Figure 2.

CORD21-ROs present with an autophagy impairment and the accumulation of CLs on TEM

(A) Diagram summarizes the effect of rapamycin and bafilomycin drugs on the autophagy pathway. Rapamycin, an mTOR inhibitor, relieves ULK1 from inhibition by mTOR. This leads to the assembly of phagophore double membrane around autophagic cargo and its subsequent maturation to a nascent autophagosome. Bafilomycin blocks the fusion of autophagosomes with lysosomes thereby preventing the formation of autolysosomes. LC3-II is attached to the double membrane of the autophagosome and serves as a marker for autophagosome turnover and degradation. An increase in pS6 expression suggested an inhibition of autophagy following bafilomycin treatment. Schematic was generated using BioRender.

(B and C) CORD21-P1 day 150 ROs show reduced rates of autophagic flux compared to isogenic control as apparent by the failure to accumulate LC3-II following single bafilomycin or rapamycin treatment. Data were normalized to GAPDH expression and presented as mean +SEM (n = 3 different differentiation experiments each consisting of 48 ROs/sample).

(D) CORD21-ROs display increased numbers of CLs (black arrowheads). By contrast no such features were present in the isogenic controls. Scale bar, 1 μm. These are representative examples from 10 ROs imaged from three different differentiation experiments/sample.

(E) Magnified images of quantified cellular structures (CL, scale bar, 500 nm; Lysosomes, scale bar, 125 nm; AVi, scale bar, 1.5 μm; 250 nm; AVd, scale bar, 500 nm, 250 nm).

(F) Bar charts showing an increase in CLs per photoreceptor in CORD21-ROs, without statistically significant changes in the numbers of lysosomes, early (AVi) and late autophagic vehicles (AVds). Data plotted as mean +SEM, assessed for normality and analyzed by non-parametric Kruskal-Wallis test (n = 10 ROs from three different differentiation experiments/sample), ^∗p <0 .05^∗∗∗p <0 .001, ^∗∗∗∗p < 0.0001.