Craig S. Atwood

Treatment with a Copper-Zinc Chelator Markedly and Rapidly Inhibits β-Amyloid Accumulation in Alzheimer's Disease Transgenic Mice

Inhibition of neocortical beta-amyloid (Abeta) accumulation may be essential in an effective therapeutic intervention for Alzheimers disease (AD). Cu and Zn are enriched in Abeta deposits in AD, which are solubilized by Cu/Zn-selective chelators in vitro. Here we report a 49% decrease in brain Abeta deposition (-375 microg/g wet weight, p = 0.0001) in a blinded study of APP2576 transgenic mice treated orally for 9 weeks with clioquinol, an antibiotic and bioavailable Cu/Zn chelator. This was accompanied by a modest increase in soluble Abeta (1.45% of total cerebral Abeta); APP, synaptophysin, and GFAP levels were unaffected. General health and body weight parameters were significantly more stable in the treated animals. These results support targeting the interactions of Cu and Zn with Abeta as a novel therapy for the prevention and treatment of AD.

DRAMATIC AGGREGATION OF ALZHEIMER ABETA BY CU(II) IS INDUCED BY CONDITIONS REPRESENTING PHYSIOLOGICAL ACIDOSIS

The cortical deposition of Aβ is an event that occurs in Alzheimer’s disease, Down’s syndrome, head injury, and normal aging. Previously, in appraising the effects of different neurochemical factors that impact upon the solubility of Aβ, we observed that Zn2+ was the predominant bioessential metal to induce the aggregation of soluble Aβ at pH 7.4 in vitro and that this reaction is totally reversible with chelation. We now report that unlike other biometals tested at maximal biological concentrations, marked Cu2+-induced aggregation of Aβ1–40 emerged as the solution pH was lowered from 7.4 to 6.8 and that the reaction was completely reversible with either chelation or alkalinization. This interaction was comparable to the pH-dependent effect of Cu2+ on insulin aggregation but was not seen for aprotinin or albumin. Aβ1–40 bound three to four Cu2+ ions when precipitated at pH 7.0. Rapid, pH-sensitive aggregation occurred at low nanomolar concentrations of both Aβ1–40 and Aβ1–42 with submicromolar concentrations of Cu2+. Unlike Aβ1–40, Aβ1–42was precipitated by submicromolar Cu2+ concentrations at pH 7.4. Rat Aβ1–40 and histidine-modified human Aβ1–40 were not aggregated by Zn2+, Cu2+, or Fe3+, indicating that histidine residues are essential for metal-mediated Aβ assembly. These results indicate that H+-induced conformational changes unmask a metal-binding site on Aβ that mediates reversible assembly of the peptide. Since a mildly acidic environment together with increased Zn2+ and Cu2+ are common features of inflammation, we propose that Aβ aggregation by these factors may be a response to local injury. Cu2+, Zn2+, and Fe3+ association with Aβ explains the recently reported enrichment of these metal ions in amyloid plaques in Alzheimer’s disease.

Zinc-induced Alzheimer’s Aβ1–40 Aggregation Is Mediated by Conformational Factors

The heterogeneous precipitates of Aβ that accumulate in the brain cortex in Alzheimer’s disease possess varying degrees of resistance to resolubilization. We previously found that Aβ1–40 is rapidly precipitated in vitro by physiological concentrations of zinc, a neurochemical that is highly abundant in brain compartments where Aβ is most likely to precipitate. We now present evidence that the zinc-induced precipitation of Aβ is mediated by a peptide dimer and favored by conditions that promote α-helical and diminish β-sheet conformations. The manner in which the synthetic peptide is solubilized was critical to its behaviorin vitro. Zinc-induced Aβ aggregation was dependent upon the presence of NaCl, was enhanced by α-helical-promoting solvents, but was abolished when the peptide stock solution was stored frozen. The Aβ aggregates induced by zinc were reversible by chelation, but could then be reprecipitated by zinc for several cycles, indicating that the peptide’s conformation is probably preserved in the zinc-mediated assembly. In contrast, Aβ aggregates induced by low pH (5.5) were not resolubilized by returning the pH milieu to 7.4. The zinc-Aβ interaction exhibits features resembling the gelation process of zinc-mediated fibrin assembly, suggesting that, in events such as clot formation or injury, reversible Aβ assembly could be physiologically purposive. Such a mechanism is contemplated in the early evolution of diffuse plaques in Alzheimer’s disease and suggests a possible therapeutic strategy for the resolubilization of some forms of Aβ deposit in the disease.

Trace metal contamination initiates the apparent auto-aggregation, amyloidosis, and oligomerization of Alzheimer’s Aβ peptides

Nucleation-dependent protein aggregation (“seeding”) and amyloid fibril-free formation of soluble SDS-resistant oligomers (“oligomerization”) by hydrophobic interaction is an in vitro model thought to propagate β-amyloid (Aβ) deposition, accumulation, and incur neurotoxicity and synaptotoxicity in Alzheimer’s disease (AD), and other amyloid-associated neurodegenerative diseases. However, Aβ is a high-affinity metalloprotein that aggregates in the presence of biometals (zinc, copper, and iron), and neocortical Aβ deposition is abolished by genetic ablation of synaptic zinc in transgenic mice. We now present in vitro evidence that trace (≤0.8xa0µM) levels of zinc, copper, and iron, present as common contaminants of laboratory buffers and culture media, are the actual initiators of the classic Aβ1–42-mediated seeding process and Aβ oligomerization. Replicating the experimental conditions of earlier workers, we found that the in vitro precipitation and amyloidosis of Aβ1–40 (20xa0µM) initiated by Aβ1–42 (2xa0µM) were abolished by chelation of trace metal contaminants. Further, metal chelation attenuated formation of soluble Aβ oligomers from a cell-free culture medium. These data suggest that protein self-assembly and oligomerization are not spontaneous in this system as previously thought, and that there may be an obligatory role for metal ions in initiating Aβ amyloidosis and oligomerization.

Neuroinflammatory Environments Promote Amyloid-ß Deposition and Posttranslational Modification

Indisputable evidence indicates that an inflammatory response is associated with neuron and neurite damage and the deposition of amyloid s (As) and neurofibrillary tangles (NFT) in Alzheimer disease (AD) (see, ref. 1 for a comprehensive review). Just as in the periphery, where degenerating tissue and insoluble materials (resulting from trauma, embolism, and rupture) promote inflammation, these classical stimulants also promote inflammation in the AD brain. From a spatio-temporal perspective, the stimuli promoting neuroinflammation are microlocalized and are present from early preclinical to the terminal stages of AD. Likewise, the upregulation of acute-phase proteins, complement, cytokines, and other inflammatory mediators also is microlocalized and chronic.