Activation of Piezo1 Mechanosensitive Channels in Retinal Ganglion Cells Leads to Cell Death

Abstract

Purpose Piezo1 mechanosensitive ion channels play an important role in mechanotransduction in all vertebrates and have been identified in a variety of tissues and cell types including neurons. Previously, we have identified Piezo1 mechanosensitive ion channels in zebrafish and mammalian retinal ganglion cells (RGCs). The principle aim of this study is to test whether activation of Piezo1 channels can produce neurodegeneration of zebrafish and mammalian RGCs. Methods For zebrafish Ca2+ imaging was performed using a genetically‐encoded Ca2+ indicator, GCaMP6f, and was targeted predominantly to RGCs using the Tg(elavl3: GCaMP6f) zebrafish line, and confirmed by backfilling the optic nerve with Texas Red hydrazide. In order to activate Piezo1 channels in RGCs, cytosolic [Ca2+]i changes were measured in response to application of the Piezo1 selective agonist, Yoda1. Immunoblotting and immunohistochemical localization for Piezo1 were used to determine the expression profile in rodent and zebrafish retina using retinal wholemounts and vertical sections. To examine apoptosis, a short‐term organotypic eyecup culture preparation was used. Eyecups were incubated in oxygenated Ringer’s solution (for 1–8 hrs) with various concentrations of Yoda1 (0.1–100μM). Following treatment with Yoda1, eyecups were fixed, sectioned, and prepared for immunohistochemistry. TUNEL assays was used to label apoptotic cells in fixed vertical retinal sections, and TUNEL‐positive cells were quantified using ImageJ software. All imaging was acquired on a Nikon C2+ confocal microscope. Results Calcium imaging experiments revealed that Yoda1 evokes a dose‐dependent activation of Piezo1 ion channels in GCAMP6f expressing RGCs, which was reduced in the presence of GsMTx‐4, a mechanosensitive channel antagonist. Immunohistochemical findings show strong Piezo1 immunoreactivity in RGCs and their axons in rodent and zebrafish retina. Moreover, Yoda1 evoked a concentration‐dependent increase in TUNEL‐positive labeling of RGCs in both zebrafish and rodent retina, implicating a common Ca2+‐mediated pathway for Piezo1 channel induced RGC death. Conclusions This study provides the first evidence for Piezo1 channel‐mediated RGC death and suggests that there is a potential link between Piezo1 channel activation on RGCs, increased [Ca2+]i influx and apoptosis. Therefore, changes in cell stiffness and pressure acting through Piezo1 mechanosensitive channels could contribute to the neurodegenerative effects observed in optic neuropathies, such as ocular injury and glaucoma. Support or Funding Information HMC CURE Zebrafish Pilot Grant, Penn State University‐Hershey College of Medicine; Pennsylvania Department of Health, Tobacco CURE Funds

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“…Support for this comes from recent literature findings, which have found that mechanosensitivity is an integral feature of rod photoreceptors 11 and its possible role in retinal GCL damage. 41,42 Consistent with these reports, studies also suggest that T1DM rats have an exaggerated mechanoreflex, which was attenuated by blocking mechano-gated Piezo channels; 43 similarly, inhibition of Piezo1 protected against thrombosis in a type 2 diabetes mellitus setting. 44 Ye and co-worker 45 recently established the role of Piezo1 as an important physiological regulator of insulin secretion.…”

“…In the present study, immunostaining revealed that STZ-induced diabetes led to the localization of Piezo1 in retinal GCL and photoreceptor layers, and the downstream mechanism involved the phototransduction signaling pathway that eventually triggers inflammation in the retina. Support for this comes from recent literature findings, which have found that mechanosensitivity is an integral feature of rod photoreceptors and its possible role in retinal GCL damage. , Consistent with these reports, studies also suggest that T1DM rats have an exaggerated mechanoreflex, which was attenuated by blocking mechano-gated Piezo channels; similarly, inhibition of Piezo1 protected against thrombosis in a type 2 diabetes mellitus setting . Ye and co-worker recently established the role of Piezo1 as an important physiological regulator of insulin secretion.…”

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“…Piezo1 has also been linked to apoptosis in cancer cells and other cell types primarily through calpain-mediated apoptosis [ 22 , 124 , 125 ]. Calpains are proteases that are activated by calcium influx and can induce apoptosis by cleaving Bid to its active form of truncated Bid (tBid) [ 126 ].…”