Robert A. Cherny

Soluble pool of Aβ amyloid as a determinant of severity of neurodegeneration in Alzheimer's disease

Genetic evidence strongly supports the view that Aβ amyloid production is central to the cause of Alzheimers disease. The kinetics, compartmentation, and form of Aβ and its temporal relation to the neurodegenerative process remain uncertain. The levels of soluble and insoluble Aβ were determined by using western blot techniques, and the findings were assessed in relation to indices of severity of disease. The mean level of soluble Aβ is increased threefold in Alzheimers disease and correlates highly with markers of disease severity. In contrast, the level of insoluble Aβ (also a measure of total amyloid load) is found only to discriminate Alzheimers disease from controls, and does not correlate with disease severity or numbers of amyloid plaques. These findings support the concept of several interacting pools of Aβ, that is, a large relatively static insoluble pool that is derived from a constantly turning over smaller soluble pool. The latter may exist in both intracellular and extracellular compartments, and contain the basic forms of Aβ that cause neurodegeneration. Reducing the levels of these soluble Aβ species by threefold to levels found in normal controls might prove to be a goal of future therapeutic intervention.

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.

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

Amyloid β peptide (Aβ) is the major constituent of extracellular plaques and perivascular amyloid deposits, the pathognomonic neuropathological lesions of Alzheimers disease. Cu2+ and Zn2+ bind Aβ, 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 Aβ is the same in both aqueous solution and lipid environments, with His6, His13, and His14 all involved. At Cu2+/peptide molar ratios >0.3, Aβ coordinated a second Cu2+ atom in a highly cooperative manner. This effect was abolished if the histidine residues were methylated at Nε 2, indicating the presence of bridging histidine residues, as found in the active site of superoxide dismutase. Addition of Cu2+ or Zn2+ to Aβ in a negatively charged lipid environment caused a conformational change from β-sheet to α-helix, accompanied by peptide oligomerization and membrane penetration. These results suggest that metal binding to Aβ generated an allosterically ordered membrane-penetrating oligomer linked by superoxide dismutase-like bridging histidine residues.

Genetic or pharmacological iron chelation prevents MPTP-induced neurotoxicity in vivo: A novel therapy for Parkinson's disease

Studies on postmortem brains from Parkinsons patients reveal elevated iron in the substantia nigra (SN). Selective cell death in this brain region is associated with oxidative stress, which may be exacerbated by the presence of excess iron. Whether iron plays a causative role in cell death, however, is controversial. Here, we explore the effects of iron chelation via either transgenic expression of the iron binding protein ferritin or oral administration of the bioavailable metal chelator clioquinol (CQ) on susceptibility to the Parkinsons-inducing agent 1-methyl-4-phenyl-1,2,3,6-tetrapyridine (MPTP). Reduction in reactive iron by either genetic or pharmacological means was found to be well tolerated in animals in our studies and to result in protection against the toxin, suggesting that iron chelation may be an effective therapy for prevention and treatment of the disease.

Rapid Restoration of Cognition in Alzheimer's Transgenic Mice with 8-Hydroxy Quinoline Analogs Is Associated with Decreased Interstitial Aβ

As a disease-modifying approach for Alzheimers disease (AD), clioquinol (CQ) targets beta-amyloid (Abeta) reactions with synaptic Zn and Cu yet promotes metal uptake. Here we characterize the second-generation 8-hydroxy quinoline analog PBT2, which also targets metal-induced aggregation of Abeta, but is more effective as a Zn/Cu ionophore and has greater blood-brain barrier permeability. Given orally to two types of amyloid-bearing transgenic mouse models of AD, PBT2 outperformed CQ by markedly decreasing soluble interstitial brain Abeta within hours and improving cognitive performance to exceed that of normal littermate controls within days. Nontransgenic mice were unaffected by PBT2. The current data demonstrate that ionophore activity, inhibition of in vitro metal-mediated Abeta reactions, and blood-brain barrier permeability are indices that predict a potential disease-modifying drug for AD. The speed of recovery of the animals underscores the acutely reversible nature of the cognitive deficits associated with transgenic models of AD.

Metalloenzyme-like Activity of Alzheimer's Disease β-Amyloid Cu-DEPENDENT CATALYTIC CONVERSION OF DOPAMINE, CHOLESTEROL, AND BIOLOGICAL REDUCING AGENTS TO NEUROTOXIC H2O2

β-Amyloid (Aβ) 1–42, implicated in the pathogenesis of Alzheimers disease, forms an oligomeric complex that binds copper at a CuZn superoxide dismutase-like binding site. Aβ·Cu complexes generate neurotoxic H2O2 from O2 through Cu2+ reduction, but the reaction mechanism has been unclear. We now report that Aβ1–42, when binding up to 2 eq of Cu2+, generates the H2O2catalytically by recruiting biological reducing agents as substrates under conditions where the Cu2+ or reducing agents will not form H2O2 themselves. Cholesterol is an important substrate for this activity, as are vitamin C,l-DOPA, and dopamine (V maxfor dopamine = 34.5 nm/min, K m = 8.9 μm). The activity was inhibited by anti-Aβ antibodies, Cu2+ chelators, and Zn2+. Toxicity of Aβ in neuronal culture was consistent with catalytic H2O2 production. Aβ was not toxic in cell cultures in the absence of Cu2+, and dopamine (5 μm) markedly exaggerated the neurotoxicity of 200 nm Aβ1–42·Cu. Therefore, microregional catalytic H2O2 production, combined with the exhaustion of reducing agents, may mediate the neurotoxicity of Aβ in Alzheimers disease, and inhibitors of this novel activity may be of therapeutic value.

Alzheimer's disease amyloid-β binds Cu and Zn to generate an allosterically-ordered membrane-penetrating structure containing SOD-like subunits

Amyloid β peptide (Aβ) is the major constituent of extracellular plaques and perivascular amyloid deposits, the pathognomonic neuropathological lesions of Alzheimers disease. Cu2+ and Zn2+ bind Aβ, 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 Aβ is the same in both aqueous solution and lipid environments, with His6, His13, and His14 all involved. At Cu2+/peptide molar ratios >0.3, Aβ coordinated a second Cu2+ atom in a highly cooperative manner. This effect was abolished if the histidine residues were methylated at Nε 2, indicating the presence of bridging histidine residues, as found in the active site of superoxide dismutase. Addition of Cu2+ or Zn2+ to Aβ in a negatively charged lipid environment caused a conformational change from β-sheet to α-helix, accompanied by peptide oligomerization and membrane penetration. These results suggest that metal binding to Aβ generated an allosterically ordered membrane-penetrating oligomer linked by superoxide dismutase-like bridging histidine residues.

Cytosolic β-amyloid deposition and supranuclear cataracts in lenses from people with Alzheimer's disease

Summary Background Pathological hallmarks of Alzheimers disease include cerebral β-amyloid (Aβ) deposition, amyloid accumulation, and neuritic plaque formation. We aimed to investigate the hypothesis that molecular pathological findings associated with Alzheimers disease overlap in the lens and brain. Methods We obtained postmortem specimens of eyes and brain from nine individuals with Alzheimers disease and eight controls without the disorder, and samples of primary aqueous humour from three people without the disorder who were undergoing cataract surgery. Dissected lenses were analysed by slit-lamp stereophotomicroscopy, western blot, tryptic-digest/mass spectrometry electrospray ionisation, and anti-Aβ surface-enhanced laser desorption ionisation (SELDI) mass spectrometry, immunohistochem-istry, and immunogold electron microscopy. Aqueous humour was analysed by anti-Aβ SELDI mass spectrometry. We did binding and aggregation studies to investigate Aβ-lens protein interactions. Findings We identified Aβ1–40 and Aβ1–42 in lenses from people with and without Alzheimers disease at concentrations comparable with brain, and Aβ1–40 in primary aqueous humour at concentrations comparable with cerebrospinal fluid. Aβ accumulated in lenses from individuals with Alzheimers disease as electron-dense deposits located exclusively in the cytoplasm of supranuclear/deep cortical lens fibre cells (n=4). We consistently saw equatorial supranuclear cataracts in lenses from people with Alzheimers disease (n=9) but not in controls (n=8). These supranuclear cataracts colocalised with enhanced Aβ immunoreactivity and birefringent Congo Red staining. Synthetic Aβ bound βB-crystallin, an abundant cytosolic lens protein. Aβ promoted lens protein aggregation that showed protofibrils, birefringent Congo Red staining, and Aβ/αB-crystallin coimmunoreactivity. Interpretation Aβ is present in the cytosol of lens fibre cells of people with Alzheimers disease. Lens Aβ might promote regionally-specific lens protein aggregation, extracerebral amyloid formation, and supranuclear cataracts.

Copper-Dependent Inhibition of Human Cytochrome c Oxidase by a Dimeric Conformer of Amyloid-β1-42

In studies of Alzheimers disease pathogenesis there is an increasing focus on mechanisms of intracellular amyloid-β (Aβ) generation and toxicity. Here we investigated the inhibitory potential of the 42 amino acid Aβ peptide (Aβ1-42) on activity of electron transport chain enzyme complexes in human mitochondria. We found that synthetic Aβ1-42 specifically inhibited the terminal complex cytochrome c oxidase (COX) in a dose-dependent manner that was dependent on the presence of Cu2+ and specific “aging” of the Aβ1-42 solution. Maximal COX inhibition occurred when using Aβ1-42 solutions aged for 3-6 h at 30°C. The level of Aβ1-42-mediated COX inhibition increased with aging time up to ∼6 h and then declined progressively with continued aging to 48 h. Photo-induced cross-linking of unmodified proteins followed by SDS-PAGE analysis revealed dimeric Aβ as the only Aβ species to provide significant temporal correlation with the observed COX inhibition. Analysis of brain and liver from an Alzheimers model mouse (Tg2576) revealed abundant Aβ immunoreactivity within the brain mitochondria fraction. Our data indicate that endogenous Aβ is associated with brain mitochondria and that Aβ1-42, possibly in its dimeric conformation, is a potent inhibitor of COX, but only when in the presence of Cu2+. We conclude that Cu2+-dependent Aβ-mediated inhibition of COX may be an important contributor to the neurodegeneration process in Alzheimers disease.

Tau deficiency induces parkinsonism with dementia by impairing APP-mediated iron export

The microtubule-associated protein tau has risk alleles for both Alzheimers disease and Parkinsons disease and mutations that cause brain degenerative diseases termed tauopathies. Aggregated tau forms neurofibrillary tangles in these pathologies, but little is certain about the function of tau or its mode of involvement in pathogenesis. Neuronal iron accumulation has been observed pathologically in the cortex in Alzheimers disease, the substantia nigra (SN) in Parkinsons disease and various brain regions in the tauopathies. Here we report that tau-knockout mice develop age-dependent brain atrophy, iron accumulation and SN neuronal loss, with concomitant cognitive deficits and parkinsonism. These changes are prevented by oral treatment with a moderate iron chelator, clioquinol. Amyloid precursor protein (APP) ferroxidase activity couples with surface ferroportin to export iron, but its activity is inhibited in Alzheimers disease, thereby causing neuronal iron accumulation. In primary neuronal culture, we found loss of tau also causes iron retention, by decreasing surface trafficking of APP. Soluble tau levels fall in affected brain regions in Alzheimers disease and tauopathies, and we found a similar decrease of soluble tau in the SN in both Parkinsons disease and the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model. These data suggest that the loss of soluble tau could contribute to toxic neuronal iron accumulation in Alzheimers disease, Parkinsons disease and tauopathies, and that it can be rescued pharmacologically.