Document Type

Thesis - University Access Only

Award Date

2005

Degree Name

Master of Science (MS)

Department / School

Biology

Abstract

Oxidative stress has been implicated in the onset of various diseases, including renal failure and neurodegenerative diseases. Increasing number of studies suggest the involvement of multiple processes in the pathogenesis of cell injury during oxidative stress. An important event caused by oxidants is the loss of cellular morphology and function, which may be due to disruption of the cytoskeleton. However, we still do not have a detailed understanding of the mechanisms by which cytoskeleton disruption takes place. Hence, an understanding of the mechanisms would be very important to explore agents that can prevent damage caused by free radicals in renal and neurodegenerative diseases. These studies were performed in monkey kidney cells (CV-1) to investigate mechanisms for kidney disorders and in human cortical neurons (HCN2) to investigate mechanisms for neurodegenerative diseases. This study has two objectives. First, is to determine effects of oxidative stress on the crosslinker protein ‘plectin’ and to visualize and compare the distribution patterns of plectin and actin distributions under conditions of oxidative stress in monkey kidney cells. Second, to determine the role of caspases in plectin breakdown in neuronal cells. Previous studies from our laboratory showed that CV-1 (monkey kidney) cells and human cortical neurons (HCN2) subjected to oxidative stress, showed damage to all the three major cytoskeletal structures. It is well understood that crosslinker proteins, like plectin, interconnect the three major cytoskeleton filaments. Hence, it is possible that early disruption of plectin results in the subsequent breakdown of all cytoskeleton components. This study examines the effect of free radical generatin toxin, tertiary-butyl hydroperoxide (t-BuOOH) on plectin in CV-1 cells, CV-1 cells were treated with t-BuOOH at times before significant cell death and apoptosis was observed. Immunofluorescence studies showed that actin co-localized with plectin in control cells. However, in cells treated with t-BuOOH for as early as 30 mins, loss of plectin was observed in areas with intact actin filaments and indicated that plectin breakdown preceded actin actin disruption. Changes in gene expression were measured by RT-PCR; proteins levels were measured by SDS-PAGE and Western blot analysis. Results showed reduced levels of plectin protein even in 30 mins-treated cells as compared to controls though there was no significant reduction of plectin gene expression. TUNEL staining studies on HCN2 cells treated with free radical toxin for various time points (30 mins, 1 or 3 hrs) showed DNA fragmentation only after 3 hrs. Previous studies in our laboratory showed breakdown of plectin proteins as early as 30 mins in neurons treated with free radical toxin (t-BuOOH). These findings suggest that plectin proteins as early as 30 mins in neurons treated with free radical toxin (t-BuOOH). These findings suggest that plectin damage takes place well before the DNA fragmentation. Further, we investigated the role of caspases in the breakdown of plectin in human neuronal cells. HCN2 cells treated with 100 uM t-BuOOH for 15 or 30 mins showed increased levels of pro-caspase-8 (active form) is seen significantly increased at 30 mins. Both caspase-9 and caspase-3 levels not altered in HCN2 cells treated with t-BuOOH for 15 and 30 mins compared to control. So cleaved products of caspase-9 or 3 were observed. These results suggested that caspase-8 is activated at 30 mins treatment even before caspase-9 and caspase-3 during t-BuOOH-induced oxidative stress in human cortical neurons. SDS-PAGE and Western blot analysis performed using pancaspase inhibitor indicated involvement of capases in plectin breakdown, studies performed using specific caspase-8 inhibitor suggested plectin cleavage by caspase-8 at 30 mins in t-BuOOH oxidative stress in human cortical neurons. In addition, the results obtained from immunofluorescence studies of plectin using specific caspase-8 inhibitor supported the biochemical analysis. This indicated that the early breakdown of plectin by caspase-8 even before activation of caspase-9 and caspase-3 could result in subsequent collapse of the cytoskeleton, ultimately cell death.

Library of Congress Subject Headings

Oxidative stress

Cytoskeletal proteins

Proteins -- Crosslinking

Format

application/pdf

Number of Pages

127

Publisher

South Dakota State University

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