Date of Degree

2008

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biochemistry

Advisor

Maria E. Figueiredo-Pereira

Committee Members

Karen Duff

Dixie Goss

Philippe Marambaud

Benjamin Ortiz

Subject Categories

Biochemistry

Abstract

Two of the hallmarks of neurodegenerative disorders, such as Alzheimer's disease (AD), are (1) the appearance of proteinaceous deposits in inclusion bodies containing aggregates of ubiquitinated proteins and (2) activated microglia and astrocytes surrounding the diseased neurons. In Alzheimer's disease, the intracellular inclusion bodies are known as neurofibrilary tangles (NFT). The mechanisms leading to inclusion body formation and their role in the progression of neurodegeneration are still largely unknown.

Many of the proteins that accumulate in inclusion bodies depend on the ubiquitin/proteasome pathway (UPP) for their degradation. This pathway is responsible for the bulk (∼80%) of intracellular protein degradation. Because of its central role in the removal of mutated and misfolded proteins by degradation, disruption of the UPP is particularly relevant to the accumulation of aberrant proteins observed in aging-related neurodegenerative disorders, such as AD. Besides containing ubiquitinated proteins, one of the major components of NFTs is the microtubule associated protein "tau". Tau protein is abundant in neurons and is a highly soluble protein. Tau must be cleaved first to function as a seed for its self aggregation.

Our main hypothesis is that toxic inflammation factors released by microglia and astrocytes will damage proteins in neurons causing protein misfolding. An abrupt or chronic increase in damaged proteins will overwhelm the proteasome, particularly in old age when proteasome activity is clearly impaired. If not resolved, the ensuing accumulation of ubiquitinated proteins is potently toxic and drives the cell to activate a death pathway, therefore launching apoptosis. Caspase activation associated with apoptosis leads to caspase-mediated proteolysis of a variety of proteins including tau, which is a microtubule stabilizing protein. Tau cleavage will destabilize microtubules and cause the collapse of the cell structure. In addition, tau cleavage will promote protein aggregation. All of these events culminate in neurodegeneration.

We tested our hypothesis by incubating neuronal cells with the cytotoxic product of inflammation prostaglandin J2 (PGJ2). As we proposed, the initial event observed upon PGJ2 treatment was the accumulation of ubiquitinated proteins. This was followed by apoptosis coinciding with caspase activation and tau cleavage, culminating in protein aggregation and cell death. In other studies, we established a direct correlation between proteasome impairment (accomplished by a genetic manipulation of its chymotrypsin-like activity) with an increased vulnerability to stress conditions induced by the heavy metal cadmium. Finally, we identified a unique aging-dependent mechanism that contributes to proteasome dysfunction in Drosophila melanogaster. Our studies were the first to show that the major proteasome form in old flies is the weakly active 20S core particle, while in younger flies the fully assembled 26S holoenzyme is the preponderant proteasome form.

In conclusion, these studies support the view that maintaining proteasome activity is critical to cell survival. If proteasome activity is disrupted by inflammation or oxidative stress or even by the build-up of mutant proteins, this will have a catastrophic effect on cell survival resulting in the induction of apoptosis. The ensuing activation of caspase-mediated proteolysis will lead to partial cleavage of a variety of proteins including tau that will in turn promote protein aggregation culminating in neurodegeneration. This neurodegenerative process is exacerbated at later stages in life, because proteasome function seems to decline abruptly at an old age. A better understanding of the mechanisms leading to the build-up of protein aggregates will open up new targets for treatment of neurodegenerative disorders, such as AD, that are associated with chronic inflammation and protein aggregation.

Comments

Digital reproduction from the UMI microform.

Included in

Biochemistry Commons

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