LAUSR

Dear Editor, Inherited and age-related retinal degenerative diseases cause progressive loss of photoreceptors, ultimately leading to blindness. Optogenetics is a promising strategy for restoring visual function through photosensitive proteins’ ectopic expression in surviving retinal neurons. Very recently, the optogenetic method with a red-shifted Channelrhodopsin was clinically applied for partial recovery of visual function in a blind patient. However, major obstacles to achieving optimal optogenetic vision restoration are either the low light sensitivity or the slow kinetics of existing rhodopsin-based optogenetic tools, which can be improved by molecular engineering to enhance the efficacy of fast Channelrhodopsins (ChRs). Here, we present a newly engineered ChR variant PsCatCh2.0, engineered from PsChR, which displays inherently high Ca and Na conductance and fast kinetics. We introduced a novel mutation PsChR L115C (PsCatCh) to enhance its Ca and Na permeability further and fused the cleavable N-terminal signal peptide Lucy-Rho (LR in Fig. 1a), in addition to a plasma membrane trafficking signal (T) and ER export signal (E), to improve its expression and plasma membrane targeting. PsCatCh2.0 exhibited significant improvements in expression levels/plasma membrane targeting efficiency and a larger photocurrent (Fig. 1a, b, e). 100-fold less light intensity is needed to generate a similar photocurrent response with PsCatCh2.0 than with CatCh (Fig. 1b), with BAPTA, Ca currents of PsCatCh2.0 were four times larger than those generated by CatCh (Fig. 1c, d), indicating that PsCatCh2.0 is a highly effective excitatory tool for future clinical applications. The photosensitivity and kinetics of PsCatCh2.0 were further investigated in vivo in rd1 mice. Notably, a low light intensity (3.7 × 10 photons/cm s) evoked a 14.5 pA (14.5 ± 7.4, n= 5) current in PsCatCh2.0-expressing RGCs in rd1 mice (Fig. 1g). It also presented a persistent periodic response that could follow up to 32 Hz light stimuli, without obvious desensitization (Fig. 1h), clearly outperformed MCO1 in kinetic aspect. Moreover, PsCatCh2.0 could reliably induce action potentials firing at 100 Hz when expressing in the hippocampal neuron (Supplementary Fig. 1). We tested whether visual information input could be transmitted from the PsCatCh2.0-treated retina to the brain in rd1 mice. We assessed the activity in the V1 cortex induced by light through c-Fos and Arc. Following 2 h of continuous light stimulation (470 nm, 4.7 × 10 photons/cm s), both IEGs c-Fos (red) and Arc (green) were expressed in the light-stimulated retina and V1 cortex of wild-type and PsCatCh2.0-expressing rd1 mice (Fig. 1j, k, m–o). In contrast, rd1 mice retina exhibited neither obvious light responses nor upregulation of IEGs in the visual cortex. Additionally, blue light flash visual evoked potential (VEP) recording in the visual cortex was performed. No obvious N1 amplitude in rd1 mice was recorded (1.6 ± 1.0 μV, n= 8) compared to the wild-type mice (−24.7 ± 6.7 μV, n= 8, Fig. 1i, l). In PsCatCh2.0-treated rd1 mice, the N1 amplitude of VEP was restored to −12.4 μV (−12.4 ± 1.8 μV, n= 8), suggesting regained visual function after optogenetic treatment of blind mice. Finally, we evaluated visually guided behavior in PsCatCh2.0treated rd1 mice. The fraction of time spent in light boxes, the distance and speed of movement for discovering the hole to the dark box were recorded. PsCatCh2.0-treated rd1 mice in the bluelight chamber could easily find the hole entering to the dark box, with similar performance as the wild-type mice (percentage of time spent in the light box: PsCatCh2.0, 40.8% ± 3.7 (n= 19); wildtype, 40.1% ± 2.1 (n= 11); rd1, 86.1% ± 4.0, (n= 13); one-way ANOVA; Fig. 1q). PsCatCh2.0 also rescued the distance and average speed performance of rd1 mice to the wild-type level (Fig. 1r: distance (cm): wild-type, 101.9 ± 25.7, n= 11; PsCatCh2.0, 82.3 ± 22.5, n= 19; rd1, 1058 ± 108.3, n= 13; Fig. 1s: average speed (cm/s): PsCatCh2.0, 7.7 ± 0.6, n= 19; wild-type, 7.6 ± 1.3, n= 11; rd1, 4.8 ± 0.5, n= 13). Especially, PsCatCh2.0-treated rd1 mice showed visual tracking behavior to the grating flash with an average peak spatial frequency of 0.22 ± 0.02 (c/d), compared to no response of the rd1 littermates, and 0.53 ± 0.02 c/d of the wildtype mice (Fig. 1t). Therefore, PsCatCh2.0-treated rd1 mice improved visual acuity dramatically. A light intensity of 4.7 × 10 photons/cm s was all present to induce retinal, cortical and behavioral responses, which is safe for light therapy. In this study, PsCatCh2.0 was expressed in retinal ganglion cells of blind rd1 mice. Visual acuity raised to 0.22 c/d, with a temporal resolution of at least 32 Hz. The faster and larger current PsCatCh2.0 may be an optimal therapeutic option for the treatment of retinal degeneration. Furthermore, the blue-shifted action spectrum of PsCatCh2.0 (Supplementary Fig. 2) provided the possibility to combine with red-shifted optogenetic tools to achieve colored vision restoration in the future.