Jack T. Rogers

Iron-export ferroxidase activity of β-amyloid precursor protein is inhibited by zinc in Alzheimer's disease.

Alzheimers Disease (AD) is complicated by pro-oxidant intraneuronal Fe(2+) elevation as well as extracellular Zn(2+) accumulation within amyloid plaque. We found that the AD β-amyloid protein precursor (APP) possesses ferroxidase activity mediated by a conserved H-ferritin-like active site, which is inhibited specifically by Zn(2+). Like ceruloplasmin, APP catalytically oxidizes Fe(2+), loads Fe(3+) into transferrin, and has a major interaction with ferroportin in HEK293T cells (that lack ceruloplasmin) and in human cortical tissue. Ablation of APP in HEK293T cells and primary neurons induces marked iron retention, whereas increasing APP695 promotes iron export. Unlike normal mice, APP(-/-) mice are vulnerable to dietary iron exposure, which causes Fe(2+) accumulation and oxidative stress in cortical neurons. Paralleling iron accumulation, APP ferroxidase activity in AD postmortem neocortex is inhibited by endogenous Zn(2+), which we demonstrate can originate from Zn(2+)-laden amyloid aggregates and correlates with Aβ burden. Abnormal exchange of cortical zinc may link amyloid pathology with neuronal iron accumulation in AD.

Redox-active metals, oxidative stress, and Alzheimer's disease pathology.

Abstract: Considerable evidence is mounting that dyshomeostasis of the redox‐active biometals, Cu and Fe, and oxidative stress contribute to the neuropathology of Alzheimers disease (AD). Present data suggest that metals can interact directly with Aβ peptide, the principal component of β‐amyloid that is one of the primary lesions in AD. The binding of metals to Aβ modulates several physiochemical properties of Aβ that are thought to be central to the pathogenicity of the peptide. First, we and others have shown that metals can promote the in vitro aggregation into tinctorial Aβ amyloid. Studies have confirmed that insoluble amyloid plaques in postmortem AD brain are abnormally enriched in Cu, Fe, and Zn. Conversely, metal chelators dissolve these proteinaceous deposits from postmortem AD brain tissue and attenuate cerebral Aβ amyloid burden in APP transgenic mouse models of AD. Second, we have demonstrated that redox‐active Cu(II) and, to a lesser extent, Fe(III) are reduced in the presence of Aβ with concomitant production of reactive oxygen species (ROS), hydrogen peroxide (H2O2) and hydroxyl radical (OH•). These Aβ/metal redox reactions, which are silenced by redox‐inert Zn(II), but exacerbated by biological reducing agents, may lead directly to the widespread oxidation damages observed in AD brains. Moreover, studies have also shown that H2O2 mediates Aβ cellular toxicity and increases the production of both Aβ and amyloid precursor protein (APP). Third, the 5′ untranslated region (5′UTR) of APP mRNA has a functional iron‐response element (IRE), which is consistent with biochemical evidence that APP is a redox‐active metalloprotein. Hence, the redox interactions between Aβ, APP, and metals may be at the heart of a pathological positive feedback system wherein Aβ amyloidosis and oxidative stress promote each other. The emergence of redox‐active metals as key players in AD pathogenesis strongly argues that amyloid‐specific metal‐complexing agents and antioxidants be investigated as possible disease‐modifying agents for treating this horrible disease.

Iron-dependent regulation of the divalent metal ion transporter

The first step in intestinal iron absorption is mediated by the H+‐coupled Fe2+ transporter called divalent cation transporter 1/divalent metal ion transporter 1 (DCT1/DMT1) (also known as natural resistance‐associated macrophage protein 2). DCT1/DMT1 mRNA levels in the duodenum strongly increase in response to iron depletion. To study the mechanism of iron‐dependent DCT1/DMT1 mRNA regulation, we investigated the endogenous expression of DCT1/DMT1 mRNA in various cell types. We found that only the iron responsive element (IRE)‐containing form, which corresponds to one of two splice forms of DCT1/DMT1, is responsive to iron treatment and this responsiveness was cell type specific. We also examined the interaction of the putative 3′‐UTR IRE with iron responsive binding proteins (IRP1 and IRP2), and found that IRP1 binds to the DCT1/DMT1‐IRE with higher affinity compared to IRP2. This differential binding of IRP1 and IRP2 was also reported for the IREs of transferrin receptors, erythroid 5‐aminolevulinate synthase and mitochondrial aconitase. We propose that regulation of DCT1/DMT1 mRNA by iron involves post‐transcriptional regulation through the binding of IRP1 to the transporters IRE, as well as other as yet unknown factors.

Translation of the alzheimer amyloid precursor protein mRNA is up-regulated by interleukin-1 through 5'-untranslated region sequences.

The amyloid precursorprotein (APP) has been associated with Alzheimer’s disease (AD) because APP is processed into the β-peptide that accumulates in amyloid plaques, and APP gene mutations can cause early onset AD. Inflammation is also associated with AD as exemplified by increased expression of interleukin-1 (IL-1) in microglia in affected areas of the AD brain. Here we demonstrate that IL-1α and IL-1β increase APP synthesis by up to 6-fold in primary human astrocytes and by 15-fold in human astrocytoma cells without changing the steady-state levels of APP mRNA. A 90-nucleotide sequence in the APP gene 5′-untranslated region (5′-UTR) conferred translational regulation by IL-1α and IL-1β to a chloramphenicol acetyltransferase (CAT) reporter gene. Steady-state levels of transfected APP(5′-UTR)/CAT mRNAs were unchanged, whereas both base-line and IL-1-dependent CAT protein synthesis were increased. This APP mRNA translational enhancer maps from +55 to +144 nucleotides from the 5′-cap site and is homologous to related translational control elements in the 5′-UTR of the light and and heavy ferritin genes. Enhanced translation of APP mRNA provides a mechanism by which IL-1 influences the pathogenesis of AD.

IRON-REGULATORY PROTEINS, IRON-RESPONSIVE ELEMENTS AND FERRITIN MRNA TRANSLATION

Iron plays a central role in the metabolism of all cells. This is evident by its major contribution to many diverse functions, such as DNA replication, bacterial pathogenicity, photosynthesis, oxidative stress control and cell proliferation. In mammalian systems, control of intracellular iron homeostasis is largely due to posttranscriptional regulation of binding by iron-regulatory RNA-binding proteins (IRPs) to iron-responsive elements (IREs) within ferritin and transferrin receptor (TfR) mRNAs. the TfR transports iron into cells and the iron is subsequently stored within ferritin. IRP binding is under tight control so that it responds to changes in intracellular iron requirements in a coordinate manner by differentially regulating ferritin mRNA translational efficiency and TfR mRNA stability. Several different stimuli, as well as intracellular iron levels and oxidative stress, are capable of regulating these RNA-protein interactions. In this mini-review, we shall concentrate on the mechanisms underlying modulation of the interaction of IRPs and the ferritin IRE and its role in regulating ferritin gene expression.

Phenserine regulates translation of β-amyloid precursor protein mRNA by a putative interleukin-1 responsive element, a target for drug development

The reduction in levels of the potentially toxic amyloid-β peptide (Aβ) has emerged as one of the most important therapeutic goals in Alzheimers disease. Key targets for this goal are factors that affect the expression and processing of the Aβ precursor protein (βAPP). Earlier reports from our laboratory have shown that a novel cholinesterase inhibitor, phenserine, reduces βAPP levels in vivo. Herein, we studied the mechanism of phenserines actions to define the regulatory elements in βAPP processing. Phenserine treatment resulted in decreased secretion of soluble βAPP and Aβ into the conditioned media of human neuroblastoma cells without cellular toxicity. The regulation of βAPP protein expression by phenserine was posttranscriptional as it suppressed βAPP protein expression without altering βAPP mRNA levels. However, phenserines action was neither mediated through classical receptor signaling pathways, involving extracellular signal-regulated kinase or phosphatidylinositol 3-kinase activation, nor was it associated with the anticholinesterase activity of the drug. Furthermore, phenserine reduced expression of a chloramphenicol acetyltransferase reporter fused to the 5′-mRNA leader sequence of βAPP without altering expression of a control chloramphenicol acetyltransferase reporter. These studies suggest that phenserine reduces Aβ levels by regulating βAPP translation via the recently described iron regulatory element in the 5′-untranslated region of βAPP mRNA, which has been shown previously to be up-regulated in the presence of interleukin-1. This study identifies an approach for the regulation of βAPP expression that can result in a substantial reduction in the level of Aβ.

Thyroid Hormone Modulates the Interaction between Iron Regulatory Proteins and the Ferritin mRNA Iron-responsive Element

The cytoplasmic iron regulatory protein (IRP) modulates iron homeostasis by binding to iron-responsive elements (IREs) in the transferrin receptor and ferritin mRNAs to coordinately regulate transferrin receptor mRNA stability and ferritin mRNA translational efficiency, respectively. These studies demonstrate that thyroid hormone (T) can modulate the binding activity of the IRP to an IRE in vitro and in vivo. T augmented an iron-induced reduction in IRP binding activity to a ferritin IRE in RNA electrophoretic mobility shift assays using cytoplasmic extracts from human liver hepatoma (HepG2) cells. Hepatic IRP binding to the ferritin IRE also diminished after in vivo administration of T with iron to rats. In transient transfection studies using HepG2 cells and a human ferritin IRE-chloramphenicol acetyltransferase (H-IRE-CAT) construct, T augmented an iron-induced increase in CAT activity by 45%. RNase protection analysis showed that this increase in CAT activity was not due to a change in the steady state level of CAT mRNA. Nuclear T-receptors may be necessary for this T-induced response, because the effect could not be reproduced by the addition of T directly to cytoplasmic extracts and was absent in CV-1 cells which lack T-receptors. We conclude that T can functionally regulate the IRE binding activity of the IRP. These observations provide evidence of a novel mechanism for T to up-regulate hepatic ferritin expression, which may in part contribute to the elevated serum ferritin levels seen in hyperthyroidism.

New Therapeutic Strategies and Drug Candidates for Neurodegenerative Diseases: p53 and TNF‐α Inhibitors, and GLP‐1 Receptor Agonists

Abstract: Owing to improving preventative, diagnostic, and therapeutic measures for cardiovascular disease and a variety of cancers, the average ages of North Americans and Europeans continue to rise. Regrettably, accompanying this increase in life span, there has been an increase in the number of individuals afflicted with age‐related neurodegenerative disorders, such as Alzheimers disease, Parkinsons disease, and stroke. Although different cell types and brain areas are vulnerable among these, each disorder likely develops from activation of a common final cascade of biochemical and cellular events that eventually lead to neuronal dysfunction and death. In this regard, different triggers, including oxidative damage to DNA, the overactivation of glutamate receptors, and disruption of cellular calcium homeostasis, albeit initiated by different genetic and/or environmental factors, can instigate a cascade of intracellular events that induce apoptosis. To forestall the neurodegenerative process, we have chosen specific targets to inhibit that are at pivotal rate‐limiting steps within the pathological cascade. Such targets include TNF‐α, p53, and GLP‐1 receptor. The cytokine TNF‐α is elevated in Alzheimers disease, Parkinsons disease, stroke, and amyotrophic lateral sclerosis. Its synthesis can be reduced via posttranscriptional mechanisms with novel analogues of the classic drug, thalidomide. The intracellular protein and transcription factor, p53, is activated by the Alzheimers disease toxic peptide, Aβ, as well as by excess glutamate and hypoxia to trigger neural cell death. It is inactivated by novel tetrahydrobenzothiazole and ‐oxazole analogues to rescue cells from lethal insults. Stimulation of the glucagon‐like peptide‐1 receptor (GLP‐1R) in brain is associated with neurotrophic functions that, additionally, can protect cells against excess glutamate and other toxic insults.

Interleukin (IL) 1β Induction of IL-6 Is Mediated by a Novel Phosphatidylinositol 3-Kinase-dependent AKT/IκB Kinase α Pathway Targeting Activator Protein-1

Here we describe a novel role for the phosphatidylinositol 3-kinase/AKT pathway in mediating induction of interleukin-6 (IL-6) in response to IL-1. Pharmacological inhibition of phosphatidylinositol 3-kinase (PI3K) inhibited IL-6 mRNA and protein production. Overexpression of either dominant-negative AKT or IκB kinase α mutant, IKKαT23A, containing a mutation in a functional AKT phosphorylation site, shown previously to be important for NFκB activation, completely abrogated IL-6 promoter activation in response to IL-1. However, mutation of the consensus NFκB site on the IL-6 promoter did not abrogate promoter activation by IL-1 in contrast to the AP-1 site mutation. IL-1 induces phosphorylation of IKKα on the NFκB inducing kinase (NIK) phosphorylation sites Ser176/Ser180 and on the Thr23 site, and although phosphorylation of IKKαT23 is inhibited both by LY294002 and wortmannin, phosphorylation of Ser176/Ser180 is not. Neither inhibition of PI 3-kinase/AKT nor IKKαT23A overexpression affected IκBα degradation in response to IL-1. Only partial inhibition by dominant-negative AKT and no inhibitory effect of IKKαT23A was observed on an IL-6 promoter-specific NFκB site in contrast to significant inhibitory effects on the AP-1 site. Taken together, we have discovered a novel PI 3-kinase/AKT-dependent pathway in response to IL-1, encompassing PI 3-kinase/AKT/IKKαT23 upstream of AP-1. This novel pathway is a parallel pathway to the PI 3-kinase/AKT upstream of NFκB and both are involved in IL-6 gene transcription in response to IL-1.

The 5′-untranslated region of Parkinson's disease α -synuclein messengerRNA contains a predicted iron responsive element

The 5′-untranslated region of Parkinsons disease α -synuclein messengerRNA contains a predicted iron responsive element