PrPC Overexpression Research Articles

Differentiated cultures of an immortalized human neural progenitor cell line do not replicate prions despite PrPC overexpression

ABSTRACT Prions are misfolded proteins that accumulate within the brain in association with a rare group of fatal and infectious neurological disorders in humans and animals. A current challenge to research is a lack of in vitro model systems that are compatible with a wide range of prion strains, reproduce prion toxicity, and are amenable to genetic manipulations. In an attempt to address this need, here we produced stable cell lines that overexpress different versions of PrPC through lentiviral transduction of immortalized human neural progenitor cells (ReN VM). Differentiated cultures made from the neural progenitor cell lines overexpressed PrPC within 3D spheroid-like structures of TUBB3+ neurons and we observed evidence that PrPC modulates formation of these structures, consistent with PrPC’s role in neurogenesis. However, through repeated measurements of amyloid seeding activity in 6-week time course experiments, we failed to observe any evidence of prion replication within the differentiated ReN cultures following challenge with four prion isolates (human sCJD subtypes MM1 and VV2, and rodent adapted scrapie strains RML and 263K). We attributed amyloid seeding activity detected within the cultures to residual inoculum and concluded that PrPC overexpression was insufficient to confer permissiveness of ReN cultures to prion infection. While our ReN cell prion infection model was unsuccessful, additional efforts to develop cellular models of human prion disease are highly warranted.

Melatonin-Assisted Cisplatin Suppresses Urinary Bladder Cancer Cell Proliferation and Growth through Inhibiting PrPC-Regulated Cell Stress and Cell Proliferation Signaling

This study investigated whether melatonin (Mel) would promote cisplatin to suppress the proliferation and growth of bladder cancer (BC) cells by inhibiting cellular prion protein (PrPC)-mediated cell stress and cell proliferation signaling. An immunohistochemical staining of tissue arrays from BC patients demonstrated that the PrPC expression was significantly upregulated from stage I to III BC (p < 0.0001). The BC cellline of T24 was categorized into G1 (T24), G2 (T24 + Mel/100 μM), G3 (T24+cisplatin/6 μM), G4 (PrPC overexpression in T24 (i.e., PrPC-OE-T24)), G5 (PrPC-OE-T24+Mel), and G6 (PrPC-OE-T24+cisplatin). When compared with a human uroepithelial cell line (SV-HUC-1), the cellular viability/wound healing ability/migration rate were significantly increased in T24 cells (G1) and further significantly increased in PrPC-OE-T24 cells (G4); and they were suppressed in Mel (G2/G5) or cisplatin (G3/G6) treatment (all p < 0.0001). Additionally, the protein expressions of cell proliferation (PI3K/p-Akt/p-m-TOR/MMP-9/PrPC), cell cycle/mitochondrial functional integrity (cyclin-D1/clyclin-E1/ckd2/ckd4/mitochondrial-cytochrome-C/PINK1), and cell stress (RAS/c-RAF/p-MEK1/2, p-ERK1/2) markers showed a similar pattern of cell viability among the groups (all p < 0.001). After the BC cell line of UMUC3 was implanted into nude mouse backs, by day 28 mthe BC weight/volume and the cellular levels of PrPC/MMP-2/MMP-9 were significantly, gradually reduced from groups one to four (all p < 0.0001). The protein expressions of cell proliferation (PI3K/p-Akt/p-m-TOR/MMP-9/PrPC), cell cycle/mitophagy (cyclin-D1/clyclin-E1/ckd2/ckd4/PINK1), and cell stress (RAS/c-RAF/p-MEK1,2/p-ERK1,2) signaling were significantly, progressively reduced from groups one to four, whereas the protein expressions of apoptotic (Mit-Bax/cleaved-caspase-3/cleaved-PARP) and oxidative stress/mitochondrial damaged (NOX-1/NOX-2/cytosolic-cytochrome-C/p-DRP1) markers expressed an opposite pattern of cell proliferation signaling among the groups (all p < 0.0001). Mel-cisplatin suppressed BC cell growth/proliferation via inhibiting the PrPC in upregulating the cell proliferation/cell stress/cell cycle signaling.

An optimized Western blot assay provides a comprehensive assessment of the physiological endoproteolytic processing of the prion protein

The prion protein (PrPC) is subjected to several conserved endoproteolytic events producing bioactive fragments that are of increasing interest for their physiological functions and their implication in the pathogenesis of prion diseases and other neurodegenerative diseases. However, systematic and comprehensive investigations on the full spectrum of PrPC proteoforms have been hampered by the lack of methods able to identify all PrPC-derived proteoforms. Building on previous knowledge of PrPC endoproteolytic processing, we thus developed an optimized Western blot assay able to obtain the maximum information about PrPC constitutive processing and the relative abundance of PrPC proteoforms in a complex biological sample. This approach led to the concurrent identification of the whole spectrum of known endoproteolytic-derived PrPC proteoforms in brain homogenates, including C-terminal, N-terminal and, most importantly, shed PrPC-derived fragments. Endoproteolytic processing of PrPC was remarkably similar in the brain of widely used wild type and transgenic rodent models, with α-cleavage-derived C1 representing the most abundant proteoform and ADAM10-mediated shedding being an unexpectedly prominent proteolytic event. Interestingly, the relative amount of shed PrPC was higher in WT mice than in most other models. Our results indicate that constitutive endoproteolytic processing of PrPC is not affected by PrPC overexpression or host factors other than PrPC but can be impacted by PrPC primary structure. Finally, this method represents a crucial step in gaining insight into pathophysiological roles, biomarker suitability, and therapeutic potential of shed PrPC and for a comprehensive appraisal of PrPC proteoforms in therapies, drug screening, or in the progression of neurodegenerative diseases.

Tau Protein as a New Regulator of Cellular Prion Protein Transcription.

Cellular prion protein (PrPC) is largely responsible for transmissible spongiform encephalopathies (TSEs) when it becomes the abnormally processed and protease resistant form PrPSC. Physiological functions of PrPC include protective roles against oxidative stress and excitotoxicity. Relevantly, PrPC downregulates tau levels, whose accumulation and modification are a hallmark in the advance of Alzheimer's disease (AD). In addition to the accumulation of misfolded proteins, in the initial stages of AD-affected brains display both increased reactive oxygen species (ROS) markers and levels of PrPC. However, the factors responsible for the upregulation of PrPC are unknown. Thus, the aim of this study was to uncover the different molecular actors promoting PrPC overexpression. In order to mimic early stages of AD, we used β-amyloid-derived diffusible ligands (ADDLs) and tau cellular treatments, as well as ROS generation, to elucidate their particular roles in human PRNP promoter activity. In addition, we used specific chemical inhibitors and site-specific mutations of the PRNP promoter sequence to analyze the contribution of the main transcription factors involved in PRNP transcription under the analyzed conditions. Our results revealed that tau is a new modulator of PrPC expression independently of ADDL treatment and ROS levels. Lastly, we discovered that the JNK/c-jun-AP-1 pathway is involved in increased PRNP transcription activity by tau but not in the promoter response to ROS.

The role of cellular prion protein in lipid metabolism in the liver

ABSTRACT Cellular prion protein (PrPC) is a plasma membrane glycophosphatidylinositol-anchored protein and it is involved in multiple functions, including neuroprotection and oxidative stress. So far, most of the PrPC functional research is done in neuronal tissue or cell lines; the role of PrPC in non-neuronal tissues such as liver is only poorly understood. To characterize the role of PrPC in the liver, a proteomics approach was applied in the liver tissue of PrPC knockout mice. The proteome analysis and biochemical validations showed an excessive fat accumulation in the liver of PrPC knockout mice with a change in mRNA expression of genes linked to lipid metabolism. In addition, the higher Bax to Bcl2 ratio, up-regulation of tgfb1 mRNA expression in PrPC knockout mice liver, further showed the evidences of metabolic disease. Over-expression of PrPC in fatty acid-treated AML12 hepatic cell line caused a reduction in excessive intracellular fat accumulation; shows association of PrPC levels and lipid metabolism. Therefore, based on observation of excessive fat globules in the liver of ageing PrPC knockout mice and the reduction of fat accumulation in AML12 cell line with PrPC over-expression, the role of PrPC in lipid metabolism is described.

Emerging Role of Cellular Prion Protein in the Maintenance and Expansion of Glioma Stem Cells.

Cellular prion protein (PrPC) is a membrane-anchored glycoprotein representing the physiological counterpart of PrP scrapie (PrPSc), which plays a pathogenetic role in prion diseases. Relatively little information is however available about physiological role of PrPC. Although PrPC ablation in mice does not induce lethal phenotypes, impairment of neuronal and bone marrow plasticity was reported in embryos and adult animals. In neurons, PrPC stimulates neurite growth, prevents oxidative stress-dependent cell death, and favors antiapoptotic signaling. However, PrPC activity is not restricted to post-mitotic neurons, but promotes cell proliferation and migration during embryogenesis and tissue regeneration in adult. PrPC acts as scaffold to stabilize the binding between different membrane receptors, growth factors, and basement proteins, contributing to tumorigenesis. Indeed, ablation of PrPC expression reduces cancer cell proliferation and migration and restores cell sensitivity to chemotherapy. Conversely, PrPC overexpression in cancer stem cells (CSCs) from different tumors, including gliomas—the most malignant brain tumors—is predictive for poor prognosis, and correlates with relapses. The mechanisms of the PrPC role in tumorigenesis and its molecular partners in this activity are the topic of the present review, with a particular focus on PrPC contribution to glioma CSCs multipotency, invasiveness, and tumorigenicity.

Prion Protein in Glioblastoma Multiforme.

The cellular prion protein (PrPc) is an evolutionarily conserved cell surface protein encoded by the PRNP gene. PrPc is ubiquitously expressed within nearly all mammalian cells, though most abundantly within the CNS. Besides being implicated in the pathogenesis and transmission of prion diseases, recent studies have demonstrated that PrPc contributes to tumorigenesis by regulating tumor growth, differentiation, and resistance to conventional therapies. In particular, PrPc over-expression has been related to the acquisition of a malignant phenotype of cancer stem cells (CSCs) in a variety of solid tumors, encompassing pancreatic ductal adenocarcinoma (PDAC), osteosarcoma, breast cancer, gastric cancer, and primary brain tumors, mostly glioblastoma multiforme (GBM). Thus, PrPc is emerging as a key in maintaining glioblastoma cancer stem cells’ (GSCs) phenotype, thereby strongly affecting GBM infiltration and relapse. In fact, PrPc contributes to GSCs niche’s maintenance by modulating GSCs’ stem cell-like properties while restraining them from differentiation. This is the first review that discusses the role of PrPc in GBM. The manuscript focuses on how PrPc may act on GSCs to modify their expression and translational profile while making the micro-environment surrounding the GSCs niche more favorable to GBM growth and infiltration.

Cellular prion protein and γ-synuclein overexpression in LS 174T colorectal cancer cell drives endothelial proliferation-to-differentiation switch.

BackgroundTumor-induced angiogenesis is an imperative event in pledging new vasculature for tumor metastasis. Since overexpression of neuronal proteins gamma-synuclein (γ-Syn) and cellular prion protein (PrPC) is always detected in advanced stages of cancer diseases which involve metastasis, this study aimed to investigate whether γ-Syn or PrPC overexpression in colorectal adenocarcinoma, LS 174T cells affects angiogenesis of endothelial cells, EA.hy 926 (EA).MethodsEA cells were treated with conditioned media (CM) of LS 174T-γ-Syn or LS 174T-PrP, and their proliferation, invasion, migration, adhesion and ability to form angiogenic tubes were assessed using a range of biological assays. To investigate plausible background mechanisms in conferring the properties of EA cells above, nitrite oxide (NO) levels were measured and the expression of angiogenesis-related factors was assessed using a human angiogenesis antibody array.ResultsEA proliferation was significantly inhibited by LS 174T-PrP CM whereas its telomerase activity was reduced by CM of LS 174T-γ-Syn or LS 174T-PrP, as compared to EA incubated with LS 174T CM. Besides, LS 174T-γ-Syn CM or LS 174T-PrP CM inhibited EA invasion and migration in Boyden chamber assay. Furthermore, LS 174T-γ-Syn CM significantly inhibited EA migration in scratch wound assay. Gelatin zymography revealed reduced secretion of MMP-2 and MMP-9 by EA treated with LS 174T-γ-Syn CM or LS 174T-PrP CM. In addition, cell adhesion assay showed lesser LS 174T-γ-Syn or LS 174T-PrP cells adhered onto EA, as compared to LS 174T. In tube formation assay, LS 174T-γ-Syn CM or LS 174T-PrP CM induced EA tube formation. Increased NO secretion by EA treated with LS 174T-γ-Syn CM or LS 174T-PrP CM was also detected. Lastly, decreased expression of pro-angiogenic factors like CXCL16, IGFBP-2 and amphiregulin in LS 174T-γ-Syn CM or LS 174T-PrP CM was detected using the angiogenesis antibody array.DiscussionThese results suggest that overexpression of γ-Syn or PrPC could possibly be involved in colorectal cancer-induced angiogenesis by inducing an endothelial proliferation–differentiation switch. NO could be the main factor in governing this switch, and modulation on the secretion patterns of angiogenesis-related proteins could be the strategy of colorectal cancer cells overexpressing γ-Syn or PrPC in ensuring this transition.

Divergent prion strain evolution driven by PrPC expression level in transgenic mice

Prions induce a fatal neurodegenerative disease in infected host brain based on the refolding and aggregation of the host-encoded prion protein PrPC into PrPSc. Structurally distinct PrPSc conformers can give rise to multiple prion strains. Constrained interactions between PrPC and different PrPSc strains can in turn lead to certain PrPSc (sub)populations being selected for cross-species transmission, or even produce mutation-like events. By contrast, prion strains are generally conserved when transmitted within the same species, or to transgenic mice expressing homologous PrPC. Here, we compare the strain properties of a representative sheep scrapie isolate transmitted to a panel of transgenic mouse lines expressing varying levels of homologous PrPC. While breeding true in mice expressing PrPC at near physiological levels, scrapie prions evolve consistently towards different strain components in mice beyond a certain threshold of PrPC overexpression. Our results support the view that PrPC gene dosage can influence prion evolution on homotypic transmission.

PrPC expression and calpain activity independently mediate the effects of closed head injury in mice

The normal cellular prion protein (PrPC) is a sialoglycoprotein with a glycophosphatidylinositol anchor and expressed in highest levels within the CNS particularly at neuronal synapses. This membrane-bound protein is involved with many cell functions including cell signaling and neuroprotection.Calpains are calcium-activated cysteine proteases that typically undergo controlled activation. PrPC is a calpain substrate and is neurotoxic if it undergoes aberrant processing with cytosol accumulation. Following traumatic brain injury (TBI), there is an abnormal influx of Ca+2 and overactivation of calpains resulting in neuronal dysfunction and cell death. We investigated whether PrPC expression and calpain activity have an effect on, or are affected by, TBI.PrPC expression in the hippocampus, cortex and cerebellum of WT and Tga20 (PrPC overexpression) mice were unchanged after closed head injury (CHI). Further, PrPC in WT and Tga20 mice was resistant to TBI-induced calpain proteolysis.CHI-induced calpain activation resulted in breakdown products (BDPs) of αII-spectrin (SBDPs) and GFAP (GBDP-44K) in all brain regions and mouse lines. CHI caused significant increases in SBDP145, GFAP and GBDP-44K when compared to sham. With few exceptions, the calpain inhibitor, SNJ-1945, reduced SBDP145 and GBDP-44K levels. Behavioral studies suggested that PrPC and calpain independently affect learning and memory. Overall, we conclude that: (i) there is SNJ-1945-sensitive calpain activation in both neuron and glial cells following CHI, (ii) closed head trauma is not affected by, nor does it have an influence on, PrPC expression, and (iii) PrPC expression plays a minor role, if any, in CHI-induced calpain activation in vivo.

LRP/LR Antibody Mediated Rescuing of Amyloid-β-Induced Cytotoxicity is Dependent on PrPc in Alzheimer's Disease.

The neuronal perturbations in Alzheimer's disease are attributed to the formation of extracellular amyloid-β (Aβ) neuritic plaques, composed predominantly of the neurotoxic Aβ42 isoform. Although the plaques have demonstrated a role in synaptic dysfunction, neuronal cytotoxicity has been attributed to soluble Aβ42 oligomers. The 37kDa/67kDa laminin receptor has been implicated in Aβ42 shedding and Aβ42-induced neuronal cytotoxicity, as well as internalization of this neurotoxic peptide. As the cellular prion protein binds to both LRP/LR and Aβ42, the mechanism underlying this cytotoxicity may be indirectly due to the PrPc-Aβ42 interaction with LRP/LR. The effects of this interaction were investigated by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide assays. PrPc overexpression significantly enhanced Aβ42 cytotoxicity in vitro, while PrP-/- cells were more resistant to the cytotoxic effects of Aβ42 and exhibited significantly less cell death than PrPc expressing N2a cells. Although anti-LRP/LR specific antibody IgG1-iS18 significantly enhanced cell viability in both pSFV1-huPrP1-253 transfected and non-transfected cells treated with exogenous Aβ42, it failed to have any cell rescuing effect in PrP-/- HpL3-4 cells. These results suggest that LRP/LR plays a significant role in Aβ42-PrPc mediated cytotoxicity and that anti-LRP/LR specific antibodies may serve as potential therapeutic tools for Alzheimer's disease.

Prion protein localizes at the ciliary base during neural and cardiovascular development, and its depletion affects α-tubulin post-translational modifications.

Although conversion of the cellular form of the prion protein (PrPC) into a misfolded isoform is the underlying cause of prion diseases, understanding PrPC physiological functions has remained challenging. PrPC depletion or overexpression alters the proliferation and differentiation properties of various types of stem and progenitor cells in vitro by unknown mechanisms. Such involvement remains uncertain in vivo in the absence of any drastic phenotype of mice lacking PrPC. Here, we report PrPC enrichment at the base of the primary cilium in stem and progenitor cells from the central nervous system and cardiovascular system of developing mouse embryos. PrPC depletion in a neuroepithelial cell line dramatically altered key cilium-dependent processes, such as Sonic hedgehog signalling and α-tubulin post-translational modifications. These processes were also affected over a limited time window in PrPC–ablated embryos. Thus, our study reveals PrPC as a potential actor in the developmental regulation of microtubule dynamics and ciliary functions.

Commentary: Advancement of Knowledge of Brucella Over the Past 50 Years.

Brucella ceti, a cetacean and zoonotic pathogen displaying a marked neurotropism in striped dolphins (Stenella coeruleoalba), appears to be the ancestor and a close relative of B. abortus, infecting cattle, as well as of B. melitensis, infecting sheep and goat (1). In addition to the elegant mechanisms and strategies utilized by Brucella genus members for entering and surviving into host cells, which are clearly explained in the aforementioned review article (2), the interaction between bacterial Hsp60 – a member of the GroEL family of chaperonins – and cellular prion protein (PrPC) has been reported to play a crucial role in B. abortus infection of murine, bone marrow-derived macrophages, with no evidence of microbial colonization and replication in cells from PrPC-deficient mice (3, 4). As “top predators,” cetaceans may accumulate within their tissues high concentrations of immunotoxic and neurotoxic “persistent organic pollutants” (POPs) like dioxins and dioxin-like compounds (DLCs) (5, 6), which have been demonstrated to exert pro-apoptotic activity on human neuronal cells (7). As a consequence, following dioxin-induced apoptosis, PrPC overexpression is likely to occur on host cell membrane, given the anti-apoptotic role of PrPC, which is consistently expressed on brain cells (8). Based upon the aforementioned model, the heavy (cerebral and extra-neural) tissue loads of lipophylic dioxins and DLCs commonly detected in stranded dolphins (5, 6) could elicit PrPC overexpression on behalf of brain cells, resulting at its turn in an amplified colonization and replication activity of B. ceti inside these cells. The recently characterized expression patterns of PrPC within the cerebral parenchyma as well as in secondary lymphoid tissues from wild dolphins (9), which resemble those previously reported in terrestrial mammals (8), provide additional support for such hypothesis, thereby making biologically plausible the occurrence of “neurobrucellosis,” a neurological disease condition frequently observed in B. ceti-affected striped dolphins (1). Indeed, high levels of dioxins and DLCs have been recently found in tissues from B. ceti meningoencephalitis-affected striped dolphins beached along the Ionian Sea coast of Italy (Antonio Petrella and Pasquale Troiano, personal communication). In this respect, it should be also emphasized that significant differences have been recently reported in the endo-cerebral expression levels of 5-lipoxygenase (5-LOX), a neurodegeneration biomarker (10), between infectious encephalitis/meningoencephalitis-affected and -unaffected striped dolphins, with the most consistent intensity of expression of the aforementioned enzyme having been detected in a B. ceti-infected individual (11). This appears to be a remarkable finding, provided that such an extensive neuroinflammatory and neurodegenerative lesions’ pattern could have acted synergistically with the high tissue concentrations of dioxins and DLCs found in this dolphin (Antonio Petrella and Pasquale Troiano, personal communication), thereby leading to PrPC overexpression, with subsequent putative increase of B. ceti colonization and replication inside brain cells. In conclusion, based upon the strong impact exerted by B. ceti infection on the health and conservation status of free-ranging cetaceans all across the globe, associated with the proven zoonotic capability of this microorganism (1, 5, 12), the study of the infection’s neuropathogenesis, including the strategies adopted by B. ceti in crossing the blood–brain barrier from striped dolphins, would deserve adequate attention. Particular emphasis should be additionally placed on the study of the infection’s epidemiology and of the evolutionary genetics of B. ceti, also in comparison with all the other known Brucella genus members, as well as on the study of host–pathogen interaction dynamics, with special reference to the “host related” (PrPC expression levels and patterns) and to the “environment related” (POPs, dioxins, DLCs, etc.) drivers and modulators of such complex interplay.

Cellular prion protein contributes to LS 174T colon cancer cell carcinogenesis by increasing invasiveness and resistance against doxorubicin-induced apoptosis

As the cellular prion protein (PrP(C)) has been implicated in carcinogenesis, we aimed to investigate the effects of cancer cell-specific PrP(C) overexpression from the invasion, metastasis, and apoptosis aspects, by performing cell motility assays, cell proliferation assays under anchorage-dependent and anchorage-independent conditions, and apoptosis evasion when subjected to multiple anti-cancer drugs. Overexpression of PrP(C) in LS 174T was achieved by stable transfection. PrP(C) overexpression was shown to increase cell proliferation in anchorage-dependent and anchorage-independent manners, as shown by more viable cells in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, more colonies formed in soft agar assay and increased resistance to anoikis in poly-2-hydroxyethyl methacrylate-coated surface. PrP(C) overexpression also increased cell motility and invasiveness of LS 174T. Cell adhesion to extracellular matrix using collagen- and fibronectin-coated surfaces revealed increased cell attachment in LS 174T cells overexpressing PrP(C). Analysis of apoptotic and necrotic cells by propidium iodide/annexin V-fluorescein isothiocyanate microscopy and 7-amino-actinomycin D/annexin V-phycoerythrin flow cytometry revealed that PrP(C) overexpression attenuated doxorubicin-induced apoptosis. Human apoptosis antibody array with 35 apoptosis-related proteins revealed that three inhibitor of apoptosis proteins (IAPs)-survivin, X-linked inhibitor of apoptosis protein (XIAP), and cellular inhibitor of apoptosis protein-1 (cIAP-1)-were upregulated in LS 174T cells overexpressing PrP(C) in doxorubicin-induced apoptosis. In conclusion, the overexpression of PrP(C) could enhance the invasiveness and survival of LS 174T colorectal cancer cells, indicating that PrP(C) plays a role in colorectal cancer biology.

Gene Expression Resulting from PrPC Ablation and PrPC Overexpression in Murine and Cellular Models

The cellular prion protein (PrP(C)) plays a key role in prion diseases when it converts to the pathogenic form scrapie prion protein. Increasing knowledge of its participation in prion infection contrasts with the elusive and controversial data regarding its physiological role probably related to its pleiotropy, cell-specific functions, and cellular-specific milieu. Multiple approaches have been made to the increasing understanding of the molecular mechanisms and cellular functions modulated by PrP(C) at the transcriptomic and proteomic levels. Gene expression analyses have been made in several mouse and cellular models with regulated expression of PrP(C) resulting in PrP(C) ablation or PrP(C) overexpression. These analyses support previous functional data and have yielded clues about new potential functions. However, experiments on animal models have shown moderate and varied results which are difficult to interpret. Moreover, studies in cell cultures correlate little with in vivo counterparts. Yet, both animal and cell models have provided some insights on how to proceed in the future by using more refined methods and selected functional experiments.

The toxicity of antiprion antibodies is mediated by the flexible tail of the prion protein

Prion infections cause lethal neurodegeneration. This process requires the cellular prion protein (PrP(C); ref. 1), which contains a globular domain hinged to a long amino-proximal flexible tail. Here we describe rapid neurotoxicity in mice and cerebellar organotypic cultured slices exposed to ligands targeting the α1 and α3 helices of the PrP(C) globular domain. Ligands included seven distinct monoclonal antibodies, monovalent Fab1 fragments and recombinant single-chain variable fragment miniantibodies. Similar to prion infections, the toxicity of globular domain ligands required neuronal PrP(C), was exacerbated by PrP(C) overexpression, was associated with calpain activation and was antagonized by calpain inhibitors. Neurodegeneration was accompanied by a burst of reactive oxygen species, and was suppressed by antioxidants. Furthermore, genetic ablation of the superoxide-producing enzyme NOX2 (also known as CYBB) protected mice from globular domain ligand toxicity. We also found that neurotoxicity was prevented by deletions of the octapeptide repeats within the flexible tail. These deletions did not appreciably compromise globular domain antibody binding, suggesting that the flexible tail is required to transmit toxic signals that originate from the globular domain and trigger oxidative stress and calpain activation. Supporting this view, various octapeptide ligands were not only innocuous to both cerebellar organotypic cultured slices and mice, but also prevented the toxicity of globular domain ligands while not interfering with their binding. We conclude that PrP(C) consists of two functionally distinct modules, with the globular domain and the flexible tail exerting regulatory and executive functions, respectively. Octapeptide ligands also prolonged the life of mice expressing the toxic PrP(C) mutant, PrP(Δ94-134), indicating that the flexible tail mediates toxicity in two distinct PrP(C)-related conditions. Flexible tail-mediated toxicity may conceivably play a role in further prion pathologies, such as familial Creutzfeldt-Jakob disease in humans bearing supernumerary octapeptides.

Hypoxia-inducible factor-1 alpha regulates prion protein expression to protect against neuron cell damage

The human prion protein fragment, PrP (106–126), may contain a majority of the pathological features associated with the infectious scrapie isoform of PrP, known as PrPSc. Based on our previous findings that hypoxia protects neuronal cells from PrP (106–126)-induced apoptosis and increases cellular prion protein (PrPC) expression, we hypothesized that hypoxia-related genes, including hypoxia-inducible factor-1 alpha (HIF-1α), may regulate PrPC expression and that these genes may be involved in prion-related neurodegenerative diseases. Hypoxic conditions are known to elicit cellular responses designed to improve cell survival through adaptive processes. Under normoxic conditions, a deferoxamine-mediated elevation of HIF-1α produced the same effect as hypoxia-inhibited neuron cell death. However, under hypoxic conditions, doxorubicin-suppressed HIF-1α attenuated the inhibitory effect on neuron cell death mediated by PrP (106–126). Knock-down of HIF-1α using lentiviral short hairpin (sh) RNA-induced downregulation of PrPC mRNA and protein expression under hypoxic conditions, and sensitized neuron cells to prion peptide-mediated cell death even in hypoxic conditions. In PrPC knockout hippocampal neuron cells, hypoxia increased the HIF-1α protein but the cells did not display the inhibitory effect of prion peptide-induced neuron cell death. Adenoviruses expressing the full length Prnp gene (Ad-Prnp) were utilized for overexpression of the Prnp gene in PrPC knockout hippocampal neuron cells. Adenoviral transfection of PrPC knockout cells with Prnp resulted in the inhibition of prion peptide-mediated cell death in these cells. This is the first report demonstrating that expression of normal PrPC is regulated by HIF-1α, and PrPC overexpression induced by hypoxia plays a pivotal role in hypoxic inhibition of prion peptide-induced neuron cell death. These results suggest that hypoxia-related genes, including HIF-1α, may be involved in the pathogenesis of prion-related diseases and as such may be a therapeutic target for prion-related neurodegenerative diseases.

Silencing of cellular prion protein (PrPC) expression by DNA-antisense oligonucleotides induces autophagy-dependent cell death in glioma cells

Malignant gliomas are the most common and lethal primary central nervous system neoplasms. Several intriguing lines of evidence have recently emerged indicating that the cellular prion protein (PrPC) may exert neuro- and cyto-protective functions: PrPC overexpression protects cultured neurons and also tumor cell lines exposed to various pro-apoptotic stimuli while, on the contrary, PrPC silencing sensitizes Adriamycin-resistant human breast carcinoma cells to TRAIL-mediated cell death. In order to determine if PrPC is involved in the resistance of glial tumors to cell death, the effects of cellular prion protein downregulation by antisense approach were investigated in different human malignant glioma cell lines. PrPC downregulation induced profound morphological changes and significant cell death. In addition, a significant tumor volume reduction was noted after PrPC silencing in a EGFP-GL261 glioma murine model. Investigations of the molecular effects induced by PrPC silencing were carried out on T98G human glioma cells by analysing autophagic as well as typical apoptotic markers (nuclear morphology, caspase-3/7, p53 and PARP-1). The results indicated that apoptosis was not induced after PrPC downregulation while, on the contrary, electron microscopy analysis, and an accumulation of GFP-LC3-II in autophagosomal membranes of GFP-LC3 transfected cells, indicated a predominant activation of autophagy. PrPC silencing also led to induction of LC3-II, increase in Beclin-1 and a concomitant decrease in p62, Bcl-2 and in the phosphorylation of 4E-BP1, a target of mTOR autophagy signaling. In conclusion, our results show for the first time that interfering with the cellular prion protein expression could modulate autophagy-dependent cell death pathways in glial tumor cells.

Cellular prion protein overexpression disturbs cellular homeostasis in SH-SY5Y neuroblastoma cells but does not alter p53 expression: a proteomic study

The definite physiological role of the cellular prion protein (PrPc) remains elusive. There is ample in vitro and in vivo evidence suggesting a neuroprotective role for PrPc. On the other hand, several in vitro and in vivo studies demonstrated detrimental effects of PrPc overexpression through activation of a p53 pathway. Recently, we reported that transient overexpression of PrPc in human embryonic kidney 293 cells elicits proteome expression changes which point to deregulation of proteins involved in energy metabolism and cellular homeostasis. Here we report proteome expression changes following stable PrPc overexpression in human neuronal SH-SY5Y cells. In total 18 proteins that are involved in diverse biological processes were identified as differentially regulated. The majority of these proteins is involved in cell signaling, cytoskeletal organization and protein folding. Annexin V exhibited a several fold up-regulation following stable PrPc overexpression in SH-SY5Y cells. This finding has been reproduced in alternative, mouse N2a and human SK-N-LO neuroblastoma cell lines transiently overexpressing PrPc. Annexin V plays an important role in maintenance of calcium homeostasis which when disturbed can activate a p53-dependent cell death. Although we did not detect changes in p53 expression between PrPc overexpressing SH-SY5Y and control cells, deregulation of several proteins including annexin V, polyglutamine tract-binding protein-1, spermine synthase and transgelin 2 indicates disrupted cellular equilibrium. We conclude that stable PrPc overexpression in SH-SY5Y cells is sufficient to perturb cellular balance but insufficient to affect p53 expression.

The CNS glycoprotein Shadoo has PrPC-like protective properties and displays reduced levels in prion infections

The cellular prion protein, PrPC, is neuroprotective in a number of settings and in particular prevents cerebellar degeneration mediated by CNS-expressed Doppel or internally deleted PrP (‘ΔPrP'). This paradigm has facilitated mapping of activity determinants in PrPC and implicated a cryptic PrPC-like protein, ‘π'. Shadoo (Sho) is a hypothetical GPI-anchored protein encoded by the Sprn gene, exhibiting homology and domain organization similar to the N-terminus of PrP. Here we demonstrate Sprn expression and Sho protein in the adult CNS. Sho expression overlaps PrPC, but is low in cerebellar granular neurons (CGNs) containing PrPC and high in PrPC-deficient dendritic processes. In Prnp0/0 CGNs, Sho transgenes were PrPC-like in their ability to counteract neurotoxic effects of either Doppel or ΔPrP. Additionally, prion-infected mice exhibit a dramatic reduction in endogenous Sho protein. Sho is a candidate for π, and since it engenders a PrPC-like neuroprotective activity, compromised neuroprotective activity resulting from reduced levels may exacerbate damage in prion infections. Sho may prove useful in deciphering several unresolved facets of prion biology.