FIGURE 4.

Immunoelectron microscopic localization of mfSOD1 on MNs undergoing vacuolar degeneration from P60 SOD1^G93A mice. Immunolabeling was visualized using a secondary antibody coupled to 12 nm gold particles. (A, B) an MN soma (shaded in red) filled with medium‐sized vacuoles (V) displaying intense immunogold labeling (detailed in b with encircled gold particles). Some altered mitochondria intermixed with the vacuoles (m, shaded in blue) and were devoid of labeling. A synaptic Bouton terminal (Sy) afferent to an MN soma was observed free of labeling. (C) Large mitochondrial‐derived vacuole showing an extension of the outer mitochondrial membrane with the corresponding expansion of the intermembrane space. The lumen of the vacuole displays intense mfSOD1 immunolabeling, but the remnants of inner mitochondrial membrane complexes (*) are not labeled. (D) Enlargement of the area delimited in (C) with encircled gold particles. (E) Mitochondria‐derived vacuole accumulated in an MN axon displaying characteristic identical labeling as described in (C). (F) Initial steps of biogenesis of mitochondrial‐derived vacuoles showing the early expansion of the outer mitochondrial membrane that displays positive C4F6 labeling (encircled gold particles). (G) High magnification detail of C4F6 immunolabeling in the lumen of an MN vacuole. Gold particles are associated with an amorphous, presumably proteinaceous, material. (H) Quantification of immunogold labeling density observed at the distinct subcellular compartments. Within the vacuolar compartment, the intermembrane mitochondrial space (IMMS) and internal mitochondrial complexes (IMMC) were analyzed separately. MIT is the number of mitochondria within vacuolated MNs but outside the vacuoles, and BKG represents the background signal. ****p < 0.0001, one‐way analysis of variance (ANOVA), Bonferroni's post hoc test (IMMS bar vs any other). Scale bar: (A) = 500 nm; (B) = 250 nm (valid for C–F); (G) = 100 nm.