In the United States, there are approximately 2.5 million people with spinal cord injuries (SCIs). Depending on the location and severity of the injury, SCIs can result in long-term motor and sensory impairment. A very promising technology in neurorehabilitation for people with SCIs is transcutaneous spinal cord stimulation (tSCS). tSCS is a novel, non-invasive technique that stimulates the spinal cord through the surface of the skin. Recent clinical studies have already shown that tSCS is effective in helping to rehabilitate people with SCIs. However, while the rehabilitation method is sound, the physiological effects of tSCS on muscle recruitment are not well understood. Therefore, we are investigating the modulation of the spinal networks after the intervention with tSCS. Six patients with cervical SCIs underwent physical training paired with tSCS. Before and after training, SCS was used to induce motor evoked potentials which were measured with electromyogram. Evoked responses were extracted and analyzed by comparing peak to peak amplitude. After training and tSCS, both motor function and motor evoked potential amplitude increased, providing evidence that tSCS improves rehabilitation outcomes by modifying spinal networks. This research could lead to innovations in neural engineering and rehabilitation medicine and could greatly improve the quality of life for many people with SCIs.

 Fluorescence resonance energy transfer (FRET) is a method used to elucidate changes in a protein’s structure (conformational changes). FRET works by measuring distances between amino acids in the presence and absence of ligand. The distance is measured by the highly distance dependent degree of energy transfer between an acceptor and a donor, typically two fluorophores which overlap in absorption and emission spectra. FRET is limited by the range of distances at which it is accurate, incomplete incorporation of acceptors, and its ease of use. The technique we will use to improve FRET is click chemistry. Click chemistry is a set of highly specific reactions that allow for the selective addition of desired functional groups to a target of interest. Our goal is to determine if FRET will be more sensitive to smaller ranges with click chemistry, which would increase measurement accuracy of conformational changes. To determine if click chemistry can be used with FRET, we first isolate maltose binding protein (MBP) with a non-canonical amino acid (ncAA) introduced at our site of interest from cells. The role of the ncAA is to “click”, or undergo a linking reaction with a specific functional group, with our selected fluorophore to ensure its attachment to the site of interest. Our modified protein will be incubated with our fluorophore, and FRET will be done on the protein under different conditions in order to test if our modified FRET works. If this method works, we expect to see distance dependent quenching of the fluorophore which would indicate that 1) the method works and 2) would serve to establish a benchmark for future experiments with different proteins. Combining click chemistry with FRET could be a powerful tool for measuring conformational dynamics in proteins.

In response to various stimuli, proteins undergo changes in shape (conformational changes) that are essential to their function in cell signalling, enzyme activity and many other crucial biological processes. Previous studies have shown that intramolecular distance changes associated with the conformational dynamics of proteins can be probed using transition-metal-ion-fluorescence-resonance-energy-transfer (tmFRET), in which a donor fluorophore transfers light energy to an acceptor metal ion in a steeply distance-dependent manner. tmFRET is both highly sensitive and highly specific: each unique donor-acceptor pair can only measure a limited range of distances reliably. Here, we expand the utility of tmFRET by employing new strategies to create a library of unique donor-acceptor pairs each sensitive to a different range of distances.To showcase the accuracy and precision of our measurements, we use a maltose-binding-protein (MBP) model system, which undergoes a well-studied conformational change upon the binding of maltose. In order to label MBP with Cu(II) ions that act as tmFRET acceptors, we utilize macrocyclic chelator-maleimides, which simultaneously coordinate Cu(II) and covalently modify a cysteine introduced into MBP. Using amber codon suppression, we introduce fluorescent non-canonical amino acids (ncAA’s) into MBP to act as tmFRET donors. Our fluorescence measurements show that tmFRET between the ncAA donor and chelated-Cu(II) acceptor accurately reports MBP’s intramolecular distance changes. We also show that by varying the exact structure of the molecule that chelates Cu(II) we can tune the sensitivity of tmFRET to distances ranging from 0.8-1.8 nanometers. Thus, this chelator-maleimide approach to labelling proteins with tmFRET acceptors could prove to be a powerful tool for researchers studying conformational motions of that scale.

Spinal cord injuries can result in devastating health consequences and impair voluntary muscle control. Animal models are invaluable for the development of new treatments to restore hand and arm function. In rats, we are developing a novel targeted, activity-dependent spinal stimulation (TADSS) therapy that promotes plasticity in spared pathways with a neuroprosthetic device after a C4-C5 spinal cord injury (SCI). Recovery is measured through training the rats in reaching tasks and comparing performance during therapy to the pre-injury scores. We have found that females show robust functional recovery with TADSS treatment, but males do not. One potential explanation for the difference in recovery is that females are more motivated to perform the behavioral tasks. Thus, we examined reaching performance of males and females prior to injury to see if there is any evidence of preexisting sex differences in reaching performance. Within groups of uninjured animals learning the reaching task, females on average had more trials than males, though both males and females had similar success rates. This may be evidence of greater motivation to perform the reaching task in females. Little research has been done on the role of sex and motivation in reaching tasks; however, other groups have shown that sex differences in performance of behavioral assays may be due to differences in motivation. We hypothesize differences in motivation may influence the level of functional recovery from SCI’s with TADSS therapy, as the intraspinal stimulation is dependent on muscle activity in impaired forelimbs. The aim of our future experiments is to determine the effect of sex on rodents’ motivation to perform reaching tasks before and after injury. By continuing to investigate the effect of sex-related motivation differences on motor tasks associated with SCI recovery, we hope to optimize TADSS therapy for clinical use in humans.