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BioLuminescent-OptoGenetics: New Approach for Restoring the Function of Severe Spinal Cord Injury

Written by: Naphat Permpredanun, '24

Edited by: Megan List, '24



3d illustration of Skull With Spinal Cord Anatomy stock photo, via iStock Getty Images.


The manipulation of specific neuronal populations of the spinal cord following spinal cord injury (SCI) could prove highly beneficial for rehabilitation in patients. This could work by maintaining and strengthening existing neuronal connections and/or facilitating neuronal growth and the formation of new synapses in a controlled, activity-dependent manner. Up until now, there have been two approaches to strengthen this neuronal connection: Electric Stimulation and Optogenetics.


For electric stimulation, the stimulation of circuits in the spinal cord would ideally be highly cell-type-specific and non-invasive. However, this is not the case and electrical stimulation excites all cells within the electrode vicinity including non-neuronal cells, potentially diluting or negating the effect of targeted stimulation of specific beneficial cell types. Optogenetics is a method for stimulating neurons of the spinal cord by allowing activation of specific channels or effectors that can be targeted to discrete, genetically unique neural sub-populations or glia. This allows treatment approaches to be tailored in highly specific and diverse ways. However, Optogenetics is dependent on high light intensity, which could interrupt the surrounding neuron and animal behavior in general.


With these limitations, researchers have come up with a new approach called “BioLuminescent-OptoGenetics”, which is the adapted approach of Optogenetics such that instead of using external sources of light, it applies a bioluminescent light internally by tethering bioluminescent luciferases to light-sensitive channelrhodopsins, also known as luminopsins (LMO). The bioluminescent light is produced by the breakdown of a specific enzymatic substrate, in this case, coelenterazine (CTZ). Stimulation only occurs when the CTZ is injected, producing bioluminescent light through catalysis by the luciferase. This Luciferase enzyme will stimulate the chemical reaction of luciferin, a molecule that, when it reacts with oxygen, produces light and oxygen. This bioluminescence then activates the opsin.

This process doesn’t penetrate through the neuron using high-intensity light, thus it’s a non-invasive stimulation and recruitment of all targeted actuators. Moreover, bioluminescence is light emitted without heat (“cold light”) and thus does not approach the damaging levels encountered for traditional optogenetics. Utilizing LMOs for neural stimulation in the spinal cord presents an innovative approach for activating neurons that could likely be therapeutically beneficial to recovery following SCI that was not previously possible with other approaches.


The initial test of Bioluminescent was on the treatment of Parkinson’s disease in mice. Parkinson’s disease. First, they used Optogenetic Stimulation of Spinal Neurons such that they apply this Bioluminescent Optogenetics (BL-OG in short) by transduced neurons of the lumbar enlargement with AAV vectors (Adeno-associated Virus) to express LMO3 (LIM domain only protein 3), which is the transcription cofactor that could enhance the gene replacement. Since AAV vectors are already a common channel to propagate the gene into the target, this approach is safe in practice. Then, the researchers implanted a lateral ventricle cannula, or a thin tube inserting into the lateral ventricle, for easy and controlled application of the luciferase substrate CTZ. The monitor of the expression is used in different measures such as checking the hSyn promoter, which is compatible with the AAV vector transplant. To ensure that viral transductions resulted in LMO3 expression at levels sufficient for neuronal activation and in the intended anatomical region, the researchers took advantage of the unique feature of LMOs allowing for in vivo bioluminescence imaging to report expression of the protein and confirm successful administration of the substrate. The result shows that light intensities over time consistently peaked between 10 and 30 min post CTZ application and decayed over the next hour (Figure 1B). Utilizing in vivo bioluminescent imaging not only allowed us to confirm LMO3 expression, the researchers were also able to verify proper cannula function.


Another research study regarding the benefits of Bioluminescent Ophthalmology is the Results in Accelerated and Enhanced Locomotor Recovery. The researchers used the measure of Basso, Beattie, and Bresnahan, which shows the sequential recovery stages and categorizes combinations of rat joint movements. In this experiment, thoracic contusion injury rats were randomly assigned to two groups with one group receiving CTZ and the other group receiving a vehicle via ventricular infusion, which is the former approach for the locomotor treatment. The researchers found that both approaches of Bioluminescent Optogenetic yield a much better recovery score (which is 13.3 for hSyn [human synapsin 1 gene] LMO3, and 11.2 for the Hb9-LMO3) compared to the traditional vehicle via ventricular infusion with the score of 7.7.


Bioluminescent Optogenetics could also increase the efficiency of recovering through the increase in neuronal plasticity. The researchers proved this by performing a qRT-PCR with tissue from the region stimulated (lumbar) at 8 days post-injury, during the treatment window. The experiment was carried out as described above with fresh tissue collection on day 8 post-injury, when rats had received four CTZ applications. The researchers chose this time point as it coincides with the first significant increase in locomotor recovery of CTZ treated vs. vehicle-treated animals.


Based on these experiments, there is a significant difference in the efficiency of using Bioluminescent Optogenetics than other traditional approaches. It was proven that there doesn’t need to be a high light intensity that could activate other neurons besides the harmed areas or a non-specific stimulation of neurons to have a decent spinal recovery measure.


References:

1. Adeno-associated virus (AAV) plasmids [Internet]. Addgene. [cited 2022Mar28]. Available from: https://www.addgene.org/viral-vectors/aav/


2. BBB Motor score [Internet]. JHU Medicine - Spinal Cord Injury. [cited 2022Mar28]. Available from: https://pages.jh.edu/SCI/characterization/bbb.shtml


3. Gomez-Ramirez M, More AI, Friedman NG, Hochgeschwender U, Moore CI. The bioluminescent-optogenetic in vivo response to coelenterazine is proportional, sensitive, and specific in neocortex [Internet]. Journal of neuroscience research. U.S. National Library of Medicine; 2019 [cited 2022Mar28]. Available from: https://pubmed.ncbi.nlm.nih.gov/31544973/


4. Mekhail NA, Mathews M, Nageeb F, Guirguis M, Mekhail MN, Cheng J. Retrospective Review of 707 cases of spinal cord stimulation: Indications and complications [Internet]. Pain practice : the official journal of World Institute of Pain. U.S. National Library of Medicine; [cited 2022Mar28]. Available from: https://pubmed.ncbi.nlm.nih.gov/21371254/

5. Parkinson's Disease [Internet]. NHS choices. NHS; 2019 [cited 2022Mar28]. Available from: https://www.nhs.uk/conditions/parkinsons-disease/causes/


6. Petersen ED, Sharkey ED, Pal A, Shafau LO, Zenchak-Petersen J, Peña AJ, et al. Restoring function after severe spinal cord injury through Bioluminescent-OptoGenetics [Internet].


7. Frontiers. Frontiers; 1AD [cited 2022Mar28]. Available from: https://www.frontiersin.org/articles/10.3389/fneur.2021.792643/full


8. [Image Citation]: 3d illustration of Skull With Spinal Cord Anatomy stock photo, iStock Getty Images. Retrieved 2021, from https://www.istockphoto.com/photo/3d-illustration-of-skull-with-spinal-cord-anatomy-gm967686594-263932138.

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