Redox-active Cu(II)-Aß causes substantial changes in axonal integrity in cultured cortical neurons in an oxidative-stress dependent manner.

BACKGROUND: The beta-amyloid (Aß) peptide comprises the amyloid plaques that characterise Alzheimer's disease (AD), and is thought to significantly contribute towards disease pathogenesis. Oxidative stress is elevated in the AD brain, and there is substantial evidence that the interaction between Aß and redox-active copper is a major contributing factor towards oxidative stress in AD. RESULTS: The major findings of this study are that redox-active Cu(II)-Aß causes pronounced axonal pathology in long-term neuronal cultures, including axonal fragmentation and the formation of hyperphosphorylated tau-immunoreactive axonal swellings. Notably, MAP-2 expressing dendritic processes remain largely un-affected by Cu(II)-Aß treatment. These dystrophic axonal manifestations resemble some of the characteristic neuritic pathology of the AD brain. We show that Cu(II)-Aß directly causes formation of intra-axonal swellings via the generation of free radicals and subsequent efflux of K+ out of neurons. CONCLUSION: In summary, we report that redox-active Cu(II)-Aß can induce substantial neurodegenerative changes in mature neurons, and may have an important role to play in the slowly progressing pathogenesis of AD.

Tg2576 cortical neurons that express human Ab are susceptible to extracellular Aß-induced, K+ efflux dependent neurodegeneration.

BACKGROUND: One of the key pathological features of AD is the formation of insoluble amyloid plaques. The major constituent of these extracellular plaques is the beta-amyloid peptide (Aß), although Aß is also found to accumulate intraneuronally in AD. Due to the slowly progressive nature of the disease, it is likely that neurons are exposed to sublethal concentrations of both intracellular and extracellular Aß for extended periods of time. RESULTS: In this study, we report that daily exposure to a sublethal concentration of Aß(1-40) (1 µM) for six days induces substantial apoptosis of cortical neurons cultured from Tg2576 mice (which express substantial but sublethal levels of intracellular Aß). Notably, untreated Tg2576 neurons of similar age did not display any signs of apoptosis, indicating that the level of intracellular Aß present in these neurons was not the cause of toxicity. Furthermore, wildtype neurons did not become apoptotic under the same chronic Aß(1-40) treatment. We found that this apoptosis was linked to Tg2576 neurons being unable to maintain K(+) homeostasis following Aß treatment. Furthermore, blocking K(+) efflux protected Tg2576 neurons from Aß-induced neurotoxicity. Interestingly, chronic exposure to 1 µM Aß(1-40) caused the generation of axonal swellings in Tg2576 neurons that contained dense concentrations of hyperphosphorylated tau. These were not observed in wildtype neurons under the same treatment conditions. CONCLUSIONS: Our data suggest that when neurons are chronically exposed to sublethal levels of both intra- and extra-cellular Aß, this causes a K(+)-dependent neurodegeneration that has pathological characteristics similar to AD.

Prolonged Abeta treatment leads to impairment in the ability of primary cortical neurons to maintain K+ and Ca2+ homeostasis.

BACKGROUND: Alzheimer's disease (AD) is a progressive neurodegenerative disease, characterised by the formation of insoluble amyloidogenic plaques and neurofibrillary tangles. Beta amyloid (Abeta) peptide is one of the main constituents in Abeta plaques, and is thought to be a primary causative agent in AD. Neurons are likely to be exposed to chronic, sublethal doses of Abeta over an extended time during the pathogenesis of AD, however most studies published to date using in vitro models have focussed on acute studies. To experimentally model the progressive pathogenesis of AD, we exposed primary cortical neurons daily to 1 muM of Abeta1-40 over 7 days and compared their survival with age-similar untreated cells. We also investigated whether chronic Abeta exposure affects neuronal susceptibility to the subsequent acute excitotoxicity induced by 10 muM glutamate and assessed how Ca2+ and K+ homeostasis were affected by either treatment. RESULTS: We show that continuous exposure to 1 muM Abeta1-40 for seven days decreased survival of cultured cortical neurons by 20%. This decrease in survival correlated with increased K+ efflux from the cells. One day treatment with 1 muM Abeta followed by glutamate led to a substantially higher K+ efflux than in the age-similar untreated control. This difference further increased with the duration of the treatment. K+ efflux also remained higher in Abeta treated cells 20 min after glutamate application leading to 2.8-fold higher total K+ effluxed from the cells compared to controls. Ca2+ uptake was significantly higher only after prolonged Abeta treatment with 2.5-fold increase in total Ca2+ uptake over 20 min post glutamate application after six days of Abeta treatment or longer (P < 0.05). CONCLUSIONS: Our data suggest that long term exposure to Abeta is detrimental because it reduces the ability of cortical neurons to maintain K+ and Ca2+ homeostasis in response to glutamate challenge, a response that might underlie the early symptoms of AD. The observed inability to maintain K+ homeostasis might furthermore be useful in future studies as an early indicator of pathological changes in response to Abeta.

The native copper- and zinc-binding protein metallothionein blocks copper-mediated Abeta aggregation and toxicity in rat cortical neurons.

BACKGROUND: A major pathological hallmark of AD is the deposition of insoluble extracellular beta-amyloid (Abeta) plaques. There are compelling data suggesting that Abeta aggregation is catalysed by reaction with the metals zinc and copper. METHODOLOGY/PRINCIPAL FINDINGS: We now report that the major human-expressed metallothionein (MT) subtype, MT-2A, is capable of preventing the in vitro copper-mediated aggregation of Abeta1-40 and Abeta1-42. This action of MT-2A appears to involve a metal-swap between Zn7MT-2A and Cu(II)-Abeta, since neither Cu10MT-2A or carboxymethylated MT-2A blocked Cu(II)-Abeta aggregation. Furthermore, Zn7MT-2A blocked Cu(II)-Abeta induced changes in ionic homeostasis and subsequent neurotoxicity of cultured cortical neurons. CONCLUSIONS/SIGNIFICANCE: These results indicate that MTs of the type represented by MT-2A are capable of protecting against Abeta aggregation and toxicity. Given the recent interest in metal-chelation therapies for AD that remove metal from Abeta leaving a metal-free Abeta that can readily bind metals again, we believe that MT-2A might represent a different therapeutic approach as the metal exchange between MT and Abeta leaves the Abeta in a Zn-bound, relatively inert form.

Neuronal growth-inhibitory factor (metallothionein-3): evaluation of the biological function of growth-inhibitory factor in the injured and neurodegenerative brain.

Neuronal growth-inhibitory factor, later renamed metallothionein-3, is one of four members of the mammalian metallothionein family. Metallothioneins are a family of ubiquitous, low-molecular-weight, cysteine-rich proteins. Although neuronal growth-inhibitory factor shares metal-binding and reactive oxygen species scavenging properties with the other metallothioneins, it displays several distinct biological properties. In this review, we examine the recent developments regarding the function of neuronal growth-inhibitory factor within the brain, particularly in response to brain injury or during neurodegenerative disease progression.

Zn(II)- and Cu(II)-induced non-fibrillar aggregates of amyloid-beta (1-42) peptide are transformed to amyloid fibrils, both spontaneously and under the influence of metal chelators.

Aggregation of amyloid-beta (Abeta) peptides is a central phenomenon in Alzheimer's disease. Zn(II) and Cu(II) have profound effects on Abeta aggregation; however, their impact on amyloidogenesis is unclear. Here we show that Zn(II) and Cu(II) inhibit Abeta(42) fibrillization and initiate formation of non-fibrillar Abeta(42) aggregates, and that the inhibitory effect of Zn(II) (IC(50) = 1.8 micromol/L) is three times stronger than that of Cu(II). Medium and high-affinity metal chelators including metallothioneins prevented metal-induced Abeta(42) aggregation. Moreover, their addition to preformed aggregates initiated fast Abeta(42) fibrillization. Upon prolonged incubation the metal-induced aggregates also transformed spontaneously into fibrils, that appear to represent the most stable state of Abeta(42). H13A and H14A mutations in Abeta(42) reduced the inhibitory effect of metal ions, whereas an H6A mutation had no significant impact. We suggest that metal binding by H13 and H14 prevents the formation of a cross-beta core structure within region 10-23 of the amyloid fibril. Cu(II)-Abeta(42) aggregates were neurotoxic to neurons in vitro only in the presence of ascorbate, whereas monomers and Zn(II)-Abeta(42) aggregates were non-toxic. Disturbed metal homeostasis in the vicinity of zinc-enriched neurons might pre-dispose formation of metal-induced Abeta aggregates, subsequent fibrillization of which can lead to amyloid formation. The molecular background underlying metal-chelating therapies for Alzheimer's disease is discussed in this light.