Abstract
Purpose :
Some glaucoma patients continue to lose visual function long after therapeutic stabilization of their intraocular pressure (IOP). This suggests that injury of retinal ganglion cells (RGCs) may become independent of pressure following the activation of pressure-sensitive trigger mechanisms. We tested the hypothesis that these trigger mechanism require the involvement of glia-intrinsic mechanosensitive TRPV4 channels.
Methods :
Intraocular pressure in C57BL/6, TRPV4flox/CRALBP-/- and TRPV4flox/Thy1-/- mouse retinas was elevated through injection of polystyrene microbeads in the ipsilateral eye. RGCs and Müller cells were isolated by magnetic-activated cell sorting (MACS). Immunolabeling, Ca-imaging and qRT-PCR were utilized to determine the changes in gene and protein expression, and localization. Pressure-evoked currents and calcium signals in isolated Müller cells and endfeet from intact glia were measured with high-speed pressure clamp combined with optical imaging.
Results :
cKO retinas showed ~50% reduction in Trpv4 mRNA, absence of TRPV4 immunoreactivity in glutamine synthetase-immunopositive compartments and total loss of glial but not neuronal, currents and calcium signals induced by the selective agonist GSK1016790A. Pressure clamp experiments revealed that TRPV4 mediates a substantial fraction of the Müller glial pressure response, which was reduced in cKO and pan-KO glia. Glaucomatous eyes exhibited elevated levels of transcripts encoding proinflammatory and proapoptic signals including calcium-dependent calpain, caspase and ER stress mechanisms. Expression of ER stress (CHOP, pIRE1a, etc.) and inflammatory (GFAP, MAPK, etc.) markers was significantly lower in retinas from mice with conditionally ablated TRPV4 channels.
Conclusions :
We identify the mechanosensitive TRPV4 channel as a Müller glial pressure sensor, and show that its activation is required for IOP-dependent retinal inflammation in mouse glaucoma. Conditional channel ablation from Müller cells reduced reactive gliosis and protected RGCs from pressure-induced ER stress and apoptotic signaling. Our data thus provide experimental support for the hypothesis that elevated IOP acts on nonaxonal targets within the retina, and identify a novel potential mechanism that could drive functional loss of vision in glaucoma patients with therapeutically stabilized IOP.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.