Reduced Retinal Pigment Epithelial Autophagy Due to Loss of Rab12 Prenylation in a Human iPSC-RPE Model of...

Area of Science:

• Genetics and Molecular Biology
• Ophthalmology
• Cell Biology

Background:

• Choroideremia is an X-linked disorder caused by mutations in the CHM gene, affecting Rab escort protein 1 (REP-1).
• This leads to the under-prenylation of Rab GTPases (Rabs), crucial for cellular function.
• While CHM is widely expressed, the disease specifically impacts the retina, retinal pigment epithelium (RPE), and choroid, suggesting primary RPE cell pathology.

Purpose of the Study:

• To investigate the spectrum of under-prenylated Rabs in RPE cells in choroideremia.
• To elucidate the mechanisms by which Rab dysfunction contributes to RPE cell dysfunction.
• To explore potential therapeutic targets by understanding the molecular pathways involved.

Main Methods:

• Generation of a CRISPR/Cas-9-edited CHM knockout induced pluripotent stem cell-derived RPE (iPSC-RPE) model with isogenic controls.
• In vitro biotinylation of unprenylated Rabs followed by tandem mass tag (TMT) spectrometry for identification.
• Assessment of mTORC1 signaling and autophagic flux in CHM iPSC-RPE cells and controls, including gene replacement and siRNA knockdown experiments.

Main Results:

• Rab12 was identified as one of the least prenylated Rabs in CHM iPSC-RPE cells.
• CHM iPSC-RPE cells exhibited increased mTORC1 signaling and reduced autophagic flux, consistent with Rab12 dysfunction.
• Gene replacement therapy rescued autophagic flux in CHM cells, while Rab12 knockdown impaired it in control cells.

Conclusions:

• Rab12 under-prenylation is a significant contributor to RPE cell dysfunction in choroideremia.
• Elevated mTORC1 signaling and diminished autophagy are key pathological pathways in the disease.
• These findings provide a basis for developing targeted therapies for choroideremia.