Neena Singh - US grants

membrane proteins; intermolecular interaction; cell membrane; membrane lipids; cerebrohepatorenal syndrome; achondroplasia; glycolipids; peroxisome; membrane activity; human genetic material tag; human tissue; transfection; tissue /cell culture;

DESCRIPTION: (From the Applicant's proposal): Gerstmann-Straussler-Schienker syndrome (GSS) is an inherited human prion disease with spongiform degeneration, astrocylic gliosis, prion protein (PrP) amyloid plaques, and neurofibrillary tangles. Point mutations at condons 102, 105, 117, 145, 198 and 217 of the prion protein gene (PRNP) segregate with this disorder. Amyloid plaque cores include internal PrP fragments of 7, 11, 15-20, and 25-30 kDa that polymerize into insoluble fibrils. The subcellular site and mechanism of production of these fragments is not known. PI's initial studies of an in vitro transfected cell model of GSS subtype with the Q217R mutation show that the localization, processing, and fate of mutant PrP (PrPM) and its fragments differ from those of normal PrP (PrPC) in the following ways: a) About 50 percent of PrPC is normally truncated at a site that disrupts the amyloidogenic region, but much smaller amounts of PrPM are truncated, and a significant proportion is cleaved at a unique site that does not disrupt the amyloidogenic region. b) All three forms of full-length and truncated PrPC are expressed at the cell surface, but only one truncated form, and small amounts of full-length PrPM are at the surface. Thus, cleavage of PrPC and PrPM may occur in different subcellular compartments, or the cleaved fragments are routed differentially. It is therefore important to identify the subcellular site(s) of cleavage of PrP. c) Significant quantities of PrPC and PrPM are released extracellularly into the culture medium. Their differential processing by microglia may also contribute to the amyloidogenic process. d) A proportion of PrPM is synthesized as an abnormal, relatively stable isoform that does not leave the endoplasmic reticulum (ER). The mechanisms of its retention and eventual degradation are not clear. ER-resident chaperones may be involved, and compromise of the cellular quality-control with age may affect its accumulation, extent of intracellular aggregation, and eventual processing by microglia after cell-death. The proposed study will evaluate: a) the subcellular site(s) of cleavage of PrPC and PrPM, b) whether secreted PrP is processed extracellularly, c) which ER-chaperones associate with the abnormal isoform of PrPM, and d) the distribution of normal and mutant PrP and its fragments in polarized cells. The experimental approach involves: a) identification of site(s) of cleavage by blocking transport of PrP at different sites, b) characterization of secreted PrP and its processing by neuroblastoma cells or primary cultures of microglia, c) evaluation of polarized distribution of PrPC, PrPM, and their fragments in polarized CaCo2 cells, and c) co-immunoprecipitation to check for associated ER-resident chaperones.

DESCRIPTION (Adapted from the Applicant's Abstract): Neuronal death in prion disorders is believed to result from a conformationally transformed, scrapie isoform (PrPSc) of the normal host prion protein (PrPC). The high correlation between PrPSc deposits and neurodegeneration has led to a cause and effect hypothesis. However, presence of neurodegeneration and transmission of prion diseases without detectable PrPSc suggest the presence of alternative mechanisms of neuronal death. Our long-term goal is to investigate potentially neurotoxic pathways of metabolism of normal and mutant PrP that initiate neurotoxicity without significant prpSC deposition. Recently, we have identified novel pathways of processing and turnover of mutant PrP with a stop codon at residue 145 (PrP'45), associated with a familial prion disorder. We believe that PrP'45 is neurotoxic through intracellular pathways. In particular, our data show that PrPi45 is degraded by the proteasomal pathway, and aggregates intracellularly. Surprisingly, a significant amount of PrP'45 is also rnistargeted to the nucleus. We hypothesize that cytotoxicity in this case is caused by perturbation of cellular metabolism by these unconventional pathways of PrP metabolism. Since fragments similar to PrP'45 are also generated by atypical processing of prpc and other mutant PrPs, the central goal of the present proposal is to analyze the cellular events leading to neurotoxicity by the abnormal accumulation of PrPt4s in the nucleus, and pathways of generation of similar PrP fragments in neuronal cells. In the first aim, we will identify specific nuclear localization signal(s) and the mechanism of transport of PrP'45 to the nucleus. The second aim will focus on whether accumulated PrP in the nucleus alters transcriptional activity that is physiologically relevant. In the third aim, we will determine if rnistargeted PrP is bound to specific nuclear proteins, and whether this association is biologically significant, and finally, we will analyze the mechanism(s) of generation of PrP fragments similar to PrPt45 from prpc or other mutant PrPs, since these would cause neurotoxicity in a manner similar to PrP'45. The experiments designed will use a variety of cell- and molecular biology techniques. In vitro nuclear transport, cell viability assays, in vitro translation, co-immunoprecipitation, Western blots, confocal immunomicroscopy and cell sorting will be used for the studies proposed in specific aims 1 and 2. For aims 3 and 4, Far Western analysis, electrophoretic mobility shift assay, in vitro transcriptional run-off assay, and differential mRNA display will be carried out. Our studies will provide a cell biological explanation for neurotoxicity of PrP that operates either concomitant with, or prior to PrPSc deposition, and help in developing strategies to disrupt these abnormal pathways of PrP metabolism.

[unreadable] DESCRIPTION (provided by applicant): Prion disorders manifest when a normal cell surface glycoprotein, the prion protein (PrPC), undergoes a conformational change from an a-helical to a 13-sheet rich structure (PrPSc) that is pathogenic. Deposits of PrPSC in the brain parenchyma are considered the principal cause of neurotoxicity in prion disorders. In familial forms of these disorders, a point mutation in the prion protein gene (PRNP) is thought to mediate the conformational change of mutant PrP to PrPSc, resulting in pathogenicity. Despite the reported high co-relation between PrPSc deposits and clinical disease, neurodegeneration in prion disorders is often seen without detectable PrPS, indicating the presence of alternative mechanisms of cellular toxicity. Results from my laboratory show that in familial prion disorders, abnormal metabolism of the mutant prion protein leads to neuronal toxicity by distinct cellular pathways, unique to each mutation. The stop codon mutation at residue 145 (PrP145) leads to accumulation of PrP145 in the endoplasmic reticulum (ER) and in the nucleus, while the Q21 7R mutation (PrP217) causes accumulation of PrP217 in the ER and in the lysosomal compartment. The P102L mutation (PrP'[unreadable]2), on the other hand, results in the accumulation of an amyloidogenic fragment of PrP'[unreadable]2 on the cell surface. Based on these observations, we hypothesize that point mutations in different region(s) of PrP alter PrP metabolism and initiate neuronal death through distinct intracellular pathways unique to each mutation (or a group of mutations), and not by the common pathway of PrPSc accumulation. In this proposal, we will investigate this hypothesis by analyzing the biogenesis of mutant PrP with point mutations in three distinct regions of PrP: (1) the glycosylphosphatidyl inositol anchor signal peptide (GPI-SP) of PrP, (2) up to twenty-five amino acids upstream from the GPI addition site, and (3) in the vicinity of PrP glycosylation sites. [unreadable] [unreadable] We will use transfected human neuroblastoma cells to investigate the processing, transport, and turnover of wild type and mutant PrP using immunoprecipitation, Western blotting, and other cell and molecular biology techniques. Sub-cellular localization of PrP will be examined by confocal and electron microscopy. The results obtained will be confirmed in differentiated NT-2N cells and human embryonic neurons in culture. A C-terminal Flag-tagged PrP and a PrP-GFP construct developed in my laboratory will be used to complement the above methods. The Flag tag will allow specific identification of the GPI-SP, and the PrP-GFP construct will enable analysis of mutant PrP in living cells. This study will uncover alternative pathways of neuronal toxicity by mutant PrP besides conversion to PrPSc, and help in developing rational therapeutic strategies.

DESCRIPTION (provided by applicant): The transmission of variant Creutzfeldt-Jakob disease (vCJD) to humans from bovine-spongiform encephalopathy (BSE)-contaminated meat, and the transmission of BSE by intra-venous inoculation of peripheral blood to experimental animals raises two important questions: 1) how are prions from food transported across the intestinal epithelial barrier, and 2) how do prions in the peripheral blood cross the endothelial blood brain barrier (BBB). These questions have gained increasing importance with the realization that close to one million BSE infected cows may have entered the human food chain. An emerging concern is the spread of Chronic Wasting Disease (CWD), a prion disease of the deer and elk in certain parts of USA, and the uncertainties regarding its transmission to livestock and humans. Despite these concerns, surprisingly little is known about the mechanism(s) by which the infectious prion or PrP-scrapie (PrPsc), a protein of 27-30kDa, is transported from the intestine or peripheral blood to the central nervous system. Preliminary data from my laboratory demonstrate that PrPsc in sporadic CJD (sCJD) brain homogenates is transported across epithelial cells in association with ferritin. When considered in context with additional data indicating an upregulation of brain ferritin levels in response to redox active iron in the brain parenchyma of sCJD cases, this observation raises important questions. We hypothesize that the transport of PrPsc across epithelial and endothelial cell barriers is facilitated by proteins like ferritin that have a defined transcytotic route, and that imbalance of brain iron homeostasis contributes directly to the pathogenesis of certain prion disorders, and indirectly by promoting infectivity through ferritin. Thus, the central goal of this proposal is to investigate the role of PrPs-associated proteins including ferritin and transferrin in facilitating its transport across the intestinal epithelium and the BBB, and to evaluate the role of redox active iron in the pathogenesis of prion disorders. The proposed studies will be carried out in three specific aims. In aim 1, the role of ferritin, transferrin, and other PrPsc-associated proteins in the transport of PrPsc across in vitro models of human intestinal epithelial cell barrier and the BBB will be evaluated. In aim 2, the results obtained from in vitro models in aim 1 will be confirmed in vivo in transgenic mice expressing human PrP. In aim 3, the role of redox active iron in the pathogenesis of priori disorders will be investigated using ferritin over-expressing and H-ferritin deletion transgenic mice. These studies will help in evaluating the risk of human population to BSE, vCJD, and CWD infection, and help in understanding the mechanism of prion disease pathogenesis.

DESCRIPTION (provided by applicant): The complexity and multiplicity of factors involved in prion disease pathogenesis has hampered the search for an effective therapeutic strategy. Though each report has improved our understanding of prion disease pathogenesis, neither the normal function of prion protein (PrPC) nor the mechanism of toxicity by the disease associated conformer PrPSc is entirely clear. Reports of abnormal iron metabolism in prion infected neuroblastoma cells and mouse models implicate redox-iron mediated neurotoxicity in these disorders as has been proposed for several other neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, Huntington's disease, and others. Recent data from my laboratory indicate that PrPC functions as an iron sensing, uptake, or transport protein in human neuroblastoma cells, and pathogenic mutations in PrPC alter cellular iron status in ways that are specific to each mutation. We have also noted evidence of abnormal iron metabolism in prion disease affected hamster and human brains, leading us to hypothesize that PrPC modulates cellular iron status by regulating iron uptake or transport, and alteration of this function due to point mutations or aggregation in the diseased state induces neurotoxicity. In the proposed studies we will check this hypothesis in two specific aims. In aim 1 we will investigate whether PrPC is involved in cellular iron uptake, efflux, or transfer to ferritin, and identify PrP-interacting proteins or factors responsible for its iron modulating function. In aim 2, the influence of pathogenic mutations of PrP on cellular iron status and susceptibility to free radical damage will be assessed. Cell lines exhibiting increased susceptibility to free radicals will be used to evaluate the protective effect of iron chelators and free radical scavenging compounds. These studies will improve understanding of prion disease pathogenesis in two distinct areas: 1) clarify the role of PrP in altering brain iron homeostasis during prion disease progression, and 2) validate the effectiveness of iron chelation in alleviating prion disease associated neurotoxicity. PUBLIC HEALTH RELEVANCE: Prion diseases are fatal neurodegenerative disorders for which there is currently no treatment. These disorders are infectious, familial, and sporadic in nature. The principal infectious and pathogenic agent in all prion disorders comprises of a beta-sheet rich isoform of the prion protein termed PrP-scrapie (PrPSc). Our data suggest that imbalance in brain iron homeostasis helps in the generation and propagation of PrPSc and contributes to the neurodegeneration observed in these disorders. In the proposed studies we will test this hypothesis in cell models, and investigate whether iron chelation can be used as a therapeutic measure to alleviate prion disease associated neurotoxicity.

DESCRIPTION (provided by applicant): Prion protein (PrPC) is a normal glycoprotein implicated in the pathogenesis of prion disorders, a group of fatal neurodegenerative conditions of humans and animals. A change in the conformation of PrPC to an aggregated, PrP-scrapie (PrPSc) form is believed to be the principal cause of neurotoxicity in these disorders. Recently, we demonstrated that PrPC plays a functional role in cellular iron uptake and transport, and selective deletion of PrPC in PrP knock-out (PrPKO) mice induces a state of systemic iron deficiency in these animals relative to wild-type (wt) controls. Specifically, PrPKO mice show impaired transport of orally introduced iron from the duodenum to the blood stream, and inefficient uptake of plasma iron by hematopoietic precursor cells and parenchymal cells of major organs. Together with our recent report demonstrating a phenotype of iron deficiency that correlates with PrPSc deposits in prion disease affected brains, these observations suggest that loss of normal function of PrPC due to aggregation to the PrPSc form may be responsible for brain iron dys- homeostasis in diseased brains. In this application, we will focus on the mechanism of iron modulation by PrPC, and hypothesize that PrPC is a novel iron uptake and transport protein, and modulates cellular iron metabolism either directly or by interacting with other iron transport protein(s). The proposed studies will test this hypothesis using two complementary approaches: 1) by evaluating the transport of different sources of iron that utilize distinct pathways of transport in the same tissue, and 2) by investigating the transport of same source of iron across tissues that utilize distinct pathways of uptake and transport. Mouse models that express no PrPC (PrPKO), wt levels (wt), and 10-fold higher than wt levels of PrPC (PrPOV) will be used for this analysis. This approach will allow identification of pathways of iron transport by PrPC, and the point where PrPC intersects with known pathways of iron metabolism. Three specific aims are proposed to accomplish these goals. In aim 1, the functional role of PrPC in brain iron metabolism will be evaluated in wt, PrPKO, and PrPOV mice, and the underlying mechanism will be investigated in neuroblastoma cells expressing normal and mutant forms of PrP defective in iron transport. In addition, the correlation between PrPC, PrPSc, and brain iron status will be assessed in scrapie infected wt and PrPOV mice. In aim 2, the transport of different sources of iron will be checked in hematopoietic and reticuloendothelial cells isolated from wt, PrPKO, and PrPOV mice, and the underlying mechanism will be investigated in K562 erythroleukemia cells transfected with normal and mutant PrP forms. In aim 3, the uptake and transport of different sources of iron across the intestinal epithelium will be checked in wt, PrPKO, and PrPOV mice, and compared with aim 2 above. Based on in vivo results, polarized Caco-2 cells transfected to express PrPC or mutant PrP forms will be used as models of absorptive enterocytes to understand the mechanism of iron transport by PrPC. Successful completion of these studies will uncover novel pathway(s) of iron modulation by PrPC, and improve our understanding of the mechanism(s) underlying brain iron imbalance and associated neurotoxicity in prion disorders. PUBLIC HEALTH RELEVANCE: The long term goal of this application is to understand the role of prion protein (PrPC) in iron uptake, transport, and utilization. Preliminary data from my laboratory demonstrate that PrPC is involved in iron uptake and transport, and loss of this function contributes to brain iron dyshomeostasis in prion disorders. Successful completion of the proposed studies will improve our understanding of the functional role of PrPC in iron metabolism, and the underlying cause of brain iron imbalance in prion disorders.

DESCRIPTION (provided by applicant): Several neurodegenerative conditions such as sporadic Creutzfeldt-Jakob disease (sCJD), Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD) are associated with imbalance of iron homeostasis in diseased brains, raising the possibility that redox-iron induced oxidative damage plays a significant role in the neurotoxicity associated with these disorders. Recent evidence from the Singh laboratory indicates a significant reduction of ferroxidase activity in sCJD affected human and scrapie infected mouse brains. Combined with the fact that sCJD brains show a phenotype of 'apparent' iron deficiency despite the presence of normal or increased brain iron levels, these observations suggest dysregulation of the normal iron homeostatic machinery in diseased brains. A recent report demonstrates decreased ferroxidase activity leading to iron accumulation in AD brains, suggesting that this phenomenon is shared by neurodegenerative disorders of disparate etiology. In an independent set of studies, the Mukhopadhayay laboratory reported that ceruloplasmin (Cp), a major ferroxidase in the brain, is down regulated by reactive oxygen species (ROS). Since iron is highly redox-active and a major contributor of ROS if mismanaged, it is likely that once initiated by a specific disease process, iron imbalance is perpetuated by ROS through down regulation of major brain ferroxidases. Based on these observations, we hypothesize that ROS mediated misregulation of brain specific ferroxidases contributes to iron imbalance in AD and sCJD. The proposed studies will test this hypothesis in two specific aims. In aim 1, the role of ROS in regulating specific ferroxidases will be investigated in cell models o AD and prion disease. Once the ferroxidases have been identified, their regulation will be compared with Cp which is known to be regulated by an mRNA decay mechanism in response to ROS. Subsequently, the minimal region of 3' UTR and binding proteins responsible for regulating ROS-mediated Cp activity will be identified. In aim 2, the ferroxidases identified in ai 1 will be evaluated for their expression and activity in mouse models of AD and scrapie infection during disease progression. The results will be compared with human brain tissue from AD and sCJD cases using ferroxidase assay and immunohistochemistry as the read-out. Successful completion of these studies will clarify the role of major brain ferroxidases in iron dyshomeostasis associated with AD and sCJD brains, and provide the ground-work for future studies on the mechanism of brain iron dyshomeostasis in PD, HD, and other neurodegenerative conditions associated with brain iron imbalance.

DESCRIPTION (provided by applicant): Sporadic Creutzfeldt-Jakob disease (sCJD) is a fatal prion disorder of humans that escapes detection until autopsy. The principal cause of neurotoxicity in this and other prion disorders is accumulation of PrP-scrapie (PrPSc), a ¿-sheet rich isoform of prion protein (PrPC) in the brain parenchyma. Participation of other processes is suspected, but their mechanism of action is unclear. Emerging evidence indicates that imbalance of iron homeostasis is a consistent feature of affected brains, implicating redox-iron in disease associated neurotoxicity. Unlike Alzheimer's disease (AD) where diseased brains accumulate iron, brains from sCJD and scrapie infected animal models reveal a phenotype of 'functional' iron deficiency as suggested by a significant increase in the iron uptake protein transferrin (Tf) despite minimal change in brain iron levels. Moreover, Tf levels increase with disease progression in direct correlation with PrPSc, suggesting a cause and effect relationship with disease pathogenesis. Surprisingly, instead of a compensatory increase, levels of Tf are significantly decreased in the cerebrospinal-fluid (CSF) of sCJD cases much before end-stage disease, indicating mis-regulation of signaling between iron starved brain parenchymal cells and the blood-brain and brain-CSF barriers involved in iron transport from the peripheral circulation to the brain and secretion of brain Tf respectively. Based on these observations, we hypothesize that sCJD associated disruption of brain iron regulation causes specific changes in CSF levels of iron management proteins that correlate with disease progression, providing disease specific biomarker(s) for sCJD. Two specific aims are proposed to test this hypothesis. In aim 1, the mechanism underlying decreased CSF Tf in sCJD brains will be explored using scrapie infected mice as models. Specifically, change in brain iron levels during scrapie infection, correlation between brain iron and PrPSc levels, and change in iron transport from the peripheral circulation to the brain will be monitored by tracking transport of radiolabeled iron (59Fe) from the tail vein to the brain and CSF compartments during prion disease progression. 59Fe counts in the brain and CSF will be correlated with levels of iron management proteins to evaluate the integrity of brain iron signaling mechanisms. In aim 2, the specificity and sensitivity of CSF Tf and other iron management proteins, the 'new' biomarkers, will be compared with 'current' surrogate CSF biomarkers of sCJD. The accuracy of new biomarkers in differentiating sCJD from rapidly progressive dementia and AD, potentially treatable causes of dementias often misdiagnosed as sCJD, will be emphasized. The prognostic reliability of new biomarkers will be assessed in CSF samples collected at different time points during disease progression from scrapie infected mice and Chronic Wasting Disease infected cervids, and the earliest time-point of a significant change will be identified. Successful completion of these studies will improve our understanding of the mechanism leading to decreased CSF Tf in sCJD, and facilitate the development of a rapid, specific, and sensitive pre-mortem diagnostic test for sCJD.

Prion disorders are infectious and invariably fatal neurodegenerative conditions associated with accumulation of PrP-scrapie (PrPSc), a ?-sheet rich isoform of the normal prion protein (PrPC), in the brain and retina of humans and certain animal species. Sporadic Creutzfeldt-Jakob-disease (sCJD) is the most common human prion disorder, and PrPSc-infected animal models are used to understand the mechanism of infectivity and toxicity. Neuroinflammation and iron accumulation are consistent features of these disorders, the latter contributing to neurotoxicity by iron-catalyzed reactive oxygen species (ROS). The cause of iron accumulation, however, has remained elusive. Recent data from my laboratory suggest cytokine-mediated upregulation of hepcidin synthesized by astrocytes as a significant cause. Hepcidin regulates iron by downregulating ferroportin (Fpn), the only known iron export protein. Under normal conditions, hepcidin is upregulated when iron saturation of transferrin (Tf-Fe) is low. Upregulation by cytokines, however, supersedes the signal from Tf-Fe. It is likely that cytokine-mediated upregulation of hepcidin by astrocytes is the cause of iron neuronal accumulation that express Fpn on their plasma membrane, and toxicity by ROS. In support of this hypothesis, sCJD brain homogenates show upregulation of hepcidin mRNA and protein, downregulation of Fpn, and increase in ferritin. Likewise, brain homogenates from PrPSc-infected mice show upregulation of hepcidin mRNA, and retinal sections from PrPSc- infected hamsters show activation of microglia before or concomitant with upregulation of ferritin during disease progression. Based on these observations, we hypothesize that iron accumulation in sCJD and PrPSc-infected brains and retina results from cytokine-mediated upregulation of local hepcidin. Two specific aims are proposed to test this hypothesis. In aim 1, additional sCJD brain and retinal tissue will be checked for increase in hepcidin mRNA and accumulation of iron, and correlated with neuronal and retinal ganglion cell (RGC) death in immunostained sections. In addition, the brain and retina of PrPSc-infected mice will be examined during disease progression to explore if increase in cytokines precedes upregulation of hepcidin and iron accumulation, and whether retinal degeneration precedes neurodegeneration. This will pave the way for retinal imaging a pre- clinical diagnostic test for sCJD. In aim 2, the role of hepcidin in brain and retinal iron accumulation will be further explored using hepcidin knock-out (hepc-/-) and littermate (hepc+/+) control mice inoculated with PrPSc. A significant decrease in iron accumulation in hepc-/- mice relative to hepc+/+ controls despite similar increase in cytokines and PrPSc load with disease progression will suggest local hepcidin as the cause of iron accumulation. Moreover, a marked reduction in neuronal and RGC death in hepc-/- mice will suggest a significant role of iron in inducing neurotoxicity, and justify the use of hepcidin antagonists and Fpn stabilizing agents to reduce accumulated iron as therapeutic options. No change in iron levels in hepc-/- mice will refute our hypothesis, and suggest iron as an epiphenomenon of the disease process.