Irene Volitakis

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.

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.

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.

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.

Hypoxia-inducible Factor Prolyl 4-Hydroxylase Inhibition A TARGET FOR NEUROPROTECTION IN THE CENTRAL NERVOUS SYSTEM

Hypoxia-inducible factor (HIF) prolyl 4-hydroxylases are a family of iron- and 2-oxoglutarate-dependent dioxygenases that negatively regulate the stability of several proteins that have established roles in adaptation to hypoxic or oxidative stress. These proteins include the transcriptional activators HIF-1α and HIF-2α. The ability of the inhibitors of HIF prolyl 4-hydroxylases to stabilize proteins involved in adaptation in neurons and to prevent neuronal injury remains unclear. We reported that structurally diverse low molecular weight or peptide inhibitors of the HIF prolyl 4-hydroxylases stabilize HIF-1α and up-regulate HIF-dependent target genes (e.g. enolase, p21waf1/cip1, vascular endothelial growth factor, or erythropoietin) in embryonic cortical neurons in vitro or in adult rat brains in vivo. We also showed that structurally diverse HIF prolyl 4-hydroxylase inhibitors prevent oxidative death in vitro and ischemic injury in vivo. Taken together these findings identified low molecular weight and peptide HIF prolyl 4-hydroxylase inhibitors as novel neurological therapeutics for stroke as well as other diseases associated with oxidative stress.

Degradation of the Alzheimer Disease Amyloid β-Peptide by Metal-dependent Up-regulation of Metalloprotease Activity

Biometals play an important role in Alzheimer disease, and recent reports have described the development of potential therapeutic agents based on modulation of metal bioavailability. The metal ligand clioquinol (CQ) has shown promising results in animal models and small phase clinical trials; however, the actual mode of action in vivo has not been determined. We now report a novel effect of CQ on amyloid β-peptide (Aβ) metabolism in cell culture. Treatment of Chinese hamster ovary cells overexpressing amyloid precursor protein with CQ and Cu2+ or Zn2+ resulted in an ∼85–90% reduction of secreted Aβ-(1–40) and Aβ-(1–42) compared with untreated controls. Analogous effects were seen in amyloid precursor protein-overexpressing neuroblastoma cells. The secreted Aβ was rapidly degraded through up-regulation of matrix metalloprotease (MMP)-2 and MMP-3 after addition of CQ and Cu2+. MMP activity was increased through activation of phosphoinositol 3-kinase and JNK. CQ and Cu2+ also promoted phosphorylation of glycogen synthase kinase-3, and this potentiated activation of JNK and loss of Aβ-(1–40). Our findings identify an alternative mechanism of action for CQ in the reduction of Aβ deposition in the brains of CQ-treated animals and potentially in Alzheimer disease patients.

Mitochondrial oxidative stress causes hyperphosphorylation of tau.

Age-related neurodegenerative disease has been mechanistically linked with mitochondrial dysfunction via damage from reactive oxygen species produced within the cell. We determined whether increased mitochondrial oxidative stress could modulate or regulate two of the key neurochemical hallmarks of Alzheimers disease (AD): tau phosphorylation, and ß-amyloid deposition. Mice lacking superoxide dismutase 2 (SOD2) die within the first week of life, and develop a complex heterogeneous phenotype arising from mitochondrial dysfunction and oxidative stress. Treatment of these mice with catalytic antioxidants increases their lifespan and rescues the peripheral phenotypes, while uncovering central nervous system pathology. We examined sod2 null mice differentially treated with high and low doses of a catalytic antioxidant and observed striking elevations in the levels of tau phosphorylation (at Ser-396 and other phospho-epitopes of tau) in the low-dose antioxidant treated mice at AD-associated residues. This hyperphosphorylation of tau was prevented with an increased dose of the antioxidant, previously reported to be sufficient to prevent neuropathology. We then genetically combined a well-characterized mouse model of AD (Tg2576) with heterozygous sod2 knockout mice to study the interactions between mitochondrial oxidative stress and cerebral Aß load. We found that mitochondrial SOD2 deficiency exacerbates amyloid burden and significantly reduces metal levels in the brain, while increasing levels of Ser-396 phosphorylated tau. These findings mechanistically link mitochondrial oxidative stress with the pathological features of AD.

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 β-peptide (Aβ) burden and dementia severity. Methods: The authors measured zinc, copper, iron, manganese, and aluminum and Aβ 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/mm2. 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 β-peptide accumulation and dementia severity in AD.

Neurotoxic, Redox-competent Alzheimer's β-Amyloid Is Released from Lipid Membrane by Methionine Oxidation

The amyloid β peptide is toxic to neurons, and it is believed that this toxicity plays a central role in the progression of Alzheimers disease. The mechanism of this toxicity is contentious. Here we report that an Aβ peptide with the sulfur atom of Met-35 oxidized to a sulfoxide (Met(O)Aβ) is toxic to neuronal cells, and this toxicity is attenuated by the metal chelator clioquinol and completely rescued by catalase implicating the same toxicity mechanism as reduced Aβ. However, unlike the unoxidized peptide, Met(O)Aβ is unable to penetrate lipid membranes to form ion channel-like structures, and β-sheet formation is inhibited, phenomena that are central to some theories for Aβ toxicity. Our results show that, like the unoxidized peptide, Met(O)Aβ will coordinate Cu2+ and reduce the oxidation state of the metal and still produce H2O2. We hypothesize that Met(O)Aβ production contributes to the elevation of soluble Aβ seen in the brain in Alzheimers disease.