All ETDs from UAB

Advisory Committee Chair

Gautam Bijur

Advisory Committee Members

Rosalinda Roberts

John Hablitz

Harald Sontheimer

Linda Wadiche

Document Type

Dissertation

Date of Award

2011

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

The widely distributed serine/threonine kinase glycogen synthase kinase 3beta is well known for its multiple functions in the health brain tissue, which include cell fate, neuronal growth and remodeling, synaptic plasticity, and neuroinflammation. However much of GSK3beta focuses on its abnormal functions in neurological disorders like Alzheimer's Disease and psychiatric disorders. To maintain normal GSK3beta function in the brain, this constitutively active kinase must be strictly regulated. Many factors within the cell work together to influence GSK3beta activity. Two ways to regulate GSK3beta activity are through phosphorylation by upstream kinases or the formation of protein complexes that block substrate binding. Additionally, GSK3beta activity in the brain can also be controlled by intracellular localization. While GSK3beta is mainly a cytosolic protein, it can also exist in discrete subcellular pools that restrict its access to compartment specific substrates. Of these forms of regulation, little has been shown concerning GSK3beta intracellular distribution in the brain. Therefore, the first goals of this research were to visualize GSK3beta intracellular distribution in both healthy mouse brain tissue and in diseased mouse brain tissue. Finally, a specific pool of GSK3beta in the mitochondria was examined for its role in binding and targeting mitochondrial complex-I of the electron transport chain. Previous studies had demonstrated through biochemical separation that GSK3beta exists in the brain within the cytoplasm, mitochondria, and nucleus yet none had actually visualized this distribution. Using light and electron microscopy of healthy mouse brain tissue, we found that GSK3beta is widely distributed in neurons throughout brain regions and is localizes to the rough endoplasmic reticulum, ribosomes, mitochondria, and post synaptic densities. We also found inactive GSK3beta, phosphorylated at its Serine-9 residue, to be highly expressed in microglia and also present in these same intracellular areas. An examination of GSK3beta distribution in the brain was also performed in a transgenic mouse called BiAT that mimics the characteristics of Alzheimer's Disease. In old BiAT mice, a population of neurons show increased GSK3beta expression only in brain regions where pathology develop as compared to the modest GSK3beta staining in these regions in control mice. Intracellularly, more robust GSK3beta labeling was found in the rough endoplasmic reticulum, ribosomes, and microtubules of old BiAT mice as compared to age matched control mice. Younger BiAT mice were identical to age matched control mice indicating the increase in GSK3beta accumulation is age dependent. Mitochondrial complex-I of the electron transport chain was previously shown to be regulated by mitochondrial GSK3beta activity, yet little was known about GSK3beta interaction with complex-I. Examination of isolated mitochondrial complex-I showed that GSK3beta associates with complex-I and that overactive mitochondrial GSK3beta increases serine phosphorylation of a complex-I subunit. This data provides new insights into the intracellular distribution of GSK3beta in both healthy and diseased brain tissue, which can provide a foundation for investigating GSK3beta function within these subcellular regions.

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