We will develop a powerful analytical method to determine the activity of ectopeptidases in vivo and to determine the fate of peptides in the extracellular space of selected areas of the brain. 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 for others their activity is changed by ectopeptidases. These phenomena are implicated in stroke (hypoxia/ischemia) and its effect on cognition, and in degenerative diseases of the brain. Certain peptides can protect neurons against damage, or can do the opposite by increasing inflammation which follows injury from hypoxia/ischemia. In fact, certain peptides are thought to play an important role in preconditioning the brain to recover better from stroke or to slow the rate of decline in degenerative diseases. The problem is that current capabilities to learn about what actually happens to peptides in vivo are not adequate. We propose to use electroosmotic flow to push substrate peptide solutions through small, sub-mm regions of the hippocampus, collect substrate and products with microdialysis, and analyze the samples quantitatively using capillary liquid chromatography with dynamic temperature control followed by mass spectrometry. We will apply this to the determination of differences in extracellular processing of neuroprotective and pro-inflammatory peptides in the dorsal (cognition) and ventral (behavior) hippocampus in vivo in both male and female adult rats, to understanding whether the activity of the ectopeptidase known as IRAP, which contributes to brain damage in hypoxia/ischemia, is decreased by hypoxic preconditioning, and to determine whether IRAP activity in hippocampus increases in neonates following systemic inflammation.
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 neuropeptides in brain. The health focus in this project is how enzymes in the brain act to preserve brain health or to harm the brain after stroke and in neurodegenerative diseases, such as Alzheimer's, Parkinson's, and ALS. The enzymes in question can abolish, augment, or alter the effect of neuropeptides and thus they determine how peptides act to protect (or not) neurons from damage. No adequate methods to determine how these enzymes alter peptide actions in vivo exist. The proposed methods will be applied to problems related to stroke and cognition as well as neuroinflammation. Based on our work, new directions for drug development to control these enzymes' activity can be envisioned.
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