Improved Locomotor Recovery in a Rat Model of Spinal Cord Injury by BioLuminescent-OptoGenetic (BL-OG) Stimulation with an Enhanced Luminopsin

Abstract

Irrespective of the many strategies focused on dealing with spinal cord injury (SCI), there is still no way to restore motor function efficiently or an adequate regenerative therapy. One promising method that could potentially prove highly beneficial for rehabilitation in patients is to re-engage specific neuronal populations of the spinal cord following SCI. Targeted activation may maintain and strengthen existing neuronal connections and/or facilitate the reorganization and development of new connections. BioLuminescent-OptoGenetics (BL-OG) presents an avenue to non-invasively and specifically stimulate neurons; genetically targeted neurons express luminopsins (LMOs), light-emitting luciferases tethered to light-sensitive channelrhodopsins that are activated by adding the luciferase substrate coelenterazine (CTZ). This approach employs ion channels for current conduction while activating the channels through treatment with the small molecule CTZ, thus allowing non-invasive stimulation of all targeted neurons. We previously showed the efficacy of this approach for improving locomotor recovery following severe spinal cord contusion injury in rats expressing the excitatory luminopsin 3 (LMO3) under control of a pan-neuronal and motor-neuron-specific promoter with CTZ applied through a lateral ventricle cannula. The goal of the present study was to test a new generation of LMOs based on opsins with higher light sensitivity which will allow for peripheral delivery of the CTZ. In this construct, the slow-burn Gaussia luciferase variant (sbGLuc) is fused to the opsin CheRiff, creating LMO3.2. Taking advantage of the high light sensitivity of this opsin, we stimulated transduced lumbar neurons after thoracic SCI by intraperitoneal application of CTZ, allowing for a less invasive treatment. The efficacy of this non-invasive BioLuminescent-OptoGenetic approach was confirmed by improved locomotor function. This study demonstrates that peripheral delivery of the luciferin CTZ can be used to activate LMOs expressed in spinal cord neurons that employ an opsin with increased light sensitivity.

Bioluminescent Genetically Encoded Glutamate Indicators for Molecular Imaging of Neuronal Activity

“…Furthermore, since these reporters produce their own light, they can potentially be used as activators for light-sensitive proteins to carry out a variety of downstream functions within a cell. For example, sensors such as BLING can replace the intact luciferases in current BioLuminescent-OptoGenetic (BL-OG) constructs ,, so the light-sensitive ion channels open in response to glutamate resulting in activity-dependent excitation or inhibition. Additionally, now that we have created BLING variants with diverse properties such as varying levels of background luminescence and responses to their neurotransmitter (Figure ) we can create a variety of neuromodulators based on these sensors tailored to specific applications.…”

“…Additionally, now that we have created BLING variants with diverse properties such as varying levels of background luminescence and responses to their neurotransmitter (Figure ) we can create a variety of neuromodulators based on these sensors tailored to specific applications. These can then be used to improve our prior and ongoing work in neurodegenerative disorders such as spinal cord injury and Parkinson’s disease by allowing the noninvasive current stimulation of neurons to be dependent on endogenous activity. ,,− These new BLINGs also present ample opportunity as multiple starting points for further evolution to create new BLINGs with greater responses to glutamate and lower background. Specifically improving BLINGs to have higher affinity to glutamate, in the 100–500 micromolar range to respond to fluctuations in glutamate on the lower end of the physiological spectrum and by improving membrane trafficking and trafficking the sensor specifically to the synaptic cleft as was recently done with fluorescent glutamate sensors…”

“…Since glutamatergic neurotransmission is directly implicated in behavior, movement, mental health, pain perception, and addiction, BLING can potentially be used in a variety of drug discovery applications and used to record glutamatergic activity in experimental animal models. In the future, the design and engineering approach we describe here can be expanded to encompass other neurotransmitters and small-molecule analytes as has been done with fluorescent neurotransmitter sensors in recent years by substituting various analyte binding domains. ,, Moreover, we expect BLING can be adapted to act on light-sensitive proteins (optogenetic actuators) for BioLuminescent-OptoGenetics (BL-OG) …”

Coelenterazine-Type Bioluminescence-Induced Optical Probes for Sensing and Controlling Biological Processes

“…Recently, LMO3.2 was designed by replacing the Volvox channel used in LMO3 with a more sensitive blue-shifted opsin from Scherffelia, which resulted in a four-fold higher response after being induced by CTZ. With the increased light sensitivity and accelerated response time, LMO3.2 has the capacity to efficiently control neuronal activity by noninvasive modulation and improve locomotor function after thoracic spinal cord injury [ 63 ]. Moreover, luminopsin 4 (LMO4) was created by fusing the brighter Gaussia luciferase mutant GlucM23 with the optogenetic element Volvox channelrhodopsin-1 (VChR1).…”

“…Furthermore, the optimization of CTZ analogs is helpful for achieving more efficient substrates so as to produce brighter emissions with luciferase, which may result in more efficient photoswitching of the optogenetic elements. CTZ-type luciferins, which are popular chemical substrates, have the ability of crossing the blood–brain barrier into brain to trigger the luminopsins to control neuronal modulation in freely moving animals [ 54 , 55 , 57 , 61 , 62 , 63 , 66 , 67 ]. Only CTZ, CTZ h, furimazine, and its analog FFz are applied in bioluminescence-aided optogenetics, even though many CTZ analogs have been developed.…”