ABSTRACT
Severe photoreceptor cell death in retinal degenerative diseases leads to partial or complete blindness. Optogenetics is a promising strategy to treat blindness. The feasibility of this strategy has been demonstrated through the ectopic expression of microbial channelrhodopsins (ChRs) and other genetically encoded light sensors in surviving retinal neurons in animal models. A major drawback for ChR-based visual restoration is low light sensitivity. Here, we report the development of highly operational light-sensitive ChRs by optimizing the kinetics of a recently reported ChR variant, Chloromonas oogama (CoChR). In particular, we identified two CoChR mutants, CoChR-L112C and CoChR-H94E/L112C/K264T, with markedly enhanced light sensitivity. The improved light sensitivity of the CoChR mutants was confirmed by ex vivo electrophysiological recordings in the retina. Furthermore, the CoChR mutants restored the vision of a blind mouse model under ambient light conditions with remarkably good contrast sensitivity and visual acuity, as evidenced by the results of behavioral assays. The ability to restore functional vision under normal light conditions with the improved CoChR variants removed a major obstacle for ChR-based optogenetic vision restoration.
Subject(s)
Blindness/therapy , Channelrhodopsins/therapeutic use , Chlorophyceae/chemistry , Contrast Sensitivity/drug effects , Genetic Therapy/methods , Optogenetics/methods , Visual Acuity/drug effects , Animals , Behavior, Animal/drug effects , Channelrhodopsins/genetics , Channelrhodopsins/metabolism , Disease Models, Animal , Genetic Vectors/therapeutic use , HEK293 Cells , Humans , Light , Mice , Mice, Inbred C57BL , Mice, Transgenic , Mutant Proteins/therapeutic use , Patch-Clamp Techniques , Retina/metabolismABSTRACT
Information on the biodegradation potential of lake and river microbial communities is essential for watershed management. The water draining into the lake ecosystems often carries a significant amount of suspended sediments, which are transported by rivers and streams from the local drainage basin. The organic carbon processing in the sediments is executed by heterotrophic microbial communities, whose activities may vary spatially and temporally. Thus, to capture and apprehend some of these variabilities in the sediments, we sampled six sites: three from the Saint Clair River (SC1, SC2, and SC3) and three from Lake Saint Clair in the spring, summer, fall, and winter of 2016. Here, we investigated the shifts in metabolic profiles of sediment microbial communities, along Saint Clair River and Lake Saint Clair using Biolog EcoPlates, which test for the oxidation of 31 carbon sources. The number of utilized substrates was generally higher in the river sediments (upstream) than in the lake sediments (downstream), suggesting a shift in metabolic activities among microbial assemblages. Seasonal and site-specific differences were also found in the numbers of utilized substrates, which were similar in the summer and fall, and spring and winter. The sediment microbial communities in the summer and fall showed more versatile substrate utilization patterns than spring and winter communities. The functional fingerprint analyses clearly distinguish the sediment microbial communities from the lake sites (downstream more polluted sites), which showed a potential capacity to use more complex carbon substrates such as polymers. This study establishes a close linkage between physical and chemical properties (temperature and organic matter content) of lake and river sediments and associated microbial functional activities.
