We will develop a suite of analytical methods to determine the activity of ectopeptidases in brain tissue. Ectopeptidases are membrane-bound peptidases facing the extracellular space. They are widely understood to act as a clearance mechanism for peptides. However, recent research points to other, more subtle and important roles for these enzymes. For example, some peptides are activated, and others have their activity altered by ectopeptidases. In stroke, neurons may be damaged. Certain peptides can protect neurons in this and other neurodegenerative conditions. We hypothesize that when neurons are damaged, ectopeptidase activity is changed in order to support the neuroprotective functions of particular neuropeptides (galanin, dynorphins and enkephalins). However, the tools that can test this idea do not exist. The methods that we will create largely focus on obtaining samples. Our experimental model is the organotypic culture of the hippocampal formation. We have learned how to withdraw fluid from the extracellular space in these preparations using electroosmotic flow. Electroosmotic flow results from the interaction of an externally applied electric field and the natural charges on the surfaces of cells. By using this approach, we can pull peptide solutions through small, few hundred Methods developed in this project will be widely applicable in- and outside of neuroscience for detailed investigations of the concentrations and changes in concentrations of substances in the extracellular space of tissues. The health focus in this project is particularly stroke, however findings will apply to other neurodegenerative diseases, such as Alzheimer's, Parkinson's, and ALS. These new methods will, in this project, be used to determine how certain peptides act to protect neurons from damage in stroke. The project will establish that the role of ectopeptidases, enzymes that can abolish, augment, or alter the effect of neuropeptides, in protecting neurons is more important than is currently appreciated. A plausible mechanism is that neuroprotective peptides exert their protective effect by reducing the inflammatory response of microglia. There are currently no drugs used clinically that reduce neuroinflammation effectively.Public Health Relevance
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