WO2012027794A2 - Method of treatment and agents useful for same - Google Patents

Abstract

The present invention relates generally to a method of treating conditions characterised by aberrant amyloid precursor protein ferroxidase activity and agents useful for same. More particularly, the present invention relates to a method of treating conditions characterised by aberrant amyloid precursor protein ferroxidase activity by modulating amyloid precursor protein ferroxidase activity, in particular modulating the interaction of zinc or GFD with amyloid precursor protein. The method of the present invention is useful, inter alia, in the treatment and/or prophylaxis of conditions including, but not limited to, Alzheimer's disease.

Description

METHOD OF TREATMENT AND AGENTS USEFUL FOR SAME

FIELD OF THE INVENTION

The present invention relates generally to a method of treating conditions characterised by aberrant amyloid precursor protein ferroxidase activity and agents useful for same. More particularly, the present invention relates to a method of treating conditions characterised by aberrant amyloid precursor protein ferroxidase activity by modulating amyloid precursor protein ferroxidase activity, in particular modulating the interaction of zinc or GFD with amyloid precursor protein. The method of the present invention is useful, inter alia, in the treatment and/or prophylaxis of conditions including, but not limited to, Alzheimer^" s disease.

BACKGROUND OF THE INVENTION

Bibliographic details of the publications referred to by author in this specification are col lected alphabetically at the end of the description.

The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publ ication (or information derived from it) or known matter forms part of the common general knowledge i n the field of endeavour to which this spec i fication relates.

A lzheimer's disease is a progressive neurodegenerative disorder that is characterised by synaptic and neuronal loss (Whitehouse et ai , 1 982, Science 21 5 : 1237- 1 239) and the deposition of protein aggregates in the intracellular and extracellular compartments of the brain, leading to the loss of memory, cognitive disturbances and behav ioural changes. The extracellular deposits or amyloid plaques consist primarily of the β-amyloid protein (Αβ) (Glenner et al. , 1984, Biochem. Biophys. Res. Commun. 1 20:885-89), whereas the intracel lular deposits or neurofibrillary tangles contain the microtubule-associated protein tau (Grundke- Iqbal et ai , 1986, J. Biol. Chem. 26 1 :6084-6089). Alzheimer' s disease is the fourth largest cause of death in the United States and affects five percent of people over age 65 and 20 percent of people over age 80. To date, there has been no established treatment developed which w ill prevent the onset of or significantly delay the progression of Alzheimer's disease.

The most characteristic neuropathological feature of Alzheimer' s disease is the deposition of β amyloid peptide (herein referred to as 'Άβ") into plaques in the brain parenchyma and cerebral blood vessels leading to neuronal loss and cerebral atrophy (Terry R.D. et al., 198 1 ). Ap is proteolytically derived from a large membrane-spanning glycoprotein known as β amyloid precursor protein (herein referred to as "APP") ( ang J. et al, 1987). The deposition of Αβ is believed to be closely related to the pathogenesis of Alzheimer's disease. The accumulation of Αβ in diffuse plaques is one of the earliest Alzheimer-specific neuropathological changes in Down's syndrome (Mann et al, 1 989; Mann et al, 1988).

I n Alzheimer's disease, Zn²⁺ collects with 1 3-amyloid (Αβ) in these hallmark extracellular plaques (Adlard et al. , 2008; Cherny et al. , 1999; Lee et al.. 2002; l.ovel l et al. , 1 998; M i ller et al. , 2006: Suh et al. , 2000), adjacent to neocortical neurons fi l led with pro- oxidant Fe²" ( Bartzokis et al. , 1 94a; Bartzokis el al.. 1 994b; Bartzokis and Tishlcr, 2000; Honda et al. , 2005). The elevated neuronal iron exacerbates the pervasive oxidative damage that characterizes Alzheimer^'s disease, 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.

Λβ is derived from a broad ly-expressed type I transmembrane protein precursor

(amyloid precursor protein) of uncertain function, and constitutively cleaved into various fragments. The 5'UTR of amyloid precursor protein mRNA possesses a functional Iron- Responsiv e Element (Iron-Responsive Element) stem loop with sequence homology to the Iron-Responsive Elements for ferritin and transferrin receptor (TfR) mRNA ( Rogers et al. , 2002). Amyloid precursor protein translation is thus responsive to cytoplasm ic free iron levels (the Labile Iron Pool, LI P), which also govern the binding of Iron Regulator)' Proteins (Iron Regulatory Proteins) to ferritin and TfR mRN A in a canonical cis-trans iron regulatory system ( lausner et al. , 1993). When cellular iron levels are high, translation of amyloid precursor protein and the iron-storage protein ferritin is increased (Rogers et al. , 2002), whi le R A for the iron importer R is degraded.

Ferroxidases prevent oxidative stress caused by Fenton and Habcr- Weiss chemistry by oxidizing Fe^{2 *} to Fe³⁺. Losses of ferroxidase activities cause pathological Fe² ' accumulation and neurodegenerative diseases, such as aceruloplasminemia where mutation of the multi- copper ferroxidase cerulopiasmin leads to glial iron accumulation and dementia (Chinnery et al. , 2007: Harris et al. , 1 95: Mantovan et al. , 2006; Patel et al. , 2002). Iron-export ferroxidases cerulopiasmin and hephaestin interact with ferroportin and faci litate the removal (e.g. by transferrin) of cytoplasmic iron translocated to the surface by ferroportin (De Domenico el ai , 2007). Their expression is cel l-speci fic (e.g. ceruloplasmin in glia. hephaestin in gut cpithelia), but an iron-export ferroxidase for neocortical neurons is unknown ( Klomp et ai.. 1 996). Accordingly, there is a need to better understand the mechanisms which underpin the oxidative stress associated with Alzheimers so as to potentially enable the development of new approaches to the therapeutic and prophylactic treatment of Alzheimer's disease. Current treatments are, at best, marginally effective and with a very large aging population, the need to develop better treatment methods is urgent.

In work leading up to the present invention it has been determined that amyloid precursor protein (APP) is a functional ferroxidase, the activity of which is mediated by a conserved H-ferritin-like active site. The activity of this site is itself downregulated by Zn² ". Since APP catalytically oxidizes Fe²⁺ to Fe³⁺, thereby mitigating the tissue damage w hich would otherwise be effected by Fe²⁺ induced oxidative stress, the determination that abnormal exchange of Zn^2† which accumulates in amyloid plaques inhibits A PP ferroxidase activ ity has now provided the basis for the development of a new treatment approach for disease conditions characterised by aberrant APP ferroxidase activity, such as A lzheimer^'s disease. Sti ll further, it has been determined that the growth factor domain (G FD) of A PP potentiates A PP ferroxidase activ ity. Accordingly, this provides a further mechanism by which to modulate APP ferroxidase activity. SUMMARY OF THE INVENTION

Throughout this spec ification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

As used herein, the term "derived from" shall be taken to indicate that a particular integer or group of integers has originated from the species specified, but has not necessari ly been obtained directly from the specified source. Further, as used herein the singular forms of "a", "and" and "the" include plural referents unless the context c learly dictates otherwise.

Unless otherwise defined, a ll technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The subject specification contains amino acid sequence information prepared using the programme Patentln Version 3.5, presented herein after the bibl iography. Each amino acid sequence is identified in the sequence listing by the numeric indicator <2 10> followed by the sequence identifier (eg. <210> 1 , <210>2, etc). The length, type of sequence (protein, etc) and source organism for each sequence is indicated by information provided in the numeric indicator fields <2 1 l >, <212> and <21 3>, respectively. Amino acid sequences referred to in the specification are identified by the indicator SEQ I D NO: followed by the sequence identifier (eg. SEQ ID NO: I . SEQ I D NO:2. etc.). The sequence identi fier referred to in the specification correlates to the information provided in numeric indicator field <400> in the sequence listing, which is followed by the sequence identifier (eg. <400> 1 , <400>2. etc). That is SEQ ID NO: l as detailed in the specification correlates to the sequence indicated as <400> 1 in the sequence listing.

One aspect of the present invention is directed to a method for the therapeutic or prophylactic treatment of a condition in a subject, which condition is characterised by aberrant APP ferroxidase activity or Fe²⁺ levels, said method comprising administering to said subject an effective amount of an agent for a time and under conditions sufficient for said agent to modulate the functional interactivity of Zn²⁺ with said APP wherein antagonising the interaction of Zn²' with said APP increases APP ferroxidase activity and facilitating the interaction of Zn²⁺ with said APP decreases APP ferro idase activity.

In a related aspect, there is provided a method for the therapeutic or prophylactic treatment of a condition in a subject, which condition is characterised by aberrant APP ferroxidase activ ity or Fe ⁺ levels, said method comprising administering to said subject an effective amount of an agent for a time and under conditions sufficient for said agent to modulate GFD potentiation of APP ferroxidase activity wherein faci litating the interaction of GFD with APP increases APP ferroxidase activity and antagonising the GFD interaction w ith APP decreases APP ferroxidase activity.

In another aspect there is provided a method for the therapeutic or prophylact ic treatment of a condition in a subject, which condition is characterised by insufficient A PP ferroxidase activity or excess Fe² ' levels, said method comprising administering to said subject an effective amount of an agent for a time and under conditions sufficient for said agent to antagonise the interaction of Zn^"+ with said APP.

In one particular embodiment, the Zn²⁺ chelator is a moderate affinity chelator which is hydrophobic. Examples include the 8-hydroxy quinol ines, such as clioquinol. P T2. M30. VK.28 or related molecules, pyrithione, diethyl py rocarbamate. 1 ,2-bis-(2-(amino- phenoxy)elhane-N.N,N ^'.N ^,-tetraacetic acid and derivatives, the bicyc lam analogue JKL I 69 ( Ι , Γ-xylyl bis- 1 ,4,8, 1 1 tetraaza cyclotctradecane), DP 1 09 and related compounds.

In still another aspect, there is provided a method for the therapeutic or prophylactic treatment of a condition in a subject, which condition is characterised by unwanted APP ferroxidase activity or insufficient Fe²⁺ levels, said method comprising increasing the level of Zn²⁺ in said subject for a time and under conditions sufficient for said Zn^{2^} to interact with said APP.

In yet another aspect, there is provided a method for the therapeutic or prophylactic treatment of a condition in a subject, which condition is characterised by insufficient APP ferroxidase activity or excess Fe²⁺ levels, said method comprising administering to said subject an effective amount of SEQ I D NO:2 or functional fragment, mimetic, analogue or homo!ogue thereof for a time and under conditions sufficient to potentiate APP ferroxidase activity.

In still another aspect there is provided a method for the therapeutic or prophylactic treatment of a condition in a subject, which condition is characterised by unwanted APP ferroxidase activity or insufficient Fe³⁺ levels, said method comprising administering to said subject an effective amount of an agent which antagonises the interaction of GFD with A PP.

In a further aspect there is provided a method for the therapeutic or prophy lactic treatment of a condition in a subject, which condition is characterised by insuffic ient central nervous system APP ferroxidase activity or excess Fe²⁺ levels, said method comprising administering to said subject an effective amount of an agent for a time and under conditions sufficient for said agent to:

(i) antagonise the functional interactivity of Zn^{2 *} with said APP; or

(ii) faci litate the interaction of GFD with APP.

In another aspect there is prov ided a method for the therapeutic or prophylactic treatment of a neurodegenerative disease in a subject, said method comprising administering to said subject an effective amount of an agent for a time and under conditions sufficient for said agent to:

(i) antagonise the functional interactivity of Zn ⁺ with said APP; or

(ii) facilitate the interaction of GFD with APP.

In yet another aspect, there is provided a method for the therapeutic or prophylactic treatment of a condition in a subject, which condition is characterised by unw anted APP ferroxidase activity or insufficient Fe^{2 t} levels, said method comprising administering to said subject an effective amount of an agent for a time and under conditions sufficient for said agent to: (i) increase the level of Zn~ ; or

(ii) antagonise the interaction of GFD with APP.

In yet still another aspect, the present invention relates to the use of an agent which:

(i) antagonises the functional interactivity of Ζη^2τ with APP; or

(ii) facilitates the interaction of GFD with APP;

in the manufacture of a medicament for the treatment of a condition characterised by insufficient APP ferroxidase activity or excess Fe~~ levels.

In yet still another aspect, the present invention relates to the use of an agent which: increases the level of Zn²' ; or

) antagonises the interaction of GFD with APP

the manufacture of a medicament for the treatment of a condition characterised by unwanted APP ferroxidase activity or insufficient Fe²¹ levels.

Yet another aspect of the present invention relates to modulatory agents, as hereinbefore defined, when used in the method of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure I . Characterization of APP695a ferroxidase activity. A, Schematic of amyloid precursor protein domains. The APP₇₇o isoform is shown. APP751 lacks the OX-2 domain, and APP695 lacks both OX-2 and unitz protease inhibitor ( PI) domains. CuBD= copper binding domain, ZnBD= zinc binding domain. B, Sequence homologies for the

REXXE motif. A sole match for the REXXE motif (in bold) of I l-ferritin is at residues 41 1 - 415 of human APP770, commencing 5 residues downstream from the RERMS neurotrophic motif (Ninomiya et ai, 1993). This is an evolutionarily-conservcd motif not present in either human APLP 1 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 amyloid precursor protein could be aligned with G l u62 of H-ferritin (in red), which is part of the ferroxidase catalytic site (Lawson el ai , 1989; Toussaint et ai, 2007) although this forces the REXXE motifs of the proteins two residues out of register. C, An overlay of the backbone atoms (^"N, Ca, C) of residues 52-67 of the known H-ferritin active site (Lawson et ai , 1 991 ) (PDB accession no. 1 FHA) with the putative ferroxidase site within residues 402-41 7 of APP695 (Wang and Ha, 2004) ( l rw6) (RMSD 0.4 A). The Fe coordinating residues of H- ferritin. E62 and H65 (shown in red) overlap with the corresponding residues E412 and E41 5 that make up the putative ferroxidase site of amyloid precursor protein (shown in green), based upon the sequence alignment in C. D & E, Kinetic values of Fe^3' formation from Fe²⁺ monitored by incorporation into transferrin, indicated within the graphs, were calculated for each protein (200 nM) incubated with various concentrations of Fe^{i 1} at pH 7,2 to reflect the normal pH of brain interstitial space, where apo-transferrin is abundant (Visser el al. , 2004). ceruloplasmin values are in close agreement with the original reports (Osaki, 1966). Data are means± SEM, n= 3 replicates, typical of 3 experiments. See also Figure 8.

Figure 2. Domains important to amyloid precursor protein ferroxidase activity and its inhibition by Zn²⁺. A, Activities of the E2 fragment of APP ± GFD-containing fragments compared to APP695a FD l ^{lEI 4N)}-APPa and APLP2a in I IBS, pU7.2. Effects of ferroxidase inhibitors NaNh ( 10 mM) for ceruloplasmin, and Zn^{2 t} ( 1 0 μΜ) for H-ferritin, are shown. FD l (^i;uN)-APP695a has the mutation in the REXXE motif shown in Figure 2B,C. B, Sequences of FD l and derived peptides used to map the active site of APP695a. The REXXE motif is in bold, and the substitution site in red. The last 3 peptides have substitutions in the putative active site that represent the homologous sequences of H-ferritin, APLP I and APLP2. respectively. C, Ferroxidase activities of a 22-residue peptide containing the REXXE consensus motif of APP ("FD l ", see B) and the same peptide where the RE WEE sequence is substituted with RE WEN ("E l 4N'\ see B). D, Ferroxidase activity of FDl is specific to the REXXE motif. Activity is retained upon deleting the first 9 residues (containing the RERMS motif), and when the H-ferritin REXXE consensus motif is substituted into the peptide

(WE 12/1 3HA). Activity is eliminated by substitution of the APLP l (EE 13/ 14AM) and APLP2 (R I OK) sequence, which disrupt the REXXE consensus sequence. All peptides were 0.5 μΜ. E, Ferroxidase activity of the E2 domain of amyloid precursor protein (0.5 μΜ) is potentiated by the El domain in a concentration-dependent manner up to a l : l stoichiometry. Values are means ± SEM, n= 3 replicates, typical of 3 experiments. See also Figure 8.

Figure 3. Amyloid precursor protein promotes iron release, lowers the labile iron pool and interacts with ferroportin in HEK293T cells. A, Iron flux was measured after incorporation of Tf(⁵⁹Fe)2. Amyloid precursor protein RNAi (vs non-specific scrambled RNAi. ^■'sham¹') induces cellular ⁵⁹Fe retention. Suppression of amyloid precursor protein, in triplicate. was confirmed by western blot (22C 1 1 ). B, APP695 (2 μΜ) added to the media promotes ⁵⁹Fe export over 6h. C & D, Western blot (as shown in Figure S2B) quantification: amyloid precursor protein RNAi increased ferritin (to ¾ 200%) and decreased TfR levels (to «50%), whi le ΑΡΡ695 partially reversed these effects. Additional iron (Fe(NH4)2(S0₄)2, Ι ΟμΜ ) raised the baseline ferritin and lowered the TfR, but the effect of adding or subtracting amy loid precursor protein was similar. Sh = "sham", non-specific scrambled RNA i. E, Interaction of amyloid precursor protein with fcrroportin using anti-Fpn for detection and anti-N-terminal amyloid precursor protein for immunoprecipitation of HEK293T cel ls treated w ith iron ( 10 μ ). No interaction with APLP2 confirmed specificity to amyloid precursor protein. Nonspecific rabbit IgG was used as a control ('"-ve"). F, Biotin-labelled APP695 , when added to the media of HEK293T cel ls treated with Fe(NH4)2(S0₄)2 ( 1 0 μΜ), is immunoprecipitated from the cell homogenate with anti-Fpn antibody. Data are means ± SEM of n=3. *= p<0.05, * *= p<0.01 , ***= p<0.001 ; A & B analysed by 2-tailed t-tests, C & D by ANOVA + Dunnet's tests. See also Figure 9.

Figure 4. Intracellular iron accumulates in APP-/- neurons. A, APP- '- primary neurons treated with Tf(⁵⁹Fe)₂ retain more ^s¾Fe after 1 2 h than cells from WT controls.

APP695a (2 μΜ) promotes ^,9Fe export into the media after 1 2 h from both WT and APP-/- neurons. In APP-/- neurons this reduces intracellular iron to approach WT levels. B, ⁵⁹l e media efflux is decreased for APP-/- compared to WT primary neurons. Data are ^,vFe counts in media expressed as a fraction of the total in culture. C. Western blot (see Figure S3D) quantification of ferritin and TfR in primary neuronal cultures from WT and APP-/- matched controls treated ± Fe( H₄)2(S0₄)2 (75 μΜ). Differences in APP-/- cells are consistent with increased retention of iron. D, amyloid precursor protein and ceru loplasrnin co- immunoprecipitate with ferroportin from human and mouse brain, but not APLP2. E.

Determination that membrane-bound full-length amyloid precursor protein interacts with ferroportin using amyloid precursor protein detection antibodies for both the N- and C-terminal ends of the protein from membrane lysate of human brain immunoprec ipitated by anti-Fpn antibody. F, amyloid precursor protein -/- neurons incubated with increasing concentrations of Fe( H₄)2(S0₄)2 are more susceptible to iron toxicity, measured by CCK-8 cell viability assay, than WT neurons. Data are means ± SEM, n=3. *= p<0.05, * *= pO.01 , ***= p<0.00 l , A - C analysed by 2- tailed t- tests, D by ANOVA + Dunnet's test compared to WT. See also Figures 10 and 1 1 .

Figure 5. Dietary iron challenge increases tissue iron in A PP-/- but not normal mice. A, 1 2 month old A PP-/- mice accumulate iron within brain (¾ I 25%), liver (¾ 1 30%) and kidney (» 1 1 5%) tissue compared to VVT matched controls. Iron levels were further increased in brain (¾140%) and l iver (=250%) of APP-/- mice fed a high iron diet for 8 days, which did not alter iron levels in WT matched controls. B-G, Labile redox-active iron detected by modified Perl's staining in hepatocytes (B, E) and cortical neurons (C-D & F-G) from ΛΡΡ-/- (F.-G) and WT matched controls (B-D) fed a high iron diet. H, Computer-assisted

quantification of modified Perl's-stained surface area of brain sections from m ice fed on a high iron diet (n=4 mice, average of 3 sections each), indicates that APP-/- mice have significantly more redox-active iron positive cells per hemisphere, and in the hippocampus, compared to WT. I , Ferroxidase activity in brain from APP-/- mice is decreased compared to WT matched controls, ceruloplasmin activity is determined after treatment of the tissue with Zn²" to inhibit the activ ity of amyloid precursor protein, amyloid precursor protein activity is determined after treatment of the tissue w ith a i to inhibit the activity of ceruloplasmin. J- . I n accord w ith increased redox-active iron in l iver and brain from APP-/- mice, signi ficantly increased protein carbonylation occurs in APP-/- mice fed on a high iron diet (J) and decreased glutathione in APP-/- high iron diet ( ). Data are means * SEM, n"4, *= p<0.05. **= pO.01 , p<0.001 , A analysed by ANOVA + Dunnet's test compared to WT, H-K by 2-tailed t-tests. See also Figure 1 1 and Table 2.

Figure 6. Decreased cortical amyloid precursor protein ferroxidase activity in Alzheimer's disease. A, Alzheimer's disease cortical tissue accumulates iron compared to age-matched non-demented (ND) samples. Iron levels were not changed in pathologically unaffected cerebel lum from the same subjects. B, amyloid precursor protein -spec i fic ferroxidase activ ity is decreased in Alzheimer^'s disease cortical tissue («75%) but not in cerebellum, consistent with the pattern of iron accumulation in A. Chelating Zn²⁺ from the tissue with TPEN restores the amyloid precursor protein ferroxidase activity in A lzheimer^'s disease sample to levels comparable to N D cortex. C, Both free Zn²\ as well as Zn^:* dissociating from washed Zn²" :A 2 aggregates, inhibit APP695a ferroxidase activ ity but not ceruloplasmin activity. D, Decrease in APP-specific ferroxidase activit correlates w ith increased Αβ content in Alzheimer^' s disease cortical tissue (pO.000 1 , r^~ 0.829). E. amyloid precursor protein ferroxidase activity is not changed in cortical tissue from ηοη-β-amy loid burdened neurodegenerative diseases such as frontotemporal dementia and Parkinson's disease. A-C & E, Data are means ±SEM, n=8, **= p<0.0 l . * **= pO.001 by 2-tai led t-tests. See also Figure 8. See also Figure 1 2 and Table 3.

Figure 7. Model for the role of amyloid precursor protein in cellular iron export and its inhibition in Alzheimer's disease. Fpn transports Fe^2÷ from the cvtosol across the plasma membrane. Fe² is then converted to Fe^3* by a membrane-bound or soluble ferroxidase such as ceruloplasmin or amyloid precursor protein (shown). The absence of the ferroxidase results in decreased iron release into the extracellular space, as Fe²' is unable to be converted into FV⁺. Amyloid precursor protein ferroxidase is inhibited by extracellular Zn²⁺ (Figures 2Λ & 6B), which can exchange from Αβ:Ζη²⁺ aggregates (Figure 6D). Free Zn²⁺ is normally buffered by the presence of ligands such as metallothioneins (including metallothionein 111 in the extracellular space), which are lost in Alzheimer's disease (Uchida el ai , 1991 ). Loss of metallothioneins and other Zn²" buffers may lie upstream in amyloid pathology, amyloid precursor protein ferroxidase inhibition and neuronal iron accumulation in Alzheimer's disease. See also Figure 1 3.

Figure 8. Ferroxidase activity of amyloid precursor protein measured by transferrin assay. A, Fe'⁺ incorporation into transferrin is catalyzed by APP695a in a pH- dependent manner as previously shown with ceruloplasmin in various pH buffers at 37 °C. B. As a control BSA does not show any ferroxidase activity compared to amyloid precursor protein and ceruloplasmin (Figure I D & E), as measured by Fe^{, T} formation from Fe^{2 ,} (50 μΜ ferrous ammonium sulphate) and incorporated into transferrin. The copper-binding domain of amyloid precursor protein (Figure 1 A), ± Cu²⁺, also was inactive (not shown). C. Ferroxidase activity of as-isolated soluble APPa isoforms has no significant difference. Values are means ± SD, n= 3 readings, typical of 3 experiments.

Figure 9. Amyloid precursor protein promotes iron release, lowers the labile iron pool and interacts with ferroportin in HEK293T cells. A, Addition of the E2 domain of amyloid precursor protein to fresh serum-free media, after pre-incubation of Tf( ⁹Fe)2 (75 g), promotes iron efflux from HE 293T cells transfected with either non-specific scrambled RNAi (sham) or RNAi specific for amyloid precursor protein suppression. B. Representative immunoblots of quantitaled proteins (see Figure 3C & D) in HEK293T cells exposed to APP695a, RNAi or Fe² ( 10 μΜ), as indicated. Amyloid precursor protein (detected by W02) was minimal in cells transfected with RNAi except where APP695cc is added exogenously. whereupon the APP695a adheres to the cells consistent with the interaction with cellular ferroportin (see Figure 3F). It is notable that in the cells that were not treated with exogenous APP695ot, treatment with iron did not increase endogenous HEK293T amyloid precursor protein levels (lanes 1 -2 vs 9- 10), even though amyloid precursor protein translation is expected to increase (Rogers at al , 2002). However, the increased amyloid precursor protein production in response to iron is only reflected in secreted amyloid precursor protein species, whose levels were elevated in the media in our experiments (not shown). This exactly replicates, using the same cell line, previous results reported where iron induced increased secreted amyloid precursor protein due to increased u-secretase processing, but no change in cellular amyloid precursor protein holoprotein (Bodovitz el al, 1995). C, amyloid precursor protein RNAi in HE 293T cells leads to reduced Iron Regulatory Protein- 1 and Iron

Regulatory Protein-2 binding to ferritin-lron-Responsive Element, which in turn leads to increased expression of ferritin and reflecting intracellular iron accumulation upon decreased amyloid precursor protein expression. Iron Regulatory Protein detection is shown after protein binding to biotinylated ferritin-lron-Responsive Element, captured with streptavidin-coated beads. D, Stably-transfected cells over-expressing wild-type APP695 (wt-APP) loaded with Tf(⁵⁹Fe)2 retained significantly less iron compared to cells transfected with empty vector. Conversely, cells transfected with inactive-ferroxidase mutant FD 1 ^{(1: MN}'-APP695 retained significantly more iron. E, In agreement with iron retention alterations, ferritin expression was significantly decreased 6h after iron loading in cells transfected with wt-APP695 but significantly increased in FD1 ^{(E I N>}-APP695 cells. F, Western blot (22C 1 1 ) showing that expression of transfected wt-APP695 or FD I ^(,';MN,-APP695 in the Η ΕΚ293Ϊ cells was comparable. G, Immunoprecipitations were performed using anti-ferroportin (Fpn) antibody to capture interacting proteins, and non-immune rabbit IgG as negative control (-ve). I n support of Figure 3E & F, anti- amyloid precursor protein anti-N-tcrminus, 22C 1 1 , and aiUi-Αβ Domain of amyloid precursor protein, W02) western blots of HEK293T cells treated with Fe(NH₄)2(S0₄)2 (10μΜ), detect amyloid precursor protein immunoprecipitated by anti-Fpn antibody. Data are means ± SEM of (A) n=5 and (C-E) n=3. * = p<0.05, ** - p<0.01. *" - p<0.001 , 2-tailed t-test, compared to control.

Figure 10. Amyloid precu rsor protein promotes iron efflux in primary neuronal cultures and rescues toxicity. A, After pre-incubalion of f(⁵ c)2 (75 ^tg) within serum-free culture media for 12h, addition of the APP695 to fresh scrum-free media for a further 6h promotes iron efflux from wild-type mouse primary cortical neurons in a concentration- dependent manner. 2 μΜ was considered the useful experimental concentration since it corresponded with previous reports of ceruloplasmin effects on other cell-types (De Domenico et al . 2007). APP695ct acting without ferroportin would not mediate iron export because this soluble spec ies does not span the membrane. B, Serum-free media from wt-APP695 HE 293T cells promoted the efflux of ^5gFe from wild-type primary neurons pre-loaded with Tf(^wl-^'e)2 whereas neurons incubated with media from FD l ^{(t l4N)}-APP695 expressing cells retained more ⁵⁹Fe compared to cel ls incubated with control empty-vector conditioned media. Western blot showed that soluble amyloid precursor protein levels in both wt-APP and FD I ^<HJN'-APP conditioned media were equivalent (data not shown). C, As with HE 293T cells (Figure S2A), the E2 domain of amyloid precursor protein (2 μΜ) added to fresh serum-free media promotes cellular iron release from wi ld-type mouse primary cortical neurons pre-loaded with Tf(⁵⁹Fe): (75 μg). Amyloid precursor protein E2 faci litated the efflux of iron into the media and cells retained less ^5yFe. D, Representative Western blots quantitatcd in Figure 4C, of wi ld-type and APP-/- primary neurons incubated ± 75 μΜ Fe²⁺ ( 1 0 pg lysates). Amyloid precursor protein ablation causes an increase in ferritin and a reduction in TfR (reflecting intracel lular iron accumulation), which is exaggerated with iron treatment. As previously reported,

ceruloplasmin was not detected in neurons. E. Purified wild-type soluble APP695a

significantly rescued mouse cortical primary neurons from glutamate toxic ity in a dose- dependent manner (p-trend <0.01 ) whereas puri fied soluble FD l ^{( MN )}-A PP695a was less effective at rescue (p-trend not significant). Data are means ± SEM of (A ) n 4. ( B) n=3. (C) n=5, (E) mean of four independent experiments, n>5 repl icates for each amyloid precursor protein concentration. A, p-trend by one-way ANOVA with post-hoc test for linear trend. B, C, 2-taiIed t-tests. E, ANOVA with post-test Dunnett's against control (no APP). * - p<0.05. ** = p<0.01 , ' = p<0.00 l .

Figure 1 1. Amyloid precursor protein, ceruloplasmin and ferroportin

interactions and levels in normal and APP-/- tissue. A-C, immunoprecipitations were performed using anti-ferroportin (Fpn) antibody to capture interacting proteins, and non- immune rabbit IgG as negative control (-ve). As in HEK293T cel ls ( Figure S2G). and complementary to Figure 4D & E, amyloid precursor protein is isolated from cortical mouse (A & B) and human (C) brain homogenate using anti-Fpn immunoprec ipitation. Speci fic ity of the immunoprec ipitation is confirmed with APP-/- mouse brain showing no detectable bands (B). D & E, Densitometric quantitation of anti-ceruloplasmin Western blot of Fpn antibody immunoprecipitations from wild-type littemiate and APP-/- brain homogenate i l lustrating increased ceruloplasmin bound to Fpn in APP-/- brain tissue. Data expressed as % of the total amount of ceruloplasmin in the brain homogenate (Start material). F, Brain and l iver tissue samples from 12-month old APP-/- and wi ld-type littermates fed a normal or iron- supplemented diet showed no significant changes in ferroportin or ceruloplasmin levels by western blot. G, amyloid precursor protein and ceruloplasmin protein levels in various tissues from 1 2-month old wild-type mice. Organs where amyloid precursor protein was expressed at levels 100 ng/mg protein accumulated iron in APP-/- m ice ( Figure 5A). Data arc means

+SEM, n=3. (E) *** = pO.001 by 2-tailed t-test. (F&G) ANOVA + Dunnet's test showed no significant differences between the genotypes.

Figure 12. Amyloid precursor protein ferroxidase activity in Alzheimer's disease and control brain. A, amyloid precursor protein ferroxidase activity in non- demented (^"N D) and Alzheimer's disease patient cortical tissue (one illustrative cortical sample assayed in triplicate) was confirmed as amyloid precursor protein by immunodepletion. Samples were assayed in the presence of azide to suppress ceruloplasmin activ ity. I lomogenate extracts depleted of amyloid precursor protein by immunoprec ipitation with 22C 1 1 retained no activity. However amyloid precursor protein eluted from the immuno-beads recovered the activ ity present in the start material (total homogenate). B, amyloid precursor protein levels

(densitometry of 22C 1 1 western blot, 10μg total protein per lane) are unchanged in ncocortical tissue from Alzheimer's disease patients compared to ND controls (n=8 each group). C. Total tissue zinc levels are unchanged in the same specimens as Figure 6A & B. D. Zn²⁺ titration to reverse TPEN-mediated release of amyloid precursor protein ferroxidase activity in cortical tissue. ND and Alzheimer's disease patient cortical tissue (3 samples each, a subset for the samples shown in Figure 6B, each sample assayed in triplicate) was assayed for amyloid precursor protein ferroxidase activity in the presence of azide. Zn^{2 t} added to N D tissue suppressed amyloid precursor protein ferroxidase activity, but only when the Zn^"

concentration was >20 μΜ and had reacted with all the TPEN (20 μΜ) present. Therefore. before titrating in Zn^{2^}, the TPEN must have been present in excess of the exchangeable tissue Zn²\ In contrast. Zn²⁺ added to Alzheimer^'s disease samples began to suppress amyloid precursor protein activity even at the lowest concentration tested (2 μΜ) indicating that the TPEN present (before the titration) was already nearly fully- bound by exchangeable tissue Zn^{2 *} E, Total and amyloid precursor protein -speci fic ferroxidase activ ity is m ildly but significantly decreased with age in wild-type mouse brain (pO.0001 ). This is greatly exaggerated in transgenic littermates (Tg2576) that carry the Swedish mutation of amyloid precursor protein and accumulate AR in brain with age (p<0.0001 ). F, amyloid precursor protein restored ferroxidase activity by TPEiN is only present in 24 month Tg2576 mice; an age consistent with high Ali plaque load. A, B, D & F. Data arc means ±SE . *** - p<0.001 by 2- tailed t-test.

Figure 13. Iron-Responsive Element motifs in ferritin subunits and the amyloid precursor protein superfamily. A, Alignment of the Iron-Responsive Element region of the amyloid precursor protein mRNA with equivalent sequences in the 5'UTRs of the ferritin L- chain and H-chain mRNAs. The canonical L- and H-ferritin Iron-Responsive Filement loop region is underlined as the CAGUGN consensus motif where the amyloid precursor protein specific CAGAGC sequence aligns with the L-mRNA CAGUGU and H-m NA CAGUGC loop sequences. These form the loop regions of the canonical ferritin Iron-Responsive HIement RNA stem loops (Rogers el l. , 2002). amyloid precursor protein possesses an Iron- Responsive Element motif with strong sequence homology to H-ferritin (also a ferroxidase) but no homology to L-ferritin, (which lacks ferroxidase activity). B. APLP I and APLP2 5'UTR specific RNA sequences respectively encode unrelated CCTGTC and CCGAGT motifs (underlined). This, and the lack of homology with either the amyloid precursor protein or the L- and H-ferritin Iron-Responsive Elements, indicates that APLP- 1 and APLP-2 mRNAs do not encode Iron-Responsive Element-like stem loops in their 5'UTRs. APL I and APLP2 lack an Iron-Responsive Element motif in this region, and neither possesses ferroxidase activity (Figure 2A). Notably, amyloid precursor protein. APLP I and APLP2 all inhibit HO. but of these, only amyloid precursor protein is a ferroxidase. C, Homology values with the amyloid precursor protein 5'UTR demonstrating that the amyloid precursor protein Iron-Responsive Element exhibits significantly greater homology with the H-subunit mRNA (61 % Overall, 72% clustered) than the L-subunit mRNA (44.2% overall, 27% clustered). Sequences were analysed by Two-way Blast alignment (NCBI ). The APLPs vs amyloid precursor protein were manually aligned so 3' ends would be 36 nt from the AUG codons.

Figure 14. Zinc inhibits ferroxidase II activity in plasma and only azide inhibitable ferroxidase I is present within APP deficient mice. (A) The effects of azide ( 10 mM), and Zn²⁺( 10 μΜ) upon odianisidine oxidation in normal adult human plasma. Azide inhibition of total ferroxidase activity leaves a residual - 1 % activity that equates to the inhibition in total activity by zinc. (B) Zn²⁺ inhibition of o-dianisidine oxidase activity in human plasma is dose dependent resulting in a near maximal inhibition at concentrations greater than 10 μΜ. (C) Transferrin ferroxidase activ ity of A PP-/- plasma is markedly decreased compared to background control and APLP2-/- mice, and is completely inhibited by azide (consistent with the activity present in APP-/- mice being CP-related ferroxidase 1). Values are means ± SEM, n= 3 readings, and are typical of 3 experiments.

Figure 15. Separation of CP and APP in plasma relates to ferroxidase I and II activity. (A & B) Protein elution profiles of APP (anti-FD l ) and CP from human serum, separated by ion exchange chromatography at pH 5.5, compared to ferroxidase activity of the same fractions ± azide or Zn²⁺. Zinc inhibitable ferroxidase I I activity is present within the void fractions and corresponds to APP immunoreactive fractions whereas azide inhibitable ferroxidase I activity is located in fractions elutcd at 0.2M NaCl and matches CP

immunoreactivity. (C) Activity eluted in the non-bound (void) fraction from ion exchange chromatography, representing ferroxidase I I. is not present in APP-/- plasma and is inhibited by Zn² ' ( 10 μ Μ) in wild-type plasma. Conversely. CP activity is present in both wi ld-type and APP-/- plasma and correlates with azide-inhibited ferroxidase I activity eluted at the higher ion exchange fraction. All human samples were tested w ith transferrin assay and mouse samples with 0-dianisidne. Data are means ±SEM, n= 3.

Figure 16. Immunodepletion of APP from human plasma ablates ferroxidase I I activity. (A) Antibody capture of CP (Sigma) and APP (22C 1 1 ) from human plasma resulted in almost complete loss in activity of ferroxidase I and II respectively from the remaining plasma. Residual activity was confirmed as ferroxidase I I (in the case of CP depletion) and ferroxidase I (in the case of APP depletion) w ith the addition of the specific inhibitors azide and zinc. (B) Further support of specificity of each ferroxidase complex was provided w ith m i ld elution of captured proteins indicating ferroxidase I activ ity in elutes from anti-CP and ferroxidase I I activity in elutes from anti-APP. Each elute showed comparable activity for their corresponding ferroxidase from total plasma. As a control, antibody capture with β-actin showed full activity of both ferroxidases in residual plasma and no activ ity in elutes. Al l assays were performed with o-dianisidine and data are means ±SEM, n= 3.

Figure 17. Both Ferroxidase I and II activity is reduced in plasma from AD patients. (A) Total ferroxidase, as measured by o-dianisidine assay, was observed to be significantly decreased in plasma from AD patients compared to age-matched healthy controls. Using azide as a specific inhibitor of ferroxidase I activity, the decreased activ ity in AD samples was found to be made up of signi ficantly reduced activity in both ferroxidase 1 (B) and ferroxidase 11 (C). Data are means ± SEM of n = 30. * = p < 0.05, * * * = p < 0.001 as analyzed by two-tailed t tests. Figure 18. Immunoblots of human plasma samples after ferroxidase complex separation and characterization of polyclonal antibody raised against the FD 1 domain.

(A) Representative immunoblots of quantitated proteins (see Fig. 2B) of human plasma samples eluted from an anion exchange chromatography at pi 15.5 using a salt gradient to resolve ferroxidase I I from ferroxidase I. APP (detected by in house anti-FD l ) was evident in void samples associated with ferroxidase I I activity, whereas CP (detected with a rabbit polyclonal antibody from Sigma) was present in samples eluted with 0.2M TMaCl that contained ferroxidase I activity. (B) Polyclona l serum ( 1 : 1 000) from a rabbit inoculated w ith the FD I peptide detects immunoreactive bands in \ 0μ of 6-month wi ld-type mouse tissue and plasma. The anti-FD l serum also detects analogous spec ific bands in aged control human cortical tissue and plasma that can be matched to similar size proteins in all mouse tissues tested. The antibody is specific to APP as no evidence of bands at the same size are seen in most age- matched tissue from APP-/- mice. Only a faint band at - 65 kDa in all tissue and a stronger band at ~ 30kDa in liver are observed in APP-/- samples and are determined as non-specific. These do not al ign with stronger immunoreactive bands in control tissue or with a band of size relative to APLP2 (~87kDa). Bands at high molecular weight (>98kDa) correspond to ful l- length APP as previously determined with other recognized antibodies to APP (not shown ).

Figure 19. DEPC treatment of ferroxidase I & II either as a recombinant protein or purified from plasma. Oxidase activity as measured by Transferrin (A) and o-dianisidine (B) assays, comparably show almost total inhibition of CP and ferroxidase 1 activity but only partial inhibition of APP and ferroxidase I I activity by 1 m DEPC. Ferroxidase 1 and II were purified from human plasma using the previously described technique (Fig. 2) modified from Topham & Frieden (2). Data are means ± SEM of n - 3. DETAILED DESCRIPTION OF THE INVENTION

The present invention is predicated, in part, on the determination that abnormal exchange of cortical zinc links amyloid pathology with neuronal ferrous (Fe^{" '} ) accumulation in Alzheimer' s disease. Specifical ly, Zn²⁺ inhibits A PP ferroxidase activity, thereby preventing the ox idation of Fe²⁴ to FV⁺. leading to neuronal ferrous accumulation and thereby tissue damage caused by the consequent oxidative stress. Still further, the GFD site of APP potentiates APP ferroxidase activity and thereby provides another means of modulating ferroxidase activity. Accordingly, these determinations have no permitted the rational design of therapeutic and prophylactic treatments for conditions characterised by aberrant APP ferroxidase activity, such as occurs in Alzheimer's disease, cardiovascular disease and anaemia.

Accordingly, one aspect of the present invention is directed to a method for the therapeutic or prophylactic treatment of a condition in a subject, which condition is characterised by aberrant APP ferroxidase activity or Fe²⁺ levels, said method comprising administering to said subject an effective amount of an agent for a time and under conditions sufficient for said agent to modulate the functional interactivity of Zn² with said APP wherein antagonising the interaction of Zn²⁺ with said APP increases APP ferroxidase activity and facilitating the interaction of Zn²⁺ with said APP decreases APP ferroxidase activity.

In a related aspect, there is provided a method for the therapeutic or prophylactic treatment of a condition in a subject, which condition is characterised by aberrant A PP ferroxidase acti v ity or Fe² ' levels, said method comprising administering to sa id subject an effective amount of an agent for a time and under conditions sufficient for said agent to modulate GFD potentiation of APP ferroxidase activity wherein facilitating the interaction of GFD with APP increases APP ferroxidase activity and antagonising the GFD interaction with APP decreases APP ferroxidase activity.

Reference to "amyloid precursor protein" (^1>ΑΡΡ") should be understood as a reference to all forms of APP including, for example, any isoforms which arise from alternative splicing of APP mRNA, allelic variants, polymorphic variants or various post translational forms of

APP which undergo modification at, for example, the level of glycosylation, phosphorylation, tyrosine sulfation and proteolytic processing. Without limiting the present invention to any one theory or mode of action, APP is an integral membrane protein expressed in many tissues and concentrated in the synapses of neurons. Its primary function is not known, though it has been implicated as a regulator of synapse formation and neural plasticity . APP is best known and most commonly studied as the precursor molecu le whose proteolysis generates beta amyloid (Αβ), a 39- to 42-amino acid peptide whose amyloid fibri l lar form is the primary component of amyloid plaques found in the brains of Alzheimer' s disease patients.

In humans, the gene for APP is located on chromosome 2 1 and contains at least 1 8 exons in 240 kilobases. Several alternative splicing isoforms of APP have been observed in humans, ranging in length from 365 to 770 amino acids, with certain isoforms preferentially expressed in neurons. Changes in the neuronal ratio of these isoforms have been associated with Alzheimer^'s disease. Homologous proteins have been identified in other organisms such as Drosophila (fruit flies), C. elegans (roundworms), and all mammals.

A number of distinct, largely independently-folding structural domains have been identified in the APP sequence. The extracellular region, much larger than the intracellular region, is divided into the E l and E2 domains, linked by an acidic domain (AcD). E l contains two subdomains including a growth factor-like domain (GFD) and a copper-binding domain (CuBD) interacting tightly together. A serine protease inhibitor domain, absent from the isoform differentially expressed in the brain, is found between the acidic region and E2 domain.

It should also be understood that amyloid precursor protein (APP) can be referred to by different names including, but not limited to amyloid beta (A4) precursor protein, A4.

A4 human, AAA, A BETA, ABPP, AD 1 , amyloid A4 protein precursor, peptidase nexin-l l, Alzheimer disease, amyloid beta-peptide, amyloid of aging and Alzheimer disease; AAA, amyloid precursor protein, APPI, cerebral vascular amyloid peptide, CVAP, P -II. PrcA4, protease nexin 2, and protease nexin-ll.

Reference to "ferroxidase activity" should be understood as a reference to the oxidation of Fe^{2 r} (ferric) to Fe³⁺ (ferrous). Without limiting the present invention to any one theory or mode of action, ferroxidases prevent oxidative stress caused by Fenton and Haber- Weiss chemistry by oxidising Fe²⁺ to Fe³\ Loss of ferroxidase activity causes pathological Fe²⁺ accumulation. APP has been determined to possess ferroxidase activity and, therefore.

reference to "APP ferroxidase activity" should be understood as a reference to ferroxidase activity of the amyloid precursor protein itself. APP possesses a REXXE ferroxidase consensus motif as found in the ferroxidase active site of H-ferritin. Both the full length and soluble APP species interact with ferroportin to facilitate iron export from cells, including neurons.

Reference to "GFD potentiation" should be understood as a reference to augmentation of APP ferroxidase activity. Without limiting the present invention to any one theory or mode of action, the ΑΡΡ77» isoform is shown in Figure I . APP751 lacks the OX-2 domain, and

APP_tws lacks both OX-2 and Kunitz protease inhibitor domains. A match for the REXXE motif (in bold) of H-ferritin occurs at residues 41 1 -4 I 5 of human APP? 7o, commencing 5 residues downstream from the RERMS neurotrophic motif (Ninomiya et al. 1993).

The ferroxidase activity of the APP is unique among its protein family and correlates with the presence of the mRNA IRE motif. The ferroxidase content of APP resides in the REXXE consensus motif of the E2 domain, with a remote potentiation domain within the GFD of E l . The GFD region corresponds to residues 28- 123 of SEQ I D NO: I (APP₇₇o) and is itsel f depicted in SEQ I D NO:2. This potentiation by heterologous components is reminiscent of augmentation of H-ferritin ferroxidase activity by L-ferritin, where the active site is on I I- ferritin yet heteropolymers of H+L subunits have a higher ferroxidase activ ity per H subunit than H homopolymers.

As detailed hereinbefore, the inhibition of APP ferroxidase activity contributes to neuronal iron accumulation and the consequent damage to the brain cortex. Equally, however, iron deficiency is also a significant problem in conditions such as anaemia. To this end, reference to "aberrant" APP ferroxidase activity should be understood as a reference to a level of APP ferroxidase activity which is problematic or otherwise not appropriate. This may b - either inadequate ferroxidase activity, thereby potentially leading to iron accumulation and localised areas of oxidative stress in tissue, or too much (ie. unwanted) ferroxidase activity, leading to iron deficiency, e.g. anaemia. It should be appreciated that in some situations, the level of APP ferroxidase activity may be physiologically normal . However, there may be other factors which are acting to cause problematic Fe²⁺ levels and in respect of which modulation of APP ferroxidase activity would nevertheless assist in normal isation. In this case, the ferroxidase activity is "aberrant" within the context of th is invention since it is an unwanted level of activity when considered in light of the individual 's overal l physiological state. The method of the present invention provides a means of modulating APP ferroxidase activity in order to improve the individual 's physiological state. I Iowever, within the context of this invention these types of conditions are also ref erred to as conditions characterised by "excess Fe^2<" levels" or "insufficient Fe² ' levels". These levels may be assessed relative to system ic Fc²⁺ levels or localised Fe²' levels, such as w ithin a speci fic tissue. I n terms of inadequate ferroxidase activity, this may take the form of either a reduction in the level of ferroxidase activity relative to normal levels or a complete ablation of ferroxidasc activ ity.

APP ferroxidase activity is inhibited by Zn²\ A treatment method has therefore been developed based on modulating the functional interactivity of Zn² ' with APP. To this end, reference to modulating the "functional interactivity" of Zn²⁺ with APP should be understood as a reference to either antagonising the subject interaction such that Zn² ' inhibition of the ferroxidase activity of APP is either minimised or entirely abrogated or else facil itating the interaction of Zn² ' with APP such that ferroxidase activity is induced . In one embodiment, said antagonism is achieved by entirely preventing the interaction of Zn^{' '} with A PP by binding an agent to Zn² ' . In another embodiment, APP ferroxidase activity can be induced by administering Zn²* to said subject.

In a related aspect, it has also been determined that GFD potentiates APP ferroxidase activity. Accordingly, a further treatment method has been developed based on modulating GFD potentiation functionality. For example, antagonising GFD interaction with APP provides a means of reducing ferroxidase activity while facil itating the interaction of GFD with APP. such as via the use of GFD mimetics. provides a means for increasing ferroxidase activity.

It should be understood that reference to antagonising "functional interactivity^" is a reference to either entirely preventing the interaction of Zn²⁺ with APP or else to sufficiently disrupting this process such that the functional outcome of inhibiting APP ferroxidase activity is either abrogated or at least reduces. A corresponding definition appl ies to antagonising "GFD potentiation^".

A lthough it is preferable that the inhibition of A PP activ ity is entirely abrogated, il would be appreciated that even reducing the extent or degree of inhibition can nevertheless produce extremely valuable therapeutic outcomes in terms of minimising Fe^{2 '} accumulation and, thereby, at least minimising, even if not entirely preventing, Fe^{2 *} induced oxidative stress. Similarly, in terms of increasing APP ferroxidase activ ity, any level of increase (even i f not restoration of normal physiological functioning) is nevertheless desirable since it at least partially restores ferroxidase activity levels.

Reference to "agent" should be understood as a reference to any proteinaceous or non- proteinaceous molecule which modulates the interaction of Zn^{2 *} with A PP or the functional ity of GFD. The subject agent may be linked, bound or otherwise associated with any

proteinaceous or non-proteinaceous molecule. For example, it may be associated with a molecule which permits targeting to a specific tissue, such as the brain .

Said proteinaceous molecule may be derived from natural, recombinant or synthetic sources including fusion proteins or following, for example, natural product screening. Said non-proteinaceous molecule may be derived from natural sources, such as for example natural product screening or may be chemically synthesised. In one embodiment, the agent is either an antagonist which interacts with Zn²⁺ to prevent its interaction with APP or is Zn²\ itself, or a molecule which results in the formation or release of Zn² ' , thereby reducing APP ferroxidase functionality. For example, the present invention contemplates Zn² ' chelators which are exemplified later in this document. Accordingly, there is provided a method for the therapeutic or prophylactic treatment of a condition in a subject, which condition is characterised by insufficient APP ferroxidase activity or excess Fe²⁺ levels, said method comprising administering to said subject an effective amount of an agent for a time and under conditions sufficient for said agent to antagonise the interaction of Zn²⁺ with said APP.

In one embodiment, said antagonist is a zinc chelator, ionophore or metal protein attenuating compound.

Zn^{2 ÷} chelators provide one method to prevent Zn²⁺ from inhibiting APP ferroxidase activity. A Zn²⁺ chelator as described herein is any compound that binds Zn² ' (whether or not it is a true chelator). Accordingly, any molecule that has the abil ity to ligand or chelate to a Zn²' molecule can be used.

In one embodiment, the Zn ⁺ chelator is any ligand that is able to form two or more coordination bonds with a zinc ion. In particular embodiments, the zinc chelator is hydrophobic and is able to pass through the blood brain barrier, and optional ly binds to zinc with moderate affinity. However, chelators that bind to zinc with high affinity may also be effective.

The zinc chelator may include a cyclic group that is substituted with tw o or more functional groups that are able to donate electrons to a coordination bond with zinc or a cycl ic group in which includes at least one heteroatom such as nitrogen, oxygen or sul fur and in which the cyclic group is substituted with one or more functional groups that are able to donate electrons to a coordination bond with zinc.

In one embodiment the cyclic group is a heteroaryl group that is substituted with one or more functional groups that are able to donate electrons to a coordination bond with zinc. The heteroaryl group is especial ly selected from quinazol inyl, quinoxal iny l, naphthy ridiny l, pyrimidopyrimidinyl, cinnolinyl. phenazinyl. acridinyl, phenanthrolinyl, pyridopyrimid inyl , pyridopyrazinyl, pyranopyridiny l, dibenzoquinolizinyl, quinolinyl, isoquinolinyl, pvridinyl and pyrimidinyl groups, especially pvridinyl and quinolyl groups. Suitable zinc chelators that include a pyridyl group include pyrithione, deferiprone and Ν,Ν,Ν',Ν'-tetrakis (2- pyridylmethyl) ethylenediamine (TPEN), espec ia lly pyrithione. Suitable quinolines may inc lude a hydroxy substituent espec ially in the 8-position. Suitable quinolines may include cl ioquinol, iodoquinol, PBT2, M30 and related molecules such as those discussed in US 7,6 1 ,091 . Further compounds that include a heteroaryl group substituted w ith a functional group that is able to carry a negative charge arc discussed in US 7,692,01 I and U S 6,855.7 1 1 . In another embodiment, the cyclic group is an aryl group that is substituted with two or more functional groups that are able to donate electrons to a coordination bond with zinc. Suitable aryl groups include phenyl and naphthyl groups. Exemplar)' compounds that include an aryl group that is substituted with a functional group that is able to donate electrons to a coordination bond with zinc are discussed in US 6.855,7 1 1 .

Functional groups able to donate electrons to a coordination bond with zinc include atoms with lone pairs of electrons. For example, such groups include heteroatoms and functional groups that are able to bear a negative charge. Suitable heteroatoms include nitrogen, oxygen and sulfur. Suitable functional groups that are able to carry a negative charge include hydroxy, mercapto, ester, carboxylate, oxime. aldehyde and ketone groups, especial ly hydroxy and mercapto groups.

Another group of zinc chelators useful in the present invention include a hclerocycl l macrocyclic group, such as a cyclam or a bicyclam. Cyclams are compounds comprising a 1 ,4,8, 1 1 -tetraazacyclotetradecane ring, which may be optionally substituted. Suitable substituents include alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocyclyl, heteroaryl and aryl groups. Bicyclams comprise two cyclam rings l inked by an aromatic or al iphatic linker. A suitable bicyclam is Ι , Γ-xylyl bis- 1 ,4.8, 1 1 -tetraazacyclotctradecane (J I . 1 69).

The zinc chelator may also be a polycarboxylic acid, such as ethy lene d iamine tetraacetic acid ( EDTA), nitrilotriacetic acid, nitri lotripropionic ac id, diethylenetriamine pentaacetic acid, 2-hydroxyethyl-ethylenediamine-triacetic acid, 1 .6-diamino-hexamethylene- tetraacetic acid, 1 ,2-diamino-cyclohexane tetraacetic acid, 0.0'-bis(2-aminoethyl)- ethyleneglycol-tetraacetic acid. 1 ,3-diaminopropane-tetraacetic acid, N,N-bis(2- hydroxybenzyl)ethylenediamine-N,N-diacetic acid, ethylenediamine-N,N'-diacetic acid.

ethylenediamine-N,N'-dipropionic acid, triethylenetetraamine hexaacetic acid, iminodiacetic acid, l ,3-diamino-2-hydroxypropane-tetraacetic acid, 1 ,2-diaminopropane-tetraacetie acid, triethylenetetramine-hexaacetic acid and l ,2-bis-(2-amino-phenoxy)elhane-N,N,N',N'- tetraacetic acid. In one embodiment, the polycarboxylic acid is 1 ,2-bis-(2-amino- phenoxy)ethane-N,N,N',N'-tetraacetic acid or ethylenediamine tetraacetic acid (EDTA), especially l ,2-bis-(2-amino-phenoxy)ethane-N.N.N',N'-tetraacetic acid. The zinc chelator may also be an ester of these polycarboxylic acids. Diesters of (HOOC-CH2-)2N-A-N(-

CH₂C()OH )2 (where A is a saturated or unsaturated, aliphatic, aromatic or heterocyclic divalent l inking radical containing, in a direct chain link between the two depicted nitrogen atoms, 2-8 carbon atoms in a continuous chain which may be interrupted by 2-4 oxygen atoms. provided that the chain members directly connected to the two depicted nitrogen atoms are not oxygen atoms) are discussed, for example, in US 6,458,837. In one embodiment, the ester of the polycarboxylic acid is an alkyl ester. In another embodiment, the zinc chelator is BAPTA- ΛΜ ( l ,2-bis-(2-amino-phenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester), DP- b99 ( 1 ,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tctraacetic acid, N,N'-di(octy loxyethyl ester), Ν,Ν'-disodium salt) or DP-109, especially DP- 109.

In another embodiment, the zinc chelator includes two carbamate groups linked by an aromatic or aliphatic linker or a heteroatom such as oxygen, nitrogen or sulfur, such as in diethylpyrocarbamate. In a further embodiment, the zinc chelator is an amino carboxy lic acid that includes a functional group that is able to donate electrons to a coordination bond with zinc. Suitable amino carboxylic acids include penicillamine, cysteine, aspartic acid and glutamic acid, and also esters of these am ino carboxyl ic acids. In one embodiment, the amino carboxyl ic acid is d-penicillamine. In one embodiment, the ester of the amino carboxyl ic acid is an alkyl ester.

In another embodiment, the zinc chelator includes a hydroxamide group, such as desferrioxamine.

The zinc chelator may also be a substituted transition metal including two or more functional groups that are able to carry a negative charge. Suitable transition metals include molybdenum, and a suitable zinc chelator is tetrathiolmolybdenate.

The term "aromatic or aliphatic linker" refers to a divalent group that connects two or more groups that are able to chelate zinc. Suitable aromatic or aliphatic linkers include optionally substituted arylene, alkylene, alkenylene, cyc loalky lene and cyc loalkenylene groups, especially optionally substituted arylene, more especially optionally substituted phenylene, most especially divalent xylene. In some embodiments, the alkylene or alkenylene groups may have one or more non-consecutive carbon atoms replaced by a heteroatom such as nitrogen, oxygen or sul ur.

As used herein, the term "alkyi'^' refers to a straight chain or branched saturated hydrocarbon group having 1 to 1 2 carbon atoms. Where appropriate, the alkyl group may have a specified number of carbon atoms, for example, Ci.₍,alky l which includes alkyl groups havi ng 1 , 2, 3, 4, 5 or 6 carbon atoms in a linear or branched arrangement. Examples of suitable alkyl groups include, but are not limited to, methyl, ethyl, ^-propyl, /-propyl, w-butyl, /-butyl, t- butyl, tt-pentyl, heptyl, octyl, nonyl and dodecyl. The tenn "alkylene" refers to a divalent alkyl group. As used herein, the term "alkenyl" refers to a straight-chain or branched hydrocarbon group having one or more double bonds between carbon atoms and having 2 to 12 carbon atoms. Where appropriate, the alkenyl group may have a specified number of carbon atoms. For example, C₂-C6 as in "C₂-C₆alkenyr includes groups having 2, 3, 4, 5 or 6 carbon atoms in a linear or branched arrangement. Examples of suitable alkenyl groups include, but are not limited to, ethenyl, propenyl, isopropenyl, buteny , butadienyl, pentenyl, pentadienyl, hexenyl, hexadienyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl and dodecenyl. The term

"alkenylenc" refers to a divalent alkylene group.

As used herein, the term "alkynyl" refers to a straight-chain or branched hydrocarbon group having one or more triple bonds between carbon atoms and having 2 to 1 2 carbon atoms. Where appropriate, the alkynyl group may have a specified number of carbon atoms. For example, r _b as in "CVC₆alkynyr includes groups having 2, 3, 4, 5 or 6 carbon atoms in a linear or branched arrangement. Examples of suitable alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, hexynyl, octynyl, nonynyl. decynyl, undecynyl and dodecynyl.

As used herein, the term "eycloalkyl" refers to a saturated cyclic hydrocarbon. The eycloalkyl ring may include a specified number of carbon atoms. For example, a 3 to 8 membered eycloalkyl group includes 3, 4, 5, 6, 7 or 8 carbon atoms. Hxamples of suitable eycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentanyl, cyclohexanyl and cycloheptanyl. The term ''cycloalkylene" refers to a divalent eycloalkyl group.

As used herein, the term "'cycloalkenyl'^* refers to a cyclic hydrocarbon having at least one double bond, which is not aromatic. The cycloalkenyl ring may include a specified number of carbon atoms. For example, a 4 to 8 membered cycloalkenyl group contains at least one double bond and 4, 5. 6, 7 or 8 carbon atoms. Hxamples of suitable cycloalkenyl groups include, but are not limited to cyclopentenyl, cyclopenta- 1 ,3-dieny , cvclohexenvi, cyclohexen- 1.3-dienyl and cyclohexen- l ,4-dienyl. The term "cycloalkenylene'^* refers to a divalent cycloalkenyl group.

As used herein, the term "aryf^" is intended to mean any stable, monocyclic, bicyclic or tricyclic carbon ring of up to 7 atoms in each ring, wherein at least one ring is aromatic. When more than one ring is present, the rings may be fused to one another. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, biphenyl.

binaphthyl, anthracenyl, phenanthrenyl, phenalenyl and fluorenyl. The term "arylene" refers to a divalent aryl group.

The term "heterocyclyl" as used herein, refers to a cycloalkyl or cycloalkenyl group in which one or more carbon atoms have been replaced by heteroatoms independently selected from N. S and O. For example, between 1 and 4 carbon atoms in each ring may be replaced by heteroatoms independently selected from N, S and O. I f the heterocyclyl group inc ludes more than one ring in a ring system, at least one ring is heterocycl ic. Examples of suitable heterocyclyl groups include tetrahydrofuranyl, tetrahydrothiophenyl, pyrrol idinyl, pyrrolinyl, dithiolyl, 1 ,3-dioxolanyl, pyrazolinyl, imidazolinyl, imidazolidonyl, dioxanyl, dioxinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, pyranyl, dithianyl, and

tetrahydropyranyl.

The term "heteroaryl" as used herein, represents a stable monocyclic, bicycl ic or tricyclic ring of up to 7 atoms in each ring, wherein at least one ring is aromatic and at least one ring contains from 1 to 4 heteroatoms selected from the group consisting of O. N and S. Examples of suitable heteroaryl groups include quinazol inyl, quinoxal inyl, naphthyrid inyl. pyrimidopyrimidinyl, cinnolinyl, phenazinyl, acridinyl, phenanthrol inyi, pyridopyrimidinyl. pyridopyrazinyl, pyranopyridinyl, dibenzoquinolizinyl, quinol inyl, isoquinol inyl. pyridinyl and pyrimidinyl.

The alky I. alkenyl, cycloalkyl, cycloalkenyl. aryl, heterocyc lyl and heteroaryl groups may be optionally substituted, for example with one or more optional substituents selected from R, R-0-(CH₂)_m-, R-S-(CH₂)_m-, HO-(CH₂)_m-, HS-(CH₂)_m-, R-C(=0)-0-(Cl l₂)_m-, R-O- C(=0)-(CH₂)_m-, R- ( 0)-(CH₂)_m- R₂N-C(=0)-(CH₂)_m-, RS(0)_n-(CH₂)_m-, R₂N-(CH₂)_m-. cyano, nitro and halo, wherein each R is independently selected from H, a lkyl, a lkenyl.

alkynyl, -(CH₂)_p-aryl, -(CH₂)_p-heteroaryl, -(CH₂)_p-cycloalkyl, -( H₂)_p-cyc loalkenyl or -(CH₂)_p- heterocyclyl; m and p are 0 or an integer from 1 to 6, and n is 0 or an integer of 1 or 2.

As used herein, the term "halo" represents fluoro, chloro. bromo or iodo.

In one particular embodiment, the Zn²⁺ chelator is a moderate affinity chelator which is hydrophobic. Examples include the 8-hydroxy quinolines, such as cl ioquinol, PBT2. M30, V 28 or related molecules, pyrithione, diethyl pyrocarbamate. l ,2-bis-(2-(amino- phenoxy)ethane-N.N.N',N'-tetraacetic acid and derivatives, the bicyclam analogue JK.L I 6 ( Ι . Γ-xyIyl bis- 1 ,4, 8, 1 1 tetraaza cyclotetradecane). DP 109 and related compounds.

In another embodiment, there is prov ided a method for the therapeutic or prophylactic treatment of a condition in a subject, which cond ition is characterised by unwanted APP ferroxidase activity or insufficient Fe ^ levels, said method comprising increasing the level of Zn²⁺ in said subject for a time and under conditions sufficient for said Zn^{2 '} to interact with said APP.

It should be understood that in the context of this aspect of the present invention, the level of Zn²⁺ can be increased in a subject either by administering Zn²⁺ itself or by

administering a compound or molecule from which Zn²⁺ is released or generated.

In yet another aspect, there is provided a method for the therapeutic or prophylactic treatment of a condition in a subject, which condition is characterised by insufficient APP ferroxidase activity or excess Fe²⁺ levels, said method comprising administering to said subject an effective amount of SEQ ID NO:2 or functional fragment, mimetic, analogue or homologue thereof for a time and under conditions sufficient to potentiate APP ferroxidase activity.

As detailed hereinbefore, SEQ ID NO:2 represents the amino acid sequence of the GFD domain of APP. Accordingly, b administering a composition comprising th is sequence, or a functional fragment, mimetic, analogue or homologue thereof, GFD potentiat ion ol^' A PP can be effectively achieved.

"Fragments" include parts and portions, mutants, variants and mimelics from natural, synthetic or recombinant sources including fusion proteins. Parts or fragments inc lude, for example, active regions of GFD. Mimetics may be derived from insertion, deletion or substitution of amino acids. Amino acid insertional derivatives include amino and/or carboxylic terminal fusions as well as intrasequence insertions of single or multiple amino acids. Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the protein although random insertion is also possible with suitable screening of the resulting product. Deletional variants are characterized by the removal of one or more amino acids from the sequence. Substitutional amino acid variants are those in which at least one residue in the sequence has been removed and a different residue inserted in its place. An example of substitutional amino acid variants are conservative amino acid substitutions. Conservative amino acid substitutions typical ly include substitutions within the following groups: glycine and alanine: val ine, isoleucine and leucine; aspartic acid and glutamic acid; asparagine and glutamine; serine and threonine; lysine and arginine; and phenylalanine and tyrosine. Additions to amino acid sequences include fusions with other peptides, polypeptides or proteins or cyclising the peptide, for example to yield a pharmacologically active form.

A "homologue" refers to a sequence in another animal or organism which has at least about 70% identity, preferably 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identify to the human GFD molecule.

Analogues include chemical and functional equivalents of GFD molecules. These should be understood as molecules exhibiting any one or more of the functional activities of GFD and may be derived from any source such as being chemically synthesized or identi fied via screening processes such as natural product screening.

The fragments may have the active sites of GFD fused to peptides, polypeptides or other proteinaceous or non-proteinaceous molecules.

Analogues contemplated herein include, but are not limited to, modi fication to side chains, incorporating of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods wh ich impose conformational constraints on the proteinaceous molecules or their analogues.

Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with aBH^ amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups ith cyanate; trinitrobenzy lation of am ino groups with 2, 4, 6-trinitrobenzene sulphonic acid (T BS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with aBH4.

The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.

The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivatisation, for example, to a corresponding amide.

Sulphydryl groups may be modified by methods such as carboxymethvlation w ith iodoacetic acid or iodoacetamide: performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide. maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4-chloromercuri- benzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2-chloromercuri-4- nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.

Tryptophan residues may be modified by, for example, oxidation with N- bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residues on the other hand, may be altered by nitration with telranitromethane to form a 3-nitrotyrosine derivative.

Modification of the imidazole ring of a histidine residue may be accompl ished by alkvlation with iodoacetic acid derivatives or N-carboethoxylation with diethylpyrocarbonate.

Examples of incorporating unnatural amino acids and derivatives during protein synthesis include, but are not limited to. use of norleucinc, 4-amino butyric acid, 4-amino-3- hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylgiycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyI alanine and/or D-isomers of amino acids. A list of unnatural amino acids contemplated herein is shown in Table 1 .

TABLE 1

Non-conventional Code Non-conventional Code am ino acid amino acid u-aminobutyric acid Abu L-N-methylalanine Nmala a-am ino-a-methylbutyrate Mgabu L-N-methylarginine Nmarg aminocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylate L-N-methylaspartic ac id Nmasp aminoisobutyric acid Aib L-N-methylcysteine mcys am inonorbornyl- Norb L-N-methylglutamine Nmgln carboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine Chexa L-N-methylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisol leucine Nmi le

D-alanine Dal L-N-methyl leucine Nmleu

D-arginine Darg L-N-methyllysine Nmlys

D-aspartic acid Dasp L-N-methylmethionine Nmmet

D-cysteine Dcys L-N-methylnorleuc ine Nmnle

D-glutamine Dgln L-N-methylnorvaline Nmnva

D-glutamic acid Dglu L-N-methylornithine Nmorn

D-histidine Dhis L-N-methy [phenylalanine Nmphe

D-isoleucine Di le L-N-methylproline Nmpro

D-leucine Dleu L-N-methylserine Nmser D-lysine Dlys L-N-methylthreonine Nmthr

D-methionine Dmet L-N-methyltryptophan Nmtrp

D-ornithine Dorn L-N-methyltyrosine Nmtyr

□-phenylalanine Dphe L-N-methylval inc Nmval

D-prol ine Dpro L-N-methylethylglycine Nmetg

D-serine Dscr L-N-methyl-t-butylglycine Nmtbug

D-threonine Dthr L-norleucrne Nle

D-tryptophan Dtrp L-norvaline Nva

D-tyrosine Dtyr -methyl-am inoisobutyrate Maib

D-valine Dval a-methyl- -am inobutyrate Mgabu

D-a-methylalanine Dmala

Mchcxa

D-cx-methylarginine Dmarg a-methylcylcopenty lalanine Mcpen

D-a-methylasparagine Dmasn -melhyl-a-napthyla lanine Manap

D-(x-methylaspartate Dmasp a-methy (penici llamine Mpen

D-a-methylcysteine Dmcys N-(4-aminobutyl)glycine Nglu

D-a-methylglutamine Dmgln N-(2-aminoethyl)glycine Naeg

D-a-methylhistidine Dmhis N-(3-aminopropyl)glycine Norn

D-(x-methylisoleucine Dmi le N-amino-a-methylbutyrate Nmaabu

D-a-methylleucine Dmleu (x-napthylalanine Anap

D- -melhyl lysine Dmlys N-benzylglycine Nphe

D-a-methylmethionine Dmmet N-(2-carbamylethyl)glycine Ngln

D-a-methylornithine Dmorn N-(carbamylmethyl)glycine Nasn

D-u-methylphenylalanine Dmphe N-(2-carboxyethyl)glycine Nglu

D-a-methylproline Dm pro N-(carboxymethyl)glycine Nasp

D-a-methylserine Dmser N-cyclobuty lglyc ine Ncbut

D-a-methylthreonine Dmthr N-cycloheptylglycine Nchep

D- -methyltryptophan Dmtrp N-cyclohexylglycinc Nchex

D-a-methyltyrosine Dmty N -eye lodecy lglyc ine Ncdec

D-a-methylvaline Dmval N-cylcododecy lglycine Ncdod

D-N-methylalanine Dnmala N-cyclooctylgrycine Ncoct

D-N-methylarginine Dnmarg N-cyclopropylglycine Ncpro

D-N-methylasparagine Dnmasn N-cycloundecylglycine Ncund

D-N-methylaspartate Dnmasp N-(2,2-dipheny lethy glycine Nbhm D-N-methylcysteine Dnmcys N-(3,3-diphenylpropy l)glycine bhe

D-N-methylglutamine Dnmgln N-(3-guanidinopropyl )glycine Narg

D-N-methylglutamate Dnmglu N-( 1 -hydroxyethyl)glyc ine Nthr

D-N-methylhistidine Dnmhis N-(hydroxyethyl))gIycine Nser

D-N-methyl isoleucine Dnmile N-(imidazolylethyl))glycine Nhis

D-N-methylleucine Dnmleu N-(3-indolylyethyl)glyc ine Nhtrp

D-N-methyllysine Dnmlys N-methyl-y-aminobutyrate N mgabu N-methylcyclohexylalanine Nmchexa D-N-methylmethionine Dnmmet

D-N-methylornithine Dnmorn N-methylcyclopenty lalanine Nmcpen N-methylglycine Nala D-N-methylphenylalanine Dnmphe N-methylaminoisobutyrate Nmaib D-N-methylprol ine Dnmpro N-( l -methylpropyl)glycine Nile D-N-methylserinc Dnmser N-(2-methylpropyl)glycine Nleu D-N-methylthreon ine Dnmthr

D-N-methyltryptophan Dnmtrp N-( l -methylethyl)glyc ine Nval

D-N-methyltyrosine Dnmtyr N-methyla-napthylalanine Nmanap

D-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acid Gabu N-(/?-hydroxyphenyI )glycinc Nhtyr

L-/-butylglycine Tbug N-(thiomethyl)glycine Ncys

L-ethylglycine Etg penicillamine Pen

L-homophenylalanine Hphe L-a-methylalanine Mala

L-a-methylarginine Marg L-a-methylasparaginc Masn

L-a-methylaspartate Masp L-a-methyl-?-butylglyc ine Mtbug

L-a-methylcysteine Mcys L-methylethylglycine Metg

L-a-methylglutamine Mgln L- -methylglulamate Mglu

L-a-methylhistidine Mhis L-a-methylhomophenylalan ineMhphe

L-a-methyl isoleucine Mile N-(2-methylthioethyl )glycine Nmet

L- -methylleucine MIeu L-a-methyllysine Mlys

L-a-methylmethionine Mmet L-a-methylnorleucine Mnle

L-a-methylnorvaline Mnva L-a-methylornithine Morn

L-a-methylphenylalanine Mphe L-a-methylproline Mpro

L-a-methylserine Mser L-a-methylthreonine Mthr

L-a-methyltryptophan Mtrp L-a-methyltyrosine Mtyr

L-a-methylval ine Mval L-N-methylhomophenylalanine Nmhphe N-(N-(2,2-diphenylethyl) Nnbhm N-(N-(3,3-diphenylpropyl)

carbamylmethyl)glycine carbamylmethyl)glycinc

1 -carboxy- 1 -(2,2-diphenyl-Nmbc ethy lamino)cyclopropane

Crosslinkers can be used, for example, to stabilise 3D conformations, using homo- bifunctional crosslinkers such as the bi functional imido esters having (CH₂)„ spacer groups with n= l to n=6, glutaraldehyde, N-hydroxysuccinimide esters and hetcro-bifunctional reagents which usually contain an amino-reactive moiety such as N-hydroxysucc inimide and another group specific-reactive moiety.

The agents which are administered to a subject in accordance with the present invention may also be linked to a targeting means, such as a monoclonal antibody, which provides specific delivery of these molecules to target tissue regions.

In still another aspect there is provided a method for the therapeutic or prophylactic treatment of a condition in a subject, which condition is characterised by unwanted A PP ferroxidase activity or insufficient Fe~ ' levels, said method comprising administering to said subject an effective amount of an agent which antagonises the interaction of GFD w ith A PP.

Methods of screening for agents which either antagonise or agonise the interaction of GFD with APP or the interaction of Zn^""* with APP would be wel l known to those of ski l l in the art. By way of example, diversity libraries, such as random combinatorial peptide or nonpeptide libraries can be screened. Many piiblically or commercial ly available libraries can be used such as chemically synthesized libraries, recombinant (e.g., phage display libraries) and in vitro translation-based libraries.

Examples of chemically synthesized libraries are described in Fodor et at.. ( 1 991 ); Houghten <?/ ø/., ( 1 991 ); Lam et al.. ( 1 991 ); Mcdynski., ( 1 994); Gallop et al.. ( 1 994 );

Ohlmeyer e/ o/., ( 1993); Erb et al., ( 1 994); Houghten et al., ( 1992); Jayavvickreme el al., ( 1994); Salmon et al., ( 1993); International Patent Publication No. WO 93/20242: and Brenner and Lerner, PNAS 89:538 1 -5383 ( 1 992).

Examples of phage display libraries are described by Scott and Smith.. ( 1 990): Dev l in et al.. { 1 990); Christian R.B et al.. ( 1992); Lenstra., ( 1 992); Kay et al.. ( 1 993) and

International Patent Publication No. WO 94/ 1 83 1 8. In vitro translation-based libraries inc lude but are not limited to those described in Mattheakis et al.. ( 1 994).

Without limiting the present invention in any way a test compound can be a macromolecule. such as biological polymer, including polypeptides or polysaccharides.

Compounds useful as potential therapeutic agents can be generated by methods well known to those skilled in the art, for example, well known methods for producing pluralities of compounds, including chemical or biological molecules such as simple or complex organic molecules, metal-containing compounds, carbohydrates, peptides, proteins, peptidomimetics. glycoproteins, lipoproteins, antibodies, and the like, are wel l known in the art and are described, for example, in Huse, U.S. Patent No. 5,264,563 ; Francis et al., Curr. Opin. Che . Biol. , 2:422-428 ( 1998): Tietze et al., Curr. Biol. , 2 :363-381 ( 1998); Sofia, Molecule. Divers. , 3 :75-94 ( 1 998); Eichler tf al., Med. Res. Rev. 15 :48 1 -496 ( 1 995); and the like. Libraries containing large numbers of natural and synthetic compounds also can be obtained from commercial sources. Combinatorial libraries of molecules can be prepared using well known combinatorial chemistry methods (Gordon et al., J. Med. Che 37: 1 233- 1 25 1 ( 1 994): Gordon et al. . Med. Chem. 37: 1 385- 1 401 ( 1 994); Gordon el al.. Acc. Chem. Res. 29: 144- 1 54 ( 1 996); Wi lson and Czarnik, eds., Combinatorial Chemistry: Synthesis and Application. John Wiley & Sons, New York ( 1 997).

Additionally, a test compound can be preselected based on a variety of criteria. For example, suitable test compounds having known modulating activity on a pathway suspected to be involved in APP ferroxidase activity can be selected for testing in the screening methods. Alternatively, the test compounds can be selected randomly and tested by the screening methods of the present invention. Test compounds can be administered to the reaction system at a single concentration or, alternatively, at a range of concentrations from about 1 nM to 1 mM .

The number of di fferent test compounds examined using the methods of the invention wi ll depend on the application of the method. It is generally understood that the larger the number of candidate compounds, the greater the likel ihood of identifying a compound having the desired activity in a screening assay. The methods can be performed in a single or multiple sample format. Large numbers of compounds can be processed in a high-throughput format which can be automated or semi-automated.

A combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis by combining a number of chemical "building blocks" such as reagents. For example, a linear combinatorial chemical l ibrary such as a polypeptide library is formed by combining a set of chemical bui lding blocks called amino acids in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks. For example, the systematic, combinatorial mixing of 100 interchangeable chemical building blocks results in the theoretical synthesis of 100 million tetrameric compounds or 10 billion pcntameric compounds (see, e.g., Gallop el al. (1994) 37(9): 1233- 1250). Preparation and screening of combinatorial chemical libraries are well known to those of skill in the art, see, e.g., U.S. Patent No.

6,004,617; 5,985,356. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Patent No.5,010.175; Furka (1991) Int. J. Pept. Prol. Res., 37:487-493, Houghton el al. (1991) Nature, 354:84-88). Other chemistries for generating chemical diversity libraries include, but are not limited to: peptoids (see, e.g.. WO 91/19735), encoded peptides (see, e.g., WO 93/20242), random bio-oligomers (see, e.g.. WO 92/000 1 ), benzodiazepines (see, e.g., U.S. Patent No.5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (see, e.g., Hobbs (1993) Proc. Nat. Acad. Sci. USA 90:6909- 6913), vinylogous polypeptides (see, e.g., Hagihara (1992) J. Amer. Chem Soc.114:6568), non-peptidal peptidomimetics with a Beta-D-Glucose scaffolding (see, e.g.. Hirschmann

(1992)./. Amer. Chem. Soc.114:9217-9218), analogous organic syntheses of small compound libraries (see. e.g.. Chen (1994)./. Amer. Chem. Soc.116:2661). oligocarbamates (see. e.g., Cho ( 1 93) Science 261 : 1303), and/or peptidyl phosphonates (see, e.g., Campbell ( 1 94) J. Org. Chem.59:658). See, e.g., U.S. Patent No.5,539,083; for antibody libraries, see, e.g.. Vaughn (1996) Nature Biotechnology 14:309-314: for carbohydrate libraries, see, e.g., Liang el al. (1996) Science 274:1520-1522, U.S. Patent No.5,593,853; for small organic molecule libraries, see, e.g., for isoprenoids U.S. Patent 5.569,588; for thiazolidinones and

metathiazanones, U.S. Patent No.5,549,974: for pyrrolidines, U.S. Patent Nos.5,525,735 and 5,519,134; for morpholino compounds, U.S. Patent No.5,506,337; for benzodiazepines U.S. Patent No.5,288,514.

Devices for the preparation of combinatorial libraries are commercially available (see. e.g., U.S. Patent No. 6,045,755; 5,792,431; 357 MPS, 390 MPS, Advanced Chem Tech, Louisville Y. Symphony, Rainin, Woburn, MA, 433A Applied Biosystems, Foster City. CA, 9050 Plus, Millipore, Bedford, MA). A number of robotic systems have also been developed for solution phase chemistries. These systems include automated workstations, e.g.. like the automated synthesis apparatus developed by Takeda Chemical Industries. LTD. (Osaka, Japan) and many robotic systems utilizing robotic arms (Zymate II, Zymark Corporation, l lopkinton, Mass.: Orca, Hewlett-Packard, Palo Alto. Calif.) which mimic the manual synthetic operations performed by a chemist. Any of the above devices are suitable for use with the present invention. The nature and implementation of modifications to these dev ices (if any) so that they can operate as discussed herein wi ll be apparent to persons skilled in the relevant art. In addition, numerous combinatorial libraries are themselves commercially available (see, e.g., ComGenex, Princeton, N.J., Asinex, Moscow. u, Tripos, I nc.. St. Louis. MO. ChemStar. Ltd. Moscow, RU, 3D Pharmaceuticals, Exton, PA, Martek Biosciences, Columbia, MD, etc.).

In practicing this aspect of the method of the invention, a variety of apparatus and methodologies can be used to in conjunction with the polypeptides of the invention (such as GFD and the APP ferroxidase site), e.g., to screen compounds as potential modulators (e.g.. inhibitors or activators).

In one aspect, the peptides and polypeptides of the invention can be bound to a sol id support. Sol id supports can include, e.g., membranes (e.g., nitrocel lu lose or nylon), a microtiter dish (e.g., PVC, polypropylene, or polystyrene), a test tube (glass or plastic ), a d i p stick (e.g., glass, PVC, polypropylene, polystyrene, latex and the like), a microfuge tube, or a glass, si lica, plastic, metallic or polymer bead or other substrate such as paper. One solid support uses a metal (e.g., cobalt or nickel)-comprising column which binds with specificity to a histidine tag engineered onto a peptide.

Adhesion of peptides to a solid support can be direct ( i .e., the protein contacts the sol id support) or indirect (a particular compound or compounds are bound to the support and the target protein binds to this compound rather than the sol id support). Peptides can be immobilized either covalently (e.g., utilizing single reactive thiol groups of cysteine residues (see, e.g., Col liuod ( 1 93) Bioconjiigate C em. 4:528-536) or non-covalently but spec i fically (e.g., via immobi lized antibodies (see, e.g., Schuhmann ( 1 991 ) Adv. Muter. 3 :388-391 ; Lu ( 1995) Anal. Chem. 67:83-87; the biotin/strepavidin system (see, e.g., I wane ( 1 97) Biophys Biochem. Res. Comm. 230:76-80); metal chelating, e.g., Langmuir-Blodgett films (see, e.g., Ng

( 1 95) Langmuir 1 1 :4048-55); metal-chelating self-assembled monolayers (see, e.g., Sigal

( 1 96) Anal. Chem. 68:490-497) for binding of polyhistidine fusions.

Indirect binding can be achieved using a variety of linkers which are commercially available. The reactive ends can be any of a variety of functionalities including, but not limited to: amino reacting ends such as N-hydroxysuccinimide ( N HS ) active esters, imidoesters, aldehydes, epoxides, sulfonyl halidcs, isocyanate. isothiocyanate. and nitroary l halides; and thiol reacting ends such as pyridyl disulfides, maleimides. ihiophthal imides, and active halogens. The heterobifunctional crossl inking reagents have two different reactive cnds, e.g., an amino-reactive end and a thiol-reactive end, while homobifunctional reagents have two similar reactive ends, e.g., bismaleimidohexane (BMH) which permits the cross- linking of sulfhydryl-containing compounds. The spacer can be of varying length and be aliphatic or aromatic. Examples of commercially available homobifunctional cross-linking reagents include, but are not limited to, the imidoesters such as dimethyl adipimidate di hydrochloride (DMA); dimethyl pimel imidate dihydrochloride ( DM P); and dimethyl suberimidate dihydrochloride (DMS). Heterobifunctional reagents include commercial ly avai lable active halogen-NHS active esters coupling agents such as N-succinimidyl bromoacetate and N-succinimidyl (4-iodoacetyl)aminobenzoate (SIA B) and the

sulfosuccinimidyl derivatives such as sulfosuccinimidyl(4-iodoacetyl)aminobenzoate (sulfo- SIAB) (Pierce). Another group of coupling agents is the heterobifunctional and thiol cleavable agents such as N-succinimidyl 3-(2-pyridyidithio)propionate (SPDP) ( Pierce Chemicals, Rockford, IL).

Antibodies can be used for binding polypeptides and peptides to a sol id support. Th is can be done directly by binding peptide-specific antibodies to the column or it can be done by creating fusion protein chimeras comprising motif-containing peptides linked to, e.g., a known epitope (e.g.. a tag (e.g., FLAG, myc) or an appropriate immunoglobu lin constant domain sequence (an ' mmunoadhesin," see, e.g., Capon ( 1989) Nature 337:525-53 1 ).

Antagonism of the interaction between GFD and ΛΡΡ may be achieved by any one of a number of techniques including, but not limited to introducing into a cel l a proteinaceous or non-proteinaceous molecule which modulates the transcriptional and/or translalional regulation of the GFD in APP or which antagonises the interaction between the G FD and APP. such as a competitive inhibitor.

Said proteinaceous molecule ma be derived from natural, recombinant or synthetic sources including fusion proteins or following, for example, natural product screening. Said non-proteinaceous molecule may be derived from natural sources, such as for example natural product screening or may be chemically synthesised. Antagonists may be any compound capable of blocking, inhibiting or otherwise preventing GFD and APP from interacting.

Antagonists include antibodies (such as monoclonal and polyclonal antibodies) specific for APP or GFD, or parts of said GFD or APP. Reference to antagonists also includes antigens which competitively inhibit GFD/APP interaction, siRNA. antisense molecules, ribozymes. DNAzymes, RNA aptamers, or molecules suitable for use in co-suppression.

In one embodiment the present invention is directed to the use of antibodies to GFD to antagonise its activity. Such antibodies may be monoclonal or polyc lonal and may be selected from naturally occurring antibodies or may be specifical ly raised. The term "antibody" ■ includes a peptide or polypeptide derived from, model led after or substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, capable of specifically binding an antigen or epitope, see, e.g. Fundamental Immunology, Third Edition, W.E. Paul, ed., Raven Press, N.Y. ( 1 993); Wilson ( 1 994) . Immunol. Methods 1 75:267-273; Yarmush ( 1992) J. Biochem. Biophys. Methods 25 :85-97. The term antibody includes antigen-binding portions, i .e., ''antigen binding sites," (e.g., fragments, subsequences, complementarity determining regions (CDRs)) that retain capacity to bind antigen, inc l uding ( i ) a Fab fragment. a monovalent fragment consisting of the VL, VH, CL and CFl 1 domains; ( ii ) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a d isul fide bridge at the hinge region; (iii) a Fd fragment consisting of the VF1 and CH I domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v ) a dAb fragment (Ward el < /., ( 1989) Nature 341 :544-546). which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Single chain antibodies are also included by reference in the term "antibody."

Antibodies may be monoclonal or polyclonal and may be selected from natural ly occurring antibodies or may be specifically raised to these polypeptide and gene products. The present invention extends to recombinant and synthetic antibodies and to antibody hybrids. A "synthetic antibody" is considered herein to include fragments and hybrids of antibodies.

In another embodiment, one may design and test molecules which can modulate the interaction of 7M² ' with the active site of APP. A three-dimensional prediction of the active site is prov ided in Figure 1 and it is with the skill of the person in the art to design, for example, in silico, agents which would appropriately interact.

The method of the present invention is directed to treating a condition characterised by aberrant APP ferroxidase activity or aberrant Fe²⁺ levels. This should be understood as a reference to any disease or other condition in respect of which aberrant APP ferroxidase activity or Fe^{2^} level is a cause, symptom or side effect. This includes, for example, conditions which occur as a side effect of a treatment regime for an unrelated disease condition. The subject disease condition may be congenital or acquired and may be in an acute or chronic phase. In one embodiment, said APP is APP of the central nervous system. In another embodiment said condition is a neurodegenerative disease.

In still another embodiment, said condition may be cardiovascular disease (which is characterised by iron buildup in the heart), hemochromatosis (which is characterised by iron buildup in the liver), aceruloplasminemia or hemosiderosis, hereditary hemochromatosis (all types, e.g. Type I or classic (HHC), Type II a. b or juveni le (Jl IC), Type II I or transferrin receptor mutation. Type IV or ferroportin mutation, neonatal (NH), African (AH) or A frican iron overload (AIO)), major thalassemia, aceruloplasminemia. atransferrinem ia,

hyperferritinemia, neuroferritinopathy; hereditary ferritinopathy. hereditary hyperferritinaemia cataract syndrome (HHCS), pantothenate kinase-2 associated neurodegeneration,

neurodegeneration with brain iron accumulation type I (NBIA type 1 ) or I lal lervorden-Spatz syndrome, phospolipase A2 associated neurodegeneration or neurodegeneration with brain iron accumulation type II (NBIA type 2), Alzheimer's disease, Park inson's disease, dementia with Lewy Bodies, Friedreich ataxia, Huntington's disease, frontotemporal dementia, progressive supranuclear palsy (PSP), amyotrophic lateral sclerosis (ALS), multiple sclerosis,

ischemic/hemorrhagic stroke, mucolipidosis type IV (ML4) neurodegenerative disease, age- related macular degeneration (AMD), liver disease (cirrhosis, liver cancer), hepatic fai lure, sickle cell disease, X-linked sideroblastic anemia, diabetes mellitus. It also may be a condition characterised by one or more of chronic fatigue, joint pain, abdominal pain, irregular heart rhythm, heart attack or heart failure, skin color changes (bronze, ashen-gray green), loss of menstrual cycle, osteoarthritis, osteoporosis, hair loss, enlarged liver or spleen, impotence, infertility, hypogonadism, hypothyroidism, hypopituitarism, depression.

In yet another example, said condition may be iron defic iency anemia, anemia of chronic disease (or anemia of inflammation), minor thalassemia, alopecia (hair loss), pruritus (itchiness), tingling, numbness, or burning sensations, glossitis (inflammation or infection of the tongue), angular cheilitis (inflammatory lesions at the mouth's corners), koi lonychia (spoon-shaped nai ls) or nai ls that are weak or brittle, Plummer-v inson syndrome - dysphagia due to formation of esophageal webs, restless legs syndrome or twitching muscles, angina, which is characterised by inadequate iron levels. It may also be a condition characterised by one or more of chronic fatigue, weakness, dizziness , headaches, sensitivity to cold ( low body temp), anxiety often resulting in obsessive compulsive disorder-type compulsions and obsessions, irritability, constipation, sleepiness, tinnitus, palpitations, fainting, depression, breathlessness on exertion, missed menstrual cycle, I leavy menstrual period, slow social development, mouth ulcers, poor appetite.

There is therefore provided, in one embodiment, a method for the therapeutic or prophylactic treatment of a condition in a subject, which condition is characterised by insufficient central nervous system APP ferroxidase activity or excess Fe^"+ levels, said method comprising administering to said subject an effective amount of an agent for a time and under conditions suffic ient for said agent to:

(i) antagonise the functional interactivity of Zn²⁺ with said APP; or

(ii) faci litate the interaction of GFD with APP.

In another embodiment there is provided a method for the therapeutic or prophylactic treatment of a neurodegenerative disease in a subject, said method comprising administering to said subject an effective amount of an agent for a time and under conditions sufficient for said agent to:

( i) antagonise the functional interactivity of Zn ^÷ with said A PP; or

(ii) faci litate the interaction of GFD with APP.

In sti ll another embodiment, said agent is a Zn²⁺ chelator, ionophore or metal protein attenuating compound as hereinbefore described.

In yet another embodiment, said agent is SEQ ID NO:2 or functional fragment, mimetic, analogue or homologue thereof.

In yet sti ll another embodiment, said neurodegenerative disease is Alzheimer's disease. Parkinson's disease, Lewy Body disease, Parkinson's dementia, neurodegeneration with brain iron accumulation, neuroferritinopathy, macular degeneration, Freidreich^'s ataxia, motor neuron disease, Huntington^' s disease, polyglutamine repeat diseases or trinucleotide repeat diseases.

In a further embodiment, said condition is characterised by insufficient A PP ferroxidase activity or excess Fe²⁺ levels outside the central nervous system. I n sti l l another embodiment, said condition is cardiovascular disease, hemochromatosis, acerulopiasminemia, beta-thalassemia or any other iron overload disorder.

In yet another embodiment, there is provided a method for the therapeutic or prophylactic treatment of a condition in a subject, which condition is characterised by unwanted APP ferroxidase activity or insufficient Fe²⁺ levels, said method comprising adm inistering to said subject an effective amount of an agent for a time and under conditions sufficient for said agent to:

(i) increase the level of Zn²⁺; or

(i i) antagonise the interaction of GFD with APP.

In one embodiment, said agent is Zn^2† or an antibody or other immunointeractive molecule directed to GFD. ln still another embodiment, said condition is anaemia.

The term "subject" as used herein includes humans, primates, livestock animals (eg. sheep, pigs, cattle, horses, donkeys), laboratory test animals (eg. mice, rabbits, rats, guinea pigs), companion animals (eg. dogs, cats), captive wild animals (eg. foxes, kangaroos, deer), aves or reptiles. Preferably, the mammal is human or a laboratory test animal Even more preferably, the mammal is a human.

An "effective amount" means an amount necessary to at least partly attain the desired response, or to delay the onset or inhibit progression or halt altogether, the onset or progression of a particular condition being treated. The amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated, the degree of protection desired, the formulation of the composition, the assessment of the medical situation, and other relevant factors. It is expected that the amount w i l l fal l in a relatively broad range that can be determined through routine trials.

The method of the present invention preferably faci litates the subject condition being reduced, retarded or otherwise inhibited. Reference to "reduced, retarded or otherwise inhibited" should be understood as a reference to inducing or facil itating the partial or complete inhibition of any one or more causes or symptoms of the subject condition. In this regard, it should be understood that conditions such as Alzheimer^" s disease arc extremely complex comprising numerous physiological events which often occur simultaneously. In terms of the object of the subject method of treatment and/or prophylaxis, it should be understood that the present invention contemplates both rel ieving any one or more symptoms of the subject condition (for example, improving one or more cogn itive functions) or facilitating retardation or cessation of the cause of the disease condition.

Administration of the agent in the form of a pharmaceutical composition may be performed by any convenient means. The agent of the pharmaceutical composition is contemplated to exhibit therapeutic activity when administered in an amount which depends on the part icular case. The variation depends, for example, on the human or animal and the form of modulatory agent chosen. A broad range of doses may be appl icable. Considering a patient, for example, from about 0.1 mg to about 1 mg of modulatory agent may be administered per kilogram of body weight per day. Dosage regimes may be adj usted to provide the optimum therapeutic response. For example, several divided doses may be administered dai ly, weekly, monthly or other suitable time intervals or the dose may be proportionally reduced as indicated by the exigencies of the situation. The agent may be coadministered in a convenient manner by any suitable route. Routes of administration include, but are not limited to, respiratorally, intratracheally, nasopharyngeal^', intravenously, intraperitoneally, subcutaneously, intracranially, intradermally, intramuscularly, intraoccularly. intrathecally, intracereberally, intranasally, infusion, orally, rectally, via IV drip patch and implant (eg. using slow release molecules).

In a related aspect of the present invention, the subject undergoing treatment or prophylaxis may be any human or animal in need of therapeutic or prophylactic treatment. In this regard, reference herein to "treatment'^" arid "'prophy laxis^" is to be considered in its broadest context. The term "treatment" does not necessarily imply that a mammal is treated until total recovery. Similarly, "prophylaxis" does not necessari ly mean that the subject wil l not eventually contract a disease condition. Accordingly, treatment and prophylaxis include amelioration of the symptoms of a particular condition or preventing or otherwise reducing the risk of developing a particular condition. The term "prophylaxis'^* may be considered as reducing the severity of the onset of a particular condition. "Treatment" may a lso reduce the severity of an existing condition.

In yet another aspect, the present invention relates to the use of an agent w hich :

(i ) antagonises the functional interactiv ity of Zn² ' with APP; or

(ii) facilitates the interaction of GFD with APP;

in the manufacture of a medicament for the treatment of a condition characterised by insufficient APP ferroxidase activity or excess Fe ⁺ levels.

In one embodiment, said APP is central nervous system APP.

In another embodiment, said agent is a Zn²⁺ chelator, ionophore or metal protein attenuating compound as hereinbefore defined.

In still another embodiment, said Zn² ' chelator is a moderate affinity chelator which is hydrophobic. Examples include the 8-hydroxy quinolines, such as clioquinol, PB^'1^'2, 30, VK.28 or related molecules, pyrithione, diethyl pyrocarbamate. 1 ,2-bis-(2-(amino- phenoxy)ethane-N,N,N ',N ^' -tetraacetic acid and derivatives, the bicyclam analogue J KL I 69 ( l , l '-xy lyl bis- 1 ,4, 8, 1 1 tetraaza cyclotetradecane), DP 1 09 and related compounds.

In still another embodiment, said agent is SliQ I D NO:2 or functional fragment.

mimetic, analogue or homologue thereof.

In yet st il l another embodiment, said condition is a neurodegenerati ve condition.

I n a further embodiment, said neurodegenerative condition is Alzheimer' s disease. Parkinson's disease, Lewy Body disease, Parkinson^'s dementia, ncurodegeneration with brain iron accumulation, neuroferritinopathy, macular degeneration, Freidreich^'s ataxia, motor neuron disease. Huntington's disease, polyglutamine repeat diseases or trinuc leotide repeat diseases.

In stil l another embodiment, said condition is characterised by insufficient APP ferroxidase activ ity or excess Fe²⁺ levels outside the central nervous system. In sti l l yet another embodiment, said condition is cardiovascular disease, hemochromatosis,

aceruloplasminemia, beta thalassemia or any other iron overload disorder.

In yet stil l another aspect, the present invention relates to the use of an agent which :

(i) increases the level of Zn²⁺; or

(ii) antagonises the interaction of GFD with APP

in the manufacture of a medicament for the treatment of a condition characterised by unwanted APP ferroxidase activity or insufficient Fe^:+ levels.

In one embodiment, said agent is Zn²⁺ or an antibody or other immunointeractivc molecule directed to GFD.

In another embodiment, said condition is anaemia.

In a related aspect of the present invention, the mammal undergoing treatment may be a human or animal in need of therapeutic or prophylactic treatment . Preferably, said mammal is a human.

In accordance with these methods, the modulatory agent defined in accordance with the present invention may be coadministered with one or more other compounds or molecules. By "coadministered" is meant simultaneous administration in the same formulation or in two different formulations via the same or different routes or sequential administration by the same or different routes. By "sequential" administration is meant a time difference of from seconds, minutes, hours or days between the administration of the two types of molecules. These molecules may be administered in any order.

The method of the present invention may also be combined with currently known methods of treatment such as, in relation to Alzheimer's disease for example, treating associated non-cognitive problems, treating patients with cholinesterase inhibitors (donepezil. rivastigmine and galantamine), which provide symptomatic treatments and have been shown to improve cognitive functioning, or treating patients with Aricept.

I n yet another aspect the present invention relates to a pharmaceutical composition comprising modulatory agent as hereinbefore defined and one or more pharmaceutical ly acceptable carriers and/or diluents. Said pharmaceutical composition may additional ly comprise molecules with which it is to be co-administered. These agents are referred to as the active ingredients.

Although the method of the present invention is preferably achieved via the intravenous or oral adm inistration of the subject agent, it should be understood that the present invention is not limited to this method of administration and may encompass any other suitable method of administration. In this regard, the pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion or may be in the form of a cream or other form suitable for topical application. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi . The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oi ls. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of superfactants. The preventions of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delay ing absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, fol lowed by filtered steri lisation. General ly, dispersions are prepared by incorporating the various sterilised active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of steri le injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which y ield a powder of the active ingredient plus any additional desired ingredient from prev iously steri le- fi ltered solution thereof.

When the active ingredients are suitably protected they may be oral ly administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 1 % by weight of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit. The amount of active compound in such therapeutically useful compositions in such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0. 1 μg and 3000 mg of active compound.

The tablets, troches, pills, capsules and the like may also contain the components as listed hereafter: a binder such as gum, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially nontoxic in the amounts employed. In addition, the active compound(s) may be incorporated into sustained-release preparations and formulations.

Yet another aspect of the present invention relates to modulatory agents, as hereinbefore defined, when used in the method of the present invention.

The present invention is further described by reference to the following non-limiting examples.

EXAMPLE 1

Experimental Procedures

Human: All human tissue cases were obtained from the Victorian Brain Bank Network. Whole brains are stored at -80°C until required. Cortical tissue from non-demented controls, Alzheimer's disease, Parkinson's disease and Frontotemporal dementia were all taken from Brodmann's area 46. Cerebellum tissue was also used from non-demented controls and Alzheimer's disease patients. For AR analysis, western blots were carried out on total brain homogenates. Ferroxidase activity was tested by transferrin ferroxidasc assay on PBS + 1 % Triton X- 100 (PBST) extracted homogenates.

Biol in labelling ofAPP695a: Sulfo-NHS-SS-Biotin (Thermo Scientific) was added to

APP695a in 20-fold excess and incubated at room temperature for 1 h in phosphate buffered saline (PBS). Removal of non-reacted sulfo-NHS-SS-Biotin was by gel filtration using a Zeba^I Desalt spi n column (Thermo Scientific).

Αβ preparation: For aggregation studies, Ι ΟΟμΜ Αβ_{) -4}2 was incubated ± 200μΜ ZnCI² for 16 h to form precipitates as previously reported (Bush el ai , 1994). Insoluble Αβ|.₄; was then centrifuged at 40,000 g for 10 min and the pellet repeatedly washed in PBS.

Aggregated Αβ|.₄₂± Zn was then added to the Tf ferroxidase assay (described below) at a final concentration of 10 μΜ Αβι.₄₂ and compared to controls including freshly prepared non- aggregated 10 μΜ Αβι.42·

Transferrin ferroxidase assay: The assay was based upon established procedures

(Bakker and Boyer. 1986), utilizing the spectroscopic change in apo-transferrin when loaded w ith Fe³' . Km and Vmax values and curve-fitting were calculated by GraphPad Prism v 5.0. In a cuvette was added (in order): 100 μί ddlF-O 200 μΐ. HBS buffer ( 1 50 mM aC 1 , 50 mM HEPES, pH 7.2, 200 μΐ of 275 μΜ apo-transferrin, 100 μΐ of sample (200 nM recombinant protein or 30 μg total tissue homogenate) and 400 μΐ. of 275 ferrous ammonium sulfate ( I l4)₂Fe(S0₄)₂. For studies of pH-dependence the buffers (50 mM) were: pi I 5 sodium acetate, pH 5.5 - 6.5 MES, pi I 7.0 - 9.0 Tris. The mixture was incubated for 5 min at 37°C with agitation, and absorbance read at 460 nm. Extinction coefficient of diferric transferrin is 4.56 mM ¹.

Immunoprecipitalion: HEK293T cells ± 3 h preincubation 2 μΜ biotin-APP695 , or brain homogenate, was extracted into PBST (or PBS and then sodium carbonate, pH 1 1 for human brain membrane homogenate), and protein assayed. 100 pg of the sample was then pre- cleared for non-specific binding with protein G agarose beads for 1 h at 4°C. The sample was then incubated with capture antibody (rabbit anti-ferroportin, 1 :200, Lifespan Biosciences). mouse anti-N-term amyloid precursor protein (22C I I ), rabbit anti-ceruloplasmin or mouse anti-APLP2 ( 1 : 1000, R&D systems) for I h (4°C) before adding fresh equilibrated protein G agarose beads and mixed for a further 2-3 h (4°C). Protein G agarose beads were then washed in PBST and bound proteins were eluted with SDS-PAGE loading buffer. The bound and unbound proteins were separated on 4-20% PAGE (Bis-Tris, Invitrogen) and visualized by western analysis with a detection antibody; mouse anti-N-term amyloid precursor protein, mouse anti-A(3 domain of amyloid precursor protein ( 1 :500, W02), rabbit anti-C-term amyloid precursor protein (1 : 10,000, Chemicon), rabbit anti-ferroportin or, in the case of biotin-labelled studies, streptavidin crosslinked to horseradish peroxidase (HRP, 1 : 1 5,000, Invitrogen).

Histochemical detection of iron by Perl's staining: For direct visualization of redox- active I-V^"1 in whole brain hemisphere and liver paraffin embedded sections, a modified Perl's technique was used, as previously described (Gonzalez-Cuyar et ai , 2008; Smith et ai , 1997). The number of iron positive structures was quantified using colour selection to separate cells from background. Deparaffinized and rehydrated tissue sections (7 pm) were incubated at 37°C for l h in 7% potassium ferrocyanide with aqueous hydrochloric acid (3%) and subsequently incubated in 0.75 mg/ml 3,3'- diaminobenzidine and 0.01 % I 1;0₂ for 5- 10 min. When required, sections were counterstaining in Mayer's haematoxy in for 2 min and washed in Scott's tap water before mounting. For brain, the number of iron positive structures was quantified using colour selection to separate cells from background as described in

supplemental methods.

Reagents: Reagents were all analytical grade and were purchased from Sigma, Australia, unless otherwise specified. Purified human ceruloplasmin (ceru!oplasmin) was purchased from Vital Products (USA). Salts were chloride unless otherwise specified. Tg2576 (Hsiao et al. , 1996) mice were from Taconic.

Recombinant Protein Production: The recombinant fragments of the human soluble APP695a, APP75 l a, APP770a, human APLP2 ectodomain. amyloid precursor protein 1:1 (28- 189, Figure 1 A), amy loid precursor protein GFD (28- 123, Figure 1 A) and amyloid precursor protein CuBD ( 124- 160, Figure I A) were all expressed in the mcthylotrophic yeast Pichia pastoris strain GS 1 1 5 and purified as previously described (Cappai et ai . 1 999; Henry et ai , 1997). Amyloid precursor protein E2 (365-495, Figure 1 A), was expressed in the E.coli BL21 (DE3) by induction with Isopropyl (3-D- l -thiogalactopyranosid (IPTG). Following induction the cells were collected by centrifugation for subsequent protein purification.

Synthetic peptide synthesis: The synthetic peptides FD 1 and variants were synthesized using solid state Fmoc chemistry, in a microwave synthesizer, using Fmoc— ΡΑΙ,-PEG-PS as resin from Applied Biosystems. All the amino acids were coupled to the resin using a 3-fold excess where 0.5 M 2-( l H-BenzotriazoIe- l -y l )- l , l ,3.3- tetramethyluronium

hexafluorophosphate and 0.5 M of Ν,Ν-diisopropylethylamine were used as activators. Once synthesis was complete, the resin was swelled in dimethylformamide (D F) and the acetylation step was performance for 30 min at room temperature. Resin was then washed with DMF and methanol, and freeze-dried. The peptide was cleaved from the resin by stirring in a solution of 1 % water, 0.5% triisopropylsilane in trifluoroacetic acid (TFA) for 3 h. HPLC of peptide was performed using a preparative C8 Vydac Column, Buffer Λ = 0. 1 % TFA in water and Buffer B = 0. 1 % TFA, 50% isopropanol in acetonitrile. The gradient used was 1 5-70% B for 30min. The peptides were collected, lyophilized and reanalyzed by analytical H PLC and MALDI-TOF MS.

Purification of recombinant fragments of amyloid precursor protein: The recombinant fragments of the human soluble APP695a, human APLP2 ectodomain, amy loid precursor protein E l , amyloid precursor protein GFD and amyloid precursor protein CuBD ( Figure I A) were all expressed in the methylotrophic yeast Pichia pastoris strain GS 1 1 5 and puri fied as previously described (Cappai et al.. 1999; Henry et al. , 1 97). amyloid precursor protein E2 (Figure 1 A), was expressed in the E.coli BL21 ( DE3) (Andersen et al. , 2006). Al l fragments required a tw o-step procedure performed using an A KTA FPLC (GE Healthcare). APP695cx and amy loid precursor protein El was purified from culture media as prev iously described (Henry et al. , 1 97) using anion exchange on a Q-Sepharose column ( 1 .6 x 25 cm column. GE Healthcare) followed by hydrophobic exchange with phenyl-Sepharose (0.5 x 5cm column. GE Healthcare). Amyloid precursor protein CuBD purification was purified using anion exchange on Q-Sepharose column ( 1 .6 x 25 cm column, GE Healthcare) followed by size exc lusion with a Superdex 75 1 0/300 GL fi ltration column (GE Healthcare) (Barnham et al. , 2003). Amyloid precursor protein E2 and amyloid precursor protein GFD purification was achieved by heparin chromatography using heparin-Sepharose ( 1 .6 x 1 2cm; GE Healthcare) and anion exchange on Q-Sepharose ( 1 .6 x 25 cm column, GE Healthcare) (Andersen el al , 2006). To el iminate any adventitial zinc within the preparation method, 20μΜ Zn²" - selective chelator N, N. Ν', N'- tetrakis (2-pyridy!methyl) ethylenediamine (TPEN) was added to exchange buffers.

HEK293T transection and culture conditions: APP695 with the inactive E 14N mutation (Figure 2A-C) of the ferroxidase domain (" D 1 ^(6MN>-APP") was generated through PCR-derived mutagenesis of wt-APP695 in pcD A3. The generation of wt-APP and

FD l ^{l,' N,}-APP (pcDNA3. 1 based ) stable transfected 1 IE 293T cel ls with the empty plasm id or

1 8 plasmid encoding wt-APP695 or FD 1 ^{(,;, 4N,}-APP was performed using FuG N ^K I I D ( Roche ) follow ing the manufacturer's recommendations. 5 pg of plasmid was transfecled into a 25 cm² flask containing HEK293T cel ls. After 24 h the media was replaced with selection media containing G41 8. Cells were maintained at 37°C and 5% C0₂ in Opti-MEM media with 1 0% foetal calf serum (PCS). Cell lines transfected with wt-APP, FD 1 ^{(EI 4N)}-APP or vector alone (pcDNA3) were cultured in medium supplemented with 600 pg/mL G41 8 to maintain transgene expression. Isolation of amyloid precursor protein excreted from stably transfected cells was performed by incubating cells in supplemented Opti-MEM media without PC for 24 h before removal and filtering. Filtered media was purified as described above for recombinant yeast amyloid precursor protein.

Mice: All mouse studies were performed with the approval of the IACUC and in accordance with statutory regulations. For iron gastrointestinal absorption and accumulation tests, APP-/- mice (Zheng et «/. , 1 95) and background C57BL6/SV 1 29 control mice aged— 1 2 months were used. Carbonyl iron was freshly prepared daily and administered at

1 20μg/g/day in an 8% sucrose solution. The dose was orally applied with the use of an oli ve- tipped oroesophageal needle. Controls for each mouse type were administered with 8% sucrose solution only. Mice were fed for 8 days and sacri ficed. Tissues were taken and stored at -80°C until required. The left hemisphere of the brain from each mouse was also fixed in 4% paraformaldehyde for 24 h, dehydrated in ascending ethanol and embedded in paraffin.

Preparation of mouse primary neuronal cultures: Primary neurons cultures from the cortices of wild-type and amyloid precursor protein knockout embryos were prepared from embryonic day 1 4 or 1 5 mice as previously described (White et ai , 1998). Cortices were removed, dissected free of meninges, and dissociated in 0.025% trypsin. Cortical cells were plated onto poly-L-Iysine (50 pg/ml)-coaled 12-well or 48-wel l plates (Nunc) at a density of 600,000 cells/cm²) in DM EM supplemented with 10% FCS, 5% HS. and 1 0pMg/m l gentamycin sulphate. The neurons were allowed to adhere for 2-3 h before the plating medium was replaced with Neurobasal supplemented medium (serum free and with B27 m inus antioxidants, 500 μΜ glutaMAX and 10 pg/ml gentamyc in sulphate). Neuronal purity of cultures was— 0-95%. On the day of iron experiments the medium was replaced with fresh Ncurobasal-supplemented medium and for all further experimentation the medium was serum- free.

Iron toxicity measurement by cell viability assays: Mouse primary neuron cultures were seeded in triplicate on a 48 well plate. (NH₄)₂Fc(SO₄)2^'6H:0 was dissolved in 1 8 mn R₂0 before being added to Neurobasal supplemented media (serum-free) with 1 0 mM L-Ascorbic acid (in order to maintain the iron in the toxic Fe^{2 <} state). The pH of the Fe- Neurobasal media solution was adjusted to 7.0 with NaOH. The cells were treated for 24 hours after which the media was removed and fresh Neurobasal media containing 1 0% v/v CCK-8 ( Dojindo

Molecular Technologies) was added to the cells. The cells were incubated at 37°C for a further 2 h before reading the absorbance at 460 nm, which is proportional to viability.

Glutamate toxicity; Mouse primary cortical neuron cultures were seeded in triplicate on a 48 wel l plate and allowed to mature for 9 days in Neurobasal supplemented with B27. At day 6 the media is replenished and cytosine 1 3-D-arabinofuranoside (2 μΜ) added to prevent astrocyte proliferation. Neurons were then pre-treated with varying concentrations of soluble wt-APP695 or FD 1 ^{(H 14N)}-APP for 6 h before 40 μΜ glutamate was added to the media and incubated for a further I h. Media was then replaced with fresh Neurobasal media for an additional 1 8 h before cell viability was measured as explained above using CCK-8 (Doj indo Molecular Technologies). MK80 I ( 10 μΜ) was added 20 minutes prior to glutamate addit ion as a control to rescue glutamate toxicity for a set of spec i fic samples in each experiment.

Tf(^5v Fe): preparation: Human apo-Transferrin (apoTf) w as treated with sod ium ascorbate to remove all trace of unlabelled Fe. ApoTf was then loaded w ith ^l-e ( Perkin F. lmer, Boston. MA ) as prev iously described (van Renswoude et al. , 1 982). Cel ls were incubated for 1 2 h with 1 .0 x 10^"6 M Tf(⁵⁹Fe)₂ unless otherwise stated.

Cellular Iron Uptake and amyloid precursor protein siRNA: Parental HEK293T cel ls, which constitutively express amyloid precursor protein (Lammich et al. , 1999), were maintained in Opti-MEM media with 10% foetal bovine serum, plated and transfected simultaneously using Lipofectamine 2000 (Invitrogen) with amyloid precursor protein

(# 1 1 8200, # 1 14057 & # 1 14058) or scrambled siRNAs (50 pmol) (Validated Si lencer® Select siRNA, and Silencer® Select Negative Control # 1 siRNA, Applied Biosystems). 36 hours after plating onto 12-well plates (4 cm²) cells were treated with serum-free Opti-MEM (SFM) ± Fe(NH₄)₂S0₄ ( 10μΜ) or Tf(⁵⁹Fe)₂ for 12- 1 8 h. Where described, cells were then washed with SFM and the medium replaced with SFM lacking tracer ± 2μΜ (unless otherwise stated) APP695a or amyloid precursor protein E2 for 6 h. prior to harvesting. Basel ine values were taken from the cells transfected with a non-specific scrambled si RNA control .

Studies w ith primary neurons were in serum-free Neurobasal B27-supplcmentcd medium. Cel ls were treated with Tflf Fe)₂ or Fe(NH._t)_(S0₄)_ (75μΜ) for 1 8-24 h. For Tf(^wFe)₂ studies, medium was removed and cells washed thoroughly before fresh media ±- 0.5- 2μΜ ΑΡΡ695α or E2, or HE 293T serum-free media containing soluble wt-APP695 or FD 1 ^{(K14I 1 )}-APP (collected after 24hrs), was added for up to 12 h. In the neuronal efflux assay cells were incubated at 37°C, media was removed at select time points and washed cel ls solubilized with PBS + 1 % Triton X- 100 + protease inhibitors (PBST). A comparable time course was carried out at 4°C to eliminate non-speci fic binding of ⁵⁹Fe to the outer membrane. All media and cell lysalcs were measured for counts/min (CPM) by y-ray counter ( W izard 3. Perkin Elmer).

Western blot analysis: 20 pg total protein from each experimental condition, or 5 pg total protein for AR detection in human samples, was separated on 4-20% PACK (Bis-Tris, Invitrogen) and transferred to nitrocellulose membrane using iBlot (Invitrogen). Primary antibodies used were rabbit anti-ferritin ( 1 : 1 0,000, #ab7332, AbCam), mouse anti-Transferrin Receptor (TfR) ( 1 :5,000, clone 1 168.4, Invitrogen), mouse anti-APP ( 1 : 100, in house 22C 1 1 ), mouse anti-Αβ ( 1 :500. 6E 10, Covance). rabbit anti-Iron Regulatory Protein I ( 1 : 1 ,000, A B I ). rabbit anti-Iron Regulatory Protein2 ( 1 : 1 ,000, ABI) and rabbit anti-ceruloplasmin ( 1 :5000. Sigma). The load control was mouse anti-(3-actin ( 1 : 10,000). Proteins were visual ized with ECL (Amersham) and a LAS-3000 Imaging suite, and analyzed using Multi Gauge (Fuji). Densitometry using Image J (NI H) of ferritin, TfR, amyloid precursor protein, AR and ceru loplasmin was performed in triplicate on 3 separate experiments. A ll quantitation was standardized against 1 3-actin levels

Iron-Responsive Element binding assay: 1 00 pg cell lysates from I IE 293T cel l ± amyloid precursor protein siRNA prepared in m idRIPA buffer (25 m Tris pH 7.4. 1 % N P40, 0.5% sodium deoxycholate, 1 5 mM Nad. protease inhibitors and RNase inhibitor), were incubated with 100 nM of biotinylated H-ferritin Iron- esponsive Element RN A

oligonucleotide (biotin-ggg uuu ecu gcu uca aca gug cuu gga egg aac cc; Sigma) for 3 h at RT. MidR IPA buffer washed paramagnetic streptavidin-conjugated Dynabeads (I nvitrogen) were added to bind to Iron Regulatory Protein( l /2)/biotinylated-R A complexes and incubated for 1 h at RT. Protein bound to the beads was repeatedly washed (midRI PA) and Iron Regulatory Protein 1 , I ron Regulatory Protein2, and biotin was visual ized by western blot analysis as described.

lmm nodepletion: Amyloid precursor protein was extracted from 30 pg human brain homogenate using the immunoprecipitation method described above using mouse anti-N- terminal amyloid precursor protein as the capture antibody. The bound and unbound proteins were tested for ferroxidase activity and compared to total brain homogenate using the transferrin ferroxidase assay described previously.

Metal Assay: Lyophilised homogenates from tissue samples were dissolved in cone.

HNO3 and H2O2 (Aristar, BDH). Metal levels were measured by inductively coupled plasma mass spectrometry with an Ultramass 700 (Varian, Victoria, Australia) as described (Maynard «^;/ «/. , 2006).

Oxyblot assay: Carbonyl groups on proteins from l iver and brain homogenates were detected using OxyBlot Protein Oxidation Detection K it (Chemicon) following the manufacturers instructions. Samples were diluted to produce a 4 pg/pl protein concentration in which two al iquots were made of 20 pg each. One aliquot was derivati/ed and the other used as a negative control. Protein was transferred to a nitrocellulose membrane by dot blot. Nonspeci fic binding was blocked by PBST containing 1 % BSA before the membrane was incubated with rabbit anti-dinitrophenyl (DNP) antibody. Immunoreactive proteins were quantified using ECL reagent as for western blots.

Glutathione assay: Glutathione (GSH) levels in tissue homogenates were detected using a GSH assay against known standard of reduced GSH. PBST soluble homogenates were added to sulfosalicyl ic acid (SSA) buffer to make a final concentration of 0.025% SSA and samples were di luted to fit within the standards used on the assay. GSH standards or di luted samples (50 pi) were added in triplicate to a 96 well plate with reaction mix ( 1 OOp 1 , 0. 1 5mM 5-5'-Dithiobis (2-nitrobenzoic acid) (DTNB). 0.2mM p-nicotinamide adenine dinucleotide phosphate (NAIzheimer's diseasePH) and 1 .0 U/ml GSH reductase) and scanned at 4 1 nm every 20 s for 3 min. The reaction rale (Vmax) was used to calculate the oxidation of GSI 1 along time.

Image analysis of histological brain sections: All sections were imaged on a Northern Light I l luminator (Imaging Research Inc, Ontario, Canada) using a Spot RT-KE 2MP digital camera (Diagnostic Instruments, MI, USA) equipped with a Nikkor 55 mm lens and 56 mm extension tube set (Nikon). Each image was analysed using ImagePro Plus 5.1 (Media Cybernetics. MD, USA). For each image an "area of interest" (AO 1 ) line was drawn around the margins of the brain occupied by the total section or hippocampus. The number of iron- positive structures present in each section was then quantitated using colour selection (brown) to separate the cells from background label l ing. This data was expressed as the total number of iron-positive counts in each section and was exported to M icrosoft Excel for analysis. Based upon the known size of each AOl, all values were normalised to allo comparisons between sections of different surface area. An average was taken from three sections from each animal.

Pathological characterization of human cortical tissue: Neuropathological diagnosis was made following macroscopic and microscopic examination of hemi-brains and examination for Αβ-immunoreactive amyloid plaques and tau-immunoreactive neurofibrillary tangles, and considering clinical history, using the consensus recommendation for the postmortem diagnosis of Alzheimer's disease (The National I nstitute on Aging, 1 97). I n line with the CERAIzheimer's disease criteria (Mirra et al. , 1991 ), a semi-quantitative analysis of the extent of plaque deposition in the frontal cortex was performed where (+) represented a mean plaque load over multiple high power fields (hpf) (x 400 magnification) of <5 plaques per hpf, (++) represented 5- 1 5/hpf, (+++) represented > 1 5 /hpf. Control cases for the study showed no evidence of any neuropathological disorder.

Results

APP695 possesses ferroxidase activity similar to ceruloplasmin

Amyloid precursor protein possesses a REXXE ferroxidase consensus motif (Gutierrez. et al. , 1 997) as found in the ferroxidase active site of H-ferritin ( Figure 1 A , B). This evolutionarily conserved motif is not present in paralogs APLP 1 or 2 (Figure I B). 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 amyloid precursor protein (Wang and Ha, 2004), with low root mean square deviation (0.4 A) when overlaying backbone atoms of the a- helical H-ferritin catalytic site (residues 52-67) with the corresponding backbone atoms of amyloid precursor protein (residues 402-41 7) (Figure 1 C). 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 E41 5 of APP695 (Figure 1 C).

Recombinant soluble APP695oc, representing the predominant neuronal amyloid precursor protein species (Rohan de Silva et al. , 1 97). possessed robust ferroxidase activ ity ( Vmax= 228.6 μΜ Ρε347ίτπη/μΜ amyloid precursor protein; Km= 48.6 μΜ; F igure 1 D). l i ke ceruloplasmin (Figure I E), as measured by the rate of Fe3+ incorporat ion into transferrin. Therefore, amyloid precursor protein is a more active ferroxidase than ferritin ( Vmax- 2.2 1 μΜ Fe3+/min^M ferritin, Km= 200 μΜ) (Bakker and Boyer, 1 986). APP695ct ferroxidase activity was maintained across a pH range 5.0-9.0 (Figure 8 A). APLP2 was inactive (Figure 2A), like the negative control albumin (Figure 8B), consistent with the absence of the REXXE motif (Figure 1 B).

Ceruloplasmin ferroxidase activity is dependent on copper and inhibited by NaN3 (Osaki, 1966). Neither NaN3 (Figure 2A) nor Cu²⁺ (2: 1 Cu:APP) altered APP695a activ ity. indicating that APP695a ferroxidase chemistry is like H-ferritin (Bakker and Boyer, 1 986), and not l ike ceruloplasmin. H-ferritin ferroxidase activity is inhibited by Zn^{2 *} (Bakker and Boyer, 1 986) and indeed, Zn²⁺ inhibited the activities of both APP695 and the E2 domain of amyloid precursor protein (Figure 2A). Inhibition was specific for Zn²⁺ among physiological divalent metal ions since Ca²⁺ (2 mM), Mg²" (0.5 mM), Cu^{2 ,} (20 μ ), Mn²⁺ ( 10 μΜ), N i² ' (20 μΜ) and Co²⁺ (20 μΜ). as chloride salts, did not inhibit APP695a ferroxidase activ ity ( not shown). The activities of the main isoforms, APP695a, APP770a. APP75 1 a were identical (Figure 8C).

A 22-residue synthetic peptide within the E2 domain (FD 1 ) (Figure 1 A & 2B), containing the putative active site of amyloid precursor protein, possessed ferroxidase activity that was r=-40% that of APP695a (Figure 2C). Mutational analysis of APP695a (Figure 2 A) and FD I (Figure 2B-D) confirmed that disruption of the REXXE moti f, by altering a single conserved amino ac id (REWEN, "El 4N" in Figure 2B, C), or substituting the homologous pentapeptide regions of APLP l (REWAM, ·'ΕΕ 1 3/ 14ΑΜ" in Figure 2D) or APLP2 (K EW EE, "R I OK'^" in ligure 2D), abolished activity. Substitution with the homologous H-ferritin sequence (REHAE, "'WE 12/ 1 3HA" in Figure 2B), which does not disrupt the consensus motif, retained activity (Figure 2D).

Like FD I peptide (Figure 2C), purified E2 polypeptide (Figure 1 A) possessed :=4()% of the ferroxidase activity of APP695a (F igure 2A). Other domains of amy loid precursor protein needed to restore ful l activity to E2 were explored for. W hile purified El domain possessed no ferroxidase activity , equimolar concentrations of El doubled E2 activ ity ( F igure 2E) to about that of APP695a (Figure 2A). This potentiation effect was mapped to the Growth Factor Domain (GFD) within El (Rossjohn et ai , 1 99) (Figure 2A). GFD did not engender activity from APLP2 (Figure 2A), consistent with the requirement for the REXXE moti f.

Amyloid precursor protein facilitates iron export and interacts with ferroportin

The impact of endogenous amyloid precursor protein suppression by RNA i on iron export w as studied in HEK293T cells, where the absence of ceruloplasm in was confirmed by western blot. Amyloid precursor protein -suppressed cells accumulated signi ficantly more (=50%, pO.01 ) radioactive iron ( Fe) than sham RNAi controls (Figure 3A & Figure 9A). Addition of APP695a (2 μΜ, Figure 3B) or the E2 domain of amyloid precursor protein (2 μΜ, Figure 9A) to the media, after incorporation of ⁵' Fe into the cells, significantly promoted the efflux of ⁵⁹Fe into the media. E2 lacks the heme-oxygenase (HO) inhibitory domain of amyloid precursor protein (Takahashi et ai , 2000) (Figure 1 A), therefore amyloid precursor protein is not promoting iron export in these cells merely through inhibition of HO.

Complementary changes in cellular levels of the iron-responsive proteins ferritin and TfR (Figure 3C & D, blots in Figure 9B), consistent with decreased Iron Regulatory Protein 1 & 2 binding to a biotinylated Iron-Rcsponsivc Element probe (Figure 9C), confirmed that amyloid precursor protein acted to lower the LIP. As a further control, the impact of stable translection of wild-type (WT) or inactive mutant (FD l ^u;MN)-APP695. Figure 2A) APP695 on iron retention in I 1 EK293T cells was studied. A PP695 signi ficantly decreased iron retention compared to cel ls transfected with vector alone, but FD 1 ^{([ I4N)}-APP695 increased iron retention, consistent with competition against endogenous amyloid precursor protein (Figure S2D-F). These data indicate that ferroxidase-active amyloid precursor protein facil itates iron export in HEK293T cells.

Ceruloplasmin co-immunoprecipitates with ferroportin in certain tissues (De Domenico et ai , 2007; Jeong and David. 2003). Analogously, most of the ferroportin in HE 293T cells co-immunoprecipitaled with endogenous amyloid precursor protein (Figure 3E, Figure 9G). Furthermore, the majority of a biotinylated A PP695a probe added to HE 293T cells co- immunoprecipitated with ferroportin (Figure 3F), consistent with exogenous APP695a promoting iron export (Figure 3 B-D) by interacting with ferroportin.

Iron transport was studied in primary cortical neurons from APP-/- m ice. ΛΡΡ v- neurons retained significantly more ⁵⁹Fe than WT neurons (+50%. p<0.01 ) (F igure 4A ). and exhibited a corresponding decrease (-60%) in the rate of iron efflux (Figure 4B ). The increased retention of iron in APP-/- neurons was comparable to that reported for ceruloplasmin-/- astrocytes over the same 12-hour incubation period (De Domenico el ai , 2007; Jeong and David, 2003). APP695a added to WT neurons induced a significant concentration-dependent decrease in ⁵⁹Fe retention (Figure S3A), and reversed much of the increased ⁵⁹Fe retention in APP-/- neurons (Figure 4A). Inactive FD l ^{tKI 4N)}- APP695cx (Figure 2A), could not promote iron efflux (Figure 10B).

The E2 domain of amyloid precursor protein also fac ilitated iron efflux in primary neuronal cultures (Figure 9C). As with amyloid precursor protein -suppressed HE 293T cells (Figure 3C&D), more ferritin and less TfR were detected in APP-/- compared to \NT neurons, exaggerated by the addition of 10 μΜ iron (Figure 4C, westerns shown in Figure 10D), consistent with increased neuronal iron. It was confirmed (Figure 1 0D) that neocortical neurons do not express ceruloplasmin (Klomp et ai , 1996). Therefore, cortical neurons may depend upon amyloid precursor protein as the ferroxidase partner for ferroportin. Consistent with this, amyloid precursor protein in human and mouse cortical tissue (including full-length membrane-bound amyloid precursor protein) had a major interaction with ferroportin in immunoprecipitation studies (Figure 4D & E, Figure 1 1 A-C). APLP2 did not co- immunoprecipitate with ferroportin from these tissues (Figure 4D).

Neocortical ferroportin also co-immunoprecipitated with ceruloplasmin (Figure 4D). This was expected, since despite being absent in cortical neurons, ceruloplasmin is expressed in glia (Klomp et ai . 1996). Co-immunoprecipitation of ceruloplasmin by anti-ferroportin was slightly but significantly increased in APP-/- brain tissue (Figure 1 1 D & K), possibly due to loss of amyloid precursor protein competition for ferroportin interaction. Therefore, ferroportin divides its interactions between amyloid precursor protein and ceruloplasmin in the brain.

However, unlike APP695a (Figs. 3B & 4A), ceruloplasmin (2 μΜ) induced no significant increase in ⁵⁹Fe efflux when added to primary neurons or HEK293T cells, consistent with previous observations that the ability of ceruloplasmin to stabilize ferroportin was cell-type specific and probably limited to cells that express ceruloplasmin (f)e Domenico et ui., 2007).

Consistent with amyloid precursor protein ferroxidase activity being protective, the LD50 for Fe"^* toxicity was 10-fold higher for primary neurons in culture from WT (2001 μΜ ) compared to amyloid precursor protein -/- mice (234 μΜ, Figure 4F). I lowever, domains and post-translational modifications outside of the ferroxidase domain, can promote protection against oxidative damage (Furukawa et ai , 1996). To appraise the contribution of amyloid precursor protein ferroxidase activity to neuroprotection against non-iron oxidative injuries, we studied the effects of APP695 compared to FD I ^{(I UN,}-APP695 in protecting primary neurons from oxidative stress induced by glutamate excitotoxicity, where sAPPa prevents intracellular Ca²' rise (Furukawa et ai, 1996; Mattson et ai , 1993). While APP695 significantly prevented glutamate toxicity under these conditions, the ferroxidase mutant did not (Figure 10E).

Amyloid precursor protein prevents iron accumulation and oxidative stress in vivo Aceruloplasminemic patients and ceruloplasmin knockout m ice exhibit marked age- related iron accumulation in liver, pancreas and brain astrocytes (Harris et ai , 1 995; Patel et ai , 2002) but not cortical neurons (Gonzalez-Cuyar et ai , 2008; Jeong and David, 2006; Patel et ai , 2002). To test whether amyloid precursor protein deficiency would cause a simi lar vulnerability, 12 month old APP-/- mice were compared to WT age-matched controls Ted a normal or high-iron diet for 8 days. Consistent with cell culture findings (Figures 3 & 4), APP- /- mice fed a normal diet had significantly more total iron in brain (+26%), liver (+3 1 %), and kidney (+ 1 5%) tissue than age-matched controls (Figure 5 A; Table 2). After chal lenge with the high-iron diet, WT mice had no significant change in tissue iron levels. In contrast, ΛΡΡ-/- mice accumulated significantly more iron in brain (+1 3%) and particularly liver (+90%) than APP-/- mice on a normal diet (Figure 5A). Ferritin levels were also increased in brain and liver tissue from APP-/- mice on the high iron diet (data not shown) consistent with increased iron content. Iron supplementation did not affect the tissue levels of other metals (Table 2).

The livers and cortex of APP-/- and WT mice were exam ined with a modi fied Perl's histological stain, which utilizes intracellular Fe²⁺ to generate H202 (Gonzalez-Cuyar et ai , 2008; Smith et al. , 1 997). This revealed elevated hepatocytic Fe^2T (Figure 5B & F). and intraneuronal Fe^2" (Figure 5C, D & F-H) of APP-/- mice compared to WT matched controls both fed iron. Fe²⁺ accumulation in the brain was confined to neocortical and hippocampal neurons (Figure 5H), while sparing microglia and astrocytes that arc known to express ceruloplasmin (Gonzalez-Cuyar et ai , 2008; Harris et ai , 1 995; Patel et ai , 2002). Assay of tissue ferroxidase activity revealed a significant =40% decrease in APP -/- brain (Figure 51). NaN3-inhibition of ceruloplasmin activity in WT brain tissue revealed rz40% residual activity, and the complete loss of ferroxidase activity in the brains of APP-/- mice (Figure 51 ). These data are consistent with amyloid precursor protein acting as a neuronal ferroxidase.

Suppression of amyloid precursor protein activity in WT brain tissue with Zn2+ revealed .=- 160% activity consistent with residual ceruloplasmin ferroxidase activity, and was slightly increased in APP-/- mice (Figure 51), perhaps reflecting homeostatic compensation. There were no conspicuous changes in ferroportin or ceruloplasmin levels in liver and brain samples from APP-/- m ice on a normal or iron-supplemented diet (Figure 1 1 F).

The constitutive abundance of amy loid precursor protein in WT l iver was found to be simi lar to that of ceruloplasmin (Figure I I G). Therefore, the increase in l iver iron in A PP-/- mice was consistent with a major loss in the total ferroxidase complement of the tissue.

Conversely. APP-/- heart and lung tissue did not show elevated iron levels even with dietary iron challenge, consistent with these organs having the lowest constitutive levels of amyloid precursor protein (Figure I I G) and expressing alternative iron-export ferroxidases, ceruloplasmin (Figure 1 I G) and hephaestin (Qian et al. , 2007). Similarly, amyloid precursor protein levels in astrocytes are much lower than in neurons (Gray and Patel, 1 93; Mita et al. , 1989; Rohan de Silva et al , 1997), and probably too low to prevent iron accumulation in ceruloplasmin-/- astrocytes. Cortical neurons have no redundancy in their export ferroxidases. and therefore accumulate iron in the absence of amy loid precursor protein (Figure 5 F-H). The likelihood that amyloid precursor protein is the unique ferroxidase of cortical neurons is supported by the lack of iron increase in the cortical neurons of ceruloplasmin-/- mice even at an age (24 months) when there is a marked increase in iron in other cells (Jeong and David, 2006; Patel et al , 2002).

Increased Fe^2* generates oxidative stress, and indeed the Fe²⁺ increase detected in iron- fed APP-/- mice was accompanied by increased protein carbonylation (indicative of hydroxy! radical damage, Figure 5J), and decreased glutathione levels (Figure 5 ) signifying depleted antioxidant reserves. Despite these signs of stress, stereological counting revealed no significant neuronal loss within the brain in iron-fed mice (data not shown). A more protracted period of iron exposure, or higher doses, may be needed to overcome survival defences. The observation that iron enters the brain neurons of iron- fed APP-/- mice (Figure 5) also indicates that either amyloid precursor protein is a component of the blood- brain barrier, or that prandial iron normally transits the blood-brain barrier to neurons where it is then exported in an amyloid precursor protein -dependent manner.

Amyloid precursor protein ferroxidase activity is inhibited by zinc in Alzheimer ^'s disease

Elevated iron and ferritin are prominent within the vicinity of amyloid plaques in both humans (Grundkc-lqbal et al , 1990; Lovell et al. , 1998; Robinson, 1995) and amyloid precursor protein transgenic mice (El Tannir El Tayara et al. , 2006: Falangola et al.. 2005; Jack et al. , 2005). A 45% increase in iron in post-mortem Alzheimer's disease cortical tissue (Brodmann area 46) was found, but no change in pathologically-unaffected cerebellum from the same patients (Figure 6A). This matched a 75% (pO.001 ) decrease in amyloid precursor protein ferroxidase activity in the same Alzheimer's disease cortical samples compared to the non-demented age-matched samples, with no difference in cerebellar tissue activities (Figure 6B). The ferroxidase activities were confirmed to be amyloid precursor protein by

immunodepletion experiments (Figure I 2A). The loss of amyloid precursor protein ferroxidase activity in Alzheimer's disease cortex was not due to decreased levels of amyloid precursor protein (Figure 12B). Therefore, a factor in Alzheimer's disease cortex appears to inhibit amyloid precursor protein. Zn²* is the only identified inhibitor of amyloid precursor protein ferroxidase activ ity (Figure 2A), but total zinc levels were not significantly elevated in the Alzheimer's disease cortical samples (Figure S5C). However, Zn² ' characteristically accumulates in extracellular amyloid in Alzheimer^' s disease (Lovell et al. , 1 998; Religa et al. , 2006; Suh et al. , 2000), which is too small a volume fraction to elevate total tissue zinc levels until the disease is advanced (Religa et al. , 2006). Indeed, treatment of the Alzheimer's disease cortical samples with the Zn² -selcctive chelator TPEN restored amyloid precursor protein ferroxidase activity to levels not signi ficantly different from non-demented samples (Figure 6B). confirming that amyloid precursor protein is inhibited by Zn²' in Alzheimer's disease tissue. TPEN did not significantly change ferroxidase activity in non-demented cortical samples (Figure 6B), indicating that Zn² ' is not inhibiting amyloid precursor protein in normal tissue. To confirm that the amyloid precursor ^" protein ferroxidase activity in A lzheimer's disease is being inhibited by Zn² ' , additional Zn^{" '} was titrated into samples that had been treated with 20 μ TPEN (Figure S5 D). Whereas Zn² ' concentrations of μ were required to suppress amyloid precursor protein ferroxidase activity in normal tissue under these conditions, far lower Zn^2* concentrations (2 μΜ ) suppressed activity in Alzheimer's disease samples ( 1050 for normal tissue = 22.6 μΜ, IC50 for

Alzheimer's disease = 1 0.2 μΜ, Figure 12D). Together these data indicate that whi le there is no clear elevation in total zinc in Alzheimer's disease tissue, there is a greater fraction of exchangeable Zn²\ which is inhibiting amyloid precursor protein ferroxidase.

Is the Zn²* trapped in extracellular Λβρ deposits suffic iently exchangeable to be the source of Zn ⁺ that inhibits amyloid precursor protein ferroxidase in Alzheimer^' s disease tissue? To test this w ashed (no free Zn²⁺) synthetic Αβ:Ζη²⁺ precipitates were prepared. It was found that they indeed inhibited APP695oc activity as efficiently as free Zn²⁺ in solution, whi le A3 alone had no effect (Figure 6C). Therefore, AR traps Zn²⁺, but can readily exchange the Zn~^r with amyloid precursor protein. Neither free Zn²⁺ nor Αβ:Ζη² * complexes inhibited ceruloplasmin activity (Figure 6C).

Consistent with Ap presenting Zn² ' to suppress amyloid precursor protein ferroxidase activity in the brain, there was a signi ficant negative correlation between A l i burden and amyloid precursor protein ferroxidase activity in a series of Alzheimer' s disease (pO.000 1 , Figure 6D) and amyloid precursor protein transgenic (Tg2576, Figure 1 2E & F) cortical samples. However, amyloid precursor protein ferroxidase activity was not diminished in cortical tissue from Frontal Temporal Dementia or Parkinson's disease (Figure 6E) that lacked amyloid pathology (Table 3), or from Tg2576 mice at an age prior to amyloid pathology (Figure 12E & F).

EXAMPLE 2

ALZHEIMER'S DISEASE β- AMYLOID PROTEIN PRECURSOR IS FERROXIDASE

II, A NON-CERULOPLASMIN ENZYME IN PLASMA Experimental Procedures

Animals. All mouse plasma studies were performed with the approval of the 1ACUC and in accordance with statutory regulations. APP knockout (APP-/-) (Zheng et al. ( 1 995) Cell 8 1 , 525-53 1 ), β-amyloid precursor-like protein 2 knockout (APLP2-/-) and background controls (C57BL6, C57BL6/SV 1 29 respectively) were thoracotomized under deep anesthesia, and heparini ed plasma obtained from the left atrium.

Human volunteers. A smal l population of plasma samples were taken from the

Australian Imaging Biomarkers & Lifestyle Flagship Study of Ageing (AI BL) (El lis et al. (2009) Jnt Psychogeriatr 2 1 , 672-687), a cohort of volunteers aged over 60 years and c lassi fied as individuals with AD as defined by N1 NCDS-ADRDA criteria (McKhann el al. ( 1 984) Neurology 34, 939-944) or cognitively healthy individuals (healthy controls, HC). The AI BL study was approved by the institutional eth ics committees of Austin Health, St, Vincent^'s

Health, Hol lywood Private Hospital, and Edith Cowan University. Written informed consent was obtained from all study participants.

Plasma sample collection. Whole blood was collected in Li Heparin Vacutainer® tubes (BD) for humans and in 1 ml LH Li Hep MiniCollect® lubes (Greiner bio-one) for mice before mixing for 10 min at room temperature. The tubes were then centrifuged at 200g and 20°C for 1 0 min. The supernatant (platelet rich plasma) was removed to a fresh tube from the red blood cell fraction and was then spun at 800g and 20°C for 1 5 m in. The platelet-depleted plasma was then aliquoted and stored in liquid nitrogen for later use. Before each assay, the plasma was thawed on ice for 1 hr and then centrifuged at 800g and 4°C for 10 m in on a bench top centrifuge before analysis.

Oxidase Activity Assays. For high throughput analysis of oxidase activity the o- Dianisidine Oxidase Activity Assay was used and based upon the procedure of Schosinsky et al (Clin Chem 20, 1 556- 1563, 1974). Samples were incubated at 37°C after being mixed with 75 mM sodium acetate buffer pH 5.0. 7.88 mM o-Dianisidine dihydrochloride substrate was added, mixed and then incubated for 5 and 60 min. 9M sulfuric acid was used to stop the reaction at these times. Oxidized product was monitored by absorbance at 540nm from where the velocity was plotted.

More specific ferroxidase activity measurements of carried out using the Transferrin assay, based upon the procedure of Bakker and Boyer { Biol Chem 26 1 , 13 1 82- 1 3 1 85, 1986). In a cuvette was added (in order): 1 OOul distilled water, 200ul of HBS buffer ( 1 50 mM NaCI, 50 mM HEPES, pH 7.2), 200μΙ of 275 μΜ transferrin, 1 00 μΐ of sample and 400 μΐ of 275 μΜ ferrous ammonium sulfate. The mixture was incubated for 5min at 37°C with agitation, and absorbance read at 460 nm. Diferric transferrin has an extinction coefficient of 4.56 mM^{' 1} ,

In both assays, select ferroxidase inhibitors were optimized for plasma samples.

Sodium azide ( 10 mM) or Diethylpyrocarbonate (DEPC; l mM) was added to abolish the activity of CP derived activity and Zinc Acetate ( Ι ΟΟμΜ) abolished the activity oi^' APP derived activity.

Separation of ferroxidase II from plasma. Non-ceruloplasmin ferroxidase was isolated from CP as previously described (Topham, R. W., and Frieden. E. ( 1 970) ,/ Biol Chem 245, 6698-6705) by anion exchange with Q-Sepharose ( 1 .6 x 25cm column. GE Healthcare). Mouse and human serum was dialyzed overnight against 0.05 M Na acetate. pFI 5.5 and protein prec ipitate was removed by centrifugation. Dialyzed scrum was appl ied to the column and protein was eluted with 0.05 M acetate, pH 5.5, and a stepwise gradient of NaCI. Fractions of 5 mL were collected and quantitated for activity ± zinc or azide with Transferrin (human) and o- Dianisidine Oxidase (mouse) assay. APP or CP protein expression in human plasma was determined by western blot.

Generation of a polyclonal antibody specific to the ferroxidase domain of APP. The

"ferroxidase domain (FD 1 ) peptide containing the putative active site of APP was coupled to Diphtheria Toxoid (DT) covalently using glutaraldehyde. Immunization of rabbits was carried out by Mimotopes (Clayton, Australia) using a standard procedure. Briefly, 1 rabbit was immunized subcutaneously at multiple sites with a total of I m L of em ulsion consisting of 200 nmol FD l conjugate with 2 volumes of Freund' s complete adjuvant. Rabbits were immunized a further 4 times over 8 weeks with 1 m l of emulsion contain ing I ^'reund' s incomplete adjuvant (without mycobacteria). The Final bleed was assessed by EI J SA and western analysis against the original peptide, recombinant APP695a, plasma and tissue homogenates to determine titer and specificity. At an optimal titer of 1 : 1000, western analysis was carried out as with other antibodies (see " western blot analysis' in methods below) with the exception of blocking conditions in 5% BSA for 2hrs before serum was added for overnight incubation.

Imm nodepletion of plasma. 1 ΟΟμί of plasma was precleared for non-specific binding with protein G agarose beads for I hr at 4°C. The sample was then incubated with capture antibody mouse anti-N-term APP ( 1 :50; in house 22C 1 1 ), rabbit anti-CP ( 1 :200; Sigma) or mouse anti-β actin ( 1 :200, R&D systems) for l hr (4°C) before adding fresh equi l ibrated protein G agarose beads and mixed for a further 2-3 hrs (4°C). Plasma supernatant ( final volume I ml.) was removed and either stored or tested immediately for oxidase activ ity.

Protein G agarose beads were then washed in PBS and bound proteins were eluted with 0.2M Glycine buffer (pH 4). The bound and unbound proteins were tested for oxidase activity and compared to total plasma using o-Dianisidine Oxidase activity assay described previously. Buffers were known not to effect assay conditions or ferroxidase activity as previous used in activity gels (Larrondo et al. (2003) Appl Environ Microbiol 69, 6257-6263). The proteins from immunodepleted plasma and immuno-captured samples were also visualized by western analysis as described below.

Western analysis. Samples containing protein from each experimental condition was separated on 4-20% PAGE (Bis-Tris. Inv itrogen) and transferred to nitrocellulose membrane using iB Iot ( Invitrogen). Primary antibodies used were mouse anti-N-term APP ( 1 : 1 00; in house 22C 1 1 ), rabbit anti-FD l of APP ( 1 : 1000; in house) and rabbit anti-CP ( 1 : 5000; Sigma). Proteins were visual ized with ECL (Amersham) and a LAS-3000 Imaging suite, and analyzed using Multi Gauge (Fuj i). Densitometry using Image J (M ill) of^' APP and C P w as performed in triplicate.

Results

Human feroxidase II is inhibited by zinc. As a multicopper ferroxidase, the activ ity of CP is dependent on copper and inhibited by azide (Erel, O. ( 1998) Clin Chem 44, 23 1 3-23 1 ). However, azide has no effect on A PP695a ferroxidase activ ity but is instead inhibited by Zn^2"* (Duce et al. (2010) Cell 142, 857-867) similar to H-ferritin (Bakker and Boyer 1986, supra).

Two major ferroxidases have been reported in human serum. CP is termed ferroxidase

1 and is measured as the fraction of total serum ferroxidase activity inhibited by azide (=90% in humans) (Erel 1998, supra). The residual azide-resistant activity is termed ferroxidase I I and while this is a smaller proportion of the total plasma ferroxidase activity in humans, it represents a greater proportion in rodents (Topham and Frieden 1 980. supra; Topham and Johnson ( 1 74 ) Arch Biochem Biophys 1 60, 647-654; Gray et al. (2009) Biochem J 41 . 237- 245). A - 1 5% residual ferroxidase II activity after azide inhibition of normal adult human serum was comparable to previously reports (Topham and Johnson 1 784. supra; Gray et al. 1973, supra) (Fig. 14 A) while the APP695a. inhibitor Zn²⁺ was found to decreased total activity by - 1 5% (Fig. 14A). The Zn²⁺ specific impairment of total ferroxidase activity closely correlating with the residual activity observed after azide inhibition and was determined to be dose dependent with maximal inhibition greater ^η Ι ΟμΜ (Fig. 14B).

APP deficient mice have reduced total ferroxidase activity. A PR' s role as ferroxidase

I I was supported in APP deficient mice compared to age- and gender-matched background controls. As expected through the increased presence of ferroxidase I I in rodent compared to human plasma (Sung and Topham ( 1973) Biochem Biophys Res Commun 53. 824-829:

Topham et al. ( 1 975) Arch Biochem Biophys 167, 129- 1 37), azide inhibition of CP in wi ld- type mice only induced a -60% drop in plasma ferroxidase activity (Fig 14C) leaving similar amounts of ferroxidase activity as previous reports on CP knockout (CP-/-) plasma (Gray et al. 2009, supra). By comparison, genetic ablation of APP caused a -40% decrease in plasma ox idase activity but this residual activity was almost completely inhibited b> azide (Fig. I C ), consistent with it being due to CP. Interestingly, the loss of APP ferroxidase activi ty in the knockout mouse did not induce increased expression of CP, suggesting that the activ ity is not redundant. No feroxidase activity loss was observed in mice deficient with the APP family homologues APLP2 (Fig. 14C).

APP co-elutes with ferroxidase II activity in plasma. The equivalence of APP to ferroxidase I I activity was confirmed in human serum by the original protocols of Topham and Frieden (Topham and Frieden 1970, supra). Whole plasma was separated by anion exchange chromatography at pH5.5 using a salt gradient to resolve ferroxidase I I from ferroxidase I ( Fig. 1 5A). Fractions were then tested for ferrox idase activity with and without the speci fic inhibitors (azide or zinc). Zinc preferentially inhibited the ferroxidase I I activity present in the void volume fractions (Fig. 15A), consistent with the inhibition of APP ferroxidase activ ity, while azide inhibited ferroxidase I activity in the fractions eluted with 0.2 NaCI, but did not inhibit activity in the void fractions, consistent with the presence of CP only in the fractions containing higher NaCI (Fig. I 5A). Ferroxidase I I fractions also correlated with A PP immunoreactivity as detect by one antibody specific for "Ferroxidase domain 1 ' (FD 1 ) (Duce et al. 201 0 supra) (Figs. 15B & 1 8A) and another against the N-termina! of A PP (22C 1 1 ; data not shown). Both epitopes are within the ectodomain of APP. Antibodies raised to F 1 were confirmed to be specific for APP using a variety of tissues and plasma from humans as wel l as APP-/- and wild-type mice (Fig. 1 8B).

In a smaller scale preparation, the same method (Topham and Frieden 1 980, supra) was utilized to determine ferroxidase 11 activity in APP-/- and background littermate controls (Fig. 1 5C). While it appears that some ferroxidase II activity was lost with this method, likely due to a dilution factor caused from running the plasma through anion exchange

chromatography, zinc inhibitable ferroxidase I I activ ity was sti ll present in the void fractions of wild-type plasma but absent in APP-/- littermates. Azide inhibited ferroxidase I activ ity was however not significantly changed in the 0.2M NaCl fraction from A PP-/- m ice compared to WT Utermates (Fig. 1 5C).

Previously, CP activity has been identified to also be inhibited by DEPC, via its abi lity to modi fy histidines required in copper binding ( ylen and Petersson ( 1 972) Eur Biochem 27. 578-584: Mukhopadhyay et al. ( 1997) Proc Natl Acad Sci U S A 94. 1 1 546- 1 1 55). To determine if disruption of histidine binding to copper may also inhibit ferroxidase I and I I activity in plasma, anion exchange fractions containing either ferroxidases were pooled and reduced in volume before assaying for absorbance changes in transferrin and o-dianisidine. DEPC treatment was able to inhibit recombinant CP and ferroxidase I almost totally, consistent with histidine binding to copper playing a major role in the proteins activity, and with ferroxidase I activity being specific to CP (Fig. 1 9). However, DEPC was also able to reduce recombinant APP and ferroxidase I I by a smal l but significant amount (Fig. 19) indicating that histidines are partially required for APP and ferroxidase I I activity.

Immunoprecipitation o f CP or APP leads to the respective loss of ferroxidase I and II activity. APP and CP's involvement as ferroxidase I I and I was confirmed by using immunodepletion as another tool for separating each complex in human plasma. After capture of APP (using 22C 1 1 ) and CP (Sigma) in plasma, the immunodepleted plasma and the captured ferroxidase complexes were tested for o-dianisidine oxidase activ ity ( F ig. 16). CP depleted plasma indicated a - 10% original activ ity that was almost total ly inhibited by zinc (Fig. 1 6A). The residual non-zinc inhibited activity was expected to be due to left over CP within the plasma sample that was not 'pulled out' by the CP antibody. This was supported by a minor additional inhibition observed when azide was added to the CP-depleted plasma. The loss of ferroxidase activity in CP-depleted plasma was found to be present in the captured elute, which was fully inhibited with azide (Fig. 16B). By adding the activity present in the captured sample to the residual activity left in the immunodepleted sample, a comparable activity was observed to that found in the control non-immunodepleted plasma.

Similar to CP-depleted human plasma, APP-depleted plasma also had a loss in activity however this was only demonstrated to be a loss of - 10% (Fig. 1 6A). This remaining activity was totally inhibited by azide, thus corresponding to ferroxidase I activ ity. Captured elutes from APP-depleted plasma was found to be azide-resistant and totally inhibited with zinc (Fig. 16B). Again, the summation of the residual activity in plasma left after CP or APP depletion was seen to parallel the activity observed after immunodepletion with a control antibody (β- actin)(black bars in Fig. I 6A).

Deficient ferroxidase II activity in Alzheime 's disease plasma. Aceruioplasminemic patients with CP deficiency in plasma and select tissues have marked reduction in ferroxidase activity within plasma and thus have an age-related iron accumulation in multiple tissues. including liver, pancreas and brain glia (Harris el al. ( 1 995 ) Proc Natl Acad Sci U S A 92. 2539-2543 : Patel et al. (2002) J Neurosci 22, 6578-6586). Similar age-related iron

accumulation has previously been noted in A D tissue corresponding with reduced APP ferroxidase activity in tissue where APP expression is high (Duce et al. 20 1 , supra). Since APP ferroxidase activity is decreased in AD tissue, we tested whether extracellular ferroxidase activity in AD patients was also reduced.

Due to the sensitivity of ferroxidase activity decay in plasma (Gutleridge el al. ( 1 85) Biochem J 230. 5 1 7-523), samples of less than 12 months that had been continuously stored in l iquid nitrogen were used to measure both ferroxidase I and 11 activity. Total oxidase activity was observed to be reduced in AD patients compared to Healthy age-matched controls (Fig 1 7A) as previously reported (Snaedal et al. ( 1998) Dement Geriatr ogn Disord 9, 239-242). From this total activity ferroxidase I and II were determined using azide inhibition. Even though CP activity was consistently observed to make up -90% of the total activity in all plasmas, the reduced total ferroxidase activity in AD patients was made up of a significant reduction in both ferroxidase I (CP) and ferroxidase 11 (APP) (Fig 1 7 B & C ).

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifica lly described. It is to be understood that the invention includes all such variations and mod i fications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

TABLE 2

Iron

Copper

Brain Liver Kidney Lung Heart

Wild-type 4.45*0.42 5.52*0.90 4.I0±0.38 1.92±0.27 4.88+0.22 Wild-type with iron 4.75*0.70 5.72±0.14 4.12*0.09 2.22*0.10 5.50*0.61 APP-/- 4.91±0.54 5.85±2.16 4.32±0.I9 2.08*0.08 5.12*0.57

APP-/- with iron 5.12*0.28 4.03±0.14 3.93*0.60 1.43±0. 3 4.99*0.15

Zinc

Brain Liver Kidney Lung Heart

Wild-type 13.44*0.93 26.94*0.89 18.18±1.46 12.40±1.40 13.35*0.56 Wild-type with iron 14.16±0.57 27.05*0.26 19.30±1.00 13.97±0.93 14.43*2.30 APP-/- 13.43*0.93 28.36±6.11 17.56*0.77 13.36±0.35 13.65*1.29

APP-/- with iron 13.I8±I.09 27.74*1.25 I7.78±0.82 13.76*1.65 13.91*0.47

Metal concentration in various tissues from amyloid precursor protein knockout and wild-type littermates, with or without a high iron diet. Iron, copper and zinc levels were determined from brain, liver, kidney, lung and heart of 12-month old APP -/- and matched WT mice (n=3 each group) either fed a normal diet or fed 120 pg Fe/g body weight for 8 days. Metal levels are given in pg/g tissue (wet weight). Mean iron levels for brain, liver and kidney were significantly higher in APP -/- mice compared to WT (APP - /- vs. WT, ^a <0.01 and p<0.05. ANOVA + Dunnet's test) and significantly enhanced when fed with iron (APP -/- with iron vs. WT, p<0.00l , APP -/- vs. APP -/- with iron, ^d p<0.05 and ^cp<0.001 ). No significant differences were observed in copper or zinc levels. TABLE 3

FrontoterriDoral dementia

03/342 63 F 5 Absent Cardiac arrest 6D Cardiac arrest

03/344 69 F 51 5 Absent RD

Bowel obstruction

03/415 76 F 26 Absent 6D

Pneumonia

04/510 73 M 65 Absent 6D

Renal Failure

04/222 69 M 65 Absent 6D

Cardiac arrest. Frontotemporal dementia

04/351 40 28 5 Absent 6D with MND type changes

Cardiac arrest

05/751 75 M 11.5 Absent 6D

Parkinson's disease

Cardiac arrest

A94-42 71 M 56 Absent 6D

PD. Cerebrovascular Infarcts

A97-43 74 M 24 5 Absent 6D

Cardiac arrest

A97-35 76 F 12 Absent 6D

Cerebrovascular Infarcts

A01-67 84 F 34 Absent 6D

05/1015 84 M 50.5 Absent Broncho pneumonia, COAD

6D

Ischemic Heart Disease, Coronary artery

06/437 74 M 23 Absent 6D atherosclerosis

Hypertensive Heart Disease

03/819 63 F 56 Absent 6D

Ischemic Heart Disease

05/738 78 F 20.5 Absent 6D

Cardiac arrest

07/294 82 F 26 Absent 6D

Pulmonary Thromboembolism, Deep Vein

05/413 72 M 45 Absent 6D

Thrombosis

Neuropathoiogical information for human cases. Tissue was provided by the Victorian Brain Bank Network. Selection criteria for most cases were on age matching, low post-mortem interval (PMl), plaque burden and cause of death. Additional Alzheimer^'s disease cases were also obtained to provide a broad range of β-amyloid burden. In line w ith the CERAlzheimer's disease criteria (Mirra el al. , 1 1 ), the extent of Αβ plaque deposition in the frontal cortex was rated (+) <5 plaques per high power field (hpf), (++) 5- 1 5/hpf, (+++) > 1 5 /hpf. Figure numbers are provided in the final column to indicate the study where the sample was used.

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Claims

CLAIMS:

1 . A method for the therapeutic or prophylactic treatment of a condition in a subject, which condition is characterised by aberrant APP ferroxidase activity or Fe²⁺ levels, said method comprising administering to said subject an effective amount of an agent for a time and under conditions sufficient for said agent to modulate the functional interactivity of Zn²⁺ with said APP wherein antagonising the interaction of Zn² ' with said APP increases APP ferroxidase activ ity and facil itating the interaction of Zn^"" with said APP decreases APP ferroxidase activity.

2. A method for the therapeutic or prophylactic treatment of a condition in a subject, which condition is characterised by aberrant APP ferroxidase activity or Fe²⁺ levels, said method comprising administering to said subject an effective amount of an agent for a time and under conditions sufficient for said agent to modulate GFD potentiation of APP ferroxidase activity wherein facilitating the interaction of GFD with APP increases APP ferroxidase activity and antagonising the GFD interaction with A PP decreases APP ferroxidase activity.

3. Use of an agent which:

( i) antagonises the functional interactivity of Zn²⁺ with A PP; or

( ii) faci litates the interaction of GFD with APP;

in the manufacture of a medicament for the treatment of a condition characterised by insufficient APP ferroxidase activity or excess Fe^{2 t} levels.

4. Use of an agent which :

(i ) increases the level of Zn^2*: or

(ii) antagonises the interaction of GFD with APP;

in the manufacture of a medicament for the treatment of a condition characterised by unwanted APP ferroxidase activity or insufficient Fe ^T levels.

5. The method according to claim 1 or the use according to claim 3 wherein said

antagonist is a zinc chelator, ionophore or metal protein attenuating compound.

6. The method or use according to claim 5 wherein said zinc chelator is a moderate affinity chelator which is hydrophobic.

7. The method or use according to claim 6 wherein said zinc chelator is any l igand which can form two or more coordination bonds with a zinc ion.

8. The method or use according to claim 6 wherein said zinc chelator includes a cyclic group that is substituted with two or more functional groups that are able to donate electrons to a coordination bond w ith zinc or a cyclic group wh ich includes at least one heteroatom such as nitrogen, oxygen or sulfur and in which the cycl ic group is substituted with one or more functional groups that are able to donate electrons to a coordination bond with zinc .

9. The method or use according to claim 8 wherein the cyclic group is a heteroaryl group that is substituted with one or more functional groups that are able to donate electrons to a coordination bond with zinc, such as quinazol inyl, quinoxal inyl, naphthyridinyl, pyrimidopyrimidinyl, cinnolinyl, phenazinyl, acridinyl, phenanthrol inyl,

pyridopyrimidinyl, pyridopyrazinyl, pyranopyridinyl, dibenzoquinolizinyl, quinoliny l. isoquinolinyl, pyridinyl and pyrimidinyl groups, especially pyridiny l and quinolyl groups such as pyrithione, deferiprone, N.N.N ' sT-tetrakis (2-pyridy lmethyl ) ethylenediamine (TPEN), pyrithione, quinolines which include a hydroxy substituent in the 8-position, cl ioquinol, iodoquinol, PBT2, 30 and related molecules.

10. The method or use according to claim 6 wherein the zinc chelator inc ludes the cycl ic group which is an aryl group that is substituted with two or more functional groups that are able to donate electrons to a coordination bond with zinc, such as phenyl and naphthyl groups.

1 1 . The method or use according to claim 6 wherein said zinc chelator includes a

heterocyclyl macrocyclic group, such as a cyc lam or a bicyc lam.

1 2. The method or use according to claim 6 wherein said zinc chelator is a polycarboxy I it- ac id, such as ethylene diamine tetraacetic acid (EDTA), nitrilotriacetic acid. nitrilotripropionic acid, diethylenetriamine pentaacetic acid, 2-hydroxyethyl- ethylenediamine-triacetic acid, 1 ,6-diamino-hexamethylene-tetraacetic acid, 1 .2- diamino-cyclohexane tetraacetic acid, O,0'-bis(2-aminoethyl)-ethyleneglycol- tetraacelic acid, 1 .3-diaminopropanc-tetraacetic acid, N,N-bis(2- hydroxybenzyl)ethylenediamine-N,N-diacetic acid, ethylcncdiamine-N,N'-diacetic acid, cthylenediamine-N,N'-dipropionic acid, tnethylenetetraamine hexaacetic acid, iminodiacetic acid. l ,3-diamino-2-hydroxypropane-tetraacetic ac id. 1 ,2- diaminopropane-tetraacetic acid, triethylenelctramine-hexaacetic acid and 1 ,2-bis-(2- amino-phenoxy)ethane-N,N,N',N'-tetraacetic acid.

1 3. The method or use according to claim 6 wherein said zinc chelator is selected from :

( i) a zinc chelator which includes two carbamate groups l inked by an aromatic or aliphatic linker or a heteroatom such as oxygen, nitrogen or su lfur, such as in diethylpyrocarbamate;

(ii) a zinc chelator which is an amino carboxylic acid that includes a functional group that is able to donate electrons to a coordination bond with zinc, such as amino carboxylic acids wh ich include penicillamine, cysteine, aspartic acid and glutam ic acid, and also esters of these am ino carboxyl ic acids:

(i ii) a zinc chelator which inc ludes a hydroxamide group, such as desferrioxam ine; and

(iv) a zinc chelator which is a substituted transition metal including two or more functional groups that are able to carry a negative charge.

14. The method or use according to claim 6 wherein the zinc chelator is 8-hydroxy

quinolines, such as ciioquinol. PBT2, M30, VK28 or related molecules, pvrithione, diethyl pyrocarbamate, 1 ,2-bis-(2-(amino-phenoxy)ethane-N,N,N^',N '-tetraacet ic ac id and derivatives, the bicyclam analogue J KL 1 69 ( 1 , 1 '-xylyl bis- 1 ,4.8, 1 1 tetraaza cyclotetradecane), DP I 09 and related compounds.

15. The method according to c laim 1 or the use according to claim 4 wherein the agent which increases zinc levels is Zn²⁺.

16. The method according to claim 2 or the use according to c laim 3 wherein said agent comprises an amino acid sequence substantially similar to SEQ ID NO:2 or functional fragment, mimetic, analogue or homologue thereof.

1 7. The method according to claim 2 or the use according to claim 4 wherein said

antagonist of the GFD/APP interaction is an antibody directed to GFD, a GFD antisense nucleic acid molecule, GFD si NA, a nucleic acid molecule suitable to induce GFD cosuppression or a GFD/APP competitive inhibitor.

1 8. The method or use according to any one of claims 1 to 3 or 5 to 1 6 wherein said

condition is characterised by insufficient APP ferroxidase acti v ity or excess Fe^{: '} levels.

1 . The method or use according to claim 1 8 wherein said condition is a condition of the central nervous system.

20. The method or use according to claim 19 wherein said condition is a neurodegenerative disease, such as Alzheimer's disease. Parkinson ^'s disease, Levvy Body disease, frontotemporal dementia, Parkinson 's dementia, neurodegenerat ion with brain iron accumulation, neuroferritinopathy, macular degeneration, Freidreich^" s ataxia, motor neuron disease, Huntington's disease, polyglulam ine repeat diseases or tri nucleotide repeat diseases.

21 . The method or use according to claim 1 8 wherein said condition is hemosiderosis, hereditary hemochromatosis (all types, e.g. Type I or classic (HHC). Type II a. b or juvenile (JHC), Type I I I or transferrin receptor mutation. Type I V or ferroportin mutation, neonatal (NH), African (AH) or African iron overload (AIO)), major thalassemia, aceruloplasminemia, atransferrinemia, hyperferritinemia.

neuroferritinopathy; hereditary ferritinopathy, hereditary hyperferritinaemia cataract syndrome (HHCS), pantothenate kinase-2 associated neurodegeneration,

neurodegeneration with brain iron accumulation type I (NBIA type 1 ) or Hallervorden- Spatz syndrome, phospolipasc A2 associated neurodegeneration or neurodegeneration with brain iron accumulation type I I (NBIA type 2), progressive supranuc lear palsy (PSP), amyotrophic lateral sclerosis (ALS), multiple sclerosis, ischem ic/hemorrhagic stroke, mucolipidosis type I V (ML4) neurodegenerative disease, l iver disease, hepatic failure, sickle cell disease, X-linked sideroblastic anemia, diabetes mellilus.

22. The method or use according to claim 1 8 wherein said condition is characterised by one or more of the symptoms chronic fatigue, joint pain, abdominal pain, irregular heart rhythm, heart attack or heart fai lure, skin color changes (bronze, ashen-gray green), loss of menstrual cycle, osteoarthritis, osteoporosis, hair loss, enlarged liver or spleen, impotence, inferti lity, hypogonadism, hypothyroidism, hypopituitarism, depression.

23. The method or use according to any one of c laims 1 , 2 or 4 wherein said condition is characterized by unwanted APP ferroxidase activity or insufficient e^2'' levels.

24. The method or use according to claim 23 wherein said condition is cardiovascular disease, hemochromatosis, aceruloplasminemia, beta-thalassemia, iron deficiency anemia, anemia of chronic disease (or anemia of inflammation), minor thalassemia, alopecia (hair loss), pruritus (itchiness), tingl ing, numbness, or burning sensations, glossitis (inflammation or infection of the tongue), angular chei l itis ( inflammatory lesions at the mouth's corners), koilonychia (spoon-shaped nai ls) or nai ls that are weak or brittle, Plummer-v inson syndrome - dysphagia due to formation of esophageal webs, restless legs syndrome or twitching muscles, angina.

25. The method or use according to claim 23 wherein said condition is characterised by one or more of the symptoms chronic fatigue, weakness, dizziness . headaches, sensitivity to cold (low body temp), anxiety often resulting in obsessive compulsive disorder-type compulsions and obsessions, irritability, constipation, sleepiness, tinnitus, palpitations, fainting, depression, breathiessness on exertion, missed menstrual cycle, Heavy menstrual period, slow social development, mouth ulcers, poor appetite.