Mechanisms of Copper Ion Mediated Huntington's Disease Progression

Huntington's disease (HD) is caused by a dominant polyglutamine expansion within the N-terminus of huntingtin protein and results in oxidative stress, energetic insufficiency and striatal degeneration. Copper and iron are increased in the striata of HD patients, but the role of these metals in HD pathogenesis is unknown. We found, using inductively-coupled-plasma mass spectroscopy, that elevations of copper and iron found in human HD brain are reiterated in the brains of affected HD transgenic mice. Increased brain copper correlated with decreased levels of the copper export protein, amyloid precursor protein. We hypothesized that increased amounts of copper bound to low affinity sites could contribute to pro-oxidant activities and neurodegeneration. We focused on two proteins: huntingtin, because of its centrality to HD, and lactate dehydrogenase (LDH), because of its documented sensitivity to copper, necessity for normoxic brain energy metabolism and evidence for altered lactate metabolism in HD brain. The first 171 amino acids of wild-type huntingtin, and its glutamine expanded mutant form, interacted with copper, but not iron. N171 reduced Cu(2+) in vitro in a 1∶1 copper∶protein stoichiometry indicating that this fragment is very redox active. Further, copper promoted and metal chelation inhibited aggregation of cell-free huntingtin. We found decreased LDH activity, but not protein, and increased lactate levels in HD transgenic mouse brain. The LDH inhibitor oxamate resulted in neurodegeneration when delivered intra-striatially to healthy mice, indicating that LDH inhibition is relevant to neurodegeneration in HD. Our findings support a role of pro-oxidant copper-protein interactions in HD progression and offer a novel target for pharmacotherapeutics

Intracellular amyloid formation in muscle cells of Aβ-transgenic Caenorhabditis elegans: determinants and physiological role in copper detoxification

Background: The amyloid β-peptide is a ubiquitous peptide, which is prone to aggregate forming soluble toxic oligomers and insoluble less-toxic aggregates. The intrinsic and external/environmental factors that determine Aβ aggregation in vivo are poorly understood, as well as the cellular meaning of this process itself. Genetic data as well as cell biological and biochemical evidence strongly support the hypothesis that Aβ is a major player in the onset and development of Alzheimer's disease. In addition, it is also known that Aβ is involved in Inclusion Body Myositis, a common myopathy of the elderly in which the peptide accumulates intracellularly. Results: In the present work, we found that intracellular Aβ aggregation in muscle cells of Caenorhabditis elegans overexpressing Aβ peptide is affected by two single amino acid substitutions, E22G (Arctic) and V18A (NIC). Both variations show decrease intracellular amyloidogenesis compared to wild type Aβ. We show that intracellular amyloid aggregation of wild type Aβ is accelerated by Cu2+ and diminished by copper chelators. Moreover, we demonstrate through toxicity and behavioral assays that Aβ-transgenic worms display a higher tolerance to Cu2+ toxic effects and that this resistance may be linked to the formation of amyloid aggregates. Conclusion: Our data show that intracellular Aβ amyloid aggregates may trap excess of free Cu2+ buffering its cytotoxic effects and that accelerated intracellular Aβ aggregation may be part of a cell protective mechanism

Decreased Serum Zinc Is An Effect Of Ageing And Not Alzheimer\u27s Disease

We examined the distribution of zinc in the periphery (erythrocytes and serum) in a large, well-characterised cohort, the Australian Imaging, Biomarkers and Lifestyle (AIBL) study, in order to determine if there is systemic perturbation in zinc homeostasis in Alzheimer’s disease (AD). We observed an age dependent decrease in serum zinc of approximately 0.4% per year. When correcting for the age dependent decline in serum zinc no significant difference between healthy controls (HC), mildly cognitively impaired (MCI) or AD subjects was observed

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

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

Selective intracellular release of copper and zinc ions from bis(thiosemicarbazonato) complexes reduces levels of Alzheimer disease amyloid-β peptide

Copper and zinc play important roles in Alzheimer disease pathology with recent reports describing potential therapeutics based on modulation of metal bioavailability. We examined the ability of a range of metal bis(thiosemicarbazonato) complexes (MII(btsc), where M = Cu II or ZnII) to increase intracellular metal levels in Chinese hamster ovary cells overexpressing amyloid precursor protein (APP-CHO) and the subsequent effect on extracellular levels of amyloid-β peptide (Aβ). The CuII(btsc) complexes were engineered to be either stable to both a change in oxidation state and dissociation of metal or susceptible to intracellular reduction and dissociation of metal. Treatment of APP-CHO cells with stable complexes resulted in elevated levels of intracellular copper with no effect on the detected levels of Aβ. Treatment with complexes susceptible to intracellular reduction increased intracellular copper levels but also resulted in a dose-dependent reduction in the levels of monomeric Aβ. Treatment with less stable ZnII(btsc) complexes increased intracellular zinc levels with a subsequent dose-dependent depletion of monomeric Aβ levels. The increased levels of intracellular bioavailable copper and zinc initiated a signaling cascade involving activation of phosphoinositol 3-kinase and c-Jun N-terminal kinase. Inhibition of these enzymes prevented Aβ depletion induced by the MII(btsc) complexes. Inhibition of metalloproteases also partially restored Aβ levels, implicating metal-driven metalloprotease activation in the extracellular monomeric Aβ depletion. However, a role for alternative metal-induced Aβ metabolism has not been ruled out. These studies demonstrate that M II(btsc) complexes have potential for Alzheimer disease therapy

Risk prediction of late-onset Alzheimer’s disease implies an oligogenic architecture

© 2020, The Author(s). Genetic association studies have identified 44 common genome-wide significant risk loci for late-onset Alzheimer’s disease (LOAD). However, LOAD genetic architecture and prediction are unclear. Here we estimate the optimal P-threshold (Poptimal) of a genetic risk score (GRS) for prediction of LOAD in three independent datasets comprising 676 cases and 35,675 family history proxy cases. We show that the discriminative ability of GRS in LOAD prediction is maximised when selecting a small number of SNPs. Both simulation results and direct estimation indicate that the number of causal common SNPs for LOAD may be less than 100, suggesting LOAD is more oligogenic than polygenic. The best GRS explains approximately 75% of SNP-heritability, and individuals in the top decile of GRS have ten-fold increased odds when compared to those in the bottom decile. In addition, 14 variants are identified that contribute to both LOAD risk and age at onset of LOAD

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 Alzheimer's 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