Ashley I. Bush, Ph.D., D.P.M., M.B.,B.S. - US grants

Alzheimer's disease is characterized by amyloidotic deposits of 39-43 residue AB peptides, as well as multiple biochemical abnormalities in the brain. Synthetic A-beta-1-40 is soluble at high concentrations, and its native species has recently been described as a CSF component, but there is no evidence to indicate that its level is increased in AD. Since forms of familial Alzheimer's disease with characteristic amyloid pathology are caused by pathogenic mutations close to and within the A-beta domain of its parent molecule (the beta-amyloid protein precursor, APP), identification and characterization of physiological factors which cause A-beta to accumulate into amyloid would appear to provide important clues to the pathogenesis of the disease. Among the biochemical abnormalities of AD is a pervasive abnormality of cerebral zinc metabolism causing intraneuronal zinc deficiency and accumulation in the interstitial fluid. We have recently shown that A-beta specifically and saturably binds zinc, and has a similar high amity for copper. We found that concentrations of zinc above 300 nM rapidly destabilize synthetic A-beta-l-40 to solutions, and induce tinctorial amyloid formation. The rat species of A-beta, however, is immune from these effects, and binds zinc less avidly, consistent with the scarcity with which these animals form cerebral A- beta amyloid. In this proposal we propose to characterize the interaction of zinc with A-beta at the ultrastructural and conformational levels to determine the extent to which the consequences of low and high affinity zinc interaction are of relevance to amyloid or preamyloid formation. Hence, we will proceed to test whether the variant forms of A-beta (eg, A-beta 1-42 and A-beta with aspartate stereoisomer substitutions) could be disproportionately enriched in amyloid deposits because they are more vulnerable to zinc-induced aggregation than the major soluble form, A- beta 1-40. If this is shown to be true, then we will assay the ratios A- beta 1-42 to A-beta 1-40 in the CSF and brain regions in AD. The rapid aggregate filtration assay employed in these studies will be used to screen neurochemical agents (such as apolipoprotein E and antioxidants) for their anti-amyloidotic properties. To complement the in vitro findings that will be assembled from the proposed studies, we intend to recruit the necessary animal and chemical data required to develop an isotopic marker that may be suitable for positron emission tomography studies of human cerebral zinc metabolism. The goal of this proposal is to develop a thorough understanding of the structural consequences of the interaction of AB and its variants with zinc, and factors that modulate it, as well as a technical basis to proceed with in vivo studies of human cerebral zinc metabolism. Collectively, these data will provide a sound platform for exploring the potentially critical role of zinc in the neuropathogenesis of Alzheimer's disease.

DESCRIPTION (From the applicant's abstract): Abeta is a metal binding protein which accumulates together with elevations of zinc, copper and iron in the brain in AD. Abeta interactions with these metals mediate the precipitation of the peptide, but binding of the redox active metal ions (Cu and Fe) engenders their reduction and the O2-dependent, cell-free generation of H2O2. H2O2 is formed most by Abeta1-42> Abeta1-40>rat Abeta, a rank order that mirrors involvement of the peptides in amyloid pathology, and also the respective H2O2-mediated toxicity of each peptide. These findings are important because there is a striking enrichment of Cu, Fe, and Zn in amyloid deposits in AD, accompanied by signs of severe oxidation stress in the neocortex, and because Abeta amyloid deposition in transgenic animals induces similar oxidation markers. We have also found that Abeta in the brain in AD bears carbonyl adducts which form as consequence of H2O2-mediated attack and may induce protease resistance. We have also found that although Zn precipitates Abeta, it also inhibits Cu reduction, H2O2 formation, and Abeta neurotoxicity, suggesting that its enrichment in plaque may represent a homeostatic defense. We hypothesize that plaque may become oxidatively inert as a consequence of concentrating Zn, and indeed may form because of the interaction of zinc with oxidized Abeta. This possibility is supported by recent data indicating an inverse correlation between plaque load and oxidation markers in AD brain. The overall goal of this competing renewal is to clarify the complex relationship between cerebral Cu, Zn, and Fe levels, amyloid formation and oxidative damage, in human post-mortem and amyloid-bearing transgenic animal brain tissue. We hypothesize that elevated Cu and Fe potentiate the oxidation damage caused by Abeta, but that Zn quenches Abeta-mediated oxidation at the expense of forming amyloid. Using inductively coupled plasma spectrometry, we will measure the enrichment of Cu, Zn, and Fe in the brains of APP transgenic animals as a consequence of developmental amyloid deposition. We will test whether targeting the metal interaction with Abeta with a bioavailable chelating compound that crosses the BBB inhibits Abeta toxicity and amyloid formation in vivo, as the basis for a potential therapeutic strategy. Finally, we will cross the ZnT3 knockout mouse that lacks vesicular zince in its neocortex with the APP2576 amyloid-bearing transgenic to determine whether this pool of brain zinc contributes to amyloid formation and if this knockout will attenuate amyloid formation.

Principal Investigator/Program Director (Last, First, Middle): Morris, Martha Clare This R01 application, entitled ?Epidemiological Neuropathologic Study of Metallomics and Alzheimer's Disease? is a new submission in response to PAR-15-356. By the year 2050 it is projected that there will be 13.5 million Americans with AD at a cost of $1.1 trillion. Recent large-scale phase III clinical trials of drugs targeting known pathways involved in AD have either failed to benefit patients, or indicated very limited efficacy. Whereas beta amyloid (A?) may be a principal driver of the disease, multiple failed clinical trials of drugs targeting this peptide suggest that A? is a poor therapeutic target for AD. Iron accumulates in the affected brain regions in AD, and has the potential to drive disease progression by causing oxidative stress and the aggregation of A? and tau, and is thus an alternate therapeutic target. In previous studies we showed that the iron burden of the brain (as reflected in CSF ferritin levels) has an effect on multiple longitudinal outcomes of AD comparable in magnitude to more established factors in the disease: tau and A?. We have preliminary data showing a strong positive association between brain iron levels and neurofibrillary tangle pathology, particularly in APOE-?4 carriers. There has not been a systematic, well powered, and detailed exploration of brain iron levels in AD. Here, we propose to use a large, well-characterized post mortem cohort study, the Rush Memory and Aging Project, to investigate brain iron concentrations in AD of 680 autopsied brains using advanced techniques. We will investigate the impact of the iron load of the brain on AD neuropathology and cognitive clinical history; investigate potential causes of iron accumulation in AD including diet, and genetic factors; and explore neurochemical mechanisms of iron elevation using advanced proteomics and metalloproteomics. This foundational study has the potential to (1) establish whether iron concentrations are related to the disease, (2) validate iron as a therapeutic target for AD, (3) discover new molecular targets for lowering iron, (4) identify whether diet influences brain iron levels and AD risk, and (6) determine whether genetic factors, including APOE allele variation, impacts on iron in AD. PHS 398/2590 (Rev. 09/04, Reissued 4/2006) Page Continuation Format Page