Investigation into the role of DRAM2 on the lysosomal activity of retinal organoids and RPE using patient-specific cell models

Abstract

Biallelic DRAM2 mutations cause an autosomal recessive cone-rod dystrophy manifesting in the third decade of life. DRAM2 is known to be expressed in the lysosomes of photoreceptors (PRs) and retinal pigment epithelium (RPE), however it remains unclear how this protein contributes to retinal disease. Induced pluripotent stem cells (iPSCs) reprogrammed from somatic tissue are successfully used for disease modelling of retinal dystrophies. Herein, we detail the generation and characterisation of retinal organoids (ROs) and RPE from two patient-specific iPSCs carrying biallelic mutations in DRAM2. To control for genetic background variation, we generated isogenic controls using CRISPR-Cas9 and differentiated them to ROs and RPE alongside wild-type controls. Preliminary data showed a fundamental difference in the ability of both iPSC-derived patient ROs to initiate autophagic processes. Complete loss of DRAM2 in ROs resulted in a failure to initiate autophagy, as opposed to minimal DRAM2 expression, which was sufficient to maintain basal levels of autophagic activity. Characterisation of ROs by immunofluorescence did not show a reduction in either rods or cones consistent with an adult-onset of the disease. Patient ROs and RPE, however, presented with a marked accumulation of lipid deposits on electron microscopy suggesting inefficient degradation of cellular waste. A combined proteomics and western blot approach revealed a cytosolic reduction and increased media expression of key lysosomal degradative enzymes in both cell models. A significant decrease in lysosomal enzyme activity corroborated a putative lysosomal deficiency. Altered lipid composition of ROs indicated that aberrant membrane dynamics may exacerbate the vesicular trafficking defect. Co-expression of DRAM2 with AP-1 (Adaptor Related Protein Complex 1) and AP-3 (Adaptor Related Protein Complex 3) provided further evidence for a role in clathrinmediated transport. Collectively, these data suggest an indispensable role for DRAM2 in the maintenance of PRs and RPE in overseeing the proper transport of lysosomal enzymes.