ABSTRACT
Optogenetic strategies to restore vision in patients blind from end-stage retinal degenerations aim to render remaining retinal neurons light-sensitive. We present an innovative combination of multi-electrode array recordings together with a complex pattern-generating light source as a toolset to determine the extent to which neural retinal responses to complex light stimuli can be restored following viral delivery of red-shifted channelrhodopsin in the retinally degenerated mouse. Our data indicate that retinal output level spatiotemporal response characteristics achieved by optogenetic gene therapy closely parallel those observed for normal mice but equally reveal important limitations, some of which could be mitigated using bipolar-cell targeted gene-delivery approaches. As clinical trials are commencing, these data provide important new information on the capacity and limitations of channelrhodopsin-based gene therapies. The toolset we established enables comparing optogenetic constructs and stem-cell-based techniques, thereby providing an efficient and sensitive starting point to identify future approaches for vision restoration.
Subject(s)
Genetic Therapy , Neurons/metabolism , Retina/metabolism , Retinal Degeneration/therapy , Animals , Channelrhodopsins/genetics , Channelrhodopsins/therapeutic use , Clinical Trials as Topic , Gene Transfer Techniques/trends , Genetic Vectors/therapeutic use , Humans , Light , Mice , Neurons/pathology , Optogenetics , Retina/pathology , Retinal Degeneration/genetics , Retinal Degeneration/pathologyABSTRACT
Cold stimuli and the subsequent activation of ß-adrenergic receptor (ß-AR) potently stimulate adipose tissue thermogenesis and increase whole-body energy expenditure. However, systemic activation of the ß3-AR pathway inevitably increases blood pressure, a significant risk factor for cardiovascular disease, and, thus, limits its application for the treatment of obesity. To activate fat thermogenesis under tight spatiotemporal control without external stimuli, here, we report an implantable wireless optogenetic device that bypasses the ß-AR pathway and triggers Ca2+ cycling selectively in adipocytes. The wireless optogenetics stimulation in the subcutaneous adipose tissue potently activates Ca2+ cycling fat thermogenesis and increases whole-body energy expenditure without cold stimuli. Significantly, the light-induced fat thermogenesis was sufficient to protect mice from diet-induced body-weight gain. The present study provides the first proof-of-concept that fat-specific cold mimetics via activating non-canonical thermogenesis protect against obesity.
Subject(s)
Adipocytes/metabolism , Adipose Tissue/metabolism , Channelrhodopsins/metabolism , Obesity/therapy , Optogenetics/instrumentation , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism , Thermogenesis/radiation effects , Adipocytes/radiation effects , Adipose Tissue/radiation effects , Animals , Body Weight/physiology , Body Weight/radiation effects , Calcium/metabolism , Cells, Cultured , Channelrhodopsins/radiation effects , Channelrhodopsins/therapeutic use , Diet , Energy Metabolism/radiation effects , Locomotion , Male , Mice , Mice, Knockout , Obesity/metabolism , Optogenetics/methods , Oxygen Consumption , Receptors, Adrenergic, beta/metabolism , Sarcoplasmic Reticulum Calcium-Transporting ATPases/genetics , Thermogenesis/physiologyABSTRACT
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
The clinical success of gene replacement therapies in recent years has served as a proof of concept for the treatment of inherited retinal degenerations using adeno-associated virus (AAV) as viral vector. However, inherited retinal degenerative diseases showcase a broad genetic and mechanistic heterogeneity, challenging the development of mutation-specific therapies for each specific mutation. Mutation-independent approaches must be developed to slow down retinal degeneration regardless of the underlying genetic mutation and onset of the disease. New understanding of cell death mechanisms in rod-cone dystrophies have led to promising rescue of photoreceptor cell death by virally mediating expression of anti-apoptotic factors and secretion of retinal neurotrophic factors. Optogenetic therapies are also able to restore light sensitivities in blind retinas.
