Abstract
Purpose :
As glaucoma occurs from multifactorial causes, studying glaucoma causative mutation leading to a monogenic form of glaucoma would help better understand glaucoma development. Here, we tested if glaucoma may sensitize cells to death induced by cell stressors.
Methods :
Gene editing
CRISPR/Cas9-mediated deletion of genes was performed by electroporation of Cas9 RNP in 661w.
OPTN E50K cell line generation
To generate cell lines harboring Optn-WT and Optn-E50K, PhiC31 integrase system was adapted following the manufacturer’s protocol.
Ischemia-reperfusion injury model
The needle was connected to a 0.9 % NaCl reservoir and hung it up to sustain the pressure of the mice eye. After 45 minutes of ischemia induction by pressure.
Results :
Optn-KO and Optn-E50K sensitize cells to TNFα-dependent necroptosis.
Optn-KO and Optn-E50K are more susceptible to necroptosis stimuli TBE (TNFα, BV6, Emricasan) and TTaE (TNFα, Takinib, Emricasan) confirmed by lactate dehydrogenase (LDH) release. GNE684, a small molecule Ripk1 inhibitor, suppressed LDH release. WB analysis confirmed that the phosphorylation of Ripk1, Ripk3, and Mlkl, the core axis of necroptosis, were induced more prominently in Optn-KO and Optn-E50K under necroptosis stimuli also in the glaucoma experimental mice model. Only necroptosis conferred this susceptibility of Optn-KO and Optn-E50K but not apoptosis such as doxorubicin or staurosporine. Thus, we confirmed that Optn can protect cells against TNFα-mediated necroptosis, and Optn-E50K is a loss-of-function mutation in the cell death context.
mRNA levels of TNFα and RIPK3 are elevated in the retina from glaucoma patients.
TNFα and RIPK3 showed a well correlated transcript-to-translation ratio. The transcript level of TNFα and RIPK3, a critical component of necroptosis, are upregulated in the glaucomatous retina compared to the non-glaucomatous retina. These data could underline the connection to necroptotic cell death in glaucoma pathogenesis.
Conclusions :
Here, we showed that the Optn-E50K variant, a glaucoma causative mutation in an autosomal dominant pattern sensitizes cells to TNFα-mediated necroptosis. This study may suggest implications of TNFα in glaucoma pathogenesis in both subtypes, intraocular pressure (IOP) –related and non-IOP-related glaucoma, and inform possible future therapy for neuropathies in the retina.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.