[Show abstract][Hide abstract] ABSTRACT: Alzheimer's disease (AD) is a multifactorial neurodegenerative disease. It begins years prior to the onset of clinical symptoms, such as memory loss and cognitive decline. Pathological hallmarks of AD include the accumulation of β-amyloid in plaques and hyperphosphorylated tau in neurofibrillary tangles. Copper, iron, and zinc are abnormally accumulated and distributed in the aging brain. These metal ions can adversely contribute to the progression of AD. Dysregulation of cholesterol metabolism has also been implicated in the development of AD pathology. To date, large bodies of research have been carried out independently to elucidate the role of metals or cholesterol on AD pathology. Interestingly, metals and cholesterol affect parallel molecular and biochemical pathways involved in AD pathology. The possible links between metal dyshomeostasis and altered brain cholesterol metabolism in AD are reviewed.
Full-text · Article · May 2014 · Frontiers in Aging Neuroscience
[Show abstract][Hide abstract] ABSTRACT: The loss of NPC1 protein function is the predominant cause of Niemann-Pick type C1 disease (NP-C1), a systemic and neurodegenerative disorder characterized by late-endosomal/lysosomal accumulation of cholesterol and other lipids. Limited evidence from post-mortem human tissues, an Npc1(-/-) mouse model, and cell culture studies also suggest failure of metal homeostasis in NP-C1. To investigate these findings, we performed a comprehensive transition metal analysis of cerebrospinal fluid (CSF), plasma and tissue samples from human NP-C1 patients and an Npc1(-/-) mouse model. NPC1 deficiency in the Npc1(-/-) mouse model resulted in a perturbation of transition metal homeostasis in the plasma and key organs (brain, liver, spleen, heart, lungs, and kidneys). Analysis of human patient CSF, plasma and post-mortem brain tissues also indicated disrupted metal homeostasis. There was a disparity in the direction of metal changes between the human and the Npc1(-/-) mouse samples, which may reflect species-specific metal metabolism. Nevertheless, common to both species is brain zinc accumulation. Furthermore, treatment with the glucosylceramide synthase inhibitor miglustat, the only drug shown in a controlled clinical trial to have some efficacy for NP-C1, did not correct the alterations in CSF and plasma transition metal and ceruloplasmin (CP) metabolism in NP-C1 patients. These findings highlight the importance of NPC1 function in metal homeostasis, and indicate that metal-targeting therapy may be of value as a treatment for NP-C.
[Show abstract][Hide abstract] ABSTRACT: Over the past two decades there have been significant advances in our understanding of copper homeostasis and the pathological consequences of copper dysregulation. Cumulative evidence is revealing a complex regulatory network of proteins and pathways that maintain copper homeostasis. The recognition of copper dysregulation as a key pathological feature in prominent neurodegenerative disorders such as Alzheimer's, Parkinson's, and prion diseases has led to increased research focus on the mechanisms controlling copper homeostasis in the brain. The copper-transporting P-type ATPases (copper-ATPases), ATP7A and ATP7B, are critical components of the copper regulatory network. Our understanding of the biochemistry and cell biology of these complex proteins has grown significantly since their discovery in 1993. They are large polytopic transmembrane proteins with six copper-binding motifs within the cytoplasmic N-terminal domain, eight transmembrane domains, and highly conserved catalytic domains. These proteins catalyze ATP-dependent copper transport across cell membranes for the metallation of many essential cuproenzymes, as well as for the removal of excess cellular copper to prevent copper toxicity. A key functional aspect of these copper transporters is their copper-responsive trafficking between the trans-Golgi network and the cell periphery. ATP7A- and ATP7B-deficiency, due to genetic mutation, underlie the inherited copper transport disorders, Menkes and Wilson diseases, respectively. Their importance in maintaining brain copper homeostasis is underscored by the severe neuropathological deficits in these disorders. Herein we will review and update our current knowledge of these copper transporters in the brain and the central nervous system, their distribution and regulation, their role in normal brain copper homeostasis, and how their absence or dysfunction contributes to disturbances in copper homeostasis and neurodegeneration.
Full-text · Article · Aug 2013 · Frontiers in Aging Neuroscience
[Show abstract][Hide abstract] ABSTRACT: Altered copper homeostasis and hypercholesterolemia have been identified independently as risk factors for Alzheimer's disease (AD). Abnormal copper and cholesterol metabolism are implicated in the genesis of amyloid plaques and neurofibrillary tangles (NFT), which are two key pathological signatures of AD. Amyloidogenic processing of a sub-population of amyloid precursor protein (APP) that produces Aβ occurs in cholesterol-rich lipid rafts in copper deficient AD brains. Co-localization of Aβ and a paradoxical high concentration of copper in lipid rafts fosters the formation of neurotoxic Aβ:copper complexes. These complexes can catalytically oxidize cholesterol to generate H2O2, oxysterols and other lipid peroxidation products that accumulate in brains of AD cases and transgenic mouse models. Tau, the core protein component of NFTs, is sensitive to interactions with copper and cholesterol, which trigger a cascade of hyperphosphorylation and aggregation preceding the generation of NFTs. Here we present an overview of copper and cholesterol metabolism in the brain, and how their integrated failure contributes to development of AD.
Full-text · Article · May 2013 · Frontiers in Physiology
[Show abstract][Hide abstract] ABSTRACT: Inhibition of GSL (glycosphingolipid) synthesis reduces Aβ (amyloid β-peptide) production in vitro. Previous studies indicate that GCS (glucosylceramide synthase) inhibitors modulate phosphorylation of ERK1/2 (extracellular-signal-regulated kinase 1/2) and that the ERK pathway may regulate some aspects of Aβ production. It is not clear whether there is a causative relationship linking GSL synthesis inhibition, ERK phosphorylation and Aβ production. In the present study, we treated CHO cells (Chinese-hamster ovary cells) and SH-SY5Y neuroblastoma cells, that both constitutively express human wild-type APP (amyloid precursor protein) and process this to produce Aβ, with GSL-modulating agents to explore this relationship. We found that three related ceramide analogue GSL inhibitors, based on the PDMP (D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol) structure, reduced cellular Aβ production and in all cases this was correlated with inhibition of pERK (phosphorylated ERK) formation. Importantly, the L-threo enantiomers of these compounds (that are inferior GSL synthesis inhibitors compared with the D-threo-enantiomers) also reduced ERK phosphorylation to a similar extent without altering Aβ production. Inhibition of ERK activation using either PD98059 [2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one] or U0126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio] butadiene) had no impact on Aβ production, and knockdown of endogenous GCS using small interfering RNA reduced cellular GSL levels without suppressing Aβ production or pERK formation. Our data suggest that the alteration in pERK levels following treatment with these ceramide analogues is not the principal mechanism involved in the inhibition of Aβ generation and that the ERK signalling pathway does not play a crucial role in processing APP through the amyloidogenic pathway.
[Show abstract][Hide abstract] ABSTRACT: Accumulation of the amyloid β peptide in the cortical and hippocampal regions of the brain is a major pathological feature
of Alzheimer disease. Amyloid β peptide is generated from the sequential protease cleavage of the amyloid precursor protein
(APP). We reported previously that copper increases the level of APP at the cell surface. Here we report that copper, but
not iron or zinc, promotes APP trafficking in cultured polarized epithelial cells and neuronal cells. In SH-SY5Y neuronal
cells and primary cortical neurons, copper promoted a redistribution of APP from a perinuclear localization to a wider distribution,
including neurites. Importantly, a change in APP localization was not attributed to an up-regulation of APP protein synthesis.
Using live cell imaging and endocytosis assays, we found that copper promotes an increase in cell surface APP by increasing
its exocytosis and reducing its endocytosis, respectively. This study identifies a novel mechanism by which copper regulates
the localization and presumably the function of APP, which is of major significance for understanding the role of APP in copper
homeostasis and the role of copper in Alzheimer disease.
No preview · Article · Dec 2010 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Copper is a cofactor for many essential enzymes in aerobic organisms. When intracellular copper levels are elevated, the Menkes (ATP7A) P-Type ATPase traffics from the trans-Golgi network (TGN) towards the plasma membrane to facilitate copper efflux. The ADP-ribosylation factor 1 (Arf1) is required for maintenance of Golgi architecture and for vesicular trafficking, including the copper-responsive trafficking of ATP7A. Here we report an ATP7A-independent role of Arf1 in copper homeostasis. Whilst the loss of ATP7A function increased copper levels, RNA interference mediated Arf1 knockdown reduced copper accumulation in HeLa cells as well as in both wild-type and ATP7A-null cultured fibroblasts. Arf1 therefore affected copper levels independently of ATP7A mediated copper efflux. Knockdown of Arf79F, the Drosophila melanogasterArf1 orthologue, also reduced copper accumulation in cultured Drosophila S2 cells, indicating an evolutionarily conserved role for this protein in cellular copper homeostasis. Whereas severe Arf1 inhibition with brefeldin A caused fragmentation and dispersal of the TGN resident protein Golgin 97, the peri-nuclear localisation of the Golgin 97 was retained following Arf1 knockdown, consistent with a moderate reduction in Arf1 activity. Ctr1 levels at the plasma membrane of cultured fibroblast cells were reduced following Arf1 knockdown, indicating an Arf1-dependent trafficking pathway is required for correct distribution of this copper uptake protein. Arf1-dependent trafficking pathways are therefore required for optimal copper uptake efficiency in cultured human and Drosophila cells.
No preview · Article · Oct 2010 · The international journal of biochemistry & cell biology
[Show abstract][Hide abstract] ABSTRACT: The primary constituent of the amyloid plaque, beta-amyloid (Abeta), is thought to be the causal "toxic moiety" of Alzheimer's disease. However, despite much work focused on both Abeta and its parent protein, amyloid precursor protein (APP), the functional roles of APP and its cleavage products remain to be fully elucidated. Protein-protein interaction networks can provide insight into protein function, however, high-throughput data often report false positives and are in frequent disagreement with low-throughput experiments. Moreover, the complexity of the CNS is likely to be under represented in such databases. Therefore, we curated the published work characterizing both APP and Abeta to create a protein interaction network of APP and its proteolytic cleavage products, with annotation, where possible, to the level of APP binding domain and isoform. This is the first time that an interactome has been refined to domain level, essential for the interpretation of APP due to the presence of multiple isoforms and processed fragments. Gene ontology and network analysis were used to identify potentially novel functional relationships among interacting proteins.
[Show abstract][Hide abstract] ABSTRACT: Alzheimer's disease (AD) is the most common form of neurodegenerative disease. The brain is particularly vulnerable to oxidative damage induced by unregulated redox-active metals such as copper and iron, and the brains of AD patients display evidence of metal dyshomeostasis and increased oxidative stress. The colocalisation of copper and amyloid beta (Abeta) in the glutamatergic synapse during NMDA-receptor-mediated neurotransmission provides a microenvironment favouring the abnormal interaction of redox-potent Abeta with copper under conditions of copper dysregulation thought to prevail in the AD brain, resulting in the formation of neurotoxic soluble Abeta oligomers. Interactions between Abeta oligomers and copper can further promote the aggregation of Abeta, which is the core component of extracellular amyloid plaques, a central pathological hallmark of AD. Copper dysregulation is also implicated in the hyperphosphorylation and aggregation of tau, the main component of neurofibrillary tangles, which is also a defining pathological hallmark of AD. Therefore, tight regulation of neuronal copper homeostasis is essential to the integrity of normal brain functions. Therapeutic strategies targeting interactions between Abeta, tau and metals to restore copper and metal homeostasis are discussed.
No preview · Article · Oct 2009 · European Journal of Biochemistry
[Show abstract][Hide abstract] ABSTRACT: Redox-active copper is implicated in the pathogenesis of Alzheimer disease (AD), β-amyloid peptide (Aβ) aggregation, and amyloid
formation. Aβ·copper complexes have been identified in AD and catalytically oxidize cholesterol and lipid to generate H2O2 and lipid peroxides. The site and mechanism of this abnormality is not known. Growing evidence suggests that amyloidogenic
processing of the β-amyloid precursor protein (APP) occurs in lipid rafts, membrane microdomains enriched in cholesterol.
β- and γ-secretases, and Aβ have been identified in lipid rafts in cultured cells, human and rodent brains, but the role of
copper in lipid raft amyloidogenic processing is presently unknown. In this study, we found that copper modulates flotillin-2
association with cholesterol-rich lipid raft domains, and consequently Aβ synthesis is attenuated via copper-mediated inhibition
of APP endocytosis. We also found that total cellular copper is associated inversely with lipid raft copper levels, so that
under intracellular copper deficiency conditions, Aβ·copper complexes are more likely to form. This explains the paradoxical
hypermetallation of Aβ with copper under tissue copper deficiency conditions in AD.
[Show abstract][Hide abstract] ABSTRACT: The MNK (Menkes disease protein; ATP7A) is a major copper- transporting P-type ATPase involved in the delivery of copper to cuproenzymes in the secretory pathway and the efflux of excess copper from extrahepatic tissues. Mutations in the MNK (ATP7A) gene result in Menkes disease, a fatal neurodegenerative copper deficiency disorder. Currently, detailed biochemical and biophysical analyses of MNK to better understand its mechanisms of copper transport are not possible due to the lack of purified MNK in an active form. To address this issue, we expressed human MNK with an N-terminal Glu-Glu tag in Sf9 [Spodoptera frugiperda (fall armyworm) 9] insect cells and purified it by antibody affinity chromatography followed by size-exclusion chromatography in the presence of the non-ionic detergent DDM (n-dodecyl beta-D-maltopyranoside). Formation of the classical vanadate-sensitive phosphoenzyme by purified MNK was activated by Cu(I) [EC50=0.7 microM; h (Hill coefficient) was 4.6]. Furthermore, we report the first measurement of Cu(I)-dependent ATPase activity of MNK (K0.5=0.6 microM; h=5.0). The purified MNK demonstrated active ATP-dependent vectorial 64Cu transport when reconstituted into soya-bean asolectin liposomes. Together, these data demonstrated that Cu(I) interacts with MNK in a co-operative manner and with high affinity in the sub-micromolar range. The present study provides the first biochemical characterization of a purified full-length mammalian copper-transporting P-type ATPase associated with a human disease.
Full-text · Article · Feb 2007 · Biochemical Journal