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.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
5R01GM044842-23
Application #
8654336
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
23
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Pittsburgh
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Patil, Jaspal; Matte, Ashok; Nissbrandt, Hans et al. (2016) Sustained Effects of Neonatal Systemic Lipopolysaccharide on IL-1β and Nrf2 in Adult Rat Substantia Nigra Are Partly Normalized by a Spirulina-Enriched Diet. Neuroimmunomodulation :
D'Angelo, Barbara; Ek, C Joakim; Sun, Yanyan et al. (2016) GSK3β inhibition protects the immature brain from hypoxic-ischaemic insult via reduced STAT3 signalling. Neuropharmacology 101:13-23
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
Groskreutz, Stephen R; Horner, Anthony R; Weber, Stephen G (2015) Temperature-based on-column solute focusing in capillary liquid chromatography reduces peak broadening from pre-column dispersion and volume overload when used alone or with solvent-based focusing. J Chromatogr A 1405:133-9
Sandberg, Mats; Patil, Jaspal; D'Angelo, Barbara et al. (2014) NRF2-regulation in brain health and disease: implication of cerebral inflammation. Neuropharmacology 79:298-306
Ou, Yangguang; Wu, Juanfang; Sandberg, Mats et al. (2014) Electroosmotic perfusion of tissue: sampling the extracellular space and quantitative assessment of membrane-bound enzyme activity in organotypic hippocampal slice cultures. Anal Bioanal Chem 406:6455-68
Groskreutz, Stephen R; Weber, Stephen G (2014) Temperature-assisted on-column solute focusing: a general method to reduce pre-column dispersion in capillary high performance liquid chromatography. J Chromatogr A 1354:65-74
Rupert, Amy E; Ou, Y; Sandberg, M et al. (2013) Electroosmotic push-pull perfusion: description and application to qualitative analysis of the hydrolysis of exogenous galanin in organotypic hippocampal slice cultures. ACS Chem Neurosci 4:838-48
Correa, Fernando; Ljunggren, Elin; Patil, Jaspal et al. (2013) Time-dependent effects of systemic lipopolysaccharide injection on regulators of antioxidant defence Nrf2 and PGC-1α in the neonatal rat brain. Neuroimmunomodulation 20:185-93
Rupert, Amy E; Ou, Y; Sandberg, M et al. (2013) Assessment of tissue viability following electroosmotic push-pull perfusion from organotypic hippocampal slice cultures. ACS Chem Neurosci 4:849-57

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