DEVELOPMENTAL CHANGES IN THE EXPRESSION OF ATP7A DURING A CRITICAL PERIOD IN POSTNATAL NEURODEVELOPMENT M. J. NICIU, a X.-M. MA, a R. EL MESKINI, a,b G. V. RONNETT, b,c R. E. MAINS a AND B. A. EIPPER a * a University of Connecticut Health Center, Department of Neuro- science, Academic Research Building (E)-4047, 263 Farmington Av- enue, Farmington, CT 06030, USA b Department of Neuroscience, The Johns Hopkins University School of Medicine, 1006B Preclinical Teaching Building, 725 North Wolfe Street, Baltimore, MD 21205, USA c Department of Neurology , 1006B Preclinical Teaching Building, 725 North Wolfe Street, Baltimore, MD 21205, USA Abstract—ATP7A is a P-type ATPase that transports copper from cytosol into the secretory pathway for loading onto cuproproteins or efflux. Mutations in Atp7a cause Menkes disease, a copper-deficiency disorder fatal in the postnatal period due to severe neurodegeneration. Early postnatal cop- per injections are known to diminish degenerative changes in some human patients and mice bearing mutations in Atp7a. In situ hybridization studies previously demonstrated that ATP7A transcripts are expressed widely in the brain. ATP7A- specific antibody was used to study the neurodevelopmental expression and localization of ATP7A protein in the mouse brain. Based on immunoblot analyses, ATP7A expression is most abundant in the early postnatal period, reaching peak levels at P4 in neocortex and cerebellum. In the developing and adult brain, ATP7A levels are greatest in the choroid plexus/ependymal cells of the lateral and third ventricles. ATP7A expression decreases in most neuronal subpopula- tions from birth to adulthood. In contrast, ATP7A expression increases in CA2 hippocampal pyramidal and cerebellar Pur- kinje neurons. ATP7A is expressed in a subset of astrocytes, microglia, oligodendrocytes, tanycytes and endothelial cells. ATP7A is largely localized to the trans-Golgi network, adopt- ing the cell-specific and developmentally-regulated morphol- ogy of this organelle. The presence of ATP7A in the axons of postnatal, but not adult, optic nerve suggests stage-specific roles for this enzyme. In sum, the precisely-regulated neuro- developmental expression of ATP7A correlates well with the limited therapeutic window for effective treatment of Menkes disease. © 2006 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: copper, Menkes disease, neurodegeneration, hip- pocampus, Purkinje cells, ependymal cells. Copper is a transition metal required from dietary sources for normal development and function. In the CNS, copper is utilized as a cofactor for oxidation–reduction reactions catalyzed by cuproenzymes such as peptidylglycine -amidating monooxygenase (PAM), dopamine -mono- oxygenase, type I copper–zinc superoxide dismutase (SOD1), cytochrome c oxidase and ceruloplasmin (Suzuki and Gitlin, 1999; Waggoner et al., 1999; Prigge et al., 2000; Puig and Thiele, 2002; Hellman and Gitlin, 2002; Mercer and Llanos, 2003). Excess copper is deleterious, participating in non-enzymatic reactions that lead to free radical generation damaging DNA, lipid and protein (Ca- makaris et al., 1999; Pena et al., 1999). An elaborate and highly conserved set of copper transporters and chaper- ones delivers copper to target cuproproteins with specific subcellular localizations. ATP7A, a P-type ATPase, is in- volved in copper delivery to secretory pathway cuproen- zymes such as PAM and tyrosinase (Petris et al., 2000; Lutsenko and Petris, 2003; Steveson et al., 2003). In ad- dition, ATP7A is necessary for copper secretion to avoid toxic intracellular accumulation. ATP7A is also thought to be required for copper to cross the blood– brain barrier and enter the underlying neuropil (Qian et al., 1998). ATP7A is an eight transmembrane domain protein (Fig. 1A) with multiple copper-binding motifs, a catalytic domain and mo- tifs that govern its intracellular cycling between the trans- Golgi network (TGN) and plasma membrane (Francis et al., 1999; Petris et al., 2002). ATP7A bears close homology to ATP7B. Mutations in Atp7a and Atp7b are responsible for Menkes disease and Wilson disease, respectively (Bull et al., 1993; Petrukhin et al., 1993; Tanzi et al., 1993; Vulpe et al., 1993; Yamagu- chi et al., 1993). Menkes disease is an X-linked copper- deficiency disorder that is either prenatally or postnatally lethal due to severe neocortical and cerebellar neurode- generation and connective tissue manifestations (Tumer and Horn, 1997). Wilson disease is an autosomal reces- sive condition of copper toxicosis that presents during adolescence or adulthood with hepatic and neuropsychi- atric symptoms. In addition, altered copper homeostasis has been implicated in the pathogeneses of Alzheimer disease (Bush, 2003; Barnham et al., 2004), amyotrophic lateral sclerosis (ALS) (Kiaei et al., 2004; Selverstone et al., 2005) and prion-mediated spongiform encephalopa- thies (Sigurdsson et al., 2003; Brown, 2004). Copper injections in human patients and mottled brin- dled hemizygous male (Mo Br/y ) mice, a murine model of Menkes disease, prolong survival and attenuate some of the neurological features of this disorder (Mann et al., 1979; Yamano et al., 1985; Kaler, 1998). Mottled dappled *Corresponding author. Tel: +1-860-679-8898; fax: +1-860-679-1885. E-mail address: eipper@uchc.edu (B. A. Eipper). Abbreviations: ABC, avidin– biotin complex; DAB, diaminobenzidine; DG, dentate granule; EDTA, ethylenediamine-tetraacetic acid; FITC, fluorescein isothiocyanate; GFAP, glial fibrillary acidic protein; ILK, integrin-linked kinase; MBP, myelin basic protein; Mo Br/y , mottled brin- dled hemizygous male; PAM, peptidylglycine -amidating monooxy- genase; PODG, polymorph cells in the hilar area of the dentate gyrus; PSD-95, postsynaptic density protein of 95 kDa; PVDF, polyvinyliden- edifluoride; RCA-1, Ricinus communis agglutinin-1; SDS, sodium do- decyl sulfate; SOD1, type I copper–zinc superoxide dismutase; TGN, trans-Golgi network; TTBS, 100 mM Tris.HCl, 150 mM NaCl, pH 7.5, 0.05% Tween-20. Neuroscience 139 (2006) 947–964 0306-4522/06$30.00+0.00 © 2006 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2006.01.044 947