Dihydroceramide desaturases are evolutionarily conserved enzymes that convert dihydroceramide (dhCer) to ceramide (Cer). While elevated Cer levels cause neurodegenerative diseases, the neuronal activity of its direct precursor, dhCer, remains unclear. We show that knockout of the fly dhCer desaturase gene, (), results in larval lethality with increased dhCer and decreased Cer levels. Light stimulation leads to ROS increase and apoptotic cell death in -KO photoreceptors, resulting in activity-dependent neurodegeneration. Lipid-containing Atg8/LC3-positive puncta accumulate in-KO photoreceptors, suggesting lipophagy activation. Further enhancing lipophagy reduces lipid droplet accumulation and rescues -KO defects, indicating that lipophagy plays a protective role. Reducing dhCer synthesis prevents photoreceptor degeneration and rescues-KO lethality, while supplementing downstream sphingolipids does not. These results pinpoint that dhCer accumulation is responsible for -KO defects. Human dhCer desaturase rescues-KO larval lethality, and rapamycin reverses defects caused by dhCer accumulation in human neuroblastoma cells, suggesting evolutionarily conserved functions. This study demonstrates a novel requirement for dhCer desaturase in neuronal maintenance and shows that lipophagy activation prevents activity-dependent degeneration caused by dhCer accumulation.

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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Dihydroceramide and NeurodegenerationTzou et al. Highlights  Deficiency in dihydroceramide (dhCer) desaturase induces cytoplasmic ROS elevation  dhCer alters the binding of active Rac1 to reconstituted organelle membranes  Active Rac1 is enriched in endolysosomes in ifc-KO neurons for ROS genesis  Rac1-NADPH oxidase elicits ROS, degenerating leukodystrophy-related neuronal cells Dihydroceramide and Neurodegeneration Tzou et al. Summary Disruption of sphingolipid homeostasis has been shown to cause neurological disorders. How specific sphingolipid species modulate the pathogenesis remains unknown. The last step of sphingolipid de novo synthesis is the conversion of dihydroceramide to ceramide catalyzed by dihydroceramide desaturase (human DEGS1; Drosophila Ifc). Loss of ifc leads to dihydroceramide accumulation and oxidative stress, resulting in photoreceptors degeneration, while DEGS1 variants were associated with leukodystrophy and neuropathy. Here, we demonstrated that ifc regulates Rac1 compartmentalization in fly photoreceptors and further showed that dihydroceramide alters the association of active Rac1 to membranes mimicking specific organelles. We also revealed that the major source of ROS originated from Rac1 and NADPH oxidase (NOX) in the cytoplasm, as the NOX inhibitor apocynin ameliorated the oxidative stress and functional defects in both fly ifc-KO photoreceptors and human neuronal cells with diseaseassociated variant DEGS1 H132R . Therefore, DEGS1/ifc deficiency causes dihydroceramide accumulation, resulting in Rac1 translocation and NOXdependent neurodegeneration. Dihydroceramide and Neurodegeneration Tzou et al. Graphical Abstract A DEGS1/ifc converts dihydroceramide to ceramide in neuronal cells, and the endolysosomal NOX complex is not activated. B Dihydroceramide accumulates without functional DEGS1/ifc and causes alterations in membrane microdomains and recruits active Rac1 to endolysosomes. The activation of endolysosomal Rac1-NOX complex elevates cytosolic ROS levels, causing neurodegeneration. Dihydroceramide and Neurodegeneration Tzou et al. In Brief (eTOC blurb) Deficiency in dihydroceramide desaturase causes oxidative stress-mediated neurological disorders. Tzou and Su et al. show that increased dihydroceramide causes the relocalization of active Rac1, whilst inhibition of the Rac1-NOX ameliorates the oxidative stress and neuronal defects. NOX inhibitor apocynin may provide new direction of treatments for patients with DEGS1 variants. Keywords: DEGS1/dihydroceramide/neurodegeneration/oxidative stress/Rac1 signaling Dihydroceramide and Neurodegeneration Tzou et al.