Restoration of patterned vision with an engineered photoactivatable G protein-coupled receptor

Abstract

Retinitis pigmentosa results in blindness due to degeneration of photoreceptors, but spares other retinal cells, leading to the hope that expression of light-activated signaling proteins in the surviving cells could restore vision. We used a retinal G protein-coupled receptor, mGluR2, which we chemically engineered to respond to light. In retinal ganglion cells (RGCs) of blind rd1 mice, photoswitch-charged mGluR2 (“SNAG-mGluR2”) evoked robust OFF responses to light, but not in wild-type retinas, revealing selectivity for RGCs that have lost photoreceptor input. SNAG-mGluR2 enabled animals to discriminate parallel from perpendicular lines and parallel lines at varying spacing. Simultaneous viral delivery of the inhibitory SNAG-mGluR2 and excitatory light-activated ionotropic glutamate receptor LiGluR yielded a distribution of expression ratios, restoration of ON, OFF and ON-OFF light responses and improved visual acuity. Thus, SNAG-mGluR2 restores patterned vision and combinatorial light response diversity provides a new logic for enhanced-acuity retinal prosthetics.

Optical Regulation of Class C GPCRs by Photoswitchable Orthogonal Remotely Tethered Ligands

“…BGAG n ,460 ) azobenzene core. Data on intensity and time requirements of light have been characterized in HEK 293T cells for BGAG 12 and BGAG 12,460 on SNAP-mGluR2 [44]. Similar results are expected for any PORTL with either a standard or red-shifted azobenzene core.…”

“…The bistability of standard azobenzenes, and the subsequent lack of relaxation from cis to trans on the time scale of receptor activation, allows BGAG 12 to require much lower light intensities than BGAG 12,460 . BGAG 12 produces near maximal photocurrents with light intensity levels as low as ~10 mW/cm 2 , while BGAG 12,460 requires ~1000 mW/cm 2 for maximal photocurrents [44]. Light intensities should be measured post-objective at the sample’s location with a power meter (Ex.…”

“…This approach provides a robust, repeatable, receptor-proximal readout with high temporal resolution that is orthogonal to light. These properties make such experiments ideally suited for PORTL development and refinement [30, 31], biophysical studies of receptor mechanisms [26], and is easily adapted to primary cells and tissues for studies of receptor physiology [44]. However, PORTL-mediated control of GPCRs can be applied in various experimental conditions, including imaging-based readouts and experiments in native systems, such as cultured neurons or brain slices, where endogenous reporters (i.e.…”

Branched photoswitchable tethered ligands enable ultra-efficient optical control and detection of class C G protein-coupled receptors

“…A key advantage of azobenzene-based photoswitches is the ability to tune the photochemical properties of the compound. The previously reported red-shifted BGAG 12,460 allows for visible-light induced, fast-relaxing photoactivation of mGluR2 which is advantageous in some settings, including for vision restoration applications 16 . To test if branching would also enhance PORTLs with a distinct azobenzene core, we synthesized "doubleBGAG 12,460 " and observed enhanced visible light photoactivation of SNAP-mGluR2 (Scheme S6; Fig.…”

“…These central roles in neuronal signaling have allowed the eight mGluRs to emerge as highly attractive drug targets for disorders ranging from psychiatric and neurological disease to cancer 14 , but deciphering their underlying mechanisms has proven to be challenging, limiting our understanding of both their fundamental signaling properties and the basis for their roles in disease. Azobenzene-glutamate PORTLs were initially developed for N-terminally SNAP-tagged mGluR2 9,10 where they have been shown to effectively turn mGluR2 signaling on and off with sub-second precision for applications in cultured cells 15 and in the retina of behaving mice 16 . While this system represents one of the most efficient tethered photoswitch systems characterized to date, many limits still exist including incomplete efficacy of the photoswitch relative to glutamate, the inability to visualize labeled receptors, and the lack of validation of the technique in intact brain tissue.…”