Date of Award

1-1-2012

Document Type

Masters Thesis

Degree Name

M.S.

Organizational Unit

College of Natual Science and Mathematics

First Advisor

Daniel A. Linseman, Ph.D.

Second Advisor

David Patterson

Third Advisor

Keith Miller

Fourth Advisor

Nancy Lorenzon

Keywords

Anthocyanin, Apoptosis, Cysteine, Glutathione, Neuroprotection, Nutraceutical

Abstract

Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disease that affects motor neurons of the brain and spinal cord. Many studies indicate that mitochondrial oxidative stress (MOS) is a principal mechanism underlying the pathophysiology of this and other devastating neurodegenerative diseases. Here, we investigated a unique whey protein supplement (Immunocal®) to determine its neuroprotective efficacy in several in vitro models of MOS and in an in vivo mouse model of ALS. This non-denatured whey supplement contains cystine which is an oxidized form of cysteine, an essential precursor for synthesis of the endogenous antioxidant, glutathione (GSH). In primary cultured rat cerebellar granule neurons (CGNs), pre- incubation with Immunocal® completely protected against MOS induced by HA14-1, an inhibitor of the pro-survival Bcl-2 protein. This effect was prevented by co-incubation with the gamma-glutamyl cysteine ligase inhibitor, buthionine sulfoximine, demonstrating that the de novo synthesis of GSH underlies the neuroprotective mechanism of Immunocal®. Additionally, Immunocal® displayed significant protection against an array of MOS-inducing agents, including sodium nitroprusside, copper, and aluminum, supporting its ability to upregulate mitochondrial antioxidant capacity. In accordance with these findings in CGNs, Immunocal® decreased cell death due to both H2O2 and glutamate toxicity in NSC34 motor neuron-like cells. Immunocal® also significantly protected CHO cells from MOS evoked by overexpression of amyloid precursor protein (APP). Immunocal® treatment in NSC34 motor neuron-like cells decreased cell death caused by both H2O2 and glutamate glycine. Most compelling are our findings in the hSOD1G93A mouse model of ALS. These mice were given Immunocal® (3.33% solution in drinking water) ad libitum, beginning at 60-days-old. Although no effect on overall survival was observed, Immunocal®-treated mice displayed a significant (7 ±1.08 day) delay in disease onset, compared to mutant control mice. Importantly, Immunocal®-treated mice showed a highly significant decrease in the rate of decline in grip strength. Finally, using HPLC-ECD we found that whole blood and lumbar spinal cord GSH levels were each depleted by nearly 50% in end-stage hSOD1G93A mice, and these reductions were essentially prevented in mutant mice receiving Immunocal®. These findings suggest that sustaining GSH by supplementation with Immunocal® may help to mitigate the progression of ALS through suppression of MOS.

Publication Statement

Copyright is held by the author. User is responsible for all copyright compliance.

Rights Holder

Erika Kristine Ross

Provenance

Received from ProQuest

File Format

application/pdf

Language

en

File Size

90 p.

Discipline

Biology, Neurosciences, Molecular biology

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