Characterization of the properties and trafficking of an anchorless form of the prion protein

Conversion of PrP(C) into PrP(Sc) is the central event in the pathogenesis of transmissible prion diseases. Although the molecular basis of this event and the intracellular compartment where it occurs are not yet understood, the association of PrP with cellular membranes and in particular its presence in detergent-resistant microdomains appears to be of critical importance. In addition it appears that scrapie conversion requires membrane-bound glycosylphosphatidylinositol (GPI)-linked PrP. The GPI anchor may affect either the conformation, the intracellular localization, or the association of the prion protein with specific membrane domains. However, how this occurs is not known. To understand the relevance of the GPI anchor for the cellular behavior of PrP, we have studied the biosynthesis and localization of a PrP version which lacks the GPI anchor attachment signal (PrP Delta GPI). We found that PrP Delta GPI is tethered to cell membranes and associates to membrane detergent-resistant microdomains but does not assume a transmembrane topology. Differently to PrP(C), this protein does not localize at the cell surface but is mainly released in the culture media in a fully glycosylated soluble form. The cellular behavior of anchorless PrP explains why PrP Delta GPI Tg mice can be infected but do not show the classical signs of the disorder, thus indicating that the plasma membrane localization of PrP(C) and/or of the converted scrapie form might be necessary for the development of a symptomatic disease

HIPK2 in cancer biology and therapy: Recent findings and future perspectives

Homeodomain-interacting protein kinase 2 (HIPK2) is a serine-threonine kinase that phosphorylates and regulates a plethora of transcriptional regulators and chromatin modifiers. The heterogeneity of its interactome allows HIPK2 to modulate several cellular processes and signaling pathways, ultimately regulating cell fate and proliferation. Because of its p53-dependent pro-apoptotic activity and its downregulation in many tumor types, HIPK2 is traditionally considered a bone fide tumor suppressor gene. However, recent findings revealed that the role of HIPK2 in the pathogenesis of cancer is much more complex, ranging from tumor suppressive to oncogenic, strongly depending on the cellular context. Here, we review the very recent data emerged in the last years about the involvement of HIPK2 in cancer biology and therapy, highlighting the various alterations of this kinase (downregulation, upregulation, mutations and/or delocalization) in dependence on the cancer types. In addition, we discuss the recent advancement in the understanding the tumor suppressive and oncogenic functions of HIPK2, its role in establishing the response to cancer therapies, and its regulation by cancer-associated microRNAs. All these data strengthen the idea that HIPK2 is a key player in many types of cancer; therefore, it could represent an important prognostic marker, a factor to predict therapy response, and even a therapeutic target itself

Nrf2 Pathway in Age-Related Neurological Disorders: Insights into MicroRNAs

A general hallmark of neurological diseases is the loss of redox homeostasis that triggers oxidative damages to biomolecules compromising neuronal function. Under physiological conditions the steady-state concentrations of reactive oxygen species (ROS) and reactive nitrogen species (RNS) are finely regulated for proper cellular functions. Reduced surveillance of endogenous antioxidant defenses and/or increased ROS/RNS production leads to oxidative stress with consequent alteration of physiological processes. Neuronal cells are particularly susceptible to ROS/RNS due to their biochemical composition. Overwhelming evidences indicate that nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-linked pathways are involved in protective mechanisms against oxidative stress by regulating antioxidant and phase II detoxifying genes. As such, Nrf2 deregulation has been linked to both aging and pathogenesis of many human chronic diseases, including neurodegenerative ones such as Parkinson's disease, Alzheimer's disease and amyotrophic lateral sclerosis. Nrf2 activity is tightly regulated by a fine balance between positive and negative modulators. A better understanding of the regulatory mechanisms underlying Nrf2 activity could help to develop novel therapeutic interventions to prevent, slow down or possibly reverse various pathological states. To this end, microRNAs (miRs) are attractive candidates because they are linked to intracellular redox status being regulated and, post-transcriptionally, regulating key components of ROS/RNS pathways, including Nrf2

Mitochondrial Abnormalities in Down Syndrome: Pathogenesis, Effects and Therapeutic Approaches

Down syndrome (DS) consists of a complex phenotype with constant features, such as mental retardation and hypotonia, and variable features, including heart defects and susceptibility to Alzheimer’s disease, type 2 diabetes, obesity and immune disorders. Overexpression of genes mapping to chromosome 21 (Hsa21) is directly or indirectly responsible for pathogenesis of DS phenotypic features, as overexpressed Hsa21 genes dysregulate several other genes mapping to different chromosomes. Many of these genes are involved in mitochondrial function. Recent studies highlight a link between mitochondrial dysfunction, consistently observed in DS subjects, and DS phenotype. In this review, we first provide a basic overview of mitochondrial alterations in DS in terms of mitochondrial bioenergetics, biogenesis and morphology. We then discuss how mitochondrial malfunction may contribute to the pathogenesis of clinical manifestations and how specific Hsa21 genes may cause the disruption of mitochondrial phenotype. Finally, we focus on drugs, which affect mitochondrial function and network to propose possible therapeutic approaches aimed at improving and/or preventing various aspects of the DS phenotype. Our working hypothesis is that correcting the mitochondrial defect might improve the neurological phenotype and prevent DS-associated pathologies, thus providing a better quality of life for DS individuals and their families

Lipid Rafts and Clathrin Cooperate in the Internalization of PrPC in Epithelial FRT Cells

The cellular prion protein (PrP(C)) plays a key role in the pathogenesis of Transmissible Spongiform Encephalopathies in which the protein undergoes post-translational conversion to the infectious form (PrP(Sc)). Although endocytosis appears to be required for this conversion, the mechanism of PrP(C) internalization is still debated, as caveolae/raft- and clathrin-dependent processes have all been reported to be involved. METHODOLOGY/PRINCIPAL FINDINGS: We have investigated the mechanism of PrP(C) endocytosis in Fischer Rat Thyroid (FRT) cells, which lack caveolin-1 (cav-1) and caveolae, and in FRT/cav-1 cells which form functional caveolae. We show that PrP(C) internalization requires activated Cdc-42 and is sensitive to cholesterol depletion but not to cav-1 expression suggesting a role for rafts but not for caveolae in PrP(C) endocytosis. PrP(C) internalization is also affected by knock down of clathrin and by the expression of dominant negative Eps15 and Dynamin 2 mutants, indicating the involvement of a clathrin-dependent pathway. Notably, PrP(C) co-immunoprecipitates with clathrin and remains associated with detergent-insoluble microdomains during internalization thus indicating that PrP(C) can enter the cell via multiple pathways and that rafts and clathrin cooperate in its internalization. CONCLUSIONS/SIGNIFICANCE: These findings are of particular interest if we consider that the internalization route/s undertaken by PrP(C) can be crucial for the ability of different prion strains to infect and to replicate in different cell lines

Caveolin Transfection Results in Caveolae Formation but Not Apical Sorting of Glycosylphosphatidylinositol (GPI)-anchored Proteins in Epithelial Cells

Most epithelial cells sort glycosylphosphatidylinositol (GPI)-anchored proteins to the apical surface. The “raft” hypothesis, based on data mainly obtained in the prototype cell line MDCK, postulates that apical sorting depends on the incorporation of apical proteins into cholesterol/glycosphingolipid (GSL) rafts, rich in the cholesterol binding protein caveolin/VIP21, in the Golgi apparatus. Fischer rat thyroid (FRT) cells constitute an ideal model to test this hypothesis, since they missort both endogenous and transfected GPI- anchored proteins to the basolateral plasma membrane and fail to incorporate them into cholesterol/glycosphingolipid clusters. Because FRT cells lack caveolin, a major component of the caveolar coat that has been proposed to have a role in apical sorting of GPI- anchored proteins (Zurzolo, C., W. Van't Hoff, G. van Meer, and E. Rodriguez-Boulan. 1994. EMBO [Eur. Mol. Biol. Organ.] J. 13:42–53.), we carried out experiments to determine whether the lack of caveolin accounted for the sorting/clustering defect of GPI- anchored proteins. We report here that FRT cells lack morphological caveolae, but, upon stable transfection of the caveolin1 gene (cav1), form typical flask-shaped caveolae. However, cav1 expression did not redistribute GPI-anchored proteins to the apical surface, nor promote their inclusion into cholesterol/GSL rafts. Our results demonstrate that the absence of caveolin1 and morphologically identifiable caveolae cannot explain the inability of FRT cells to sort GPI-anchored proteins to the apical domain. Thus, FRT cells may lack additional factors required for apical sorting or for the clustering with GSLs of GPI-anchored proteins, or express factors that inhibit these events. Alternatively, cav1 and caveolae may not be directly involved in these processes

Overexpression of the Hsa21 Transcription Factor RUNX1 Modulates the Extracellular Matrix in Trisomy 21 Cells

Down syndrome is a neurodevelopmental disorder frequently characterized by other developmental defects, such as congenital heart disease. Analysis of gene expression profiles of hearts from trisomic fetuses have shown upregulation of extracellular matrix (ECM) genes. The aim of this work was to identify genes on chromosome 21 potentially responsible for the upregulation of ECM genes and to pinpoint any functional consequences of this upregulation. By gene set enrichment analysis of public data sets, we identified the transcription factor RUNX1, which maps to chromosome 21, as a possible candidate for regulation of ECM genes. We assessed that approximately 80% of ECM genes overexpressed in trisomic hearts have consensus sequences for RUNX1 in their promoters. We found that in human fetal fibroblasts with chromosome 21 trisomy there is increased expression of both RUNX1 and several ECM genes, whether located on chromosome 21 or not. SiRNA silencing of RUNX1 reduced the expression of 11 of the 14 ECM genes analyzed. In addition, collagen IV, an ECM protein secreted in high concentrations in the culture media of trisomic fibroblasts, was modulated by RUNX1 silencing. Attenuated expression of RUNX1 increased the migratory capacity of trisomic fibroblasts, which are characterized by a reduced migratory capacity compared to euploid controls

Truncated Analogues of a G-Quadruplex-Forming Aptamer Targeting Mutant Huntingtin: Shorter Is Better!

Two analogues of the MS3 aptamer, which was previously shown to have an exquisite capability to selectively bind and modulate the activity of mutant huntingtin (mHTT), have been here designed and evaluated in their physicochemical and biological properties. Featured by a distinctive propensity to form complex G-quadruplex structures, including large multimeric aggregates, the original 36-mer MS3 has been truncated to give a 33-mer (here named MS3-33) and a 17-mer (here named MS3-17). A combined use of different techniques (UV, CD, DSC, gel electrophoresis) allowed a detailed physicochemical characterization of these novel G-quadruplex-forming aptamers, tested in vitro on SH-SY5Y cells and in vivo on a Drosophila Huntington’s disease model, in which these shorter MS3-derived oligonucleotides proved to have improved bioactivity in comparison with the parent aptamer

Fighting the Huntington's Disease with a G-Quadruplex-Forming Aptamer Specifically Binding to Mutant Huntingtin Protein: Biophysical Characterization, In Vitro and In Vivo Studies

A set of guanine-rich aptamers able to preferentially recognize full-length huntingtin with an expanded polyglutamine tract has been recently identified, showing high efficacy in modulating the functions of the mutated protein in a variety of cell experiments. We here report a detailed biophysical characterization of the best aptamer in the series, named MS3, proved to adopt a stable, parallel G-quadruplex structure and show high nuclease resistance in serum. Confocal microscopy experiments on HeLa and SH-SY5Y cells, as models of non-neuronal and neuronal cells, respectively, showed a rapid, dose-dependent uptake of fluorescein-labelled MS3, demonstrating its effective internalization, even in the absence of transfecting agents, with no general cytotoxicity. Then, using a well-established Drosophila melanogaster model for Huntington's disease, which expresses the mutated form of human huntingtin, a significant improvement in the motor neuronal function in flies fed with MS3 was observed, proving the in vivo efficacy of this aptamer

Wilson Disease Protein ATP7B Utilizes Lysosomal Exocytosis to Maintain Copper Homeostasis

SummaryCopper is an essential yet toxic metal and its overload causes Wilson disease, a disorder due to mutations in copper transporter ATP7B. To remove excess copper into the bile, ATP7B traffics toward canalicular area of hepatocytes. However, the trafficking mechanisms of ATP7B remain elusive. Here, we show that, in response to elevated copper, ATP7B moves from the Golgi to lysosomes and imports metal into their lumen. ATP7B enables lysosomes to undergo exocytosis through the interaction with p62 subunit of dynactin that allows lysosome translocation toward the canalicular pole of hepatocytes. Activation of lysosomal exocytosis stimulates copper clearance from the hepatocytes and rescues the most frequent Wilson-disease-causing ATP7B mutant to the appropriate functional site. Our findings indicate that lysosomes serve as an important intermediate in ATP7B trafficking, whereas lysosomal exocytosis operates as an integral process in copper excretion and hence can be targeted for therapeutic approaches to combat Wilson disease