Access Type

Dissertation/Thesis

Date of Award

January 2019

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Anatomy and Cell Biology

First Advisor

Dr. Zhuo-Hua Pan

Abstract

Discovery of channelrhodopsin (ChR), a light sensitive protein from green algae, has revolutionized the field of neuroscience research by empowering scientist to control neuron through the light, the technology popularly known as optogenetics. The ChR based optogenetics is one of the promising approaches for treating blindness caused by photoreceptor degenerative diseases such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD). Fundamentally, the approach is about imparting light sensitivity to surviving inner retinal cells by ectopic expression of genetically encoded light sensitive proteins, such as ChR2. Although the concept of optogenetic approach has been proved by using ChR2, a major obstacle of using the wild type ChR2 or in general ChRs for vision restoration is their low light-sensitivity. With the molecular engineering approach more light sensitive variants of ChR2 have been created recently, however, their light sensitivity remained 2-3 log units below the threshold of cone photoreceptors. Additionally, most of the ChR variants with improved light sensitivity are blue light sensitive. Since the longer wavelength light can have better tissue penetration and less photo-toxicity, the development of ChR with higher light-sensitivity and red-shifted peak spectra (λmax) was desired. In this study, two newly reported ChRs, the CoChR (Chloromoas oogama ChR) and the ReaChR (Red activable ChR, a chimera variant) were chosen to improve their light sensitivity by molecular engineering. The CoChR was chosen because of its larger photocurrent compared to that of ChR2, while ReaChR was selected because of its red-shifted peak spectral sensitivity (λmax). Additionally, attempts were made to shift the λmax of the CoChR towards the red. For CoChR, three different sites with specific mutations, specifically H94E (HE), L112C (LC) and K264T (KT) were identified, which together created the most light-sensitive CoChR-3Mt (CoChR-HE/LC/KT). The CoChR-3Mt markedly enhances photocurrent to low light intensity and, thus, increases operational light sensitivity in compared to the wild type CoChR (CoChR-Wt). The enhanced light sensitivity was found to be correlated with the slower off-kinetics. However, the λmax of CoChR could not shift towards longer wavelengths (red) either by site-direct mutagenesis or by chimera approaches. This suggested that the spectral sensitivity of the ChR, in general, is tightly regulated by a complex mechanism that is yet to be revealed. For ReaChR, a combination of three mutations, specifically I171M-V302L-N305H (IM-VL-NH), was identified which moderately enhanced its light sensitivity. Again, the enhanced light sensitivity was correlated with slower off-kinetics.

In conclusion, the CoChR-3Mt was found to be the most light-sensitive ChR variant that can be a better optogenetic tool for vision restoration.

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