Summary Exosomes are important for cell–cell communication. Deficiencies in the human dihydroceramide desaturase gene, DEGS1 , increase the dihydroceramide-to-ceramide ratio and cause hypomyelinating leukodystrophy. However, the disease mechanism remains unknown. Here, we developed an in vivo assay with spatially controlled expression of exosome markers in Drosophila eye imaginal discs and showed that the level and activity of the DEGS1 ortholog, Ifc, correlated with exosome production. Knocking out ifc decreased the density of the exosome precursor intraluminal vesicles (ILVs) in the multivesicular endosomes (MVEs) and reduced the number of exosomes released. While ifc overexpression and autophagy inhibition both enhanced exosome production, combining the two had no additive effect. Moreover, DEGS1 activity was sufficient to drive ILV formation in vitro . Together, DEGS1/Ifc controls the dihydroceramide-to-ceramide ratio and enhances exosome secretion by promoting ILV formation and preventing the autophagic degradation of MVEs. These findings provide a potential cause for the neuropathy associated with DEGS1-deficient mutations.

Highlights d Lack of dihydroceramide desaturase activity induces cytoplasmic ROS d Rac1-NADPH oxidase-elicited ROS mediates leukodystrophy-related neuronal death d DEGS1/ifc defects cause mislocalization of Rac1 to the endolysosomes d Dihydroceramide alters binding of active Rac1 to reconstituted organelle membranes

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Exosomes play important roles in the nervous system. Mutations in the human dihydroceramide desaturase gene, DEGS1, are recently linked to severe neurological disorders, but the cause remains unknown. Here, we show that Ifc is required for the morphology and function of Drosophila photoreceptor neurons and not in the surrounding glia, but the degeneration of ifc-KO eyes can be rescued by glial expression of ifc, possibly mediated by exosomes. We develop an in vivo assay using Drosophila eye imaginal discs and show that the level and activity of Ifc correlates with the detection of exosome-like vesicles. While ifc overexpression and autophagy inhibition both enhances exosome production, combining the two had no additive effect. Moreover, ifc-KO reduces the density of the exosome precursor intraluminal vesicles (ILVs) in vivo, and DEGS1 promotes ILV formation in vitro. In conclusion, dihydroceramide desaturase promotes exosome formation and prevents its autophagic degradation in the nervous system.