Alzheimer's disease is characterised by the accumulation of amyloid-b peptide, which is cleaved from the copper-binding amyloid-b precursor protein. Recent in vivo and in vitro studies have illustrated the importance of copper in Alzheimer's disease neuropathogenesis and suggested a role for amyloid-b precursor protein and amyloid-b in copper homeostasis. Amyloid-b precursor protein is a member of a multigene family, including amyloid precursor-like proteins-1 and -2. The copper-binding domain is similar among amyloid-b precursor protein family members, suggesting an overall conservation in its function or activity. Here, we demonstrate that double knockout of amyloid-b precursor protein and amyloid precursor-like protein-2 expression results in significant increases in copper accumulation in mouse primary cortical neurons and embryonic fibroblasts. In contrast, over-expression of amyloid-b precursor protein in transgenic mice results in significantly reduced copper levels in primary cortical neurons. These findings provide cellular neuronal evidence for the role of amyloid-b precursor protein in copper homeostasis and support the existing hypothesis that amyloid-b precursor protein and amyloid precursor-like protein-2 are copper-binding proteins with functionally interchangeable roles in copper homeostasis. Keywords: Alzheimer's disease, amyloid precursor-like protein 2, amyloid precursor protein, cortical neurons, gene knockout-out mice, neuronal copper homeostasis. The amyloid-b precursor protein (APP) of Alzheimer's disease is a type 1 transmembrane cuproprotein. The proteolytic processing of APP by secretases yields the amyloid-b peptide (Ab), the primary constituent of the amyloid plaque (Haass and Selkoe 1993). APP is a member of a multigene family that contains the paralogues amyloid precursor-like protein 1 and 2 (APLP1 and APLP2) (Wasco et al. 1992(Wasco et al. , 1993. Orthologues have been identified in a diverse range of species including Drosophila melanogaster, Xenopus laevis and Caenorhabditis elegans (Rosen et al. 1989;Okado and Okamoto 1992;Daigle and Li 1993). APP has a primary copper-binding domain, located in the N-terminal cysteinerich region next to the growth factor-like domain, and a secondary copper-binding domain, which is generated in Ab after proteolytic processing of APP (Hesse et al. 1994;Atwood et al. 2000). Both APP and Ab can strongly bind copper, as Cu(II), and reduce it to Cu(I) in vitro (Multhaup et al. 1996;Huang et al. 1999). Elevated copper concentrations reduce Ab production and increase secretion of APP in a cell line transfected with human APP cDNA (Borchardt et al. 1999). More recently, reports show that increased brain copper levels cause a decrease in Ab production in APP transgenic mouse models in vivo (Bayer et al. 2003;Phinney et al. 2003), while severely depleted cellular copper decreases APP gene expression (Bellingham et al. 2004).The copper-binding region is well conserved amongst the different APP-gene family members (Simons et al. 2002; Received Jun...

The amyloid precursor protein (APP), the source of the neurotoxic amyloid beta (A beta) peptide involved in Alzheimer's disease (AD), belongs to a conserved family of related proteins. In mammals, the APP family contains amyloid precursor-like protein 1 (APLP1) and amyloid precursor-like protein 2 (APLP2). Whilst a number of activities have been attributed to the APP family, an overall function has not been definitively established. While ablating either the APP or APLP2 gene in mice produces minimal phenotypic change, the combined knockout of these genes in mice causes postnatal mortality. Postnatal survival therefore requires a shared but unknown function of APP and APLP2. To investigate the biochemical basis for the postnatal lethality, plasma was analysed from double knockout mice (APP-/- APLP2-/-) 2 days before birth, at gestational day E17, and from mice at 12-16 h after birth. The postnatal double knockouts had 66% lower plasma glucose levels than their wild-type controls and 50% lower than their single knockout counterparts. Interestingly, the postnatal double knockouts displayed hyperinsulinaemia, as shown by inappropriate plasma insulin levels, given their degree of hypoglycaemia. The single knockout mice also showed hyperinsulinaemia and had 31% lower plasma glucose than the wild-types. While the double knockouts did not survive more than 24 h after birth, the single knockouts reached adulthood and their hypoglycaemia continued. Therefore, APP and APLP2 expression modulates plasma insulin and glucose concentrations. Plasma calcium, magnesium and phosphate were also significantly reduced in the double knockouts compared to the wild-types, and they showed distinctive growth restriction, suggesting the involvement of a metabolic impairment. These results link the expression of the APP and APLP2 genes with glucose homeostasis and growth and therefore identify a novel function for the APP family.

Processing of the recycling proteoglycan glypican-1 involves the release of its heparan sulfate chains by copper ion-and nitric oxide-catalyzed ascorbate-triggered autodegradation. The Alzheimer disease amyloid precursor protein (APP) and its paralogue, the amyloid precursor-like protein 2 (APLP2), contain copper ion-, zinc ion-, and heparan sulfate-binding domains. We have investigated the possibility that APP and APLP2 regulate glypican-1 processing during endocytosis and recycling. By using cell-free biochemical experiments, confocal laser immunofluorescence microscopy, and flow cytometry of tissues and cells from wild-type and knock-out mice, we find that (a) APP and glypican-1 colocalize in perinuclear compartments of neuroblastoma cells, (b) ascorbate-triggered nitric oxidecatalyzed glypican-1 autodegradation is zinc ion-dependent in the same cells, (c) in cell-free experiments, APP but not APLP2 stimulates glypican-1 autodegradation in the presence of both Cu(II) and Zn(II) ions, whereas the Cu(I) form of APP and the Cu(II) and Cu(I) forms of APLP2 inhibit autodegradation, (d) in primary cortical neurons from APP or APLP2 knock-out mice, there is an increased nitric oxide-catalyzed degradation of heparan sulfate compared with brain tissue and neurons from wild-type mice, and (e) in growth-quiescent fibroblasts from APLP2 knock-out mice, but not from APP knock-out mice, there is also an increased heparan sulfate degradation. We propose that the rate of autoprocessing of glypican-1 is modulated by APP and APLP2 in neurons and by APLP2 in fibroblasts. These observation identify a functional relationship between the heparan sulfate and copper ion binding activities of APP/APLP2 in their modulation of the nitroxyl anion-catalyzed heparan sulfate degradation in glypican-1. Processing of the amyloid precursor protein (APP)1 of Alzheimer disease (AD) involves several proteases and regulatory proteins, collectively designated ␣-, ␤-, and ␥-secretases (Fig. 1). ␤-and ␥-Cleavages lead to the generation of amyloid-␤ (A␤) peptides A␤1-40 and A␤1-42 (1, 2). The A␤ peptides are believed to cause the neurotoxicity associated with AD via an increase in A␤ levels (3). Eventually, A␤ accumulates into amyloid fibrils and finally senile plaques, the key pathological hallmark of AD. However, the APP paralogue amyloid precursor-like protein 2 (APLP2) does not contain the A␤ sequence and hence does not contribute to amyloid formation. Both APP and APLP2 bind metal ions and heparin, but the physiological significance of this property is unclear. Nevertheless, several in vitro studies have shown that APP and its ␣-secretory form are involved in neuronal growth and survival and in neurite outgrowth (for reviews, see Refs. 4 -7).The primary transcript of the APP gene undergoes extensive alternative splicing. The isoform APP695 is expressed at high levels in central nervous system neurons (8). APP-deficient mice (APP Ϫ/Ϫ ) show reactive gliosis, especially in the cerebral cortex and the hippocampus, and decreased locomotor activity ...