Title

Copper homeostasis in the normal and mottled brindled (MoBr/y) mouse brain

Date of Completion

January 2008

Keywords

Biology, Neuroscience|Chemistry, Biochemistry|Biophysics, Medical

Degree

Ph.D.

Abstract

Copper is an essential micronutrient for proper development and function from yeast to mammals. ATP7A is a P-type ATPase that transports copper across polarized membranes, e.g. gastrointestinal epithelium, blood-brain barrier and intracellular secretory pathway. In situ hybridization studies previously demonstrated that ATP7A transcripts are widely expressed in the developing mammalian brain, but the timing and location of its expression were unknown. Based on immunoblot analyses, ATP7A expression is most abundant in the early postnatal period, reaching peak levels at P4 in mouse neocortex and cerebellum. Although ATP7A expression decreases in most neuronal subpopulations from birth to adulthood, it increases in CA2 hippocampal pyramidal and cerebellar Purkinje neurons. ATP7A levels are greatest in the choroid plexus/ependymal cells of the lateral and third ventricles. Astrocytes, oligodendrocytes and microglia express variable levels of ATP7A. In each cell type, ATP7A is largely localized to the trans-Golgi network (TGN) in vivo. The presence of ATP7A in axons of postnatal, but not adult, optic nerve suggests stage-specific roles for this transporter. Expression of the catalytically inefficient mutant ATP7A produced in the mottled brindled hemizygous male (MoBr/y) mouse brain, a model of the human copper-deficiency disorder Menkes disease, was analyzed. Levels of mutant ATP7A protein decrease postnatally in selected neuronal subpopulations, including CA1/2 hippocampal pyramidal neurons and cerebellar Purkinje cells. Decreased mutant ATP7A expression may contribute to the postsynaptic dysfunction and axonal pathology demonstrated in cerebellar Purkinje neurons. In contrast, mutant ATP7A levels increase dramatically in the endothelial cells of small-diameter blood vessels throughout the Mo Br/y brain, presumably to increase copper penetration of the blood-brain barrier. Astrocytic end-feet and perivascular microglial processes are recruited to the MoBr/y cerebrovasculature. As expected, copper levels are depleted in the MoBr/y brain; surprisingly, MoBr/y cytosol displays the most substantial reduction. Peptide amidation can only be accomplished by a secretory pathway copper-dependent peptide amidating monooxygenase. Analysis of amidated PACAP and CCK demonstrates peptide- and area-specific deficits in the function of PAM in the MoBr/y brain. The detailed analysis of ATP7A and other cuproproteins in the normal and diseased brain sheds light into the neuropathology of Menkes disease and points to future therapeutics. ^