RESUMEN
Cells from different organs in the body experience a range of mechanical and osmotic pressures that change in various diseases, including neurological, cardiovascular, ophthalmological, and renal diseases. Here, we demonstrate the use of an engineered Sensor-Actuator-Modulator (SAM) of microbial origin derived from a mechanosensitive channel of large conductance (MscL) for sensing external mechanical stress and modulating activities of mammalian cells. SAM is reliably expressed in the mammalian cell membrane and acts as a tension-activated pressure release valve. Further, the activities of heterologously expressed SAM in mammalian cells could be modulated by osmotic pressure. A comparison of the mechanosensitive activities of SAM-variants from different microbial origins shows differential inward current and dye uptake in response to mechanical stress exerted by hypo-osmotic shock. The use of SAM channels as mechanical stress-activated modulators in mammalian cells could provide new therapeutic approaches for treating disorders related to mechanical or osmotic pressure.
RESUMEN
Stimulation and continuous monitoring of neural activities at cellular resolution are required for the understanding of the sensory processing of stimuli and development of effective neuromodulation therapies. We present bioluminescence multi-characteristic opsin (bMCOII), a hybrid optogenetic actuator, and a bioluminescence Ca2+ sensor for excitation-free, continuous monitoring of neural activities in the visual cortex, with high spatiotemporal resolution. An exceptionally low intensity (10 µW/mm2) of light could elicit neural activation that could be detected by Ca2+ bioluminescence imaging. An uninterrupted (>14 h) recording of visually evoked neural activities in the cortex of mice enabled the determination of strength of sensory activation. Furthermore, an artificial intelligence-based neural activation parameter transformed Ca2+ bioluminescence signals to network activity patterns. During continuous Ca2+-bioluminescence recordings, visual cortical activity peaked at the seventh to eighth hour of anesthesia, coinciding with circadian rhythm. For both direct optogenetic stimulation in cortical slices and visually evoked activities in the visual cortex, we observed secondary delayed Ca2+-bioluminescence responses, suggesting the involvement of neuron-astrocyte-neuron pathway. Our approach will enable the development of a modular and scalable interface system capable of serving a multiplicity of applications to modulate and monitor large-scale activities in the brain.
RESUMEN
Mouse models of inherited retinal degenerative diseases such as retinitis pigmentosa are characterized by degeneration of photoreceptors, which hinders the generation of signal to be transmitted to the visual cortex. By monitoring Ca2+-bioluminescence neural activity, we quantified changes in visual cortical activities in response to visual stimuli in RD10 mice during progression of retinal degeneration, which correlated with progressive deteriorations of electro-retinography signal from the eyes. The number of active neurons in the visual cortex, the intensity of Ca2+-bioluminescence response, and neural activation parameter showed progressive deterioration during aging. Further, we correlated the thinning of retina as measured by Optical Coherence Tomography with the decrease in visual cortical activities as retinal degeneration progressed. The present study establishes Ca2+-bioluminescence monitoring as a longitudinal imaging modality to characterize activities in visual cortex of retinal degenerative disease models and therapeutic interventions.
RESUMEN
Gene therapy of retinal diseases using recombinant adeno-associated virus (rAAV) vector-based delivery has shown clinical success, and clinical trials based on rAAV-based optogenetic therapies are currently in progress. Recently, we have developed multi-characteristic opsin (MCO), which has been shown to effectively re-photosensitize photoreceptor-degenerated retina in mice leading to vision restoration at ambient light environment. Here, we report the biodistribution of the rAAV2 carried MCO (vMCO-I) in live samples and post-mortem organs following intraocular delivery in wild-type dogs. Immunohistochemistry showed that the intravitreal injection of vMCO-I resulted in gene transduction in the inner nuclear layer (INL) but did not induce detectable inflammatory or immune reaction in the dog retina. Vector DNA analysis of live body wastes and body fluids such as saliva and nasal secretions using quantitative polymerase chain reaction (qPCR) showed no correlative increase of vector copy in nasal secretions or saliva, minimal increase of vector copy in urine in the low-dose group 13 weeks after injection and in the faeces of the high-dose group at 3-13 weeks after injection suggesting clearance of the virus vector via urine and faeces. Further analysis of vector DNA extracted from faeces using PCR showed no transgene after 3 weeks post-injection. Intravitreal injection of vMCO-I resulted in few sporadic off-target presences of the vector in the mesenteric lymph node, liver, spleen and testis. This study showed that intravitreal rAAV2-based delivery of MCO-I for retinal gene therapy is safe.
