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. Author manuscript; available in PMC: 2020 Nov 24.
Published in final edited form as: N Engl J Med. 2019 Sep 11;381(15):1422–1433. doi: 10.1056/NEJMoa1815111

Figure 4. Effect of PPAR-α Agonist Fenofibrate on Deoxysphingolipid Toxicity in Human Photoreceptors.

Figure 4.

Panel A, subpanel a shows live bright-field image of human retinal organoid tissue showing outer segments (OS) projecting from the outer nuclear layer (ONL); subpanels b through d show representative confocal images of cell death (indicated by terminal deoxynucleotidyl transferase dUTP nick end labeling [TUNEL] staining) within the photoreceptors (indicated by the presence of α-recoverin) of the retinal organoid ONL after 4 days of treatment with control media (subpanel b), deoxysphinganine (1 μmol per liter) (subpanel c), or deoxysphinganine (1 μmol per liter) plus fenofibrate (20 μmol per liter) (subpanel d). Blue staining with 4′,6-diamidino-2-phenylindole (DAPI) indicates the presence of cell nuclei. Panel B shows the toxicity dose response of deoxysphinganine in retinal organoids. Cell death was quantified in human photoreceptors cultured in varying concentrations of deoxysphinganine for 8 days (9 per group) (Table S15 in Supplementary Appendix 1). Panel C shows pharmacologic rescue of deoxysphinganine toxicity. Cell death was quantified in human photoreceptors after treatment with control media (6 organoids), deoxysphinganine (1 μmol per liter) (22 organoids), deoxysphinganine (1 μmol per liter) plus fumonisin B1 (35 μmol per liter) (12 organoids), or deoxysphinganine (1 μmol per liter) plus fenofibrate (20 μmol per liter) (21 organoids). Bar graphs with T bars indicate the means and standard errors. P values were determined with the Wald test on a linear regression analysis. Cell death (the number of dead cells within the defined area) was normalized by a square-root transformation. Model assumptions were assessed with model diagnostics plots (Tables S16 and S17 in Supplementary Appendix 1). Panel D shows the steps in the ceramide–deoxyceramide pathway, including the interaction of fumonisin B1 and fenofibrate. Both pathways share the enzymes shown in gray. Deoxysphingosine is degraded through ω-hydroxylation; it cannot be degraded through the sphingosine-1-phosphate (S1P) pathway. Double arrows denote two sequential reactions in the biosynthetic pathway. CerS denotes ceramide synthase, and SPT serine palmitoyltransferase.