Retinal neuroprotection by the HspB1 phosphomimetic mutant

Abstract

Purpose : Small heat shock proteins inhibit stress-mediated apoptosis in cells. Previously, we have shown that AAV2-mediated expression of HspB1 in retinal ganglion cells (RGCs) protects against cell death from ocular hypertension in mice. We assessed whether a triple phosphomimetic mutant of HspB1 (S15D, S78D, S82D) (HspB1-TM) has better anti-apoptotic properties than the HspB1 wild-type protein in retinal ganglion cells (RGCs).

Methods : We constructed an AAV2 vector encoding human HspB1 or HspB1-TM under an RGC-specific promoter PLE345 for specific expression in RGCs. RGCs were isolated and cultured from human induced pluripotent stem cell-derived retinal organoids (iPSC-RGCs) on day 42. The iPSC-RGCs were transduced with either AAV2-HSPB1 or AAV2-HSPB1-TM (1x10⁹ viral genomes (vg) /mL). After ten days, cells were treated with 500 μM H₂O₂ for 24 h to induce apoptosis. The TUNEL assay was used to quantify apoptotic RGCs. Further, we tested the ability of AAV2-HSPB1 or AAV2-HSPB1-TM to protect against RGC death in the optic nerve crush (ONC) injury model in mice. AAV2-HSPB1 or AAV2-HSPB1-TM (1x10⁹ vg/mL/retina) was injected into the vitreous two weeks before ONC. One week after ONC, mice were euthanized, and the fixed retinal flatmounts were immunostained with RBPMS antibody. The transduction of HspB1 or HspB1-TM was assessed by western blotting.

Results : The iPSC-RGCs showed a robust expression of HspB1 after transduction of AAV2-HSPB1. HspB1 and HspB1-TM significantly decreased the number of TUNEL-positive apoptotic cells upon treatment with 500 μM H₂O₂. One week after ONC, the mouse retinas showed only 37% of the RBPMS-positive RGCs compared to uninjured contralateral retinas. However, with the transduction of AAV2-HSPB1 and AAV2-HSPB1-TM, the surviving RGCs were 46% and 56%, respectively. Immunofluorescence experiments showed robust expression of HspB1 but not HspB1-TM in RGCs after three weeks of AAV2-HSPB1 and AAV2-HSPB1-TM injection, but western blotting showed the expression of HspB1 and HSPB1-TM in retinal lysates.

Conclusions : Our results showed that HspB1-TM is more efficacious than HspB1 in protecting RGCs after ONC. HspB1-TM could be further tested for its ability to protect RGCs and preserve visual functions in animal models of glaucoma.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.