Document Type

Thesis

Degree Name

Master of Science (MSc)

Department

Chemistry

Program Name/Specialization

Cognitive Neuroscience

Faculty/School

Faculty of Science

First Advisor

Lillian DeBruin

Advisor Role

Supervisor

Abstract

Myelination is the key feature of evolution in the nervous system of vertebrates. Myelin is the protrusion of glial cells. More specifically, "oligodendrocytes" in the central nervous system (CNS), and "Schwann" cells in the peripheral nervous system (PNS) form myelin membranes. Myelin remarkably, enhances the propagation of nerve impulses. However, myelin restricts the access of extracellular metabolites to the axons. A pathology called "demyelination" is associated with myelin. The myelin sheath is not only an insulator, but it is itself metabolically active. In this study it is hypothesized that the ratio of NAD(P)+/NAD(P)H and the glycolytic pathway of the myelin sheath is maintained via trans-plasma membrane electron transport system (t-PMET).

The t-PMET contains various membrane associated oxidoreductases by which cells oxidize intracellular electron donors at the expense of the extracellular acceptors. In this research two members of the t-PMET system, cytochrome b5 reductase (CB5R) and NAD(P)H: quinone oxidoreductase (NQO1), were identified through enzymatic assays and immunodetection analysis.

The CB5R was detected in the myelin membrane via Western blotting as two bands, one at 35kDa and the other at 33kDa, potentially representing the myristoylated and non-myristoylated forms, respectively. The enzymatic activity was measured as an NADH: cytochrome c reductase activity and it was inhibited by the sulfhydryl agent p-hydroxymercuribenzoic acid.

The NQO1, an important antioxidant enzyme of t-PMET, was investigated in the myelin membrane through immunodetection and kinetic analysis. Western blot analysis revealed a truncated form at ~13kDa in the myelin sheath. Furthermore, the associated activities of menadione-mediated cytochrome c reductase, water soluble tetrazolium 1 (WST-1) reductase and DCPIP reductase activity were found insensitive to the potent NQO1 inhibitor, dicoumarol.

Another facet of this research demonstrated the activity and enzyme expression level in the spontaneously demyelinating ND4 mouse model. The activity of CB5R was temporal; it increased with age, but fluctuated at earlier stages of life. Higher activity of NADH: cytochrome c reductase, and a higher rate of superoxide production were measured in the diseased myelin. The level of NQO1 also fluctuated over time. The fluctuation in the expression level may suggest its role in myelinogenesis, aging and disease. CB5R and NQO1, both, play important roles in decreasing oxidative stress by Coenzyme Q reduction, detoxification of quinones and xenobiotics, and through one and two electron reduction reactions.

Overall, this study reveals several putative components of t-PMET in the myelin membrane and provides a comprehensive insight into the activity of oxidoreductases catalyzing one and two electron transfer reduction reactions and their expression level in health and disease. The healthy and diseased myelin comparison will aid in deciphering the role of the identified proteins in the myelin energetics and the pathologies related to these enzymes.

Convocation Year

2015

Convocation Season

Spring

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