Hsp40 function in yeast prion propagation: Amyloid diversity necessitates chaperone functional complexity

It has been estimated that cerebrospinal fluid (CSF) contains approximately 80 proteins that significantly increase or decrease in response to various clinical conditions. Here we have evaluated the CSF protein PrPC (cellular prion protein) for possible increases or decreases following spinal cord injury. The physiological function of PrPC is not yet completely understood; however, recent findings suggest that PrPC may have neuroprotective properties. Our results show that CSF PrPC is decreased in spinal cord injured patients 12 hours following injury and is absent at 7 days. Given that normal PrPC has been proposed to be neuroprotective we speculate that the decrease in CSF PrPC levels may influence neuronal cell survival following spinal cord injury.     

Review of studies that have used knockout mice to assess normal function of prion protein under immunological or pathophysiological stress

Deletion of cellular isoform of prion protein (PrP^C) increases neuronal predisposition to damage by modulating apoptosis and the negative consequences of oxidative stress. In vivo studies have demonstrated that PrP^C‐deficient mice are more prone to seizure, depression, and induction of epilepsy and experience extensive cerebral damage following ischemic challenge or viral infection. In addition, adenovirus‐mediated overexpression of PrP^C reduces brain damage in rat models of cerebral ischemia. In experimental autoimmune encephalomyelitis, PrP^C‐deficient mice reportedly have a more aggressive disease onset and less clinical improvement during the chronic phase than wild‐type mice mice. In mice given oral dextran sulfate, PrP^C has a potential protective role against inflammatory bowel disease. PrP^C‐deficient mice demonstrate significantly greater increases in blood glucose concentrations after intraperitoneal injection of glucose than wild‐type mice. Further in vivo challenges to PrP gene‐deficient models and conditional knockout models with siRNA and in vivo administration of PrP‐ligating agents may assist in refining knowledge of the lymphoid function of PrP^C and predicting the effects of anti‐PrP treatment on the immune system. Together, these findings indicate that PrP^C may have multiple neuroprotective and anti‐inflammatory roles, which explains why this protein is so widely expressed.     

High levels of Cellular Prion Protein improve astrocyte development

Prion protein (PrP^C) has neuroprotective functions and herein we demonstrate that astrocytes from PrP^C-over-expressing mice are more resistant to induced cell death than wild-type astrocytes. The Stress-Inducible-Protein 1 (STI1), a PrP^C ligand, prevents cell death in both wild-type and PrP^C-over-expressing astrocytes through the activation of protein-kinase-A. Cultured embryonic astrocytes and brain extracts from PrP^C-over-expressing mice show higher glial fibrillary acidic protein expression and reduced vimentin and nestin levels when compared to wild-type astrocytes, suggesting faster astrocyte maturation in the former mice. Our data indicate that PrP^C levels modulate astrocyte development, and that PrP^C–STI1 interaction contributes to protect against astrocyte death.     

��-Cleavage of cellular prion protein

The cellular prion protein (PrP^C) is subjected to various processing under physiological and pathological conditions, of which the α-cleavage within the central hydrophobic domain not only disrupts a region critical for both PrP toxicity and PrP^C to PrP^Sc conversion but also produces the N1 fragment that is neuroprotective and the C1 fragment that enhances the pro-apoptotic effect of staurosporine in one report and inhibits prion in another. The proteases responsible for the α-cleavage of PrP^C are controversial. The effect of ADAM10, ADAM17, and ADAM9 on N1 secretion clearly indicates their involvement in the α-cleavage of PrP^C, but there has been no report of direct PrP^C α-cleavage activity with any of the three ADAMs in a purified protein form. We demonstrated that, in muscle cells, ADAM8 is the primary protease for the α-cleavage of PrP^C, but another unidentified protease(s) must also play a minor role. We also found that PrP^C regulates ADAM8 expression, suggesting that a close examination on the relationships between PrP^C and its processing enzymes may reveal novel roles and underlying mechanisms for PrP^C in non-prion diseases such as asthma and cancer.     

α-Cleavage of cellular prion protein

The cellular prion protein (PrP^C) is subjected to various processing under physiological and pathological conditions, of which the α-cleavage within the central hydrophobic domain not only disrupts a region critical for both PrP toxicity and PrP^C to PrP^Sc conversion but also produces the N1 fragment that is neuroprotective and the C1 fragment that enhances the pro-apoptotic effect of staurosporine in one report and inhibits prion in another. The proteases responsible for the α-cleavage of PrP^C are controversial. The effect of ADAM10, ADAM17, and ADAM9 on N1 secretion clearly indicates their involvement in the α-cleavage of PrP^C, but there has been no report of direct PrP^C α-cleavage activity with any of the three ADAMs in a purified protein form. We demonstrated that, in muscle cells, ADAM8 is the primary protease for the α-cleavage of PrP^C, but another unidentified protease(s) must also play a minor role. We also found that PrP^C regulates ADAM8 expression, suggesting that a close examination on the relationships between PrP^C and its processing enzymes may reveal novel roles and underlying mechanisms for PrP^C in non-prion diseases such as asthma and cancer.     

Prion Protein Protects against Renal Ischemia/Reperfusion Injury

The cellular prion protein (PrP^C), a protein most noted for its link to prion diseases, has been found to play a protective role in ischemic brain injury. To investigate the role of PrP^C in the kidney, an organ highly prone to ischemia/reperfusion (IR) injury, we examined wild-type (WT) and PrP^C knockout (KO) mice that were subjected to 30-min of renal ischemia followed by 1, 2, or 3 days of reperfusion. Renal dysfunction and structural damage was more severe in KO than in WT mice. While PrP was undetectable in KO kidneys, Western blotting revealed an increase in PrP in IR-injured WT kidneys compared to sham-treated kidneys. Compared to WT, KO kidneys exhibited increases in oxidative stress markers heme oxygenase-1, nitrotyrosine, and N^ε-(carboxymethyl)lysine, and decreases in mitochondrial complexes I and III. Notably, phosphorylated extracellular signal-regulated kinase (pERK) staining was predominantly observed in tubular cells from KO mice following 2 days of reperfusion, a time at which significant differences in renal dysfunction, histological changes, oxidative stress, and mitochondrial complexes between WT and KO mice were observed. Our study provides the first evidence that PrP^C may play a protective role in renal IR injury, likely through its effects on mitochondria and ERK signaling pathways.     

The role of the prion protein in the internalization of α-synuclein amyloids

Synucleinopathies are a group of neurodegenerative diseases characterized by the accumulation of α-synuclein amyloids in several regions of the brain. α-Synuclein fibrils are able to spread via cell-to-cell transfer, and once inside the cells, they can template the misfolding and aggregation of the endogenous α-synuclein. Multiple mechanisms have been shown to participate in the process of propagation: endocytosis, tunneling nanotubes and macropinocytosis. Recently, we published a research showing that the cellular form of the prion protein (PrP^C) acts as a receptor for α-synuclein amyloid fibrils, facilitating their internalization through and endocytic pathway. This interaction occurs by a direct interaction between the fibrils and the N-terminal domain of PrP^C. In cell lines expressing the pathological form of PrP (PrP^Sc), the binding between PrP^C and α-synuclein fibrils prevents the formation and accumulation of PrP^Sc, since PrP^C is no longer available as a substrate for the pathological conversion templated by PrP^Sc. On the contrary, PrP^Sc deposits are cleared over passages, probably due to the increased processing of PrP^C into the neuroprotective fragments N1 and C1. Starting from these data, in this work we present new insights into the role of PrP^C in the internalization of protein amyloids and the possible therapeutic applications of these findings.     

Is indeed the prion protein a Harlequin servant of "many" masters?

Tens of putative interacting partners of the cellular prion protein (PrP^C) have been identified, yet the physiologic role of PrP^C remains unclear. For the first time, however, a recent paper has demonstrated that the absence of PrP^C produces a lethal phenotype. Starting from this evidence, here we discuss the validity of past and more recent literature supporting that, as part of protein platforms at the cell surface, PrP^C may bridge extracellular matrix molecules and membrane proteins to intracellular signaling pathways.     

Cellular prion protein: A co-receptor mediating neuronal cofilin-actin rod formation induced by β-amyloid and proinflammatory cytokines

Increasing evidence suggests that proteins exhibiting “prion-like” behavior cause distinct neurodegenerative diseases, including inherited, sporadic and acquired types. The conversion of cellular prion protein (PrP^C) to its infectious protease resistant counterpart (PrP^Res) is the essential feature of prion diseases. However, PrP^C also performs important functions in transmembrane signaling, especially in neurodegenerative processes. Beta-amyloid (Aβ) synaptotoxicity and cognitive dysfunction in mouse models of Alzheimer disease are mediated by a PrP^C-dependent pathway. Here we review how this pathway converges with proinflammatory cytokine signaling to activate membrane NADPH oxidase (NOX) and generate reactive oxygen species (ROS) leading to dynamic remodeling of the actin cytoskeleton. The NOX signaling pathway may also be integrated with those of other transmembrane receptors clustered in PrP^C-enriched membrane domains. Such a signal convergence along the PrP^C-NOX axis could explain the relevance of PrP^C in a broad spectrum of neurodegenerative disorders, including neuroinflammatory-mediated alterations in synaptic function following traumatic brain injury. PrP^C overexpression alone activates NOX and generates a local increase in ROS that initiates cofilin activation and formation of cofilin-saturated actin bundles (rods). Rods sequester cofilin from synaptic regions where it is required for plasticity associated with learning and memory. Rods can also interrupt vesicular transport by occluding the neurite within which they form. Through either or both mechanisms, rods may directly mediate the synaptic dysfunction that accompanies various neurodegenerative disorders.     

Aggregation and Amyloid Fibril Formation Induced by Chemical Dimerization of Recombinant Prion Protein in Physiological-like Conditions

Prion diseases are caused by the conversion of a cellular protein (PrP^C) into a misfolded, aggregated isoform (PrP^Res). Misfolding of recombinant PrP^C in the absence of PrP^Res template, cellular factors, denaturing agents, or at neutral pH has not been achieved. A number of studies indicate that dimerization of PrP^C may be a key step in the aggregation process. In an effort to understand the molecular event that may activate misfolding of PrP^C in more relevant physiological conditions, we tested if enforced dimerization of PrP^C may induce a conformational change reminiscent of the conversion of PrP^C to PrP^Res. We used a well described inducible dimerization strategy whereby a chimeric PrP^C composed of a modified FK506-binding protein (Fv) fused with PrP^C and termed Fv-PrP is incubated in the presence of a monomeric FK506 or dimerizing AP20187 ligand. Addition of AP20187 but not FK506 to recombinant Fv-PrP (rFv-PrP) in physiological-like conditions resulted in a rapid conformational change characterized by an increase in β-sheet structure and simultaneous aggregation of the protein. Aggregates were partially resistant to proteinase K and induced the conversion of soluble rFv-PrP in serial seeding experiments. As judged from thioflavin T binding and electron microscopy, aggregates converted to amyloid fibers. Aggregates were toxic to cultured cells, whereas soluble rFv-PrP and amyloid fibers were harmless. This study strongly supports the proposition that dimerization of PrP^C is a key pathological primary event in the conversion of PrP^C and may initiate the pathogenesis of prion diseases.     

Dissecting the copper bioinorganic chemistry of the functional and pathological roles of the prion protein: Relevance in Alzheimer's disease and cancer

The cellular prion protein (PrP^C) is a metal-binding biomolecule that can interact with different protein partners involved in pivotal physiological processes, such as neurogenesis and neuronal plasticity. Recent studies profile copper and PrP^C as important players in the pathological mechanisms of Alzheimer's disease and cancer. Although the copper-PrP^C interaction has been characterized extensively, the role of the metal ion in the physiological and pathological roles of PrP^C has been barely explored. In this article, we discuss how copper binding and proteolytic processing may impact the ability of PrP^C to recruit protein partners for its functional roles. The importance to dissect the role of copper-PrP^C interactions in health and disease is also underscored.     

Quantitative and qualitative analysis of cellular prion protein (PrPC) expression in bovine somatic tissues

The host encoded cellular prion protein (PrP^C) is an N-linked glycoprotein tethered to the cell membrane by a glycophosphatidylinositol (GPI) anchor. Under certain conditions, PrP^C can undergo conversion into a conformationally-altered isoform (PrP^Sc) widely believed to be the pathogenic agent of transmissible spongiform encephalopathies (TSEs). Understanding the tissue-specific expression of PrP^C is crucial considering that cells expressing high levels of PrP^C bear a risk for conversion and accumulation of PrP^Sc. In the present study, fifteen bovine somatic tissues were analyzed for PrP^C expression by quantitative western blot and immunohistochemistry. Quantitative western blot analysis revealed highest expression of PrP^C in cerebellum, obex and spinal cord. Intermediate levels were detected in thymus, intestine, nerve, heart and spleen, and lower levels in lung, muscle, kidney, lymph node, skin, pancreas and liver. Immunohistochemical analysis detected intense cellular-specific PrP^C staining in neurons, thymocytes and lymphocytes. PrP^C was also detected in the enteric wall, pancreatic islets of langerhans, myocardium, pulmonary alveolar sacs, renal glomeruli and dermal epithelial cells. This study demonstrated the quantitatively varied, wide-spread, tissue- and cell-specific expression pattern of PrP^C in bovine somatic tissues. The importance of this study is to lay the foundation for understanding the tissue-specific expression of PrP^C and to consider the potential participation of more bovine tissues in the transmission of BSE infection.Key words: cellular prion protein (PrP^C), protein expression, bovine somatic tissues, BSE, western blot, immunohistochemistry     

Plasma Soluble Prion Protein,a Potential Biomarker for Sport-Related Concussions: A Pilot Study

Sport-related mild traumatic brain injury (mTBI) or concussion is a significant health concern to athletes with potential long-term consequences. The diagnosis of sport concussion and return to sport decision making is one of the greatest challenges facing health care clinicians working in sports. Blood biomarkers have recently demonstrated their potential in assisting the detection of brain injury particularly, in those cases with no obvious physical injury. We have recently discovered plasma soluble cellular prion protein (PrP^C) as a potential reliable biomarker for blast induced TBI (bTBI) in a rodent animal model. In order to explore the application of this novel TBI biomarker to sport-related concussion, we conducted a pilot study at the University of Saskatchewan (U of S) by recruiting athlete and non-athlete 18 to 30 year-old students. Using a modified quantitative ELISA method, we first established normal values for the plasma soluble PrP^C in male and female students. The measured plasma soluble PrP^C in confirmed concussion cases demonstrated a significant elevation of this analyte in post-concussion samples. Data collected from our pilot study indicates that the plasma soluble PrP^C is a potential biomarker for sport-related concussion, which may be further developed into a clinical diagnostic tool to assist clinicians in the assessment of sport concussion and return-to-play decision making.     

Insoluble cellular prion protein and its association with prion and Alzheimer diseases

The soluble cellular prion protein (PrP^C) is best known for its association with prion disease (PrD) through its conversion to a pathogenic insoluble isoform (PrP^Sc). However, its deleterious effects independent of PrP^Sc have recently been observed not only in PrD but also in Alzheimer disease (AD), two diseases which mainly affect cognition. At the same time, PrP^C itself seems to have broad physiologic functions including involvement in cognitive processes. The PrP^C that is believed to be soluble and monomeric has so far been the only PrP conformer observed in the uninfected brain. In 2006, we identified an insoluble PrP^C conformer (termed iPrP^C) in uninfected human and animal brains. Remarkably, the PrP^Sc-like iPrP^C shares the immunoreactivity behavior and fragmentation with a newly-identified PrP^Sc species in a novel human PrD termed variably protease-sensitive prionopathy. Moreover, iPrP^C has been observed as the major PrP species that interacts with amyloid β (Aβ) in AD. This article highlights evidence of PrP involvement in two putatively beneficial and deleterious PrP-implicated pathways in cognition and hypothesizes first, that beneficial and deleterious effects of PrP^C are attributable to the chameleon-like conformation of the protein and second, that the iPrP^C conformer is associated with PrD and AD.Key words: prion protein, prion disease, cognition, cognitive deficit, insoluble prion protein, Alzheimer disease, variably protease-sensitive prionopathy, dementia, memory     

Identification of novel putative-binding proteins for cellular prion protein and a specific interaction with the STIP1 homology and U-Box-containing protein 1

ABSTRACT

Prion diseases involve the conversion of the endogenous cellular prion protein, PrP^C, into a misfolded infectious isoform, PrP^Sc. Several functions have been attributed to PrP^C, and its role has also been investigated in the olfactory system. PrP^C is expressed in both the olfactory bulb (OB) and olfactory epithelium (OE) and the nasal cavity is an important route of transmission of diseases caused by prions. Moreover, Prnp^?/? mice showed impaired behavior in olfactory tests. Given the high PrP^C expression in OE and its putative role in olfaction, we screened a mouse OE cDNA library to identify novel PrP^C-binding partners. Ten different putative PrP^C ligands were identified, which were involved in functions such as cellular proliferation and apoptosis, cytoskeleton and vesicle transport, ubiquitination of proteins, stress response, and other physiological processes. In vitro binding assays confirmed the interaction of PrP^C with STIP1 homology and U-Box containing protein 1 (Stub1) and are reported here for the first time. Stub1 is a co-chaperone with ubiquitin E3-ligase activity, which is associated with neurodegenerative diseases characterized by protein misfolding and aggregation. Physiological and pathological implications of PrP^C-Stub1 interaction are under investigation. The PrP^C-binding proteins identified here are not exclusive to the OE, suggesting that these interactions may occur in other tissues and play general biological roles. These data corroborate the proposal that PrP^C is part of a multiprotein complex that modulates several cellular functions and provide a platform for further studies on the physiological and pathological roles of prion protein.     

Poster Session PPo5: Basic Mechanisms of Neurodegeneration and Pathology

A large number of studies have analysed the putative functions of the prion protein (PrP^C) in mammals. Although its sequence conservation over a wide range of different animals may indicate that this protein could have a key role in prion diseases, an absolutely accepted involvement has not been found so far. We have recently reported that PrP^C regulates Nanog mRNA expression, the first non-redundant function of PrP^C in embryonic stem cells (ESC), which translates into control of pluripotency and early differentiation. Contrary to what it is believed, the other two members of the prion protein family, Doppel and Shadoo, cannot replace the absence of PrP^C, causing the appearance of a new embryoid body (EB) population in our in vitro culture. The similarities between EB and an early post-implantation embryo suggest that this might also occur in vivo, enhancing the importance of this finding. On the other hand, our data may support the hypothesis of a relationship between the loss of PrP^C function and neuronal degeneration in prion diseases. A reduction in brain stem cells pluripotency after PrP^C is misfolded into the pathological conformation (PrP^Sc) could lead to a delay or a disappearance of the normal brain damage recovery.     

Mapping the interaction site of prion protein and Sho

The cellular prion protein (PrP^C) is a highly conserved protein among mammals and is considered to have important cellular functions. Despite decades of intensive research, however, the physiological function of PrP^C remains unclear. Sho (Shadoo, shadow of prion protein) and PrP^C have similar N-terminals, which suggests that the two proteins share biological functions. Using truncation mutants of both proteins and yeast two-hybrid analysis, with validation by co-immunoprecipitation and surface plasmon resonance (SPR), we have identified an interaction between Sho 61–77 and PrP^C 108–126 domains. This indicates that Sho may play a role in the physiological function of PrP^C and prion pathogenesis.