Fig. 4: Sod1 accumulates in autolysosomal structures within the spinal cord of Plekhg5-deficient mice.

A Western blots showing an enrichment of Sod1 in the 25k membrane fraction upon Plekhg5 knockdown in NSC34 cells. B Quantification of the Sod1 intensities. The Sod1 intensity of the 25k pellet was normalized to γ-Adaptin, the cytosolic fraction was normalized to eIF2α. n = 3 biological replicates. Two-way ANOVA; Sidak’s multiple comparison test. C Sod1 accumulations in Plekhg5^−/− mice stained positive for the endosomal/lysosomal marker Lamp1. Co-staining of spinal cord cross sections for Sod1, Lamp1, and DAPI. The images are representative of three biological replicates. D A subset of Sod1⁺Lamp1⁺ vesicle clusters showed immunoreactivity for the lysosomal marker CathepsinD. Arrowheads point to CathepsinD⁺ vesicle clusters. The asterisk labels a CathepsinD^- vesicle cluster. The images are representative of three biological replicates. E Electron microscopic micrograph of a vesicle cluster in the spinal cord of Plekhg5^−/− mice. Postsynaptic spines adjacent to the vesicle cluster are labeled by asterisks. Electron-dense organelles are highlighted by arrowheads. The images are representative of two biological replicates. F Co-staining of Sod1 and mRFP-GFP-LC3 in spinal cord cross-sections from Plekhg5^−/− CAG:::mRFP-GFP-LC3 mice. The images are representative of three biological replicates. G Line scan of the vesicles shown in the lower panel of F. H Scheme for differential centrifugations of extracellular vesicles. I Enrichment of Sod1 in the TCA fraction of medium from NSC34 cells as shown by Western blot. The images are representative of two biological replicates. Western blots showing the knockdown of Stx17 (J), Snap29 (K), and Snap23 (L) upon simultaneous expression of two sh-RNAs in MNs. The images are representative of three biological replicates. Western blots show that the Sod1 secretion is blocked upon knockdown of Stx17 (M), Snap29 (N), and Snap23 (O). P Quantification of the Sod1 intensities. sh-Stx17, n = 4; sh-Snap29, n = 6; sh-Snap23, n = 6, biological replicates. One-sample t-test, two-tailed. Quantification of Sod1 secretion was calculated as the ratio between the amount of Sod1 in the medium and in the lysate. Sod1 levels in the lysates were adjusted to Tuj1. The normalized Sod1 intensities were set to 1 in each experiment. Data are mean ± SEM. Source data are provided as a Source Data file.