Deferiprone Treatment in Aged Transgenic Tau Mice Improves Y-Maze Performance and Alters Tau Pathology.

The accumulation of neurofibrillary tangles (NFTs), which is composed of abnormally hyperphosphorylated tau aggregates, is the classic neuropathology associated with cognitive dysfunction in tauopathies such as Alzheimer's disease (AD). However, there is an emerging theory suggesting that dysregulation in cerebral iron may contribute to NFT formation. Iron is speculated to bind to tau and induce conformational changes of the protein, potentially leading to subsequent aggregation and cognitive decline. Deferiprone (DFP) is a clinically available iron chelator, which has demonstrated potential therapeutic advantages of chelating iron in neurodegenerative disorders, and is currently in clinical trials for AD. However, its effect on tau pathology remains unclear. Here, we report the effects of short-term DFP treatment (4 weeks, 100 mg/kg/daily, via oral gavage) in a mixed-gender cohort of the rTg(tauP301L)4510 mouse model of tauopathy. Our results revealed that DFP improved Y-maze and open field performance, accompanied by a 28% decrease in brain iron levels, measured by inductively coupled plasma mass spectrometry (ICP-MS) and reduced AT8-labeled p-tau within the hippocampus in transgenic tau mice. This data supports the notion that iron may play a neurotoxic role in tauopathies and may be a potential therapeutic target for this class of disorders that can be modulated by the clinically available metal chelator DFP.

Copper Ionophores as Novel Antiobesity Therapeutics.

The therapeutic utility of the copper ionophore disulfiram was investigated in a diet-induced obesity mouse model (C57BL/6J background), both through administration in feed (0.05 to 1% (w/w)) and via oral gavage (150 mg/kg) for up to eight weeks. Mice were monitored for body weight, fat deposition (perigonadal fat pads), metabolic changes (e.g., glucose dyshomeostasis) and pathologies (e.g., hepatic steatosis, hyperglycaemia and hypertriglyceridemia) associated with a high-fat diet. Metal-related pharmacological effects across major organs and serums were investigated using inductively coupled plasma mass spectrometry (ICP-MS). Disulfiram treatments (all modes) augmented hepatic copper in mice, markedly moderated body weight and abolished the deleterious systemic changes associated with a high-fat diet. Likewise, another chemically distinct copper ionophore H2(gtsm), administered daily (oral gavage), also augmented hepatic copper and moderated mouse body weight. Postmortem histological examinations of the liver and other major organs, together with serum aminotransferases, supported the reported therapeutic safety of disulfiram. Disulfiram specifically altered systemic copper in mice and altered hepatic copper metabolism, perturbing the incorporation of copper into ceruloplasmin (holo-ceruloplasmin biosynthesis) and subsequently reducing serum copper concentrations. Serum ceruloplasmin represents a biomarker for disulfiram activity. Our results establish copper ionophores as a potential class of antiobesity agents.

CuII (atsm) inhibits ferroptosis: Implications for treatment of neurodegenerative disease.

BACKGROUND AND PURPOSE: Diacetyl-bis(4-methyl-3-thiosemicarbazonato)copperII (CuII (atsm)) ameliorates neurodegeneration and delays disease progression in mouse models of amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD), yet the mechanism of action remains uncertain. Promising results were recently reported for separate Phase 1 studies in ALS patients and PD patients. Affected tissue in these disorders shares features of elevated Fe, low glutathione and increased lipid peroxidation consistent with ferroptosis, a novel form of regulated cell death. We therefore evaluated the ability of CuII (atsm) to inhibit ferroptosis. EXPERIMENTAL APPROACH: Ferroptosis was induced in neuronal cell models by inhibition of glutathione peroxidase-4 activity with RSL3 or by blocking cystine uptake with erastin. Cell viability and lipid peroxidation were assessed and the efficacy of CuII (atsm) was compared to the known antiferroptotic compound liproxstatin-1. KEY RESULTS: CuII (atsm) protected against lipid peroxidation and ferroptotic lethality in primary and immortalised neuronal cell models (EC50 : ≈130 nM, within an order of magnitude of liproxstatin-1). NiII (atsm) also prevented ferroptosis with similar potency, whereas ionic CuII did not. In cell-free systems, CuII (atsm) and NiII (atsm) inhibited FeII -induced lipid peroxidation, consistent with these compounds quenching lipid radicals. CONCLUSIONS AND IMPLICATIONS: The antiferroptotic activity of CuII (atsm) could therefore be the disease-modifying mechanism being tested in ALS and PD trials. With potency in vitro approaching that of liproxstatin-1, CuII (atsm) possesses favourable properties such as oral bioavailability and entry into the brain that make it an attractive investigational product for clinical trials of ferroptosis-related diseases.

Regional iron distribution and soluble ferroprotein profiles in the healthy human brain.

Iron is essential for brain development and health where its redox properties are used for a number of neurological processes. However, iron is also a major driver of oxidative stress if not properly controlled. Brain iron distribution is highly compartmentalised and regulated by a number of proteins and small biomolecules. Here, we examine heterogeneity in regional iron levels in 10 anatomical structures from seven post-mortem human brains with no apparent neuropathology. Putamen contained the highest levels, and most case-to-case variability, of iron compared with the other regions examined. Partitioning of iron between cytosolic and membrane-bound iron was generally consistent in each region, with a slightly higher proportion (55 %) in the 'insoluble' phase. We expand on this using the Allen Human Brain Atlas to examine patterns between iron levels and transcriptomic expression of iron regulatory proteins and using quantitative size exclusion chromatography-inductively coupled plasma-mass spectrometry to assess regional differences in the molecular masses to which cytosolic iron predominantly binds. Approximately 60 % was associated with ferritin, equating to approximately 25 % of total tissue iron essentially in storage. This study is the first of its kind in human brain tissue, providing a valuable resource and new insight for iron biologists and neuroscientists, alike.

Characterising the brain metalloproteome in Down syndrome patients with concomitant Alzheimer's pathology.

Down syndrome (DS) is a common intellectual disability, with an incidence of 1 in 700 and is caused by trisomy 21. People with DS develop Alzheimer's disease (AD)-like neuropathology by the age of 40. As metal ion dyshomeostasis (particularly zinc, iron and copper) is one of the characteristics of AD and is believed to be involved in the pathogenesis of disease, we reasoned that it may also be altered in DS. Thus, we used inductively coupled plasma mass spectrometry to examine metal levels in post-mortem brain tissue from DS individuals with concomitant AD pathology. Size exclusion-ICPMS was also utilised to characterise the metalloproteome in these cases. We report here for the first time that iron levels were higher in a number of regions in the DS brain, including the hippocampus (40%), frontal cortex (100%) and temporal cortex (34%), compared to controls. Zinc and copper were also elevated (both 29%) in the DS frontal cortex, but zinc was decreased (23%) in the DS temporal cortex. Other elements were also examined, a number of which also showed disease-specific changes. The metalloproteomic profile in the DS brain was also different to that in the controls. These data suggest that metals and metal:protein interactions are dysregulated in the DS brain which, given the known role of metals in neurodegeneration and AD, is likely to contribute to the pathogenesis of disease. Interrogation of the underlying cellular mechanisms and consequences of this failure in metal ion homeostasis, and the specific contributions of the individual DS and AD phenotypes to these changes, should be explored.

The Effects of Clioquinol on P-glycoprotein Expression and Biometal Distribution in the Mouse Brain Microvasculature.

Previous studies have demonstrated that the ionophore clioquinol (CQ), in conjunction with the biometals copper and zinc, increases the expression of P-glycoprotein (P-gp) in human cerebral microvascular endothelial (hCMEC/D3) cells. As P-gp expression and function at the blood-brain barrier (BBB) is of great interest regarding CNS drug access and endogenous toxin trafficking (e.g., amyloid beta), the present study assessed the in vivo translation of these previous in vitro findings. Swiss outbred mice received an 11-day treatment of CQ (30 mg/kg) by oral gavage, after which brain microvessel-enriched fractions (MEFs) and surrounding interfaces (subcortical brain tissue and plasma) were extracted. P-gp expression was quantified in the MEF, and biometal concentrations in all 3 compartments were assessed via inductively coupled plasma mass spectrometry. CQ treatment did not modify the expression of P-gp, nor copper or zinc concentrations in the brain MEF under this treatment regime. Metallomic analysis revealed, however, that CQ reduced potassium and magnesium levels in the brain MEF and also lowered brain iron levels. This study has shown that under this dosing regimen, CQ does not increase BBB P-gp expression in Swiss outbred mice, but that CQ facilitates redistribution of certain metal ions within the brain MEF, plasma, and brain parenchyma.

Zn-DTSM, A Zinc Ionophore with Therapeutic Potential for Acrodermatitis Enteropathica?

Acrodermatitis enteropathica (AE) is a rare disease characterised by a failure in intestinal zinc absorption, which results in a host of symptoms that can ultimately lead to death if left untreated. Current clinical treatment involves life-long high-dose zinc supplements, which can introduce complications for overall nutrient balance in the body. Previous studies have therefore explored the pharmacological treatment of AE utilising metal ionophore/transport compounds in an animal model of the disease (conditional knockout (KO) of the zinc transporter, Zip4), with the perspective of finding an alternative to zinc supplementation. In this study we have assessed the utility of a different class of zinc ionophore compound (zinc diethyl bis(N4-methylthiosemicarbazone), Zn-DTSM; Collaborative Medicinal Development, Sausalito, CA, USA) to the one we have previously described (clioquinol), to determine whether it is effective at preventing the stereotypical weight loss present in the animal model of disease. We first utilised an in vitro assay to assess the ionophore capacity of the compound, and then assessed the effect of the compound in three in vivo animal studies (in 1.5-month-old mice at 30 mg/kg/day, and in 5-month old mice at 3 mg/kg/day and 30 mg/kg/day). Our data demonstrate that Zn-DTSM has a pronounced effect on preventing weight loss when administered daily at 30 mg/kg/day; this was apparent in the absence of any added exogenous zinc. This compound had little overall effect on zinc content in various tissues that were assessed, although further characterisation is required to more fully explore the cellular changes underlying the physiological benefit of this compound. These data suggest that Zn-DTSM, or similar compounds, should be further explored as potential therapeutic options for the long-term treatment of AE.

Ionophore and Biometal Modulation of P-glycoprotein Expression and Function in Human Brain Microvascular Endothelial Cells.

PURPOSE: Biometals such as zinc and copper have been shown to affect tight junction expression and subsequently blood-brain barrier (BBB) integrity. Whether these biometals also influence the expression and function of BBB transporters such as P-glycoprotein (P-gp) however is currently unknown. METHODS: Using the immortalised human cerebral microvascular endothelial (hCMEC/D3) cell line, an in-cell western assay (alongside western blotting) assessed relative P-gp expression after treatment with the metal ionophore clioquinol and biometals zinc and copper. The fluorescent P-gp substrate rhodamine-123 was employed to observe functional modulation, and inductively coupled plasma mass spectrometry (ICP-MS) provided information on biometal trafficking. RESULTS: A 24-h treatment with clioquinol, zinc and copper (0.5, 0.5 and 0.1 µM) induced a significant upregulation of P-gp (1.7-fold) assessed by in-cell western and this was confirmed with western blotting (1.8-fold increase). This same treatment resulted in a 23% decrease in rhodamine-123 accumulation over a 1 h incubation. ICP-MS demonstrated that while t8his combination treatment had no effect on intracellular zinc concentrations, the treatment significantly enhanced bioavailable copper (4.6-fold). CONCLUSIONS: Enhanced delivery of copper to human brain microvascular endothelial cells is associated with enhanced expression and function of the important efflux pump P-gp, which may provide therapeutic opportunities for P-gp modulation.

Effect of Structural Modifications to Glyoxal-bis(thiosemicarbazonato)copper(II) Complexes on Cellular Copper Uptake, Copper-Mediated ATP7A Trafficking, and P-Glycoprotein Mediated Efflux.

Bis(thiosemicarbazonato)copper(II) complexes are of interest as potential therapeutics for cancer and neurodegenerative diseases as well as imaging agents for positron emission tomography (PET). The cellular uptake of six bis(thiosemcarbazonato)copper(II)complexes derived from glyoxal, with different functional groups Cu(gtsx) where x = different functional groups, was investigated in SKOV-3, HEK293, and HEK293 P-gp cell lines. Treatment of the cells with the copper complexes increased intracellular copper and increased levels of p-ERK due to activation of the Ras-Raf-MEK-ERK pathway. Treatment of SKOV-3 cells with low concentrations (µM) of two of the copper complexes led to trafficking of the endogenous copper transporter ATP7A from the Golgi network to the cell membrane. Experiments in HEK293 and HEK293-P-gp cells suggest that Cu(gtsm) and Cu(gtse) are substrates for the P-gp efflux protein but the complex with a pyrrolidine functional group, Cu(gtspyr), is not. A PET experiment in mice showed that [64Cu]Cu(gtspyr) has reasonable brain uptake but high liver uptake.

The APOE ε4 Allele Is Associated with Lower Selenium Levels in the Brain: Implications for Alzheimer's Disease.

The antioxidant activity of selenium, which is mainly conferred by its incorporation into dedicated selenoproteins, has been suggested as a possible neuroprotective approach for mitigating neuronal loss in Alzheimer's disease. However, there is inconsistent information with respect to selenium levels in the Alzheimer's disease brain. We examined the concentration and cellular compartmentalization of selenium in the temporal cortex of Alzheimer's disease and control brain tissue. We found that Alzheimer's disease was associated with decreased selenium concentration in both soluble (i.e., cytosolic) and insoluble (i.e., plaques and tangles) fractions of brain homogenates. The presence of the APOE ε4 allele correlated with lower total selenium levels in the temporal cortex and a higher concentration of soluble selenium. Additionally, we found that age significantly contributed to lower selenium concentrations in the peripheral membrane-bound and vesicular fractions. Our findings suggest a relevant interaction between APOE ε4 and selenium delivery into brain, and show changes in cellular selenium distribution in the Alzheimer's disease brain.

Rubidium and potassium levels are altered in Alzheimer's disease brain and blood but not in cerebrospinal fluid.

Loss of intracellular compartmentalization of potassium is a biochemical feature of Alzheimer's disease indicating a loss of membrane integrity and mitochondrial dysfunction. We examined potassium and rubidium (a biological proxy for potassium) in brain tissue, blood fractions and cerebrospinal fluid from Alzheimer's disease and healthy control subjects to investigate the diagnostic potential of these two metal ions. We found that both potassium and rubidium levels were significantly decreased across all intracellular compartments in the Alzheimer's disease brain. Serum from over 1000 participants in the Australian Imaging, Biomarkers and Lifestyle Flagship Study of Ageing (AIBL), showed minor changes according to disease state. Potassium and rubidium levels in erythrocytes and cerebrospinal fluid were not significantly different according to disease state, and rubidium was slightly decreased in Alzheimer's disease patients compared to healthy controls. Our data provides evidence that contrasts the hypothesized disruption of the blood-brain barrier in Alzheimer's disease, with the systemic decrease in cortical potassium and rubidium levels suggesting influx of ions from the blood is minimal and that the observed changes are more likely indicative of an internal energy crisis within the brain. These findings may be the basis for potential diagnostic imaging studies using radioactive potassium and rubidium tracers.

Copper as a target for prostate cancer therapeutics: copper-ionophore pharmacology and altering systemic copper distribution.

Copper-ionophores that elevate intracellular bioavailable copper display significant therapeutic utility against prostate cancer cells in vitro and in TRAMP (Transgenic Adenocarcinoma of Mouse Prostate) mice. However, the pharmacological basis for their anticancer activity remains unclear, despite impending clinical trails. Herein we show that intracellular copper levels in prostate cancer, evaluated in vitro and across disease progression in TRAMP mice, were not correlative with copper-ionophore activity and mirrored the normal levels observed in patient prostatectomy tissues (Gleason Score 7 & 9). TRAMP adenocarcinoma cells harbored markedly elevated oxidative stress and diminished glutathione (GSH)-mediated antioxidant capacity, which together conferred selective sensitivity to prooxidant ionophoric copper. Copper-ionophore treatments [CuII(gtsm), disulfiram & clioquinol] generated toxic levels of reactive oxygen species (ROS) in TRAMP adenocarcinoma cells, but not in normal mouse prostate epithelial cells (PrECs). Our results provide a basis for the pharmacological activity of copper-ionophores and suggest they are amendable for treatment of patients with prostate cancer. Additionally, recent in vitro and mouse xenograft studies have suggested an increased copper requirement by prostate cancer cells. We demonstrated that prostate adenocarcinoma development in TRAMP mice requires a functional supply of copper and is significantly impeded by altered systemic copper distribution. The presence of a mutant copper-transporting Atp7b protein (tx mutation: A4066G/Met1356Val) in TRAMP mice changed copper-integration into serum and caused a remarkable reduction in prostate cancer burden (64% reduction) and disease severity (grade), abrogating adenocarcinoma development. Implications for current clinical trials are discussed.

Lead and manganese levels in serum and erythrocytes in Alzheimer's disease and mild cognitive impairment: results from the Australian Imaging, Biomarkers and Lifestyle Flagship Study of Ageing.

We examined serum and erythrocyte lead and manganese levels in the Australian Imaging, Biomarkers and Lifestyle Flagship Study of Ageing (AIBL), which contains over 1000 registrants including over 200 cases of Alzheimer's disease (AD) and 100 mildly cognitively impaired (MCI) individuals. After correcting for confounding effects of age, collection site and sex, we found a significant decrease in serum manganese levels in AD subjects compared to healthy controls. Analysis of smaller subset of erythrocytes revealed no difference in either lead or manganese levels in AD. Although lead and manganese have neurotoxic effects and may be involved in AD pathology, our results showed that neither metal in serum nor erythrocytes are suitable biomarkers in our cohort. However, prospective studies might reveal whether the burden of either metal modifies disease outcomes.

Effects of Neonatal Iron Feeding and Chronic Clioquinol Administration on the Parkinsonian Human A53T Transgenic Mouse.

Increased nigral iron (Fe) is a cardinal feature of Parkinson's disease, as is the accumulation of aggregates comprising α-synuclein. We used wild-type mice and transgenic mice overexpressing the human A53T mutation to α-synuclein to examine the influence of increased Fe (days 10-17 postpartum) on the parkinsonian development phenotype of these animals (including abnormal nigral Fe levels and deficits in both cell numbers and locomotor activity), and to explore the impact of the Fe chelator clioquinol in the model. Both untreated and Fe-loaded A53T mice showed similar levels of nigral cell loss, though 5 months of clioquinol treatment was only able to prevent the loss in the non-Fe-loaded A53T group. Iron levels in the Fe-loaded A53T mice returned to normal at 8 months, though effects of dopamine denervation remained, demonstrated by limited locomotor activity and sustained neuron loss. These data suggest that Fe exposure during a critical developmental window, combined with the overexpression mutant α-synuclein, presents a disease phenotype resistant to intervention using clioquinol later in life.

Novel Fluorinated 8-Hydroxyquinoline Based Metal Ionophores for Exploring the Metal Hypothesis of Alzheimer's Disease.

Zinc, copper, and iron ions are involved in amyloid-beta (Aß) deposition and stabilization in Alzheimer's disease (AD). Consequently, metal binding agents that prevent metal-Aß interaction and lead to the dissolution of Aß deposits have become well sought therapeutic and diagnostic targets. However, direct intervention between diseases and metal abnormalities has been challenging and is partially attributed to the lack of a suitable agent to determine and modify metal concentration and distribution in vivo. In the search of metal ionophores, we have identified several promising chemical entities by strategic fluorination of 8-hydroxyquinoline drugs, clioquinol, and PBT2. Compounds 15-17 and 28-30 showed exceptional metal ionophore ability (6-40-fold increase of copper uptake and >2-fold increase of zinc uptake) and inhibition of zinc induced Aß oligomerization (EC50s < ∼5 µM). These compounds are suitable for further development as drug candidates and/or positron emission tomography (PET) biomarkers if radiolabeled with (18)F.

Enduring Elevations of Hippocampal Amyloid Precursor Protein and Iron Are Features of ß-Amyloid Toxicity and Are Mediated by Tau.

The amyloid cascade hypothesis of Alzheimer's disease (AD) positions tau protein as a downstream mediator of ß-amyloid (Aß) toxicity This is largely based on genetic cross breeding, which showed that tau ablation in young (3-7-month-old) transgenic mice overexpressing mutant amyloid precursor protein (APP) abolished the phenotype of the APP AD model. This evidence is complicated by the uncertain impact of overexpressing mutant APP, rather than Aß alone, and for potential interactions between tau and overexpressed APP. Cortical iron elevation is also implicated in AD, and tau promotes iron export by trafficking APP to the neuronal surface. Here, we utilized an alternative model of Aß toxicity by directly injecting Aß oligomers into the hippocampus of young and old wild-type and tau knockout mice. We found that ablation of tau protected against Aß-induced cognitive impairment, hippocampal neuron loss, and iron accumulation. Despite injected human Aß being eliminated after 5 weeks, enduring changes, including increased APP levels, tau reduction, tau phosphorylation, and iron accumulation, were observed. While the results from our study support the amyloid cascade hypothesis, they also suggest that downstream effectors of Aß, which propagate toxicity after Aß has been cleared, may be tractable therapeutic targets.

Clioquinol rescues Parkinsonism and dementia phenotypes of the tau knockout mouse.

Iron accumulation and tau protein deposition are pathological features of Alzheimer's (AD) and Parkinson's diseases (PD). Soluble tau protein is lower in affected regions of these diseases, and we previously reported that tau knockout mice display motor and cognitive behavioral abnormities, brain atrophy, neuronal death in substantia nigra, and iron accumulation in the brain that all emerged between 6 and 12 months of age. This argues for a loss of tau function in AD and PD. We also showed that treatment with the moderate iron chelator, clioquinol (CQ) restored iron levels and prevented neuronal atrophy and attendant behavioral decline in 12-month old tau KO mice when commenced prior to the onset of deterioration (6 months). However, therapies for AD and PD will need to treat the disease once it is already manifest. So, in the current study, we tested whether CQ could also rescue the phenotype of mice with a developed phenotype. We found that 5-month treatment of symptomatic (13 months old) tau KO mice with CQ increased nigral tyrosine hydroxylase phosphorylation (which induces activity) and reversed the motor deficits. Treatment also reversed cognitive deficits and raised BDNF levels in the hippocampus, which was accompanied by attenuated brain atrophy, and reduced iron content in the brain. These data raise the possibility that lowering brain iron levels in symptomatic patients could reverse neuronal atrophy and improve brain function, possibly by elevating neurotrophins.

Neonatal iron supplementation potentiates oxidative stress, energetic dysfunction and neurodegeneration in the R6/2 mouse model of Huntington's disease.

Huntington's disease (HD) is a progressive neurodegenerative disorder caused by a CAG repeat expansion that encodes a polyglutamine tract in huntingtin (htt) protein. Dysregulation of brain iron homeostasis, oxidative stress and neurodegeneration are consistent features of the HD phenotype. Therefore, environmental factors that exacerbate oxidative stress and iron dysregulation may potentiate HD. Iron supplementation in the human population is common during infant and adult-life stages. In this study, iron supplementation in neonatal HD mice resulted in deterioration of spontaneous motor running activity, elevated levels of brain lactate and oxidized glutathione consistent with increased energetic dysfunction and oxidative stress, and increased striatal and motor cortical neuronal atrophy, collectively demonstrating potentiation of the disease phenotype. Oxidative stress, energetic, and anatomic markers of degeneration were not affected in wild-type littermate iron-supplemented mice. Further, there was no effect of elevated iron intake on disease outcomes in adult HD mice. We have demonstrated an interaction between the mutant huntingtin gene and iron supplementation in neonatal HD mice. Findings indicate that elevated neonatal iron intake potentiates mouse HD and promotes oxidative stress and energetic dysfunction in brain. Neonatal-infant dietary iron intake level may be an environmental modifier of human HD.