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2.  Correction: Iron Accumulates in Huntington’s Disease Neurons: Protection by Deferoxamine  
PLoS ONE  2013;8(11):10.1371/annotation/67f555f5-35b7-4468-8bab-26d518942803.
doi:10.1371/annotation/67f555f5-35b7-4468-8bab-26d518942803
PMCID: PMC3829977  PMID: 24250777
3.  Decreased Copper in Alzheimer's Disease Brain Is Predominantly in the Soluble Extractable Fraction 
Alzheimer's disease (AD) is the leading cause of dementia and represents a significant burden on the global economy and society. The role of transition metals, in particular copper (Cu), in AD has become of significant interest due to the dyshomeostasis of these essential elements, which can impart profound effects on cell viability and neuronal function. We tested the hypothesis that there is a systemic perturbation in Cu compartmentalization in AD, within the brain as well as in the periphery, specifically within erythrocytes. Our results showed that the previously reported decrease in Cu within the human frontal cortex was confined to the soluble (P < 0.05) and total homogenate (P < 0.05) fractions. No differences were observed in Cu concentration in erythrocytes. Our data indicate that there is a brain specific alteration in Cu levels in AD localized to the soluble extracted material, which is not reflected in erythrocytes. Further studies using metalloproteomics approaches will be able to elucidate the metabolic mechanism(s) that results in the decreased brain Cu levels during the progression of AD.
doi:10.1155/2013/623241
PMCID: PMC3818847  PMID: 24228186
4.  Iron Accumulates in Huntington’s Disease Neurons: Protection by Deferoxamine  
PLoS ONE  2013;8(10):e77023.
Huntington’s disease (HD) is a progressive neurodegenerative disorder caused by a polyglutamine-encoding CAG expansion in the huntingtin gene. Iron accumulates in the brains of HD patients and mouse disease models. However, the cellular and subcellular sites of iron accumulation, as well as significance to disease progression are not well understood. We used independent approaches to investigate the location of brain iron accumulation. In R6/2 HD mouse brain, synchotron x-ray fluorescence analysis revealed iron accumulation as discrete puncta in the perinuclear cytoplasm of striatal neurons. Further, perfusion Turnbull’s staining for ferrous iron (II) combined with transmission electron microscope ultra-structural analysis revealed increased staining in membrane bound peri-nuclear vesicles in R6/2 HD striatal neurons. Analysis of iron homeostatic proteins in R6/2 HD mice revealed decreased levels of the iron response proteins (IRPs 1 and 2) and accordingly decreased expression of iron uptake transferrin receptor (TfR) and increased levels of neuronal iron export protein ferroportin (FPN). Finally, we show that intra-ventricular delivery of the iron chelator deferoxamine results in an improvement of the motor phenotype in R6/2 HD mice. Our data supports accumulation of redox-active ferrous iron in the endocytic / lysosomal compartment in mouse HD neurons. Expression changes of IRPs, TfR and FPN are consistent with a compensatory response to an increased intra-neuronal labile iron pool leading to increased susceptibility to iron-associated oxidative stress. These findings, together with protection by deferoxamine, support a potentiating role of neuronal iron accumulation in HD.
doi:10.1371/journal.pone.0077023
PMCID: PMC3795666  PMID: 24146952
5.  Therapeutics for Alzheimer’s Disease Based on the Metal Hypothesis 
SUMMARY
Alzheimer’s disease (AD), the most common form of dementia in the elderly, is characterized by elevated brain iron levels and accumulation of copper and zinc in cerebral β-amyloid deposits; e.g., senile plaques. Both ionic zinc and copper are able to accelerate the aggregation of Aβ, the principle component of β-amyloid deposits. Copper (and iron) can also promote the neurotoxic redox activity of Aβ and induce oxidative cross-linking of the peptide into stable oligomers. Recent reports have documented the release of Aβ together with ionic zinc and copper in cortical glutamatergic synapses following excitation. This, in turn, leads to the formation of Aβ oligomers, which, in turn, modulate long-term potentiation (by controlling synaptic levels of the NMDA receptor). The excessive accumulation of Aβ oligomers in the synaptic cleft would then be predicted to adversely affect synaptic neurotransmisson. Based on these findings, we have proposed the “Metal Hypothesis of Alzheimer’s Disease” which stipulates that the neuropathogenic effects of Aβ in AD are promoted by, and possibly even dependent upon Aβ-metal interactions. Increasingly sophisticated pharmaceutical approaches are now being implemented to attenuate abnormal Aβ-metal interactions without causing systemic disturbance of essential metals. Small molecules targeting Aβ–metal interactions, e.g. PBT2, are currently advancing through clinical trials and show increasing promise as disease-modifying agents for AD based on the “metal hypothesis”.
doi:10.1016/j.nurt.2008.05.001
PMCID: PMC2518205  PMID: 18625454
copper; zinc; amyloid; free radical; oxidation; PBT2
6.  Links between copper and cholesterol in Alzheimer's disease 
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.
doi:10.3389/fphys.2013.00111
PMCID: PMC3655288  PMID: 23720634
copper; cholesterol; Alzheimer's disease; ApoE; amyloid precursor protein; Aβ; tau; lipid rafts
7.  Presenilin Promotes Dietary Copper Uptake 
PLoS ONE  2013;8(5):e62811.
Dietary copper is essential for multicellular organisms. Copper is redox active and required as a cofactor for enzymes such as the antioxidant Superoxide Dismutase 1 (SOD1). Copper dyshomeostasis has been implicated in Alzheimer’s disease. Mutations in the presenilin genes encoding PS1 and PS2 are major causes of early-onset familial Alzheimer’s disease. PS1 and PS2 are required for efficient copper uptake in mammalian systems. Here we demonstrate a conserved role for presenilin in dietary copper uptake in the fly Drosophila melanogaster. Ubiquitous RNA interference-mediated knockdown of the single Drosophila presenilin (PSN) gene is lethal. However, PSN knockdown in the midgut produces viable flies. These flies have reduced copper levels and are more tolerant to excess dietary copper. Expression of a copper-responsive EYFP construct was also lower in the midgut of these larvae, indicative of reduced dietary copper uptake. SOD activity was reduced by midgut PSN knockdown, and these flies were sensitive to the superoxide-inducing chemical paraquat. These data support presenilin being needed for dietary copper uptake in the gut and so impacting on SOD activity and tolerance to oxidative stress. These results are consistent with previous studies of mammalian presenilins, supporting a conserved role for these proteins in mediating copper uptake.
doi:10.1371/journal.pone.0062811
PMCID: PMC3646984  PMID: 23667524
8.  The Neurophysiology and Pathology of Brain Zinc 
Our understanding of the roles played by zinc in the physiological and pathological functioning of the brain is rapidly expanding. The increased availability of genetically modified animal models, selective zinc-sensitive fluorescent probes, and novel chelators is producing a remarkable body of exciting new data that clearly establishes this metal ion as a key modulator of intracellular and intercellular neuronal signaling. In this Mini-Symposium, we will review and discuss the most recent findings that link zinc to synaptic function as well as the injurious effects of zinc dyshomeostasis within the context of neuronal death associated with major human neurological disorders, including stroke, epilepsy, and Alzheimer’s disease.
doi:10.1523/JNEUROSCI.3454-11.2011
PMCID: PMC3223736  PMID: 22072659
9.  Dissociation of ERK signalling inhibition from the anti-amyloidogenic action of synthetic ceramide analogues 
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.
doi:10.1042/CS20110257
PMCID: PMC3259697  PMID: 22103431
Alzheimer's disease; amyloid β-peptide; amyloid precursor protein; extracellular-signal-regulated kinase (ERK); glycosphingolipid; 2AA, 2-anthranilic acid; Aβ, amyloid β-peptide; Aβ40, Aβ-(1–40); Aβ42, Aβ-(1–42); AD, Alzheimer's disease; APP, amyloid precursor protein; BACE-1, β-site APP-cleaving enzyme; CHO cell, Chinese-hamster ovary cell; DAPT, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-(S)-phenylglycine t-butyl ester; ERK, extracellular-signal-regulated kinase; EtDO-P4, D-threo-ethylenedioxy-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GCS, glucosylceramide synthase; GlcCer, glucosylceramide; GSL, glycosphingolipid; LacCer, lactosylceramide; MAPK, mitogen-activated protein kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-2H-tetrazolium bromide; PDMP, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol; pERK, phosphorylated ERK; PPMP, D-threo-1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol; PS, presenilin; sAPPα, soluble APPα fragment; siRNA, small interfering RNA
10.  Disturbed Copper Bioavailability in Alzheimer's Disease 
Recent data from in vitro, animal, and human studies have shed new light on the positive roles of copper in many aspects of AD. Copper promotes the non-amyloidogenic processing of APP and thereby lowers the Aβ production in cell culture systems, and it increases lifetime and decreases soluble amyloid production in APP transgenic mice. In a clinical trial with Alzheimer patients, the decline of Aβ levels in CSF, which is a diagnostic marker, is diminished in the verum group (8 mg copper/day), indicating a beneficial effect of the copper treatment. These observations are in line with the benefit of treatment with compounds aimed at normalizing metal levels in the brain, such as PBT2. The data reviewed here demonstrate that there is an apparent disturbance in metal homeostasis in AD. More research is urgently needed to understand how this disturbance can be addressed therapeutically.
doi:10.4061/2011/345614
PMCID: PMC3227474  PMID: 22145082
11.  Insulin-like Signaling Determines Survival During Stress via Post Transcriptional Mechanisms in C. elegans 
Cell metabolism  2010;12(3):260-272.
Summary
The insulin-like signaling (ILS) pathway regulates metabolism and is known to modulate adult lifespan in C. elegans. Altered stress responses and resistance to a wide range of stressors are also associated with changes in ILS and contribute to enhanced longevity. The transcription factors DAF-16 and HSF-1 are key effectors of the longevity phenotype. We demonstrate that increased intrinsic thermotolerance, due to lower ILS, is not dependent on stress induced transcriptional responses but instead requires active protein translation. Translation profiling experiments reveal genes that are post-transcriptionally regulated in response to altered ILS during heat shock in a DAF-16-dependent manner. Furthermore, several novel proteins are specifically required for ILS effects on thermotolerance. We propose that lowered-ILS results in metabolic and physiological changes. These DAF-16-induced changes precondition a translational response under acute stress to modulate survival.
doi:10.1016/j.cmet.2010.08.004
PMCID: PMC2945254  PMID: 20816092
12.  ALTERED MICROGLIAL COPPER HOMEOSTASIS IN A MOUSE MODEL OF ALZHEIMER’S DISEASE 
Journal of neurochemistry  2010;114(6):1630-1638.
Alzheimer’s disease (AD) is characterized by progressive neurodegeneration associated with the aggregation and deposition of β-amyloid (Aβ40 and Aβ42) peptide in senile plaques. Recent studies suggest that copper may play an important role in AD pathology. Copper concentrations are elevated in amyloid plaques and copper binds with high affinity to the Aβ peptide and promotes Aβ oligomerization and neurotoxicity. Despite this connection between copper and AD, it is unknown whether the expression of proteins involved in regulating copper homeostasis is altered in this disorder. In this study we demonstrate that the copper transporting P-type ATPase, ATP7A, is highly expressed in activated microglial cells that are specifically clustered around amyloid plaques in the TgCRND8 mouse model of AD. Using a cultured microglial cell line, ATP7A expression was found to be increased by the pro-inflammatory cytokine IFN-γ, but not by TNFα or IL-1β. IFN-γ also elicited marked changes in copper homeostasis, including copper-dependent trafficking of ATP7A from the Golgi to cytoplasmic vesicles, increased copper uptake and elevated expression of the CTR1 copper importer. These findings suggest that pro-inflammatory conditions associated with AD cause marked changes in microglial copper trafficking, which may underlie the changes in copper homeostasis in AD. It is concluded that copper sequestration by microglia may provide a neuroprotective mechanism in AD.
doi:10.1111/j.1471-4159.2010.06888.x
PMCID: PMC2945454  PMID: 20626553
Alzheimer’s disease; copper homeostasis; ATP7A; microglia; inflammation
13.  Copper Modulation as a Therapy for Alzheimer's Disease? 
The role of metals in the pathophysiology of Alzheimer's disease (AD) has gained considerable support in recent years, with both in vitro and in vivo data demonstrating that a mis-metabolism of metal ions, such as copper and zinc, may affect various cellular cascades that ultimately leads to the development and/or potentiation of AD. In this paper, we will provide an overview of the preclinical and clinical literature that specifically relates to attempts to affect the AD cascade by the modulation of brain copper levels. We will also detail our own novel animal data, where we treated APP/PS1 (7-8 months old) mice with either high copper (20 ppm in the drinking water), high cholesterol (2% supplement in the food) or a combination of both and then assessed β-amyloid (Aβ) burden (soluble and insoluble Aβ), APP levels and behavioural performance in the Morris water maze. These data support an interaction between copper/cholesterol and both Aβ and APP and further highlight the potential role of metal ion dyshomeostasis in AD.
doi:10.4061/2011/370345
PMCID: PMC3162974  PMID: 21876828
14.  GSK-3 in Neurodegenerative Diseases 
Glycogen synthase kinase-3 (GSK-3) regulates multiple cellular processes, and its dysregulation is implicated in the pathogenesis of diverse diseases. In this paper we will focus on the dysfunction of GSK-3 in Alzheimer's disease and Parkinson's disease. Specifically, GSK-3 is known to interact with tau, β-amyloid (Aβ), and α-synuclein, and as such may be crucially involved in both diseases. Aβ production, for example, is regulated by GSK-3, and its toxicity is mediated by GSK-induced tau phosphorylation and degeneration. α-synuclein is a substrate for GSK-3 and GSK-3 inhibition protects against Parkinsonian toxins. Lithium, a GSK-3 inhibitor, has also been shown to affect tau, Aβ, and α-synuclein in cell culture, and transgenic animal models. Thus, understanding the role of GSK-3 in neurodegenerative diseases will enhance our understanding of the basic mechanisms underlying the pathogenesis of these disorders and also facilitate the identification of new therapeutic avenues.
doi:10.4061/2011/189246
PMCID: PMC3100544  PMID: 21629738
15.  Metal Ionophore Treatment Restores Dendritic Spine Density and Synaptic Protein Levels in a Mouse Model of Alzheimer's Disease 
PLoS ONE  2011;6(3):e17669.
We have previously demonstrated that brief treatment of APP transgenic mice with metal ionophores (PBT2, Prana Biotechnology) rapidly and markedly improves learning and memory. To understand the potential mechanisms of action underlying this phenomenon we examined hippocampal dendritic spine density, and the levels of key proteins involved in learning and memory, in young (4 months) and old (14 months) female Tg2576 mice following brief (11 days) oral treatment with PBT2 (30 mg/kg/d). Transgenic mice exhibited deficits in spine density compared to littermate controls that were significantly rescued by PBT2 treatment in both the young (+17%, p<0.001) and old (+32%, p<0.001) animals. There was no effect of PBT2 on spine density in the control animals. In the transgenic animals, PBT2 treatment also resulted in significant increases in brain levels of CamKII (+57%, p = 0.005), spinophilin (+37%, p = 0.04), NMDAR1A (+126%, p = 0.02), NMDAR2A (+70%, p = 0.05), pro-BDNF (+19%, p = 0.02) and BDNF (+19%, p = 0.04). While PBT2-treatment did not significantly alter neurite-length in vivo, it did increase neurite outgrowth (+200%, p = 0.006) in cultured cells, and this was abolished by co-incubation with the transition metal chelator, diamsar. These data suggest that PBT2 may affect multiple aspects of snaptic health/efficacy. In Alzheimer's disease therefore, PBT2 may restore the uptake of physiological metal ions trapped within extracellular β-amyloid aggregates that then induce biochemical and anatomical changes to improve cognitive function.
doi:10.1371/journal.pone.0017669
PMCID: PMC3055881  PMID: 21412423
16.  A Copper Binding Site within the Pathological Conformer Epitope of Mutant SOD1 
doi:10.3389/fnins.2011.00097
PMCID: PMC3154295  PMID: 21886603
17.  Syntaxin 5 Is Required for Copper Homeostasis in Drosophila and Mammals 
PLoS ONE  2010;5(12):e14303.
Copper is essential for aerobic life, but many aspects of its cellular uptake and distribution remain to be fully elucidated. A genome-wide screen for copper homeostasis genes in Drosophila melanogaster identified the SNARE gene Syntaxin 5 (Syx5) as playing an important role in copper regulation; flies heterozygous for a null mutation in Syx5 display increased tolerance to high dietary copper. The phenotype is shown here to be due to a decrease in copper accumulation, a mechanism also observed in both Drosophila and human cell lines. Studies in adult Drosophila tissue suggest that very low levels of Syx5 result in neuronal defects and lethality, and increased levels also generate neuronal defects. In contrast, mild suppression generates a phenotype typical of copper-deficiency in viable, fertile flies and is exacerbated by co-suppression of the copper uptake gene Ctr1A. Reduced copper uptake appears to be due to reduced levels at the plasma membrane of the copper uptake transporter, Ctr1. Thus Syx5 plays an essential role in copper homeostasis and is a candidate gene for copper-related disease in humans.
doi:10.1371/journal.pone.0014303
PMCID: PMC3004795  PMID: 21188142
18.  Clioquinol Inhibits Zinc-Triggered Caspase Activation in the Hippocampal CA1 Region of a Global Ischemic Gerbil Model 
PLoS ONE  2010;5(7):e11888.
Background
Excessive release of chelatable zinc from excitatory synaptic vesicles is involved in the pathogenesis of selective neuronal cell death following transient forebrain ischemia. The present study was designed to examine the neuroprotective effect of a membrane-permeable zinc chelator, clioquinol (CQ), in the CA1 region of the gerbil hippocampus after transient global ischemia.
Methodology/Principal Findings
The common carotid arteries were occluded bilaterally, and CQ (10 mg/kg, i.p.) was injected into gerbils once a day. The zinc chelating effect of CQ was examined with TSQ fluorescence and autometallography. Neuronal death, the expression levels of caspases and apoptosis inducing factor (AIF) were evaluated using TUNEL, in situ hybridization and Western blotting, respectively. We were able to show for the first time that CQ treatment attenuates the ischemia-induced zinc accumulation in the CA1 pyramidal neurons, accompanied by less neuronal loss in the CA1 field of the hippocampus after ischemia. Furthermore, the expression levels of caspase-3, -9, and AIF were significantly decreased in the hippocampus of CQ-treated gerbils.
Conclusions/Significance
The present study indicates that the neuroprotective effect of CQ is related to downregulation of zinc-triggered caspase activation in the hippocampal CA1 region of gerbils with global ischemia.
doi:10.1371/journal.pone.0011888
PMCID: PMC2912365  PMID: 20686690
19.  Zinc and Copper Modulate Alzheimer Aβ Levels in Human Cerebrospinal Fluid 
Neurobiology of aging  2007;30(7):1069-1077.
Abnormal interaction of β-amyloid 42 (Aβ42) with copper, zinc and iron induce peptide aggregation and oxidation in Alzheimer's disease (AD). However, in health, Aβ degradation is mediated by extracellular metalloproteinases, neprilysin, insulin degrading enzyme (IDE) and matrix metalloproteinases. We investigated the relationship between levels of Aβ and biological metals in CSF. We assayed CSF copper, zinc, other metals and Aβ42 in ventricular autopsy samples of Japanese American men (N= 131) from the population-based Honolulu–Asia Aging Study. There was a significant inverse correlation of CSF Aβ42 with copper, zinc, iron, manganese and chromium. The association was particularly strong in the subgroup with high levels of both zinc and copper. Selenium and aluminum levels were not associated to CSF Aβ42. In vitro, the degradation of synthetic Aβ substrate added to CSF was markedly accelerated by low levels (2 μM) of exogenous zinc and copper. While excessive interaction with copper and zinc may induce neocortical Aβ precipitation in AD, soluble Aβ degradation is normally promoted by physiological copper and zinc concentrations.
doi:10.1016/j.neurobiolaging.2007.10.012
PMCID: PMC2709821  PMID: 18068270
amyloid; Alzheimer's disease; metalloproteinase; cerebrospinal fluid; zinc; copper; iron; manganese; chromium
20.  DISC1 Regulates Primary Cilia That Display Specific Dopamine Receptors 
PLoS ONE  2010;5(5):e10902.
Background
Mutations in the DISC1 gene are strongly associated with major psychiatric syndromes such as schizophrenia. DISC1 encodes a cytoplasmic protein with many potential interaction partners, but its cellular functions remain poorly understood. We identified a role of DISC1 in the cell biology of primary cilia that display disease-relevant dopamine receptors.
Methodology/Principal Findings
A GFP-tagged DISC1 construct expressed in NIH3T3 cells and rat striatal neurons localized near the base of primary cilia. RNAi-mediated knockdown of endogenous DISC1 resulted in a marked reduction in the number of cells expressing a primary cilium. FLAG-tagged versions of the cloned human D1, D2 and D5 dopamine receptors concentrated highly on the ciliary surface, and this reflects a specific targeting mechanism specific because D3 and D4 receptors localized to the plasma membrane but were not concentrated on cilia.
Conclusions/Significance
These results identify a role of DISC1 in regulating the formation and/or maintenance of primary cilia, and establish subtype-specific targeting of dopamine receptors to the ciliary surface. Our findings provide new insight to receptor cell biology and suggest a relationship between DISC1 and neural dopamine signaling.
doi:10.1371/journal.pone.0010902
PMCID: PMC2878344  PMID: 20531939
21.  Heme Mediates Cytotoxicity from Artemisinin and Serves as a General Anti-Proliferation Target 
PLoS ONE  2009;4(10):e7472.
Heme (Fe2+ protoporphyrin IX) is an essential molecule that has been implicated the potent antimalarial action of artemisinin and its derivatives, although the source and nature of the heme remain controversial. Artemisinins also exhibit selective cytotoxicity against cancer cells in vitro and in vivo. We demonstrate that intracellular heme is the physiologically relevant mediator of the cytotoxic effects of artemisinins. Increasing intracellular heme synthesis through the addition of aminolevulinic acid, protoporphyrin IX, or transferrin-bound iron increased the cytotoxicity of dihydroartemisinin, while decreasing heme synthesis through the addition of succinyl acetone decreased its cytotoxic activity. A simple and robust high throughput assay was developed to screen chemical compounds that were capable of interacting with heme. A natural products library was screened which identified the compound coralyne, in addition to artemisinin, as a heme interacting compound with heme synthesis dependent cytotoxic activity. These results indicate that cellular heme may serve a general target for the development of both anti-parasitic and anti-cancer therapeutics.
doi:10.1371/journal.pone.0007472
PMCID: PMC2764339  PMID: 19862332
22.  Gene Expression Profiling in Cells with Enhanced γ-Secretase Activity 
PLoS ONE  2009;4(9):e6952.
Background
Processing by γ-secretase of many type-I membrane protein substrates triggers signaling cascades by releasing intracellular domains (ICDs) that, following nuclear translocation, modulate the transcription of different genes regulating a diverse array of cellular and biological processes. Because the list of γ-secretase substrates is growing quickly and this enzyme is a cancer and Alzheimer's disease therapeutic target, the mapping of γ-secretase activity susceptible gene transcription is important for sharpening our view of specific affected genes, molecular functions and biological pathways.
Methodology/Principal Findings
To identify genes and molecular functions transcriptionally affected by γ-secretase activity, the cellular transcriptomes of Chinese hamster ovary (CHO) cells with enhanced and inhibited γ-secretase activity were analyzed and compared by cDNA microarray. The functional clustering by FatiGO of the 1,981 identified genes revealed over- and under-represented groups with multiple activities and functions. Single genes with the most pronounced transcriptional susceptibility to γ-secretase activity were evaluated by real-time PCR. Among the 21 validated genes, the strikingly decreased transcription of PTPRG and AMN1 and increased transcription of UPP1 potentially support data on cell cycle disturbances relevant to cancer, stem cell and neurodegenerative diseases' research. The mapping of interactions of proteins encoded by the validated genes exclusively relied on evidence-based data and revealed broad effects on Wnt pathway members, including WNT3A and DVL3. Intriguingly, the transcription of TERA, a gene of unknown function, is affected by γ-secretase activity and was significantly altered in the analyzed human Alzheimer's disease brain cortices.
Conclusions/Significance
Investigating the effects of γ-secretase activity on gene transcription has revealed several affected clusters of molecular functions and, more specifically, 21 genes that hold significant potential for a better understanding of the biology of γ-secretase and its roles in cancer and Alzheimer's disease pathology.
doi:10.1371/journal.pone.0006952
PMCID: PMC2739295  PMID: 19763259
23.  Hypoxia-inducible Factor Prolyl 4-Hydroxylase Inhibition A TARGET FOR NEUROPROTECTION IN THE CENTRAL NERVOUS SYSTEM* 
The Journal of biological chemistry  2005;280(50):41732-41743.
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.
doi:10.1074/jbc.M504963200
PMCID: PMC2586128  PMID: 16227210
24.  Interaction of C-Terminal Truncated Human αA-Crystallins with Target Proteins 
PLoS ONE  2008;3(9):e3175.
Background
Significant portion of αA-crystallin in human lenses exists as C-terminal residues cleaved at residues 172, 168, and 162. Chaperone activity, determined with alcohol dehydrogenase (ADH) and βL-crystallin as target proteins, was increased in αA1–172 and decreased in αA1–168 and αA1–162. The purpose of this study was to show whether the absence of the C-terminal residues influences protein-protein interactions with target proteins.
Methodology/Principal Findings
Our hypothesis is that the chaperone-target protein binding kinetics, otherwise termed subunit exchange rates, are expected to reflect the changes in chaperone activity. To study this, we have relied on fluorescence resonance energy transfer (FRET) utilizing amine specific and cysteine specific fluorescent probes. The subunit exchange rate (k) for ADH and αA1–172 was nearly the same as that of ADH and αA-wt, αA1–168 had lower and αA1–162 had the lowest k values. When βL-crystallin was used as the target protein, αA1–172 had slightly higher k value than αA-wt and αA1–168 and αA1–162 had lower k values. As expected from earlier studies, the chaperone activity of αA1–172 was slightly better than that of αA-wt, the chaperone activity of αA1–168 was similar to that of αA-wt and αA1–162 had substantially decreased chaperone activity.
Conclusions/Significance
Cleavage of eleven C-terminal residues including Arg-163 and the C-terminal flexible arm significantly affects the interaction with target proteins. The predominantly hydrophilic flexible arm appears to be needed to keep the chaperone-target protein complex soluble.
doi:10.1371/journal.pone.0003175
PMCID: PMC2527529  PMID: 18779867
25.  Impairment of Adolescent Hippocampal Plasticity in a Mouse Model for Alzheimer's Disease Precedes Disease Phenotype 
PLoS ONE  2008;3(7):e2759.
The amyloid precursor protein (APP) was assumed to be an important neuron-morphoregulatory protein and plays a central role in Alzheimer's disease (AD) pathology. In the study presented here, we analyzed the APP-transgenic mouse model APP23 using 2-dimensional gel electrophoresis technology in combination with DIGE and mass spectrometry. We investigated cortex and hippocampus of transgenic and wildtype mice at 1, 2, 7 and 15 months of age. Furthermore, cortices of 16 days old embryos were analyzed. When comparing the protein patterns of APP23 with wildtype mice, we detected a relatively large number of altered protein spots at all age stages and brain regions examined which largely preceded the occurrence of amyloid plaques. Interestingly, in hippocampus of adolescent, two-month old mice, a considerable peak in the number of protein changes was observed. Moreover, when protein patterns were compared longitudinally between age stages, we found that a large number of proteins were altered in wildtype mice. Those alterations were largely absent in hippocampus of APP23 mice at two months of age although not in other stages compared. Apparently, the large difference in the hippocampal protein patterns between two-month old APP23 and wildtype mice was caused by the absence of distinct developmental changes in the hippocampal proteome of APP23 mice. In summary, the absence of developmental proteome alterations as well as a down-regulation of proteins related to plasticity suggest the disturption of a normally occurring peak of hippocampal plasticity during adolescence in APP23 mice. Our findings are in line with the observation that AD is preceded by a clinically silent period of several years to decades. We also demonstrate that it is of utmost importance to analyze different brain regions and different age stages to obtain information about disease-causing mechanisms.
doi:10.1371/journal.pone.0002759
PMCID: PMC2447155  PMID: 18648492

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