Changing serotonin receptor 2C splice variants to combat spasticity after spinal cord injury

University Of Kentucky, Lexington KY

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Abstract

Project Summary Spinal cord injury (SCI) affects approximately 300,000 Americans resulting in devastating neurological and physical limitations. SCI in the chronic phase is complicated by muscle spasms, which are to a large degree caused by hyperactivation of the serotonin receptor 2C (5HT2C) caudal to the injury. Through a combination of alternative splicing and editing, the G-protein coupled receptor generates at least 25 isoforms with different regulatory properties: one intracellular truncated receptor 5HT2C_tr, one non-edited full-length receptor 5HT2C_Fl_INI and 23 full length edited receptors 5HT2C_FL_ed. The ratio of these isoforms wherein the 5HT2C_FL shows constitutive activity plays a major part in the maladaptation after injury. SCI leads to a loss of editing activity of ADAR2 (adenosine deaminase acting on RNA), which results in a downregulation of 5HT2C_FL_ed isoforms, relative to the 5HT2C_FL_INI. Extending our joint published work (Zhang et al., 2016, EMBO Mol. Med)1, we concentrated on the novel 5HT2C_tr isoform that sequesters the 5HT2C_FL intracellularly. We found an increase of the 5HT2C_tr isoform after SCI, possibly as an adaptation to prevent the constitutively active 5HT2C_FL_INI from hyperactivity. To intervene with the isoform ratios, we developed a series of oligonucleotides that either increase or decrease the 5HT2C_tr /5HT2C_FL_INI ratio, as well as an antiserum that is specific for the 5HT2C_tr protein. Using these tools, we will test the hypothesis that SCI leads to an increase in both constitutively active 5HT2C_FL_INI and its downregulating 5HT2C_tr isoform. This change deregulates spinal cord neuronal activity leading to spasms, likely due to insufficient 5HT2C_tr isoform to stop the 5HT2C_FL_INI activity. We will thus further test whether these spasms can be mitigated using our developed oligonucleotides that promote 5HT2C_tr. Using an established S2 spinal cord transection rat model, we will map the spatial and temporal changes in receptor isoforms and test the influence of splicing-changing oligonucleotides on the development of tail spasms. The oligonucleotides interfere with the serotonin system by changing the surface localization of constitutive active 5HT2C_FL, which is a novel approach compared to ligand- based drugs activating surface receptors. Using this innovative approach targeting the pre-mRNA, these studies are significant with great potential to treat spasticity, which is a major co-morbidity of SCI.