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

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 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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Enabling Bioanalytical and Imaging Technologies Study Section (EBIT)
Program Officer
Edmonds, Charles G
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of Pittsburgh
Schools of Arts and Sciences
United States
Zip Code
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

Showing the most recent 10 out of 60 publications