Human glaucoma is associated with sphingomyelin depletion and acid sphingomyelinase elevation in retinal ganglion cells

Glaucoma is linked to altered phospholipid metabolism, which may contribute to the death of retinal ganglion cells (RGC). Here, we investigate the impact of glaucoma on the phospholipid sphingomyelin (SM) and the mechanisms that mediate SM biosynthesis and degradation in human RGCs. Using retinas from donors with or without a glaucoma diagnosis, we assessed RGC-associated SM with enhanced green fluorescent protein conjugated to non-toxic lysenin (EGFP-Lysenin), which specifically binds SM, combined with a detergent-free histochemistry method to identify RNA-binding protein with multiple splicing (RBPMS)-positive RGCs. We further performed immunohistochemistry to evaluate enzymes that catabolize SM - neutral and acid sphingomyelinases (NSM, ASM) - and enzymes that synthesize SM - sphingomyelin synthases 1 and 2 (SMS1, SMS2) - and we measured ASM catalytic activity and ceramide, the direct product of SM catabolism, in retinal tissue. RGC density was significantly reduced in donor eyes diagnosed with glaucoma. Glaucoma significantly reduced EGFP-Lysenin labeling of SM in RGCs, a finding we confirmed in flash-frozen retinal homogenates by dot blot. In parallel, ASM protein expression was significantly increased within RGCs, and the Zn 2+ -independent lysosomal pool of ASM activity was significantly elevated in glaucomatous retinas, whereas the Zn 2+ -dependent secretory pool was unchanged. Glaucoma was not associated with significant changes in the expression of NSM, SMS1, or SMS2. Although overall RGC ceramide was not significantly altered, glaucoma increased the incidence of RGCs harboring ceramide aggregates. Our results suggest that increased lysosomal ASM expression and activity drives the reduction of RGC-associated SM and may promote ceramide clustering in RGCs marked for degeneration in glaucoma.