Year of Award

2008

Document Type

Dissertation

Degree Type

Doctor of Philosophy (PhD)

Degree Name

Pharmaceutical Sciences

Other Degree Name/Area of Focus

Pharmacology

Department or School/College

College of Health Professions and Biomedical Sciences

Committee Chair

Diana I. Lurie

Commitee Members

Charles M. Thompson, Darrell Jackson, Michael Kavanaugh, Edward Rubel

Keywords

Auditory system, Pb, proteomics

Abstract

Low level lead (Pb) exposure is a risk factor for neurological dysfunction including ADHD. How Pb produces these behavioral deficits is unknown, but low-level exposure during development is associated with auditory temporal processing deficits, even though hearing remains normal. Pb disrupts cellular energy metabolism and efficient energy production is crucial for auditory neurons to maintain their high rates of synaptic activity. The voltage dependent ion channel (VDAC) is an ion channel involved in the regulation of mitochondrial physiology and is a critical component in controlling mitochondrial energy production. No studies to date have investigated the effect of Pb on VDAC, therefore the current series of studies examines the interactions between Pb and VDAC. In-vitro studies were used to delineate the effects of Pb on VDAC expression. Both differentiated SH-SY5Y cells and PC-12 cells exposed to 10 μM Pb for 48 h result in a significant decrease in VDAC expression. Exposure to 24 h of hypoxia fails to decrease VDAC expression, suggesting this is a specific effect of Pb. In addition, a corresponding decrease in cellular ATP that is correlated with decreased VDAC expression occurs with Pb. Real-time RT-PCR demonstrated a significant decrease in mRNA levels for VDAC1 isoform, suggesting that Pb decreases VDAC protein expression through decreased transcription. A proteomics approach was then used to confirm that Pb exposure during development results in changes in proteins involved in energy metabolism in auditory regions of the brainstem. CBA mice were exposed to 0 mM (control), 0.01 mM (low), or 2 mM (high) Pb acetate during development. At P21, the ventral brainstem region containing several auditory nuclei, including the Medial Nucleus of the Trapezoid Body, and the medial and lateral superior olivary nuclei, was separated from the total brainstem. Proteomic analysis (isolation and separation of proteins by 2D-PAGE; analysis by MALDI-MS) revealed that chronic Pb exposure alters the expression of proteins involved in the regulation of cellular energy metabolism including VDAC and creatine kinase B. Immunohistochemistry confirms that Pb exposure results in decreased expression of VDAC in auditory nuclei, supporting the hypothesis that Pb disrupts energy metabolism in auditory neurons.

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© Copyright 2008 John Prins