Mechanosensitive PIEZO1 and PIEZO2 channels in the RPE and in axons of RGCs may initiate cellular responses to tissue stretch

Abstract

Purpose : Excessive mechanical strain can initiate pathological changes throughout the nervous system. Several disorders of the inner and outer retina involve abnormal tissue stretch; the stretch induced by increased intraocular pressure (IOP) is known to damage retinal ganglion cells (RGCs), but hypertrophy of retinal pigmented epithelial (RPE) cells and stretch of the tissue is emerging as an issue. The recent identification of PIEZO ion channels as bona fide mechanosensitive channels suggests their contribution to this stretch. Here, PIEZO channels were identified in the retina using molecular, functional, and histochemical approaches, and the effect of in vivo stretch on sustained PIEZO expression was examined.

Methods : Expression of Piezo transcripts was examined with semi-quantitative and quantitative PCR in mouse tissues. Immunohistochemistry localized PIEZO channels in mouse retina and human iPSC-RGCs; siRNA knockdown reduced expression in ARPE-19 cells, supporting antibody specificity. Ca²⁺-reporter Fura-2 indicated cytoplasmic Ca²⁺ levels. In vivo stretch was produced by elevation of IOP to 58-60 mmHg for 4h.

Results : Expression of Piezo1 transcript was greatest in the RPE/choroid of the mouse, while Piezo2 was highest in the optic nerve; both channels were also expressed in the retina. Retinal expression of Piezo1 and Piezo2 transcripts increased in both 1 and 10 days after 4h elevation of IOP. The specific PIEZO1 agonist Yoda1 increased cytoplasmic Ca²⁺ in the soma and neurites of primary mouse RGCs cultures and in RPE cells. PIEZO1 and 2 were both expressed primarily on the ganglion cell layer in the retina. PIEZO1 staining overlapped with Brn3a in RGC soma and axon-marker β-tubulin III in the optic nerve head. PIEZO2 was detected in the soma and dendrites of mouse RGCs and with β-tubulin III in the nerve fiber layer and optic nerve head. Human iPSC-RGCs expressed PIEZO1 and PIEZO2 in soma and in axons. Both PIEZO isoforms were also stained brightly in the RPE in vitro and in vivo.

Conclusions : Molecular, live-cell imaging, and immunohistochemical approaches suggest that PIEZO1 and PIEZO2 are predominantly expressed in the RPE and RGCs. The expression of both channels in RGC axons was particularly strong. The sustained rise in Piezo expressions after a single instance of IOP elevation suggests an enhanced contribution may follow repeated tissue stretch.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.