Date of Completion

5-9-2013

Embargo Period

5-9-2013

Major Advisor

Richard Mains

Associate Advisor

Eric Levine

Associate Advisor

Douglas Oliver

Associate Advisor

Jonathan Covault

Associate Advisor

Elizabeth Eipper

Field of Study

Biomedical Science

Degree

Doctor of Philosophy

Open Access

Open Access

Abstract

Peptidylglycine α-amidating monooxygenase (PAM) is a secretory granule membrane protein whose luminal enzymatic domains catalyze the Cu-dependent amidation biosynthetic step common to many neuroactive peptides. Aside from its essential role as the sole mammalian amidating enzyme, PAM alters Cu homeostasis, modulates transcription and regulates secretory granule trafficking. The model of PAM haploinsufficiency employs mice heterozygous for the Pam gene (PAM+/-); the mice show anxiety-like disorders and deficiencies in fear learning and memory. We performed whole-cell recordings of pyramidal neurons in the PAM+/- amygdala to elucidate neurophysiological correlates of the fear behavioral phenotypes. Consistent with these observations, thalamic afferent synapses in the PAM+/- lateral nucleus were deficient in long-term potentiation (LTP). This deficit was apparent in the absence and presence of the GABAA receptor antagonist picrotoxin and was abolished when both GABAA and GABAB receptors were blocked. Dietary Cu supplementation rescued the cued learned-fear deficits of PAM+/- mice, with little effect on the behaviors of wildtype mice. Dietary Cu supplementation also corrected the LTP deficit of PAM+/- mice in vitro. Bath application of the extracellular specific Cu chelator bathocuproine disulfonate abolished LTP in wildtype and PAM+/- amygdalae, demonstrating a vital role for Cu in amygdalar synaptic plasticity. Dietary Cu supplementation had no effect on brain Cu or PAM levels, therefore PAM+/- behavioral deficiencies do not result from insufficient Cu and/or PAM. Localization of the major neuronal Cu transporter, ATP7A, was altered in the PAM+/- brain. In addition, quantitative PCR revealed region-specific deficits in Atox-1 and ATP7A that may account for the physiological and behavioral defects associated with PAM heterozygosity. These data indicate that Cu is necessary for normal amygdalar synaptic function, suggesting the PAM+/- behavioral and physiological phenotype stems from dysregulated Cu secretion. Additional studies include analyses of serum Cu and PAM in a population of frail elderly men and electrophysiological study of hippocampal and nucleus accumbens neuronal membrane and synaptic properties in Kalirin7 null mice. Future directions to test the hypothesis of a direct role for PAM in regulating neuronal Cu secretion and explore the essential role of Cu at amygdala afferent synapses are outlined.

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