This grant proposal requests equipment for state-of-the-art rodent motor assessments and single-cell proteomics to provide users with objective techniques for fine motor behavior, which can then be strategically evaluated on a molecular level by evaluating multiple proteins on different cells types in one experiment. The requested motor assessment instrumentation includes a Noldus Catwalk XT, which uses Illuminated footprints technology, i.e., LED lighting on a glass stepping surface to record paw placement, providing a highly-sensitive method to assess gait and locomotion. To further quantify more specific changes in forelimb motor function, the Mototrak system is requested with computerized evaluation of three motor assessments: lever press, dynamic/isometric pull, and supination/pronation. Pairing objective motor assessments with the requested Fluidigm single-cell proteomic instrumentation will not only provide an innovative approach for investigators to evaluate neurological changes, but it will also deliver an unparalleled strategy for leveraging the recently-acquired Fluidigm single-cell genomics platform to validate transcriptional changes at the protein level. The requested workflow will not replace or make any existing technologies at the Lerner Research Institute obsolete, but rather it will complement and advance our currently-available tools. Investigators currently employ subjective measures of motor function at important developmental time points in disease progression. This includes subjective motor assessments in rodent models of multiple sclerosis (Drs. Trapp, DeSilva, Min, Li, Bergmann, Davalos, Dutta, and Williams), spinal cord injury (Drs. Lee and Labhasetwar), stroke (Drs. Machado and Baltan), Parkinson's disease (Dr. Taylor), pulmonary arterial hypertension (Dr. Erzurum), chronic alcohol consumption (Dr. Suh) and glioma (Dr. Lathia). The CatWalk XT and Mototrak systems not only provide objective assessments, but also afford a more sensitive analysis of fine motor deficits that can be important predictors of disease progression. The goal of the aforementioned research programs is to determine the underlying mechanisms responsible for generating motor deficits in order to develop therapeutic targets for intervention. In this regard, all of our investigators currently utilize antibody- based technologies to detect proteins on specific cell populations using immunocytochemistry and fluorescent imaging or fluorescent activated cell sorting (FACS) analysis, therefore possessing the tools necessary to transition into CyTOF technology. CyTOF (cytometry by time of flight) identifies antibody-conjugated metal isotopes providing high-content protein detection in single cells while minimizing spectral overlap. CyTOF technology is capable of analyzing 37 different proteins in 20 different samples simultaneously, due to channels reserved for bar-coding technology, augmenting multi-parameter analysis. The integrated behavior-single cell proteomics workflow provides a pioneering system to interrogate neurological deficits in rodent models of disease to facilitate translational potential for human therapeutic interventions. The Cleveland Clinic has made a strong financial commitment to ensuring the long-term maintenance and operation of these instruments.
Integrating state-of-the-art motor assessments with single-cell proteomics will provide an unprecedented workflow for interrogating neurological deficits in rodent models of disease, with the goal of providing investigators with a more direct pathway from basic biomedical research to clinical application.
