showing no evidence of misSOD1. Scale bars are 50 μm (a, b), 30 μm (c, d), 20 μm (e, f) or 50 μm (g, h, i)

Fig. 2

MisSOD1 staining is seen in human spinal cord ventral horn motoneurons and glial cells of ALS patients. All sections were stained with the Ra 57–72 ab. Presence of misSOD1 in glial cell nuclei (arrowheads) in a familial ALS (FALS) patient with the G127X SOD1 mutation (a), in a sporadic ALS (SALS) patient (b), and in a FALS patient not linked to SOD1 mutation (c). Higher magnification of different misSOD1 staining patterns in motoneurons from two SALS cases. The nuclear staining appears as granular aggregates approximately 0.5–3 μm in size, often seen in association with the nucleolus (d). In motoneurons with nuclear staining, the cytoplasm appears to contain less of the small misSOD1 inclusions. In panels b and e, misSOD1 aggregates can be seen scattered throughout the cytoplasm of motoneurons, and are particularly abundant in the somal area, while not penetrating the nuclear envelope (arrows). f MisSOD1 staining in a sample from a neurodegenerative control patient with Alzheimer’s disease. Glial cell nuclei and motoneurons lack misSOD1 aggregates/inclusions. Scale bars are 40 μm (a, b), 50 μm (c), 20 μm (d, e) or 30 μm (f)

Fig. 3

Aggregates of misfolded SOD1 are found in astrocytes, microglia and oligodendrocytes. Micrographs of lumbar ventral horn glial cells from two SALS patients (a–c and g–i) and a FALS patient homozygous for the D90A mutation (d–f) labeled with the Chi 131–153 ab (a, g) and the Ra 131–153 ab (d). (a–c) Double immunofluorescence labeling showing misSOD1 (green) and the astrocyte marker GFAP (red). In panel c, most of the glial cell nuclei containing misSOD1 are astrocytes. d–f Double immunofluorescence labeling showing misSOD1 (green) and the microglial marker Iba1 (red). In panel f, some of the glial cells with nuclear misSOD1 staining are microglia. g–i Double immunofluorescence labeling showing misSOD1 (green) and the oligodendroglial marker Olig 2 (red). In panel i, misSOD1 is present in the nuclei of oligodendrocytes. Scale bars are 20 μm (a–c) and 10 μm (d–i)

Fig. 4

Aggregates of misfolded SOD1 in glial cell nuclei occasionally co-localize with ubiquitin, but not with TDP-43. Micrographs of cervical and lumbar spinal cord ventral horn sections from a SALS patient stained with the Chi 57–72 ab for misSOD1 (green) and with antibodies to ubiquitin and TDP-43 (red). MisSOD1 could be seen in glial cell nuclei (a) and in the cytoplasm of a motoneuron (d). Staining with the anti-ubiquitin antibody showed that some glial cell nuclei contained ubiquitin (b, arrows) and the merged picture (c) revealed some co-localization of ubiquitin and misSOD1 (arrows), although misSOD1 could also be seen separate from ubiquitin (arrowheads). Staining with anti-TDP-43 antibody revealed that some glial nuclei contained TDP-43 (e, arrows). Occasionally, misSOD1 and TDP-43 were present in the same glial cell nuclei (f) but no distinct co-localization of misSOD1 and TDP-43 was apparent. Scale bars are 20 μm (a–f)