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. 2022 Mar 21;31(16):2693–2710. doi: 10.1093/hmg/ddac063

Figure 2.

Figure 2

Lysosomal aberrations are an early event in disease pathogenesis. (A) Lysosomes are swollen in 15 month old diseased mice. The SPG15 KO brain cortex of aged mice shows significantly enlarged LAMP2-positive organelles when compared to controls. Scale bar = 100 pixels. N = 3 (brains, with >200 LAMP2 organelles analyzed per brain). Mann–Whitney test, error bars represent SEM. **** corresponds to P < 0.0001. Lysosomes located (B) in the soma and (C) along the axons of cortical neurons derived from SPG15 KO mouse embryos are enlarged. Axons were separated from cell bodies by means of a microfluidic system. Confocal micrographs highlighting Lysotracker Red DND-99 (LTR)-positive organelles show a significant increase in the size of these organelles in SPG15 KO neurons. N = 3 (independent experiments, each evaluating up to 200 lysosomes). Scale bar = 10 μm (for soma). Scale bar = 100 pixels (for axonal network). Mann–Whitney test, error bars represent SEM. **** corresponds to P < 0.0001. (D) Electron micrographs of primary cortical neurons derived from SPG15 KO and control embryos. Snapshots are taken from both cell body and axonal network. Note that the soma of SPG15 KO neurons accumulates distinct organelles consistent with ALs, harboring undigested material (black arrows) and multilamellar bodies (black arrowheads). SPG15 KO neurons also exhibit lysosomal traffic jams (TJs) along their axons. Scale bar = 500 nm. (E) P62 accumulates in SPG15 KO neurons. Immunoblotting shows a significant increase in diseased cells as compared to controls, suggesting a block in autophagic flux. N = 5. Unpaired t-test, error bars represent SD. * corresponds to P < 0.05.