Lithium suppression of tau induces brain iron accumulation and neurodegeneration.

Lithium is a first-line therapy for bipolar affective disorder. However, various adverse effects, including a Parkinson-like hand tremor, often limit its use. The understanding of the neurobiological basis of these side effects is still very limited. Nigral iron elevation is also a feature of Parkinsonian degeneration that may be related to soluble tau reduction. We found that magnetic resonance imaging T2 relaxation time changes in subjects commenced on lithium therapy were consistent with iron elevation. In mice, lithium treatment lowers brain tau levels and increases nigral and cortical iron elevation that is closely associated with neurodegeneration, cognitive loss and parkinsonian features. In neuronal cultures lithium attenuates iron efflux by lowering tau protein that traffics amyloid precursor protein to facilitate iron efflux. Thus, tau- and amyloid protein precursor-knockout mice were protected against lithium-induced iron elevation and neurotoxicity. These findings challenge the appropriateness of lithium as a potential treatment for disorders where brain iron is elevated (for example, Alzheimer's disease), and may explain lithium-associated motor symptoms in susceptible patients.

X-ray fluorescence imaging reveals subcellular biometal disturbances in a childhood neurodegenerative disorder.

Biometals such as zinc, iron, copper and calcium play key roles in diverse physiological processes in the brain, but can be toxic in excess. A hallmark of neurodegeneration is a failure of homeostatic mechanisms controlling the concentration and distribution of these elements, resulting in overload, deficiency or mislocalization. A major roadblock to understanding the impact of altered biometal homeostasis in neurodegenerative disease is the lack of rapid, specific and sensitive techniques capable of providing quantitative subcellular information on biometal homeostasis in situ. Recent advances in X-ray fluorescence detectors have provided an opportunity to rapidly measure biometal content at subcellular resolution in cell populations using X-ray Fluorescence Microscopy (XFM). We applied this approach to investigate subcellular biometal homeostasis in a cerebellar cell line isolated from a natural mouse model of a childhood neurodegenerative disorder, the CLN6 form of neuronal ceroid lipofuscinosis, commonly known as Batten disease. Despite no global changes to whole cell concentrations of zinc or calcium, XFM revealed significant subcellular mislocalization of these important biological second messengers in cerebellar Cln6nclf (CbCln6nclf ) cells. XFM revealed that nuclear-to-cytoplasmic trafficking of zinc was severely perturbed in diseased cells and the subcellular distribution of calcium was drastically altered in CbCln6nclf cells. Subtle differences in the zinc K-edge X-ray Absorption Near Edge Structure (XANES) spectra of control and CbCln6nclf cells suggested that impaired zinc homeostasis may be associated with an altered ligand set in CbCln6nclf cells. Importantly, a zinc-complex, ZnII(atsm), restored the nuclear-to-cytoplasmic zinc ratios in CbCln6nclf cells via nuclear zinc delivery, and restored the relationship between subcellular zinc and calcium levels to that observed in healthy control cells. ZnII(atsm) treatment also resulted in a reduction in the number of calcium-rich puncta observed in CbCln6nclf cells. This study highlights the complementarities of bulk and single cell analysis of metal content for understanding disease states. We demonstrate the utility and broad applicability of XFM for subcellular analysis of perturbed biometal metabolism and mechanism of action studies for novel therapeutics to target neurodegeneration.

Abeta produced as a fusion to maltose binding protein can be readily purified and stably associates with copper and zinc.

The 42 amino acid Alzheimer's Abeta peptide has been produced in E. coli as a soluble fusion to maltose binding protein (MBP). Affinity purification on amylose columns of MBP-Abeta and MBP led to the recovery of proteins at purities that were suited for physicochemical analyses. MBP-Abeta was able to bind approximately 2 mole equivalents of copper or 4 mole equivalents of zinc, while MBP alone bound negligible amounts of zinc or copper. We conclude that Abeta can bind 2 copper or 4 zinc ions in its fusion format. Because MBP-Abeta is a convenient protein to work with, this system is well suited for further studies on the structure of Abeta and its interactions with metals.

Elevated cortical zinc in Alzheimer disease.

OBJECTIVE: To determine whether changes in brain biometals in Alzheimer disease (AD) and in normal brain tissue are tandemly associated with amyloid beta-peptide (Abeta) burden and dementia severity. METHODS: The authors measured zinc, copper, iron, manganese, and aluminum and Abeta levels in postmortem neocortical tissue from patients with AD (n = 10), normal age-matched control subjects (n = 14), patients with schizophrenia (n = 26), and patients with schizophrenia with amyloid (n = 8). Severity of cognitive impairment was assessed with the Clinical Dementia Rating Scale (CDR). RESULTS: There was a significant, more than twofold, increase of tissue zinc in the AD-affected cortex compared with the other groups. Zinc levels increased with tissue amyloid levels. Zinc levels were significantly elevated in the most severely demented cases (CDR 4 to 5) and in cases that had an amyloid burden greater than 8 plaques/mm(2). Levels of other metals did not differ between groups. CONCLUSIONS: Brain zinc accumulation is a prominent feature of advanced Alzheimer disease (AD) and is biochemically linked to brain amyloid beta-peptide accumulation and dementia severity in AD.

Copper and zinc binding modulates the aggregation and neurotoxic properties of the prion peptide PrP106-126.

The abnormal form of the prion protein (PrP) is believed to be responsible for the transmissible spongiform encephalopathies. A peptide encompassing residues 106-126 of human PrP (PrP106-126) is neurotoxic in vitro due its adoption of an amyloidogenic fibril structure. The Alzheimer's disease amyloid beta peptide (Abeta) also undergoes fibrillogenesis to become neurotoxic. Abeta aggregation and toxicity is highly sensitive to copper, zinc, or iron ions. We show that PrP106-126 aggregation, as assessed by turbidometry, is abolished in Chelex-100-treated buffer. ICP-MS analysis showed that the Chelex-100 treatment had reduced Cu(2+) and Zn(2+) levels approximately 3-fold. Restoring Cu(2+) and Zn(2+) to their original levels restored aggregation. Circular dichroism showed that the Chelex-100 treatment reduced the aggregated beta-sheet content of the peptide. Electron paramagnetic resonance spectroscopy identified a 2N1S1O coordination to the Cu(2+) atom, suggesting histidine 111 and methionine 109 or 112 are involved. Nuclear magnetic resonance confirmed Cu(2+) and Zn(2+) binding to His-111 and weaker binding to Met-112. An N-terminally acetylated PrP106-126 peptide did not bind Cu(2+), implicating the free amino group in metal binding. Mutagenesis of either His-111, Met-109, or Met-112 abolished PrP106-126 neurotoxicity and its ability to form fibrils. Therefore, Cu(2+) and/or Zn(2+) binding is critical for PrP106-126 aggregation and neurotoxicity.

Treatment with a copper-zinc chelator markedly and rapidly inhibits beta-amyloid accumulation in Alzheimer's disease transgenic mice.

Inhibition of neocortical beta-amyloid (Abeta) accumulation may be essential in an effective therapeutic intervention for Alzheimer's 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.

Alzheimer's disease amyloid-beta binds copper and zinc to generate an allosterically ordered membrane-penetrating structure containing superoxide dismutase-like subunits.

Amyloid beta peptide (Abeta) is the major constituent of extracellular plaques and perivascular amyloid deposits, the pathognomonic neuropathological lesions of Alzheimer's disease. Cu(2+) and Zn(2+) bind Abeta, inducing aggregation and giving rise to reactive oxygen species. These reactions may play a deleterious role in the disease state, because high concentrations of iron, copper, and zinc have been located in amyloid in diseased brains. Here we show that coordination of metal ions to Abeta is the same in both aqueous solution and lipid environments, with His(6), His(13), and His(14) all involved. At Cu(2+)/peptide molar ratios >0.3, Abeta coordinated a second Cu(2+) atom in a highly cooperative manner. This effect was abolished if the histidine residues were methylated at N(epsilon)2, indicating the presence of bridging histidine residues, as found in the active site of superoxide dismutase. Addition of Cu(2+) or Zn(2+) to Abeta in a negatively charged lipid environment caused a conformational change from beta-sheet to alpha-helix, accompanied by peptide oligomerization and membrane penetration. These results suggest that metal binding to Abeta generated an allosterically ordered membrane-penetrating oligomer linked by superoxide dismutase-like bridging histidine residues.

Chelation and intercalation: complementary properties in a compound for the treatment of Alzheimer's disease.

Selective application of metal chelators to homogenates of human Alzheimer's disease (AD) brain has led us to propose that the architecture of aggregated beta-amyloid peptide, whether in the form of plaques or soluble oligomers, is determined at least in part by high-affinity binding of transition metals, especially copper and zinc. Of the two metals, copper is implicated in reactive oxygen species generating reactions, while zinc appears to be associated with conformational and antioxidant activity. We tested the copper chelators trientine, penicillamine, and bathophenanthroline for their ability to mobilize brain Abeta as measured against our benchmark compound bathocuproine (BC). All of these agents were effective in solubilizing brain Abeta, although BC was the most consistent across the range of AD brain tissue samples tested. Similarly, all of the copper chelators depleted copper in the high-speed supernatants. BC alone had no significant effect upon zinc levels in the soluble fraction. BC extraction of brain tissue from C100 transgenic mice (which express human Abeta but do not develop amyloid) revealed SDS-resistant dimers as Abeta was mobilized from the sedimentable to the soluble fraction. NMR analysis showed that, in addition to its copper chelating properties, BC interacts with Abeta to form a complex independent of the presence of copper. Such hybrid copper chelating and "chain breaking" properties may form the basis of a rational design for a therapy for Alzheimer's disease.