All ETDs from UAB

Advisory Committee Chair

Gautam N Bijur

Advisory Committee Members

Scott Wilson

John Hablitz

Anne Theibert

Richard Jope

Document Type

Dissertation

Date of Award

2010

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

The ubiquitously expressed kinase Akt is a known survival protein, and is involved in multiple cell signaling cascades, notably the phosphatidylinositol 3-kinase (PI3K) pathway. Active Akt translocates from the plasma membrane to different subcellular compartments, including mitochondria, where it phosphorylates compartment-specific substrates. The mechanism of this translocation and the specific function of Akt within the mitochondria remains a mystery. The goals of this study were to elucidate the mechanism by which Akt enters the mitochondria and examine the role of Akt in mitochondrial function. Finally, the possibilities of using post-mortem human brain tissue to study mitochondrial function in normal and diseased states were considered. Heat shock protein-90 (HSP90) is a major player in translocation of proteins into mitochondria, and Akt is a known client protein of HSP90. Knockdown or pharmacological inhibition of HSP90 caused decreases in mitochondrial Akt levels, as well as inhibition of Akt protein translocation into isolated mitochondria. This indicates HSP90 mediates mitochondrial accumulation of Akt. Furthermore, incubation of constitutively active Akt with isolated mitochondria caused alterations in mitochondrial morphology which are indicative of increased respiration. An ultrastructural study revealed widespread Akt labeling in neurons. Surprisingly, mitochondria in neuronal processes (pMito) contained the highest levels of Akt compared to mitochondria in the soma (cMito). Functional studies of the two mitochondrial populations revealed that pMito produce ATP at higher rates than cMito, and rates of ATP production in cortical mitochondria can be modulated via activation or inhibition of PI3K signaling. These data support the notion that levels of Akt in mitochondria can influence morphology and energy production. To test if mitochondrial function could be examined in human brain, it was necessary to determine if it is possible to obtain structurally and functionally intact mitochondria from post-mortem brain. Studies in human and rodent models indicate that functional mitochondria can be isolated from post-mortem brain up to 10 hours after expiration, and these samples withstand cryopreservation for later testing. These data provide novel insights into mitochondrial translocation and function of Akt, as well as new frontiers for the study of mitochondria in human brain in health and disease.

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