Document Type

Thesis - University Access Only

Award Date


Degree Name

Master of Science (MS)

Department / School

Pharmaceutical Sciences

First Advisor

Manisha Sonee


Oxidative stress is the major cause of injury that leads to a number of neurodegenerative diseases. Increasing number of studies suggest the involvement of multiple processes in the pathogenesis of cell injury during oxidative stress. Cytoskeleton disruption is known to be one such process by which oxidative stress disrupts cellular function. However we still do not have a detailed understanding of the mechanism by which the cytoskeletaJ disruption takes place. Hence an understanding of this mechanism would be very important to explore agents that can prevent the damage caused by free radicals in neurodegenerative diseases. This study has two objectives, First, is to compare and contrast the effect of oxidative stress on the three major cytoskeleton filaments, actin, microtubule and vimentin. Second, is to determine the effect of oxidative stress on the crosslinker protein 'plectin' and also to visualize and compare the distribution patterns of plectin and actin distributions under conditions of oxidative stress in neuronal cells. Human cortical neuronal cell line (HCN 2) was treated with 100 µM tertiary butylhydroperoxide, a free radical generating toxin, for 30 min, 1, 3 or 6 hrs. The IV cytoskeletal filament distribution and structure were studied by immunofluorescence studies and the protein levels were quantitated by SDS-PAGE and Western Blot assay. Immunofluorescence studies showed that in neurons subjected to oxidative stress for 30min and 1hr there were no major changes in the microfilament distribution though there was altered distribution of microtubules and vimentin observed as compared to control cells. However, loss and disruption of all the 3 cytoskeletaJ filaments was observed at later times (3 and 6hr), which was confirmed by Western blot analysis. This study indicates that free radical generation in human neurons results in loss of all the three cytoskeletal structures and also that there is a difference in susceptibility of the filaments to oxidative stress.

Plectin is a crosslinker protein, which binds and stabilizes all these three cytoskeletal structure. An early breakdown of plectin could indicate a subsequent damage to all the major cytoskeletal structures. Results have shown that plectin gene is downregulated in HCN2 cells subjected to oxidative stress at earlier time points (30mins). Both protein quantification and structural studies have also shown a down regulation of plectin as early as 30min. This indicates that plectin disruption is an early event in human neurons subjected to oxidative stress. Colocalization studies of actin and plectin distribution have shown that loss of plectin precedes actin disruption in HCN2 cells subjected to oxidative stress. This indicates that the downregulation of plectin and decrease in the levels of plectin protein may be one of the early events that take place in human neuronal cells subjected to oxidative stress.

Library of Congress Subject Headings

Oxidative stress.
Cytoplasmic filaments.
Proteins -- Crosslinking.
Nervous system -- Degeneration.


South Dakota State University



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