Iron-responsive elements (IREs) are the RNA stem loops that control cellular iron homeostasis by regulating ferritin translation and transferrin receptor mRNA stability. We mapped a novel iron-responsive element (IRE-Type II) within the 5-untranslated region (5-UTR) of the Alzheimer's amyloid precursor protein (APP) transcript (؉51 to ؉94 from the 5-cap site). The APP mRNA IRE is located immediately upstream of an interleukin-1 responsive acute box domain (؉101 to ؉146). APP 5-UTR conferred translation was selectively downregulated in response to intracellular iron chelation using three separate reporter assays (chloramphenicol acetyltransferase, luciferase, and red fluorescent protein reflecting an inhibition of APP holoprotein translation in response to iron chelation. Iron influx reversed this inhibition. As an internal control to ensure specificity, a viral internal ribosome entry sequence was unresponsive to intracellular iron chelation with desferrioxamine. Using RNA mobility shift assays, the APP 5-UTRs, encompassing the IRE, bind specifically to recombinant iron-regulatory proteins (IRP) and to IRP from neuroblastoma cell lysates. IRP binding to the APP 5-UTR is reduced after treatment of cells with desferrioxamine and increased after interleukin-1 stimulation. IRP binding is abrogated when APP cRNA probe is mutated in the core IRE domain (⌬4 bases:⌬83AGAG86). Iron regulation of APP mRNA through the APP 5-UTR points to a role for iron in the metabolism of APP and confirms that this RNA structure can be a target for the selection of small molecule drugs, such as desferrioxamine (Fe chelator) and clioquinol (Fe, Cu, and Zn chelator), which reduce A␤ peptide burden during Alzheimer's disease. The amyloid precursor protein (APP)1 is cleaved into the 40 -42-amino acid A␤ peptides that constitute the main component of the neurotoxic amyloid plaques formed during the progression of Alzheimer's disease (AD) and Down's syndrome (1, 2). In healthy individuals, APP holoprotein is expressed ubiquitously as a protein resembling a type I transmembrane receptor and metal-binding protein (3-6). Secreted APP (APP(s)) is neurotrophic (7).There are now several reports supporting an important role for translational regulatory mechanisms to control APP synthesis and probably A␤ peptide secretion in biologically relevant circumstances (8). First, interleukin-1 (IL-1), the first cytokine released during the acute phase response, significantly increases APP protein synthesis in astrocytes without altering APP mRNA levels (9). IL-1 acts by regulating APP and ferritin genes at the level of message translation (9). Second, reversible ischemic assault significantly increases APP levels without any alteration in the steady-state levels of APP mRNA in rabbit spinal cord neurons (10). Third, APP mRNA 3Ј-UTR sequences located between alternative poly(A) selection sites maintain efficient translation of microinjected APP in Xenopus oocytes and in Chinese hamster ovary transfectants (11).Iron-responsive elements (IREs) are RNA stem loops...

Calcineurin (CN), a calcium-and calmodulin-dependent protein phosphatase, plays a significant role in the central nervous system. Previously, we reported that forebrain-specific CN knockout mice (CN mutant mice) have impaired working memory. To further analyze the behavioral effects of CN deficiency, we subjected CN mutant mice to a comprehensive behavioral test battery. Mutant mice showed increased locomotor activity, decreased social interaction, and impairments in prepulse inhibition and latent inhibition. In addition, CN mutant mice displayed an increased response to the locomotor stimulating effects of MK-801. Collectively, the abnormalities of CN mutant mice are strikingly similar to those described for schizophrenia. We propose that alterations affecting CN signaling could comprise a contributing factor in schizophrenia pathogenesis.C alcineurin (CN), also called protein phosphatase 2B, is a heterodimeric Ca 2ϩ ͞calmodulin-dependent serine͞threonine protein phosphatase composed of CNB regulatory and CNA catalytic subunits (1). Originally identified in the brain, CN was later found to play a critical role in T cell function, through activation of nuclear factor of activated T cell-mediated transcription of cytokine genes, including the IL-2 gene (2, 3). This action of CN comprises a target for the immunosuppressants, cyclosporin A and FK506, which associate with immunophilins and bind to and inactivate CN. More recently, CN has been shown to play an important role in CNS functions, including neurite extension, synaptic plasticity and learning and memory (4, 5).We previously reported a severe and specific working memory deficit of forebrain specific CNB-deficient mice (CN mutant mice) as assessed by delayed matching to place Morris water maze and eight-arm radial maze paradigms (5). To further investigate the behavioral significance of CN, CN mutant mice were subjected to a comprehensive behavioral test battery (6, 7). CN mutant mice display a spectrum of abnormalities that is strikingly similar to those observed in schizophrenia patients. In addition, a number of supporting lines of evidence are consistent with the possibility that alterations in CN function occur in schizophrenia. Furthermore, in our accompanying paper, we investigated the potential involvement of altered CN signaling in genetic susceptibility to schizophrenia, and we report evidence supporting association of the PPP3CC gene encoding the CNA␥ catalytic subunit with disease (8). Based on these findings, we propose that alterations affecting CN signaling could comprise an important contributing factor in schizophrenia pathogenesis. Materials and MethodsAnimals and Experimental Design. The generation of the CN mutants is detailed elsewhere (5). The background strain used to generate the mutation was C57BL͞6J. All mutant and control mice were in a pure C57BL͞6J background. All CN mutant mice consisted of homozygous floxed, heterozygous Cre recombinase transgenic mice. All control mice consisted of homozygous or heterozygous floxed, Cre transg...

The amyloid precursor protein (APP) has been associated with Alzheimer's disease (AD) because APP is processed into the ␤-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-1␣ and IL-1␤ 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-1␣ and IL-1␤ to a chloramphenicol acetyltransferase (CAT) reporter gene. Steady-state levels of transfected APP(5-UTR)/CAT mRNAs were unchanged, whereas both baseline 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.

We have developed methods to use anticyclin A, B, and E antibodies as reagents to specifically detect proliferating cells in specific phases of the cell cycle in formalin-fixed, paraffin-embedded sections of tissues and cells. Staining of 48 archival cases of breast cancer showed that these antibodies estimate the tumor proliferation fraction and therefore are potentially useful for the prediction of prognosis. A subset of cancers had a high frequency of tumor cells expressing cyclins A and E, out of proportion to other proliferation markers, suggesting that these tumors may have deregulated cyclin expression. In addition to recognizing authentic cyclin E in the nucleus of proliferating cells, anticyclin E antibody cross-reacted with a cytoplasmic protein in nonproliferating endothelial cells. This cross-reaction allows the simultaneous visualization and quantitation of microvessels in the tumors, measuring a second potential predictor of breast cancer prognosis, tumor angiogenesis.Cyclins are proteins that vary in abundance and associate with and activate different cyclin-dependent kinases (cdk) at different stages of the cell cycle, a given cyclin-cdk complex being essential for passage through a specific stage in the cycle. The periodic appearance of the cyclins in distinct phases of the cell cycle suggests that they can be used as markers for proliferation of tissues. Because the three different cyclins mark different phases of the cell cycle-E for G1 and early S, A for S and G2, and B for late G2 (1-3)-the fraction of cells positive for a given cyclin should predict the fraction in a given phase of the cell cycle. Further, increased expression of various cyclins has been noted in extracts of human cancers, cyclin E in particular being markedly elevated in multiple tumors of different origins (4, 5), and an easy method for detecting such tumors will allow their biological behavior to be separately followed in clinical studies. Estimation of the proliferative behavior of a tumor is important for the management and prognosis of breast cancers. After local excision of node-negative tumors, two-thirds of the patients are expected to do well without further adjuvant therapy, and one-third are expected to relapse. Since chemotherapy is most effective before widespread metastases, it is useful to be able to predict which cancers are likely to recur in order to avoid undertreating these patients or overtreating patients whose tumors are not expected to recur (6, 7). One such prognostic factor is the S phase fraction (SPF) of the tumor estimated by fluorescence-activated cell sorting (FACS) analysis for DNA content of singly suspended tumor cells (8-10). Patients with a large fraction of tumor cells in S and G2 have a poorer prognosis and SPF has been used to guide the selection of patients for adjuvant therapy.Despite its value, flow cytometry of breast cancers is not performed widely, mostly because such analyses require fresh, relatively large tumors with enough tumor left over after tissueThe publication ...