Alterations in mTOR Signaling Result in Glaucomatous Neurodegeneration from Human Pluripotent Stem Cell-Derived Retinal Ganglion Cells

Abstract

Purpose : Optineurin (OPTN) mutations contribute to neurodegenerative diseases including glaucoma, with the more severe forms resulting from an E50K mutation in the OPTN gene. However, mechanisms underlying how the OPTN(E50K) mutation leads to glaucomatous retinal ganglion cell (RGC) neurodegeneration have not been fully discovered. Here, we used human pluripotent stem cell (hPSC)-derived RGCs with the OPTN(E50K) mutation as a model to explore cellular pathways that contribute to glaucomatous neurodegeneration.

Methods : The OPTN(E50K) hPSC line was established utilizing clustered regularly-interspaced short palindromic repeats (CRISPR)/Cas9 gene editing approach, in which a knock-in E50K mutation was inserted into a wild-type H7 hPSC line. The OPTN(E50K) and isogenic control hPSCs were differentiated to retinal organoids, followed by dissociation and purification of RGCs. Morphological, functional and protein expression differences in OPTN(E50K) RGCs were identified in comparison to isogenic controls.

Results : OPTN(E50K) RGCs exhibited neurodegenerative phenotypes including reduced soma size, decreased branching and length of neurites, as well as increased excitability compared to isogenic controls. To identify mechanisms triggering disease phenotypes, western blotting demonstrated reduced expression of the OPTN protein in OPTN(E50K) RGCs, indicating a lack of OPTN protein adversely affects autophagy processing. Considering that mTOR activation promotes neural development and regulates autophagy, a downregulation of mTOR signaling was observed as a possible compensatory mechanism for autophagy deficits. To provide direct evidence that mTOR inhibition leads to degenerative phenotypes, pharmacological inhibition of mTor in wild-type RGCs resulted in shorter neurites, a smaller cell body size, and fewer primary neurites than controls. Additionally, these disease-related phenotypes were observed more rapidly in OPTN(E50K) RGCs grown in the absence of insulin, a known mTOR activator, suggesting a role for mTOR signaling deficits in neurodegeneration.

Conclusions : The results of this study indicate that deficiencies in OPTN lead to autophagy deficits and decreased mTOR activation, leading to RGC disease phenotypes. Thus, further studies of this pathway may lead to the identification of new targets for therapeutic approaches to glaucomatous neurodegeneration.

This is a 2021 ARVO Annual Meeting abstract.