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.

Public Health Relevance

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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Enabling Bioanalytical and Imaging Technologies Study Section (EBIT)
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Edmonds, Charles G
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University of Pittsburgh
Schools of Arts and Sciences
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Ou, Yangguang; Wilson, Rachael E; Weber, Stephen G (2018) Methods of Measuring Enzyme Activity Ex Vivo and In Vivo. Annu Rev Anal Chem (Palo Alto Calif) 11:509-533
Tecuatl, Carolina; Herrrera-López, Gabriel; Martín-Ávila, Alejandro et al. (2018) TrkB-mediated activation of the phosphatidylinositol-3-kinase/Akt cascade reduces the damage inflicted by oxygen-glucose deprivation in area CA3 of the rat hippocampus. Eur J Neurosci 47:1096-1109
Ou, Yangguang; Weber, Stephen G (2018) Higher Aminopeptidase Activity Determined by Electroosmotic Push-Pull Perfusion Contributes to Selective Vulnerability of the Hippocampal CA1 Region to Oxygen Glucose Deprivation. ACS Chem Neurosci 9:535-544
Yin, Bocheng; Barrionuevo, Germán; Weber, Stephen G (2018) Mitochondrial GSH Systems in CA1 Pyramidal Cells and Astrocytes React Differently during Oxygen-Glucose Deprivation and Reperfusion. ACS Chem Neurosci 9:738-748
Wilson, Rachael E; Jaquins-Gerstl, Andrea; Weber, Stephen G (2018) On-Column Dimethylation with Capillary Liquid Chromatography-Tandem Mass Spectrometry for Online Determination of Neuropeptides in Rat Brain Microdialysate. Anal Chem 90:4561-4568
Patil, Jaspal; Matte, Ashok; Mallard, Carina et al. (2018) Spirulina diet to lactating mothers protects the antioxidant system and reduces inflammation in post-natal brain after systemic inflammation. Nutr Neurosci 21:59-69
Ou, Yangguang; Weber, Stephen G (2017) Numerical Modeling of Electroosmotic Push-Pull Perfusion and Assessment of Its Application to Quantitative Determination of Enzymatic Activity in the Extracellular Space of Mammalian Tissue. Anal Chem 89:5864-5873
Wilson, Rachael E; Groskreutz, Stephen R; Weber, Stephen G (2016) Improving the Sensitivity, Resolution, and Peak Capacity of Gradient Elution in Capillary Liquid Chromatography with Large-Volume Injections by Using Temperature-Assisted On-Column Solute Focusing. Anal Chem 88:5112-21
Patil, Jaspal; Matte, Ashok; Nissbrandt, Hans et al. (2016) Sustained Effects of Neonatal Systemic Lipopolysaccharide on IL-1? and Nrf2 in Adult Rat Substantia Nigra Are Partly Normalized by a Spirulina-Enriched Diet. Neuroimmunomodulation 23:250-259
D'Angelo, Barbara; Ek, C Joakim; Sun, Yanyan et al. (2016) GSK3? inhibition protects the immature brain from hypoxic-ischaemic insult via reduced STAT3 signalling. Neuropharmacology 101:13-23

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