Restoring Function After Severe Spinal Cord Injury Through Bioluminescence-Driven Optogenetics

Abstract

The ability to manipulate specific neuronal populations of the spinal cord following spinal cord injury (SCI) could prove highly beneficial for rehabilitation in patients through maintaining and strengthening still existing neuronal connections and/or facilitating the formation of new connections. A non-invasive and highly specific approach to neuronal stimulation is bioluminescent-optogenetics (BL-OG), where genetically expressed light emitting luciferases are tethered to light sensitive channelrhodopsins (luminopsins, LMO); neurons are activated by the addition of the luciferase substrate coelenterazine (CTZ). This approach utilizes ion channels for current conduction while activating the channels through application of a small chemical compound, thus allowing non-invasive stimulation and recruitment of all targeted neurons. Rats were transduced in the lumbar spinal cord with AAV2/9 to express the excitatory LMO3 under control of a pan-neuronal or motor neuronspecific promoter. A day after contusion injury of the thoracic spine, rats received either CTZ or vehicle every other day for 2 weeks. Activation of either interneuron or motor neuron populations below the level of injury significantly improved locomotor recovery lasting beyond the time of stimulation. Utilizing histological and gene expression methods we identified neuronal plasticity as a likely mechanism underlying the functional recovery. These findings provide a foundation for a rational approach to spinal cord injury rehabilitation, thereby advancing approaches for functional recovery after SCI.

Bioluminescent Genetically Encoded Glutamate Indicator for Molecular Imaging of Neuronal Activity

“…For example, sensors such as BLING can replace the intact luciferases in current BioLuminescent-OptoGenetic (BL-OG) constructs 16,30 so the light sensitive ion channels open in response to glutamate resulting in activity dependent excitation or inhibition. These can then be used to improve on prior and ongoing work in neurodegenerative disorders such as spinal cord injury and Parkinson’s Disease by allowing the non-invasive current stimulation of neurons to be dependent on endogenous activity 31,32 .…”

“…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. These can then be used to improve on prior and ongoing work in neurodegenerative disorders such as spinal cord injury and Parkinson’s Disease by allowing the non-invasive current stimulation of neurons to be dependent on endogenous activity 37,38 .…”

“…All animal work was approved by Michigan State Universities Institutional Animal Care and Use Committee. BLING 1.0 was cloned into an AAV expression vector with a synapsin promoter for expression in neurons and AAV was made and purified in house as previously described 38 . Sprague Dawley rats were injected with 2μL of 0.5×1012 copies/mL at rate of 0.5μL per minute with a 33G world precision syringe at 0mm AP 3.5mm R, and 2mm ventral of the cortical surface and the needle was left in place for 5 minutes after infusion and slowly retracted.…”

Seeing (and Using) the Light: Recent Developments in Bioluminescence Technology

“…Others included Gluc fused to ChR2 harboring step-function mutations. Such probes are expanding the optogenetic toolbox for controlling neuron functions (Petersen et al, 2019;Zenchak et al, 2020).…”

The state of the art of biomedical applications of optogenetics

“…These are bioluminescent luciferases tethered to light‐sensitive channelrhodopsins. This tool eliminates the need for an external implant, which is one of the limitations of traditional optogenetic systems, and may provide a basis for a rational strategy to improve SCI recovery 39 …”