Date of Award

8-2012

Degree Name

Doctor of Philosophy

Department

Biological Sciences

First Advisor

Dr. Charles Ide

Second Advisor

Dr. Christine Byrd-Jacobs

Third Advisor

Dr. John Spitsbergen

Fourth Advisor

Dr. Robert Eversole

Keywords

Alpha-synulein, apoptosis, multiple system atrophy, FAM3B, PDCD4, Oligodendrocyte

Abstract

Multiple System Atrophy (MSA) consists of three disorders; Autonomic Dysfunction, Cerebellar Ataxia, and Parkinsonism. In MSA, the protein Alpha-Synuclein (SNCA) appears in the central nervous system as misfolded protein aggregates primarily in oligodendrocytes. This dissertation reports the results from studies examining the effect of exogenous SNCA has on a cell model: C6 oligodendrocyte-like cells. Treated cells were evaluated using western blot and DNA microarray. In addition the expression of proteins was evaluated using immunocytochemistry in MSA patient tissue.

C6 cells were shown to take up SNCA when added to the media. SNCA also underwent a truncation when taken up by C6 cells. SNCA induced cell death in C6 cells in a dose-dependent manner. SNCA also induced changes in expression of Caspase 3 (CASP3), a marker for apoptosis.

In C6 cells treated with SNCA, two apoptosis-related genes, Programmed Cell Death 4 (PDCD4) and Pancreatic Derived Factor (FAM3B) showed changes in protein expression. These changes appeared to correspond to expression of CASP3. In MSA patients, FAM3B, PDCD4 and CASP3 appeared to be expressed in fibers containing myelin basic protein, a marker for oligodendrocytes, suggesting a relationship with the disease.

Cells treated with SNCA undergo changes in gene expression. In cells treated with SNCA, 3 genes were upregulated and 20 genes were downregulated. Many of these genes were related to microtubules, apoptosis and/or the AKT/PI3K/mTOR pathway.

One gene of interest, Hook Homolog 3 (HOOK3), was evaluated via western blot. After treatment with SNCA, HOOK3 protein showed a decrease in expression that confirmed the decrease in gene expression of HOOK3 observed in the DNA microarray. In MSA patients, HOOK3 co-localized with SNCA in the granule cell layer of control patients and decreased in granule cell layer of MSA patients. Interestingly, as HOOK3 and SNCA decreased in the granule cell layer, HOOK3 co-localized with SNCA in the white matter tracks of MSA patients.

Overall these results confirm that C6 cells treated with exogenous SNCA function as a model for MSA. The treatment of SNCA also appeared to influence proteins related to the AKT/PI3K/mTOR pathway, potentially regulating apoptosis.

Access Setting

Dissertation-Open Access

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