Channelrhodopsin: Potential Applications in Vision Restoration

Abstract

Channelrhodopsins, the directly light‐gated ion channels from green algae, provide a powerful tool for basic research in neuroscience as well as potential applications for treating neurological diseases and disorders. One of the promising clinical applications is to cure blindness caused by the death of rod and cone photoreceptors. Retinitis pigmentosa (RP) is one of diseases that causes blindness. Although photoreceptor cells are degenerated in the retina of RP patients, other retinal neurons such as retinal ganglion cells and on bipolar cells still survive. Owing to the inherent characteristics of chlamydomonas‐derived channelrhodopsin‐2 (ChR2), photosensitive neurons can be produced in the retina by the transfer of the ChR2 gene into survived retinal neurons. A single injection of a virus vector into the eye can achieve the vision restoration in rodent model that will be a useful treatment for the patients with blindness. Herein, we introduce the molecular properties of ChR2 and a potential as a new strategy for restoring vision in humans.

Key Concepts

  • Channelrhodopsins, microbial retinal binding proteins, transiently induce photo currents by light stimuli.
  • Photocycle of channelrhodopsins remains preserved when they are heterologously expressed in animal and human cells.
  • Channelrhodoposins functions as a directly light‐gated cation channels with million second activation and deactivation kinetics.
  • Artificial photoreceptor cells can be produced in various types of animal neurons by transducing a single gene.
  • Converting surviving inner retinal neurons into directly photosensitive cells is a new strategy to treating blindness after the death of rod and cone photoreceptors.
  • Using delivery by adeno‐associated viral vectors, long‐term expression of channelrhodopsin‐2 can be achieved in rodent inner retinal neurons in vivo.
  • ChR2 can be expressed without immunologically harmful reactions in vivo.

Keywords: channelrhodopsins; directly light‐gated ion channels; retinal blinding diseases; vision restoration

Figure 1. Photocycle of ChR2. The D470 dark state is converted by a light‐induced retinal isomerisation into an early intermediate, P500. Thermal relaxation accompanied by transient Schiff base deprotonation (P390) and conformational changes of the protein creates the putative conducting state, P520. The D470 dark state recovers via the two P480 intermediates, P480a and P480b. The latter, via a major backbone rearrangement in P480b, leads to restoration of the original dark state. Like P520, the P480b intermediate is photoreactive and can be converted by light to the early P500 intermediate, but at lower quantum efficiency. Modified from Ritter et al. 2008 ©American Society for Biochemistry and Molecular Biology.
Figure 2. Expression of a Chop2‐GFP fusion construct in HEK 293 cells. (a) pCMV‐Chop2‐GFP expression cassette. (b) Chop2‐GFP expression in HEK 293 cells with pCMV‐Chop2‐GFP viewed under a fluorescence microscope. Scale bar: 15 µm. (c) and (d) Representative recordings of light responses from Chop2‐GFP fusion protein‐expressing HEK 293 cells. The current was recorded from a HEK cell, preincubated in the presence of 1 μM all‐trans retinal (c). The current was also observed in HEK cells in the absence of exogenous all‐trans retinal. (e) The average currents recorded from HEK cells in the absence or presence of 0.1 and 1 μM all‐trans retinal.
Figure 3. Properties of light‐evoked currents of the ChR2‐expressing retinal neurons (modified from Bi et al., ). (a) A recording of a Chop2‐expressing retinal cell response to light stimuli of wavelengths ranging from 420 to 580 nm. (b) A representative recording of the currents elicited by light stimuli at a wavelength of 460 nm with light intensities ranging from 2.2 × 1015 to 1.8 × 1018 photons cm−2 s−1. (c) Light‐intensity response curve. The data points were fitted with a single logistic function curve. Reproduced from Bi et al., 2006 © Elsevier.
Figure 4. Expression and light‐response properties of ChR2 in retinal neurons of rd1/rd1 mice (modified from Bi et al., ). Chop2‐GFP fluorescence viewed in flat retinal whole‐mount (a) and vertical sections (b). (c) Light microscope image of a semi‐thin vertical retinal section. Scale bar: 50 µm in (a) and 30 µm in (b) and (c). (d) Representative recordings of a Chop2‐GFP‐positive neuron in four incremental intensities at a wavelength of 460 nm. The relationship between the light intensity and the current amplitude (e), membrane depolarisation (f) and the number of spikes (g). Reproduced from Bi et al., 2006 © Elsevier.
Figure 5. Central projections of Chop2‐GFP‐expressing retinal ganglion cells and visual‐evoked potentials in rd1/rd1 mice (modified from Bi et al., ). GFP‐labelled terminal arbours of retinal ganglion cells in ventral lateral geniculate nucleus and dorsal lateral geniculate nucleus (a) and in superior colliculus (b). OT, optical track; vLGN, ventral lateral geniculate nucleus; dLGN, dorsal lateral geniculate nucleus; SC, superior colliculus. Scale bar: 200 µm in (a), 100 µm in (b). (c) VEPs recorded from a wild‐type mouse. The responses were observed at both wavelengths of 460 and 580 nm. (d) VEPs recorded from an rd1/rd1 mouse injected with Chop2‐GFP viral vectors. The responses were elicited only by light at the wavelength of 460 nm but not at the wavelength of 580 nm. (e) No detectable VEPs were observed from rd1/rd1 mice injected with viral vectors carrying GFP alone. Reproduced from Bi et al., 2006 © Elsevier.
Figure 6. Immune responses to the long‐term expression of ChR2 in rat retinas (modified from Sugano et al., ). Lymphocytes were isolated from the peripheral blood at the indicated time points and T‐cell ratio of CD4+CD25+ was calculated (a). Representative data of flow cytometric analysis at pre‐injection and 7 days post‐injection are indicated. An AAV‐Venus was injected into the eyes as a control. Production levels of the antibody to ChR2 were measured by the ELISA (b). At 64 weeks post‐injection, paraffin‐embedded retinal sections were stained with haematoxylin and eosin (c; left: control, right: ChR2, bar = 20μm). There is no difference between the ChR2‐injected and the age‐matched untreated RCS (rdy/rdy) rats. No inflammatory cells were observed in both retinas. Reproduced from Sugano et al., 2010 © Nature Publishing Group.
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Tomita, Hiroshi(Aug 2015) Channelrhodopsin: Potential Applications in Vision Restoration. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0021388.pub2]