Data are means SEM, n= 3 replicates, typical of 3 tests. are vulnerable to dietary iron exposure, which causes Fe2+ accumulation and oxidative stress in cortical neurons. Paralleling iron accumulation, APP ferroxidase activity in AD post-mortem neocortex is inhibited by endogenous Zn2+, which we demonstrate can originate from Zn2+-laden amyloid aggregates and correlates with A burden. Abnormal exchange of cortical zinc may link amyloid pathology with neuronal iron accumulation in AD. Introduction In Alzheimers disease (AD), Zn2+ collects with -amyloid (A) in hallmark extracellular plaques (Adlard et al., 2008; Cherny et al., 1999; Lee et al., 2002; Lovell et al., 1998; Miller et al., 2006; Suh et al., 2000), adjacent to neocortical neurons filled with pro-oxidant Fe2+ (Bartzokis et al., 1994a; Bartzokis et al., 1994b; Bartzokis and Tishler, 2000; Honda et al., 2005). The elevated neuronal iron exacerbates the pervasive oxidative damage that characterizes AD, and may foster multiple pathologies including tau-hyperphosphorylation and neurofibrillary tangle formation (Honda et al., 2005; Smith et al., 1997; Yamamoto et al., 2002), but the cause of this neuronal iron elevation is unknown. A is derived from a broadly-expressed type I transmembrane protein precursor (APP) of uncertain function, and constitutively cleaved into various fragments. The 5UTR of APP mRNA possesses a functional Iron-Responsive Element (IRE) stemloop with sequence homology to the IREs for ferritin and transferrin receptor (TfR) mRNA (Rogers et al., 2002). APP translation is thus responsive to cytoplasmic free iron levels (the Labile Iron Pool, LIP), which also govern the binding of Iron Regulatory Proteins (IRPs) to ferritin and TfR mRNA in a canonical cis-trans iron regulatory system (Klausner et al., 1993). When cellular iron LY 379268 levels are high, translation of APP and the iron-storage protein ferritin is increased (Rogers et al., 2002), while RNA for the LY 379268 iron importer TfR is degraded. Ferroxidases prevent oxidative stress caused by Fenton and Haber-Weiss chemistry by oxidizing Fe2+ to Fe3+. Losses of ferroxidase activities cause pathological Fe2+ accumulation and neurodegenerative diseases, such as aceruloplasminemia where mutation of the multi-copper ferroxidase ceruloplasmin (Cp) leads to glial iron accumulation and dementia (Chinnery et al., 2007; Harris et al., 1995; Mantovan et al., 2006; Patel et al., 2002). Iron-export ferroxidases Cp and hephaestin interact with ferroportin and facilitate the removal (e.g. by transferrin) of cytoplasmic iron translocated to the surface by ferroportin (De Domenico et al., 2007). Their expression is cell-specific (e.g. Cp in glia, hephaestin in gut epithelia), but an iron-export ferroxidase for neocortical neurons is unknown (Klomp et al., 1996). Cp is expressed in GPI-anchored and soluble forms (De Domenico et al., 2007; Jeong and David, 2003; Patel et al., 2002). APP similarly is expressed in transmembrane and secreted forms. We explored whether APP is a ferroxidase, and in turn has a role in neuronal iron export C an activity consistent with APP translation being responsive to iron levels. We also tested whether, in AD, APP ferroxidase activity is altered in a manner linked to the accumulation of its A derivative in plaque pathology. RESULTS APP695 possesses ferroxidase activity similar to ceruloplasmin We noted that APP possesses a REXXE ferroxidase consensus motif (Gutierrez et al., 1997) as found in the ferroxidase active site of H-ferritin (Figure 1A, B). This evolutionarily conserved motif is not present in paralogs APLP1 or 2 (Figure1B). There is good structural homology between the known 3D structures of H-ferritin (Lawson et al., 1991) and the REXXE-region of the E2 domain of APP (Wang and Ha, 2004), with low root mean square deviation (0.4 A) when overlaying backbone atoms of the Rabbit Polyclonal to DP-1 -helical H-ferritin catalytic site (residues 52C67) with the corresponding backbone atoms of APP (residues 402C417) (Figure 1C). The homology extends to the sidechains constituting the Fe coordinating residues of H-ferritin, E62 and H65, which overlap with potential Fe coordinating residues E412 and E415 of APP695 (Figure 1C). Open in a separate window Figure 1 Characterization of APP695 ferroxidase activityA, Schematic of APP domains. The APP770 isoform is shown, APP751 lacks the OX-2 domain, and APP695 lacks both OX-2 and Kunitz protease inhibitor (KPI) domains. CuBD= copper binding domain, ZnBD= zinc binding domain. B, Sequence homologies for the REXXE LY 379268 motif. A sole match for the REXXE motif (in bold) of H-ferritin is at residues 411C415 of human APP770, commencing 5 residues downstream from the RERMS neurotrophic motif (Ninomiya et al., 1993). This is an evolutionarily-conserved motif not present in either human APLP1 or APLP2. A consensus alignment of three glutamate residues and the ferroxidase active site of H-ferritin is underlined. The first glutamate of the REWEE motif of APP could be aligned with Glu62 of H-ferritin (in red), which is part of the ferroxidase catalytic site (Lawson et al., 1989; Toussaint et al., 2007) although this forces the REXXE motifs of the proteins two residues.