Abstract
Purpose :
Oculocutaneous albinism type 1A (OCA1A) is caused due to mutations in TYROSINASE gene and results in pigmentation defects of the skin, hair, and eyes. Retinal pigment epithelium (iRPE) derived from Oculocutaneous albinism type 1A (OCA1A) patients via induced pluripotent stem cell (iPSC) technology exhibit pigmentation defects. In contrast to pigmented iRPE, OCA1A-iRPE exhibit accumulation of pre-melanosomes, absence of mature melanosomes and increased presence of abnormal and degenerating melanosomes. Here we studied the role of autophagy in clearing of abnormal melanosomes in pigmented (CTRL) and OCA1A- iRPE.
Methods :
All human iPSC work was approved by the NIH Institutional Review Board, protocol
#11-E1-0245 (NCT01432847). Three control and two OCA1A iPSC lines used in the study have been reported earlier (George e al., 2022). An isogenic pair of iPSC comprising of normal TYR gene and CRISPR/Cas9 mediated TYR knockout were also generated. Directed differentiation of iPSCs towards RPE was performed as described earlier (Sharma et al., 2019). Morphology of melanosomes, autophagosomes and lysosomes was studied using transmission electron microscopy (TEM) and immunofluorescence staining. Protein expression of autophagy related genes was studied using Western blotting.
Results :
OCA1A patient derived iRPE exhibited lack of pigmentation and mature melanosomes, as reported earlier. Lysosome number was significantly increased by TEM and immunofluorescence staining in all three OCA1A-iRPE lines as compared to CTRL-iRPE. However, no changes were observed in the protein levels of LAMP2, a marker of lysosomes. The number and size of autophagosomes in OCA1A-iRPE were also increased, although protein levels of ATG5 and ATG7 were similar in OCA1A and CTRL-iRPE. The autophagy marker protein LC3b remained unchanged between OCA1A and CTRL-iRPE, but p62 levels were significantly upregulated in OCA1A-iRPE, as studied by Western blotting post Bafilomycin A treatment.
Conclusions :
OCA1A iRPE exhibits increased build-up of cellular debris in the form of enlarged autophagosomes and increased protein levels of p62, which could be a result of abnormal melanosomes accumulation. Autophagic flux remained unchanged in OCA1A-iRPE compared to CTRL-iRPE as measured by LC3b protein levels.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.