Abstract
Purpose :
Up to one-third of POAG patients develop glaucomatous optic neuropathy at a normal or below average (<21 mmHg) intraocular pressure (IOP), a condition called normal tension glaucoma (NTG). Mutations of the optineurin (OPTN) gene cause NTG in human patients, but, how that leads to retinal ganglion cell (RGC) degeneration is unknown. To elucidate the molecular mechanism of OPTN-associated glaucomatous neurodegeneration, we used spatially and temporally controlled genetic manipulation of mouse RGCs in vivo by AAV to compare the effects of different forms of OPTN (wild type (WT), E50K mutation and C-terminus truncation) on RGC survival and function.
Methods :
We used an RGC-specific promoter, mouse gamma synuclein (mSncg), packaged into AAV2 to overexpress OPTN-WT, OPTN-E50K mutant and Cre specifically and efficiently in murine RGCs. WT C57BL/6 mice and OPTN-C-terminus floxed transgenic mice received intravitreal injection of 3x109 vector genomes in 2uL. We used multiple behavioral assays to measure RGC glaucomatous degeneration in living mice, followed by quantification of RGC somata and axons in the enucleated eyes. Optical coherence tomography (OCT), pattern electroretinography (PERG), and optokinetic response (OKR) assays were performed on each animal at baseline and every 2 weeks after injection. Global ganglion cell complex thickness (GCC), P1-N2 amplitude, and maximum spatial frequency are presented as % of that in contralateral control eyes. We counted and quantified RGC somata and axons at the end of each experiment. Each treatment group contained 8-12 mice.
Results :
In WT mice, neither AAV-mSncg-OPTN-WT nor AAV-mSncg-OPTN-E50K alone affected any in vivo or post-mortem assay readout for at least 34 weeks post-injection. Surprisingly, in the OPTN floxed mice, truncation of the OPTN gene C-terminus by intravitreal injection of AAV-mSncg-Cre led to significant RGC degeneration in all in vivo and post-mortem assays by 8 weeks. Interestingly, in these OPTN-truncated mice, both OPTN-WT and OPTN-E50K overexpression rescued P1-N2 amplitude of PERG, GCC thickness and RGC soma counts 8 weeks post-injection.
Conclusions :
Truncation of OPTN C-terminus, but not OPTN-E50K overexpression, rapidly causes significant RGC degeneration. OPTN-E50K at least partially preserves OPTN C-terminus function. The important role of OPTN C-terminus in RGC integrity indicates a previously unrecognized mechanism of OPTN-induced NTG.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.