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 ␤-peptide (A␤) metabolism in cell culture. Treatment of Chinese hamster ovary cells overexpressing amyloid precursor protein with CQ and Cu 2؉ or Zn 2؉ resulted in an ϳ85-90% reduction of secreted A␤-(1-40) and A␤-(1-42) compared with untreated controls. Analogous effects were seen in amyloid precursor protein-overexpressing neuroblastoma cells. The secreted A␤ was rapidly degraded through up-regulation of matrix metalloprotease (MMP)-2 and MMP-3 after addition of CQ and Cu 2؉ . MMP activity was increased through activation of phosphoinositol 3-kinase and JNK. CQ and Cu 2؉ also promoted phosphorylation of glycogen synthase kinase-3, and this potentiated activation of JNK and loss of A␤-(1-40). Our findings identify an alternative mechanism of action for CQ in the reduction of A␤ deposition in the brains of CQ-treated animals and potentially in Alzheimer disease patients. Alzheimer disease (AD)4 is characterized by progressive neuronal dysfunction, reactive gliosis, and the formation of amyloid plaques in the brain. The major constituent of AD plaques is the amyloid ␤-peptide (A␤), which is cleaved from the membrane-bound amyloid precursor protein (APP) (1). Aggregated or oligomeric A␤ can induce neurotoxicity through pathways involving free radical production and increased neuronal oxidative stress (2). Among the factors capable of promoting A␤ aggregation in vivo, recent evidence supports a central role for biometals such as Cu 2ϩ and Zn 2ϩ in this process (3). An important factor in controlling A␤ accumulation in AD patients is the activity of A␤-degrading enzymes. Recent studies have identified several candidate proteases that may contribute to catabolism of A␤ in the brain. Neprilysin, insulin-degrading enzyme, angiotensin-converting enzyme, and matrix metalloproteases (MMPs) have all demonstrated A␤-degrading activity in vitro and/or in vivo (4 -6). Reduced activity of these or other A␤-degrading proteases with age may play a role in promoting accumulation and deposition of A␤ in AD patients. Development of strategies to enhance clearance of A␤ may lead to novel therapeutic treatments for AD patients.Promoting A␤ clearance may be achieved through modulating metal sequestration or metal-protein interactions. 5-Chloro-7-iodo-8-hydroxyquinoline or clioquinol (CQ), a disused antibiotic, has received considerable attention as a potential metal ligand in AD and Parkinson disease patients (7-9). Preliminary studies revealed that CQ rapidly and potently dissolved aggregates of synthetic or AD brain-derived A␤ in vitro (10). In subsequent animal studies, a 9-week oral treatment with CQ resulted in a 49% reduction of...

Cognitive decline in Alzheimer's disease (AD) involves pathological accumulation of synaptotoxic amyloid-␤ (A␤) oligomers and hyperphosphorylated tau. Because recent evidence indicates that glycogen synthase kinase 3␤ (GSK3␤) activity regulates these neurotoxic pathways, we developed an AD therapeutic strategy to target GSK3␤. The strategy involves the use of copper-bis(thiosemicarbazonoto) complexes to increase intracellular copper bioavailability and inhibit GSK3␤ through activation of an Akt signaling pathway. Our lead compound Cu II (gtsm) significantly inhibited GSK3␤ in the brains of APP/PS1 transgenic AD model mice. Cu II (gtsm) also decreased the abundance of A␤ trimers and phosphorylated tau, and restored performance of AD mice in the Y-maze test to levels expected for cognitively normal animals. Improvement in the Y-maze correlated directly with decreased A␤ trimer levels. This study demonstrates that increasing intracellular copper bioavailability can restore cognitive function by inhibiting the accumulation of neurotoxic A␤ trimers and phosphorylated tau.Alzheimer's disease ͉ bioinorganic chemistry ͉ glycogen synthase kinase ͉ therapeutic ͉ animal model A lzheimer's disease (AD) is a neurodegenerative disorder characterized clinically by impaired cognitive performance and pathologically by cerebral deposition of extracellular amyloid plaques and intracellular neurofibrillary tangles. Amyloid plaques in AD contain aggregated forms of the 39-to 43-aa amyloid-␤ peptide (A␤) and A␤ is strongly implicated as a causative agent responsible for cognitive failure in AD. A diverse range of mechanisms for A␤ toxicity has been reported (1). A␤ is produced from the amyloid precursor protein (APP) (2-5) and readily aggregates to form insoluble, high-molecular-mass amyloid structures. Intermediates on the A␤ aggregation pathway, primarily low-molecular-mass oligomers such as dimers and trimers, exhibit the greatest neurotoxicity (6-8). In addition to A␤ oligomers, aberrantly phosphor ylated microtubuleassociated protein tau is also associated with cognitive decline in AD (9). Intracellular neurofibrillary tangles in the AD brain contain hyperphosphorylated tau, and A␤ induced cognitive deficits characteristic of AD transgenic mice are attenuated by decreasing levels of endogenous tau (10).It is now widely recognized that a truly effective therapeutic compound for treating AD needs to attenuate both the A␤-and tau-mediated pathologies. Recent positive outcomes for PBT2 in clinical and preclinical trials are therefore pertinent. Lannfelt et al.(11) demonstrated in phase IIa clinical trials that PBT2 lowers plasma A␤ levels and attenuates cognitive decline, and Adlard et al. (12) have shown that PBT2 decreases interstitial A␤ and phosphorylated tau in the brains of AD model mice. PBT2 is a secondgeneration 8-OH quinoline, which, unlike its predecessor clioquinol, lacks iodine and was selected for clinical development because of its easier chemical synthesis, higher solubility, and increased blood-brain barrier perme...

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Accumulation of neurotoxic amyloid‐β (Aβ) is central to the pathology of Alzheimer’s disease (AD). Elucidating the mechanisms of Aβ accumulation will therefore expedite the development of Aβ‐targeting AD therapeutics. We examined activity of an Aβ‐degrading protease (matrix metalloprotease 2) to investigate whether biochemical factors consistent with conditions in the AD brain contribute to Aβ accumulation by altering Aβ sensitivity to proteolytic degradation. An Aβ amino acid mutation found in familial AD, Aβ interactions with zinc (Zn), and increased Aβ hydrophobicity all strongly prevented Aβ degradation. Consistent to all of these factors is the promotion of specific Aβ aggregates where the protease cleavage site, confirmed by mass spectrometry, is inaccessible within an amyloid structure. These data indicate decreased degradation due to amyloid formation initiates Aβ accumulation by preventing normal protease activity. Zn also prevented Aβ degradation by the proteases neprilysin and insulin degrading enzyme. Treating Zn‐induced Aβ amyloid with the metal‐protein attenuating compound clioquinol reversed amyloid formation and restored the peptide’s sensitivity to degradation by matrix metalloprotease 2. This provides new data indicating that therapeutic compounds designed to modulate Aβ‐metal interactions can inhibit Aβ accumulation by restoring the catalytic potential of Aβ‐degrading proteases.

BackgroundDengue remains an important public health problem in Timor-Leste, with several major epidemics occurring over the last 10 years. The aim of this study was to identify dengue clusters at high geographical resolution and to determine the association between local environmental characteristics and the distribution and transmission of the disease.MethodsNotifications of dengue cases that occurred from January 2005 to December 2013 were obtained from the Ministry of Health, Timor-Leste. The population of each suco (the third-level administrative subdivision) was obtained from the Population and Housing Census 2010. Spatial autocorrelation in dengue incidence was explored using Moran’s I statistic, Local Indicators of Spatial Association (LISA), and the Getis-Ord statistics. A multivariate, Zero-Inflated, Poisson (ZIP) regression model was developed with a conditional autoregressive (CAR) prior structure, and with posterior parameters estimated using Bayesian Markov chain Monte Carlo (MCMC) simulation with Gibbs sampling.ResultsThe analysis used data from 3206 cases. Dengue incidence was highly seasonal with a large peak in January. Patients ≥ 14 years were found to be 74% [95% credible interval (CrI): 72–76%] less likely to be infected than those < 14 years, and females were 12% (95% CrI: 4–21%) more likely to suffer from dengue as compared to males. Dengue incidence increased by 0.7% (95% CrI: 0.6–0.8%) for a 1 °C increase in mean temperature; and 47% (95% CrI: 29–59%) for a 1 mm increase in precipitation. There was no significant residual spatial clustering after accounting for climate and demographic variables.ConclusionsDengue incidence was highly seasonal and spatially clustered, with positive associations with temperature, precipitation and demographic factors. These factors explained the observed spatial heterogeneity of infection.Electronic supplementary materialThe online version of this article (10.1186/s13071-017-2588-4) contains supplementary material, which is available to authorized users.

Biometals have an important role in AD (Alzheimer's disease) and metal ligands have been investigated as potential therapeutic agents for treatment of AD. In recent studies the 8HQ (8-hydroxyquinoline) derivative CQ (clioquinol) has shown promising results in animal models and small clinical trials; however, the actual mode of action in vivo is still being investigated. We previously reported that CQ-metal complexes up-regulated MMP (matrix metalloprotease) activity in vitro by activating PI3K (phosphoinositide 3-kinase) and JNK (c-jun N-terminal kinase), and that the increased MMP activity resulted in enhanced degradation of secreted Abeta (amyloid beta) peptide. In the present study, we have further investigated the biochemical mechanisms by which metal ligands affect Abeta metabolism. To achieve this, we measured the effects of diverse metal ligands on cellular metal uptake and secreted Abeta levels in cell culture. We report that different classes of metal ligands including 8HQ and phenanthroline derivatives and the sulfur compound PDTC (pyrrolidine dithiocarbamate) elevated cellular metal levels (copper and zinc), and resulted in substantial loss of secreted Abeta. Generally, the ability to inhibit Abeta levels correlated with a higher lipid solubility of the ligands and their capacity to increase metal uptake. However, we also identified several ligands that potently inhibited Abeta levels while only inducing minimal change to cellular metal levels. Metal ligands that inhibited Abeta levels [e.g. CQ, 8HQ, NC (neocuproine), 1,10-phenanthroline and PDTC] induced metal-dependent activation of PI3K and JNK, resulting in JNK-mediated up-regulation of metalloprotease activity and subsequent loss of secreted Abeta. The findings in the present study show that diverse metal ligands with high lipid solubility can elevate cellular metal levels resulting in metalloprotease-dependent inhibition of Abeta. Given that a structurally diverse array of ligands was assessed, the results are consistent with the effects being due to metal transport rather than the chelating ligand interacting directly with a receptor.

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Biometals such as copper and zinc have an important role in Alzheimer's disease (AD). Accumulating evidence indicates that copper homeostasis is altered in AD brain with elevated extracellular and low intracellular copper levels. Studies in animals and cell cultures have suggested that increasing intracellular copper can ameliorate AD-like pathology including amyloid deposition and tau phosphorylation. Modulating copper homeostasis can also improve cognitive function in animal models of AD. Treatments are now being developed that may result in redistribution of copper within the brain. Metal ligands such as clioquinol (CQ), DP-109 or pyrrolidine dithiocarbamate (PDTC) have shown promising results in animal models of AD, however, the actual mode of action in vivo has not been fully determined. We previously reported that CQ-metal complexes were able to increase intracellular copper levels in vitro. This resulted in stimulation of phosphoinositol-3-kinase activity and mitogen activated protein kinases (MAPK). Increased kinase activity resulted in up-regulated matrix metalloprotease (MMP2 and MMP3) activity resulting in enhanced degradation of secreted A beta. These findings are consistent with previous studies reporting metal-mediated activation of MAPKs and MMPs. How this activation occurs is unknown but evidence suggests that copper may be able to activate membrane receptors such as the epidermal growth factor receptor (EGFR) and result in downstream activation of MAPK pathways. This has been supported by studies showing metal-mediated activation of EGFR through ligand-independent processes in a number of cell-types. Our initial studies reveal that copper complexes can in fact activate EGFR. However, further studies are necessary to determine if metal complexes such as CQ-copper induce up-regulation of A beta-degrading MMP activity through this mechanism. Elucidation of this pathway may have important implications for the development of metal ligand based therapeutics for treatment of AD and other neurodegenerative disorders.