Differential solubility of prions is associated in manifold phenotypes

The main feature of prion diseases is the accumulation of infectious proteins (PrP^Sc). Since PrP^Sc results from conversion of cellular prion proteins (PrP^C), differential expressed PrP^C types may play an important role in the formation and conversion efficiency to specific PrP^Sc forms. However, little is known about the PrP^C expression, regulation and differentiation. Here, we demonstrate a new type of differentiation of overlapping PrP^C isoforms in brain homogenates using differential SDS solubility. Low and highly soluble PrP^C were detected along with various types of protein which are present in the brain of non-infected humans, sheep and cattle. Our findings provide evidence for the existence of several overlapping PrP^C proteins exhibiting distinct glycotypes. The selection of defined PrP^C types offers new possibilities for identifying highly efficient converting proteins and provides the potential for disease control.     

Distribution of the cellular prion protein (PrPC) in brains of livestock and domesticated species

In transmissible spongiform encephalopathies (TSEs) the prion protein (PrP) plays a central role in pathogenesis. The PrP gene (Prnp) has been described in a number of mammalian and avian species and its expression product, the cellular prion protein (PrP^C), has been mapped in brains of different laboratory animals (rodent and non-human primates). However, mapping of PrP^C expression in mammalian species suffering from natural (bovine and ovine) and experimental (swine) TSE or in species in which prion disease has never been reported (equine and canine) deserves further attention. Thus, localising the cellular prion protein (PrP^C) distribution in brain may be noteworthy for the understanding of prion disease pathogenesis since lesions seem to be restricted to particular brain areas. In the present work, we analysed the distribution of PrP^C expression among several brain structures of the above species. Our results suggest that the expression of PrP^C, within the same species, differs depending on the brain structure studied, but no essential differences between the PrP^C distribution patterns among the studied species could be established. Positive immunoreaction was found mainly in the neuropil and to a lesser extent in neuronal bodies which occasionally appeared strongly stained in discrete regions. Overall, the expression of PrP^C in the brain was significantly higher in grey matter areas than in white matter, where accumulation of PrP^Sc is first observed in prion diseases. Therefore, other factors besides the level of expression of cellular PrP may account for the pathogenesis of TSEs     

Expression and localization of the prion protein PrPC in the olfactory system of the mouse

The normal physiological function of the prion protein PrP^C remains elusive despite its widespread expression, particularly throughout the nervous system. A critical step toward identifying its function is to precisely localize its pattern of expression. Historically, the immunolocalization of PrP^C has proved to be notoriously difficult and nonconsensual. We have thus undertaken a detailed expression analysis by means of a combination of in situ hybridization, knockout mice, and immunohistochemistry, using recently generated highly specific antibodies. We have attempted to accurately localize PrP^C expression in a tissue that is highly structured and of crucial behavioral importance to mice, the olfactory system. We found that PrP^C was expressed in both peripheral and central neurons of the olfactory system and that its distribution was axonal‐specific in both olfactory sensory neurons of the olfactory epithelium and mitral cells of the olfactory bulb. Our detailed expression analysis and the axonal localization we observed may provide important hints toward potential functions of PrP^C. J. Comp. Neurol. 508:487–499, 2008. © 2008 Wiley‐Liss, Inc.     

Transport of prion protein across the blood-brain barrier

The cellular form of the prion protein (PrP^c) is necessary for the development of prion diseases and is a highly conserved protein that may play a role in neuroprotection. PrP^c is found in both blood and cerebrospinal fluid and is likely produced by both peripheral tissues and the central nervous system (CNS). Exchange of PrP^c between the brain and peripheral tissues could have important pathophysiologic and therapeutic implications, but it is unknown whether PrP^c can cross the blood-brain barrier (BBB). Here, we found that radioactively labeled PrP^c crossed the BBB in both the brain-to-blood and blood-to-brain directions. PrP^c was enzymatically stable in blood and in brain, was cleared by liver and kidney, and was sequestered by spleen and the cervical lymph nodes. Circulating PrP^c entered all regions of the CNS, but uptake by the lumbar and cervical spinal cord, hypothalamus, thalamus, and striatum was particularly high. These results show that PrP^c has bidirectional, saturable transport across the BBB and selectively targets some CNS regions. Such transport may play a role in PrP^c function and prion replication.     

The Role of the Prion Protein in the Molecular Basis for Synaptic Plasticity and Nervous System Development

The cellular prion protein (PrP^C) is found prominently at the synapse. However, its role at the nerve termini and elsewhere is unknown. Here we discuss research presented at the 2005 International Institute for Complex Adaptive Matter (I2CAM) first Annual Amyloid Conference that provides insight into the role of synaptic PrP^C. The prion protein can interact and facilitate copper uptake at the synapse, is presumed to oligodimerize to facilitate putative cell-cell adhesion, and it transports toward the synapse by fast microtubule-based anterograde transport. While PrP^C appears to be involved in all these processes, the mechanisms of PrP^C function in each of them remain unclear. A role for PrP^C in these distinct processes suggests a complex role for this protein at the synapse. Unraveling PrP^C function will likely entail employing combined approaches that take into account its possible multifaceted functions.     

Cellular prion protein is present in dopaminergic neurons and modulates the dopaminergic system

Cellular prion protein (PrP^C) is widely expressed in the brain. Although the precise role of PrP^C remains uncertain, it has been proposed to be a pivotal modulator of neuroplasticity events by regulating the glutamatergic and serotonergic systems. Here we report the existence of neurochemical and functional interactions between PrP^C and the dopaminergic system. PrP^C was found to co‐localize with dopaminergic neurons and in dopaminergic synapses in the striatum. Furthermore, the genetic deletion of PrP^C down‐regulated dopamine D₁ receptors and DARPP‐32 density in the striatum and decreased dopamine levels in the prefrontal cortex of mice. This indicates that PrP^C affects the homeostasis of the dopaminergic system by interfering differently in different brain areas with dopamine synthesis, content, receptor density and signaling pathways. This interaction between PrP^C and the dopaminergic system prompts the hypotheses that the dopaminergic system may be implicated in some pathological features of prion‐related diseases and, conversely, that PrP^C may play a role in dopamine‐associated brain disorders.     

Increased PrPC expression correlates with endoglin (CD105) positive microvessels in advanced carotid lesions

Normal cellular prion protein (PrP^C) has multiple functions but its role in the development of atherosclerosis has not been studied. Our pilot microarray data showed increased expression of PrP^C in tissue samples of complicated carotid lesions. Therefore in this study, we aimed to investigate its localisation within atherosclerotic arteries and its concentration in patient plasma. PrP^C expression was examined using an enzyme immunometric assay (EIA) in plasma from patients undergoing endarterectomy. Carotid specimens and control vascular transplants were studied for PrP^C and CD105 (endoglin, a marker of active vessels) expression by immunohistochemistry and real-time PCR. Patients with carotid disease had higher levels of plasma PrP^C than the control group [4.35 ng/ml (n = 22; 3.1–5.3) vs. 1.95 ng/ml (n = 21; 1.1–2.5), P < 0.001]. Furthermore, CD105-positive plaques had higher PrP^C expression which colocalized with CD105 in neovessels. There was a significant correlation between mRNA expression of PrP^C and CD105 in tested plaques (P < 0.001; r = 0.7) supporting our immunohistochemical findings. We conclude that PrP^C is expressed in carotid specimens and may be associated with neovessel growth or survival in these plaques. Our results suggest a role for PrP^C in modulating neovessel formation in complicated plaques.     

Endogenous cellular prion protein regulates contractility of the mouse ileum

Background Cellular prion protein (PrP^C) is expressed in the enteric nervous system (ENS), however, its physiological role has not been identified. Studies suggest that PrP^C can function as a metal‐binding protein, as absence of the protein has been linked to altered copper metabolism and atypical synaptic activity. Because copper is known to modulate smooth muscle relaxation, we tested the hypothesis that PrP^C deficiency would alter intestinal contractility. Methods We examined electrically evoked ileal contractility in Prnp^?/? or wild type littermate mice and the effects of copper or copper chelation. PrP^C expression was studied in whole mount ileal preparations of mice and guinea pigs by immunohistochemistry. Key Results Relative to wild type mice, ileal tissues of Prnp^?/? mice exhibited reduced electrical field stimulation (EFS)‐evoked contractility. Furthermore, EFS‐induced relaxation, as a percentage of that induced by a nitric oxide donor, was enhanced. Addition of a copper donor to the organ bath increased, whereas the addition of a copper chelator inhibited, nitric oxide donor‐induced ileal relaxation in Prnp^?/? mice. PrP^C was expressed on nerve fibers or terminals, and some cell bodies in the myenteric and submucosal plexuses of wild type mice. PrP^C colocalized with a neuron‐specific ectonucleotidase, nucleoside triphosphate diphosphohydrolase 3 (NTPDase3), but to only a limited extent with GFAP, a marker of enteric glia. Guinea pigs expressed PrP^C in nerve fibers or terminals and enteric glia in the myenteric and submucosal plexuses. Conclusions & Inferences Our findings suggest that PrP^C, which is abundant in the ENS, has a role in the regulation of ileal contractility.     

Developmental expression of prion protein and its ligands stress‐inducible protein 1 and vitronectin

Prion protein (PrP^C) is the normal isoform of PrP^Sc, a protein involved in neurodegenerative disorders. PrP^C participates in neuritogenesis, neuroprotection, and memory consolidation through its interaction with the secreted protein stress‐inducible protein 1 (STI1) and the extracellular matrix protein vitronectin (Vn). Although PrP^C mRNA expression has been documented during embryogenesis, its protein expression patterns have not been evaluated. Furthermore, little is known about either Vn or STI protein expression. In this study, PrP^C, STI1, and Vn protein expression was explored throughout mouse embryonic life. We found that the distributions of the three proteins were spatiotemporally related. STI1 and Vn expression became evident at E8, earlier than PrP^C, in the nervous system and heart. At E10, we observed, in the spinal cord, a gradient of expression of the three proteins, more abundant in the notochord and floor plate, suggesting that they can have a role in axonal growth. As development proceeded, the three proteins were detected in other organs, suggesting that they may play a role in the development of nonneural tissues as well. Finally, although STI1 and Vn are PrP^C ligands, their expression was not altered in PrP^C‐null mice. J. Comp. Neurol. 517:371–384, 2009. © 2009 Wiley‐Liss, Inc.     

Subtyping of human cellular prion proteins and their differential solubility

A human form of a prion disorder is the Creutzfeldt-Jakob disease. A hallmark of the disease is the accumulation of misfolded prion proteins (PrP^Sc), which exist as heterogeneous subtypes. PrP^Sc is formed by protein conversion from the host-encoded cellular prion (PrP^C), which is expressed and modified to various isoforms. Little is known about variation in PrP^C; however, it is assumed that PrP^C types play important roles in the formation of PrP^Sc. In this study, we separated distinct human PrP^C subtypes on the basis of differential protein solubilities in detergent solutions. Single and sequential application of the detergents Triton X-100, octyl-glucopyranoside and CHAPS facilitated high solubility of glycosylated PrP^C isoforms, whereas high proportions of nonglycosylated PrP^C remained non-soluble. Most proteins became highly soluble with laurylsarcosine and sodium dodecyl sulphate. Our findings demonstrate that the solubility characteristics of heterogeneous PrP^C overlap in human brains and convey distinct solubility subtypes. Differentiation by solubility experiments can therefore provide valuable information on prion protein composition, facilitate the separation of subtypes, and offer new prospects for conversion specificity of distinct isoforms.     

Age‐dependent neuromuscular impairment in prion protein knockout mice

Introduction: The cellular prion protein (PrP^C) is commonly recognized as the precursor of prions, the infectious agents of the fatal transmissible spongiform encephalopathies, or prion diseases. Despite extensive effort, the physiological role of PrP^C is still ambiguous. Evidence has suggested that PrP^C is involved in different cellular functions, including peripheral nerve integrity and skeletal muscle physiology. Methods: We analyzed the age‐dependent influence of PrP^C on treadmill test–based aerobic exercise capacity and on a series of morphological and metabolic parameters using wild‐type and genetically modified mice of different ages expressing, or knockout (KO) for, PrP^C. Results: We found that aged PrP‐KO mice displayed a reduction in treadmill performance compared with PrP‐expressing animals, which was associated with peripheral nerve demyelination and alterations of skeletal muscle fiber type. Conclusion: PrP‐KO mice have an age‐dependent impairment of aerobic performance as a consequence of specific peripheral nerve and muscle alterations. Muscle Nerve 53: 269–279, 2016     

Persistent retroviral infection with MoMuLV influences neuropathological signature and phenotype of prion disease

A fundamental step in pathophysiology of prion diseases is the conversion of the host encoded prion protein (PrP^C) into a misfolded isoform (PrP^Sc) that accumulates mainly in neuronal but also non-neuronal tissues. Prion diseases are transmissible within and between species. In a subset of prion diseases, peripheral prion uptake and subsequent transport to the central nervous system are key to disease initiation. The involvement of retroviruses in this process has been postulated based on the findings that retroviral infections enhance the spread of prion infectivity and PrP^Sc from cell to cell in vitro. To study whether retroviral infection influences the phenotype of prion disease or the spread of prion infectivity and PrP^Sc in vivo, we developed a murine model with persistent Moloney murine leukemia retrovirus (MoMuLV) infection with and without additional prion infection. We investigated the pathophysiology of prion disease in MoMuLV and prion-infected mice, monitoring temporal kinetics of PrP^Sc spread and prion infectivity, as well as clinical presentation. Unexpectedly, infection of MoMuLV challenged mice with prions did not change incubation time to clinical prion disease. However, clinical presentation of prion disease was altered in mice infected with both pathogens. This was paralleled by remarkably enhanced astrogliosis and pathognomonic astrocyte morphology in the brain of these mice. Therefore, we conclude that persistent viral infection might act as a disease modifier in prion disease.     

On the biology of prions

Summary Prions cause scrapie and Creutzfeldt-Jakob disease (CJD); these infectious pathogens are composed largely, if not entirely, of protein molecules. No prion-specific polynucleotide has been identified. Purified preparations of scrapie prions contain high titers (10^9.5 ID₅₀/ml), one protein (PrP 27-30) and amyloid rods (10–20 nm in diameter ×100–200 nm in length). Considerable evidence indicates that PrP 27-30 is required for and inseparable from scrapie infectivity. PrP 27-30 is encoded by a cellular gene and is derived from a larger protein, denoted PrP^Sc or PrP 33-35^Sc, by protease digestion. A cellular isoform, designated PrP^C or PrP 33-35^C, is encoded by the same gene as PrP^Sc and both proteins appear to be translated from the same 2.1 kb mRNA. Monoclonal antibodies to PrP 27-30, as well as antisera to PrP synthetic peptides, specifically react with both PrP^C and PrP^Sc, establishing their relatedness. PrP^C is digested by proteinase K, while PrP^Sc is converted to PrP 27-30 under the same conditions. Prion proteins are synthesized with signal peptides and are integrated into membranes. Detergent extraction of microsomal membranes isolated from scrapie-infected hamster brains solubilizes PrP^C but induces PrP^Sc to polymerize into amyloid rods. This procedure allows separation of the two prion protein isoforms and the demonstration that PrP^Sc accumulates during scrapie infection, while the level of PrP^C does not change. The prion amyloid rods generated by detergent extraction are identical morphologically, except for length, to extracellular collections of prion amyloid filaments which form plaques in scrapie- and CJD-infected brains. The prion amyloid plaques stain with antibodies to PrP 27-30 and PrP peptides. PrP 33-35^C does not accumulate in the extracellular space. Prion rods composed of PrP 27-30 can be dissociated into phospholipid vesicles with full retention of scrapie infectivity. The murine PrP gene (Prn-p) is linked to thePrn-i gene which controls the length of the scrapie incubation period. Prolonged incubation times are a cardinal feature of scrapie and CJD. While the central role of PrP^Sc in scrapie pathogenesis is well established, the chemical as well as conformational differences between PrP^C and PrP^Sc are unknown but probably arise from post-translational modifications.Supported by research grants from the National Institutes of Health (AG 02132 and NS 14069) and a Senator Jacob Javits Center of Excellence in Neuroscience (NS 22786) as well as by gifts from RJR-Nabisco, Inc. and Sherman Fairchild FoundationThis review is based upon a plenary lecture entitled Biology and Neuropathology of Prions presented at the Xth International Congress of Neuropathology, Stockholm, Sweden, September 11, 1986, and is dedicated to the memory of Peter Wilhelm Lampert (1929–1986)     

Cellular Prion Protein Participates in the Regulation of Inflammatory Response and Apoptosis in BV2 Microglia During Infection with Mycobacterium bovis

The cellular prion protein (PrP^C) is a glycoprotein anchored by glycosylphosphatidylinositol to the cell surface and is abundantly expressed in the central nervous system. A previous study has shown that PrP^C contributes to the establishment of infections with intracellular bacteria in macrophages. In the present work, we investigated the role of PrP^C in the response of BV2 microglia to Mycobacterium bovis infection. For this purpose, we examined the mRNA expression of prion protein gene (PRNP) upon M. bovis infection and analyzed the effect of siRNA-mediated disruption of PRNP on different parameters of microglial activation and apoptosis in M. bovis-infected microglia. We found that M. bovis infection induced a gradual increase in PRNP mRNA level and that siRNA-mediated silencing of PRNP in M. bovis-infected microglia reduced M. bovis-induced upregulation of pro-inflammatory factors, increased the rate of apoptosis in infected microglia, promoted the intrinsic apoptotic pathway, and downregulated the extrinsic apoptotic pathway. We conclude that PrP^C participates in the regulation of the response of microglia to M. bovis infection through the upregulation of pro-inflammatory cytokines and the modulation of apoptosis by interference with the intrinsic apoptotic pathway.     

Cellular and sub-cellular pathology of animal prion diseases: relationship between morphological changes,accumulation of abnormal prion protein and clinical disease

The transmissible spongiform encephalopathies (TSEs) or prion diseases of animals are characterised by CNS spongiform change, gliosis and the accumulation of disease-associated forms of prion protein (PrP^d). Particularly in ruminant prion diseases, a wide range of morphological types of PrP^d depositions are found in association with neurons and glia. When light microscopic patterns of PrP^d accumulations are correlated with sub-cellular structure, intracellular PrP^d co-localises with lysosomes while non-intracellular PrP^d accumulation co-localises with cell membranes and the extracellular space. Intracellular lysosomal PrP^d is N-terminally truncated, but the site at which the PrP^d molecule is cleaved depends on strain and cell type. Different PrP^d cleavage sites are found for different cells infected with the same agent indicating that not all PrP^d conformers code for different prion strains. Non-intracellular PrP^d is full-length and is mainly found on plasma-lemmas of neuronal perikarya and dendrites and glia where it may be associated with scrapie-specific membrane pathology. These membrane changes appear to involve a redirection of the predominant axonal trafficking of normal cellular PrP and an altered endocytosis of PrP^d. PrP^d is poorly excised from membranes, probably due to increased stabilisation on the membrane of PrP^d complexed with other membrane ligands. PrP^d on plasma-lemmas may also be transferred to other cells or released to the extracellular space. It is widely assumed that PrP^d accumulations cause neurodegenerative changes that lead to clinical disease. However, when different animal prion diseases are considered, neurological deficits do not correlate well with any morphological type of PrP^d accumulation or perturbation of PrP^d trafficking. Non-PrP^d-associated neurodegenerative changes in TSEs include vacuolation, tubulovesicular bodies and terminal axonal degeneration. The last of these correlates well with early neurological disease in mice, but such changes are absent from large animal prion disease. Thus, the proximate cause of clinical disease in animal prion disease is uncertain, but may not involve PrP^d.     

A neurotoxic prion protein fragment enhances proliferation of microglia but not astrocytes in culture

The scrapie isoform of the prion protein (PrP^Sc) induces pathological changes in the central nervous system including neurodegeneration and gliosis. A synthetic prion protein (PrP) peptide corresponding to amino acid residues 106–126 has been shown to be toxic to neurons that express PrP^C, the cellular isoform of PrP. Here we show that in mixed glial cultures PrP106–126 induces astroglial proliferation that is dependent on cellular PrP^c expression. In purified cultures of glial subtypes only microglia proliferated in response to PrP106–126. This effect was independent of PrP expression. Destruction of microglia in mixed glial cultures by L-leucine methyl ester (LLME) treatment abolished enhanced proliferation caused by PrP106–126. This proliferative effect can be restored by co-culturing LLME-treated astrocytes with microglia. Microglia therefore seem to mediate the proliferative effect exerted by PrP106–126 on astrocytes. © 1996 Wiley-Liss, Inc.     

α-cleavage of the prion protein occurs in a late compartment of the secretory pathway and is independent of lipid rafts

Endoproteolysis of the cellular prion protein (PrP^C) modulates both the normal function of the protein and the pathogenesis of the neurodegenerative prion diseases. PrP^C undergoes α-cleavage to generate the N-terminally truncated fragment C1. Utilizing various constructs of PrP^C expressed in human neuroblastoma cells we investigated the subcellular compartment where α-cleavage occurs. C1 was detected at the cell surface and the generation of C1 occurred in mutants of PrP^C incapable of Cu²⁺-mediated endocytosis. A transmembrane-anchored form that is not lipid raft-localised, as well as a secreted construct lacking the glycosyl-phosphatidylinositol membrane anchor, were also subject to α-cleavage. However, when this transmembrane-anchored form was modified with an endoplasmic reticulum retention motif, C1 was not formed. Inhibition of protein export from the Golgi by temperature block increased the amount of C1. Our data thus demonstrate that the α-cleavage of PrP^C occurs predominantly in a raft-independent manner in a late compartment of the secretory pathway.     

Abnormal Activation of Microglia Accompanied with Disrupted CX3CR1/CX3CL1 Pathway in the Brains of the Hamsters Infected with Scrapie Agent 263K

Microglial cells are resident mononuclear phagocytes of the central nervous system (CNS). Active proliferation of microglia in the brain has been identified in neurodegenerative disorders, including some kinds of prion disease. However, the detailed regional distribution between microglia and PrP^Sc deposition has not been presented, and investigation of fractalkine signaling which is involved in the regulation of activation of microglia in prion disease is not well documented. In this study, the disease phenomenon of microglial accumulation in the CNS was thoroughly analyzed using a scrapie-infected experimental model. Western blots of microglia-specific markers Iba1 and CD68, immunohistochemical and immunofluorescent assays demonstrated obviously activation of microglia in almost whole brain regions in the infected animals. Under the dynamic analysis on hallmarks of activation of microglia, a time-dependent increase of Iba1 and CD68 was detected, accompanied by accumulation of PrP^Sc and progression of neurodegenerative symptoms. With serial brain sections and double staining of Iba1 and PrP^Sc, we observed that the microglia distributed around PrP^Sc deposits in 263K-infected hamsters’ brains, proposing PrP^Sc phagocytosis. Flow cytometry assays with the single-cell suspensions prepared from the cortical region of the infected brains verified an activation of microglial population. ELISA assays of the cytokines in brain homogenates revealed significant upregulations of interleukin (IL)-1β, IL-6 and TNF-α when infected. Evaluation of fractalkine signaling in the infected hamsters’ brains showed progressively downregulation of CX3CL1 during the incubation. Prion peptide PrP106-126 also disrupted fractalkine and evoked microglial activation in rat primary neuron–glia mixed cultures. Our data here demonstrate an activated status of microglia in CNS tissues of infectious prion disease, possibly through fractalkine signaling deficiency.     

Pathogenesis of prion diseases

Prion diseases are rare neurological disorders that may be of genetic or infectious origin, but most frequently occur sporadically in humans. Their outcome is invariably fatal. As the responsible pathogen, prions have been implicated. Prions are considered to be infectious particles that represent mainly, if not solely, an abnormal, protease-resistant isoform of a cellular protein, the prion protein or PrP^C. As in other neurodegenerative diseases, aggregates of misfolded protein conformers are deposited in the CNS of affected individuals. Pathogenesis of prion diseases comprises mainly two equally important, albeit essentially distinct, topics: first, the mode, spread, and amplification of infectivity in acquired disease, designated as peripheral pathogenesis. In this field, significant advances have implicated an essential role of lymphoid tissues for peripheral prion replication, before a likely neural spread to the CNS. The second is the central pathogenesis, dealing, in addition to spread and replication of prions within the CNS, with the mechanisms of nerve cell damage and death. Although important roles for microglial neurotoxicity, oxidative stress, and complement activation have been identified, we are far from complete understanding, and therapeutic applications in prion diseases still need to be developed.