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.     

Anti-prion Drugs Targeting the Protein Folding Activity of the Ribosome Reduce PABPN1 Aggregation

Prion diseases are caused by the propagation of PrP^Sc, the pathological conformation of the PrP^C prion protein. The molecular mechanisms underlying PrP^Sc propagation are still unsolved and no therapeutic solution is currently available. We thus sought to identify new anti-prion molecules and found that flunarizine inhibited PrP^Sc propagation in cell culture and significantly prolonged survival of prion-infected mice. Using an in silico therapeutic repositioning approach based on similarities with flunarizine chemical structure, we tested azelastine, duloxetine, ebastine, loperamide and metixene and showed that they all have an anti-prion activity. Like flunarizine, these marketed drugs reduced PrP^Sc propagation in cell culture and in mouse cerebellum organotypic slice culture, and inhibited the protein folding activity of the ribosome (PFAR). Strikingly, some of these drugs were also able to alleviate phenotypes due to PABPN1 nuclear aggregation in cell and Drosophila models of oculopharyngeal muscular dystrophy (OPMD). These data emphasize the therapeutic potential of anti-PFAR drugs for neurodegenerative and neuromuscular proteinopathies.Supplementary InformationThe online version contains supplementary material available at 10.1007/s13311-020-00992-6.Key Words: Prions, PrP^Sc, drug repositioning, PFAR, OPMD, PABPN1     

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)     

Molecular biology and pathology of prion strains in sporadic human prion diseases

Prion diseases are believed to propagate by the mechanism involving self-perpetuating conformational conversion of the normal form of the prion protein, PrP^C, to the misfolded, pathogenic state, PrP^Sc. One of the most intriguing aspects of these disorders is the phenomenon of prion strains. It is believed that strain properties are fully encoded in distinct conformations of PrP^Sc. Strains are of practical relevance to human prion diseases as their diversity may explain the unusual heterogeneity of these disorders. The first insight into the molecular mechanisms underlying heterogeneity of human prion diseases was provided by the observation that two distinct disease phenotypes and their associated PrP^Sc conformers co-distribute with distinct PrP genotypes as determined by the methionine/valine polymorphism at codon 129 of the PrP gene. Subsequent studies identified six possible combinations of the three genotypes (determined by the polymorphic codon 129) and two common PrP^Sc conformers (named types 1 and 2) as the major determinants of the phenotype in sporadic human prion diseases. This scenario implies that each 129 genotype–PrP^Sc type combination would be associated with a distinct disease phenotype and prion strain. However, notable exceptions have been found. For example, two genotype–PrP^Sc type combinations are linked to the same phenotype, and conversely, the same combination was found to be associated with two distinct phenotypes. Furthermore, in some cases, PrP^Sc conformers naturally associated with distinct phenotypes appear, upon transmission, to lose their phenotype-determining strain characteristics. Currently it seems safe to assume that typical sporadic prion diseases are associated with at least six distinct prion strains. However, the intrinsic characteristics that distinguish at least four of these strains remain to be identified.     

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.     

Virus-induced alterations of membrane lipids affect the incorporation of PrP Sc into cells

Prion diseases are fatal neurodegenerative disorders characterized by long incubation periods. To investigate whether concurrent diseases can modify the clinical outcome of prion‐affected subjects, we tested the effect of viral infection on the binding and internalization of PrP^Sc, essential steps of prion propagation. To this effect, we added scrapie brain homogenate or purified PrP^Sc to fibroblasts previously infected with minute virus of mice (MVM), a mouse parvovirus. We show here that the rate of incorporation of PrP^Sc into MVM‐infected cells was significantly higher than that observed for naïve cells. Immunostaining of cells and immunoblotting of subcellular fractions using antibodies recognizing PrP and LysoTracker, a lysosomal marker, revealed that in both control and MVM‐infected cells the incorporated PrP^Sc was associated mostly with lysosomes. Interestingly, floatation gradient analysis revealed that the majority of the PrP^Sc internalized into MVM‐infected cells shifted toward raft‐containing low‐density fractions. Concomitantly, the MVM‐infected cells demonstrated increased levels of the glycosphingolipid GM1 (an essential raft lipid component) throughout the gradient and a shift in caveolin 1 (a raft protein marker) toward lighter membrane fractions compared with noninfected cells. Our results suggest that the effect of viral infection on membrane lipid composition may promote the incorporation of exogenous PrP^Sc into rafts. Importantly, membrane rafts are believed to be the conversion site of PrP^C to PrP^Sc; therefore, the association of exogenous PrP^Sc with such membrane microdomains may facilitate prion infection. © 2008 Wiley‐Liss, Inc.     

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     

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.     

Intracellular mechanisms mediating the neuronal death and astrogliosis induced by the prion protein fragment 106–126

Prion encephalopathies include fatal diseases of the central nervous system of men and animals characterized by nerve cell loss, glial proliferation and deposition of amyloid fibrils into the brain. During these diseases a cellular glycoprotein (the prion protein, PrP^C) is converted, through a not yet completely clear mechanism, in an altered isoform (the prion scrapie, PrP^Sc) that accumulates within the brain tissue by virtue of its resistance to the intracellular catabolism. PrP^Sc is believed to be responsible for the neuronal loss that is observed in the prion disease. The PrP 106–126, a synthetic peptide that has been obtained from the amyloidogenic portion of the prion protein, represents a suitable model for studying the pathogenic role of the PrP^Sc, retaining, in vitro, some characteristics of the entire protein, such as the capability to aggregate in fibrils, and the neurotoxicity. In this work we present the results we have recently obtained regarding the action of the PrP 106–126 in different cellular models. We report that the PrP 106–126 induces proliferation of cortical astrocytes, as well as degeneration of primary cultures of cortical neurons or of neuroectodermal stable cell lines (GH₃ cells). In particular, these two opposite effects are mediated by the same attitude of the peptide to interact with the L-type calcium channels: in the astrocytes, the activity of these channels seems to be activated by PrP 106–126, while, in the cortical neurons and in the GH₃ cells, the same treatment causes a blockade of these channels causing a toxic effect.     

Neurodevelopmental expression and localization of the cellular prion protein in the central nervous system of the mouse

Transmissible spongiform encephalopathies (TSEs) are neurodegenerative disorders caused by PrP^Sc, or prion, an abnormally folded form of the cellular prion protein (PrP^C). The abundant expression of PrP^C in the central nervous system (CNS) is a requirement for prion replication, yet despite years of intensive research the physiological function of PrP^C still remains unclear. Several routes of investigation point out a potential role for PrP^C in axon growth and neuronal development. Thus, we undertook a detailed analysis of the spatial and temporal expression of PrP^C during mouse CNS development. Our findings show regional differences of the expression of PrP, with some specific white matter structures showing the earliest and highest expression of PrP^C. Indeed, all these regions are part of the thalamolimbic neurocircuitry, suggesting a potential role of PrP^C in the development and functioning of this specific brain system. J. Comp. Neurol. 518:1879–1891, 2010. © 2010 Wiley‐Liss, Inc.     

Using Protein Misfolding Cyclic Amplification Generates a Highly Neurotoxic PrP Dimer Causing Neurodegeneration

Under the “protein-only” hypothesis, prion-based diseases are proposed to result from an infectious agent that is an abnormal isoform of the prion protein in the scrapie form, PrP^Sc. However, since PrP^Sc is highly insoluble and easily aggregates in vivo, this view appears to be overly simplistic, implying that the presence of PrP^Sc may indirectly cause neurodegeneration through its intermediate soluble form. We generated a neurotoxic PrP dimer with partial pathogenic characteristics of PrP^Sc by protein misfolding cyclic amplification in the presence of 1-palmitoyl-2-oleoylphosphatidylglycerol consisting of recombinant hamster PrP (23–231). After intracerebral injection of the PrP dimer, wild-type hamsters developed signs of neurodegeneration. Clinical symptoms, necropsy findings, and histopathological changes were very similar to those of transmissible spongiform encephalopathies. Additional investigation showed that the toxicity is primarily related to cellular apoptosis. All results suggested that we generated a new neurotoxic form of PrP, PrP dimer, which can cause neurodegeneration. Thus, our study introduces a useful model for investigating PrP-linked neurodegenerative mechanisms.     

Polyunsaturated Fatty Acids Protect Against Prion-Mediated Synapse Damage In Vitro

A loss of synapses is characteristic of the early stages of the prion diseases. Here we modelled the synapse damage that occurs in prion diseases by measuring the amount of synaptophysin, a pre-synaptic membrane protein essential for neurotransmission, in cortical or hippocampal neurones incubated with the disease associated isoform of the prion protein (PrP^Sc), or with the prion-derived peptide PrP82-146. The addition of PrP^Sc or PrP82-146 caused a dose-dependent reduction in the synaptophysin content of PrP wildtype neurones indicative of synapse damage. They did not affect the synaptophysin content of PrP null neurones. The loss of synaptophysin in PrP wildtype neurones was preceded by the accumulation of PrP82-146 within synapses. Since supplements containing polyunsaturated fatty acids (PUFA) are frequently taken for their perceived health benefits including reported amelioration of neurodegenerative conditions, the effects of some common PUFA on prion-mediated synapse damage were examined. Pre-treatment of cortical or hippocampal neurones with docosahexaenoic (DHA) or eicosapentaenoic acids (EPA) protected neurones against the loss of synaptophysin induced by PrP82-146 or PrP^Sc. This effect of DHA and EPA was selective as they did not alter the loss of synaptophysin induced by a snakevenom neurotoxin. The effects of DHA and EPA were associated with a significant reduction in the amount of FITC-PrP82-146 that accumulated within synapses. Such observations raise the possibility that supplements containing PUFA may protect against the synapse damage and cognitive loss seen during the early stages of prion diseases.     

Interplay between 20S proteasomes and prion proteins in scrapie disease

Scrapie is a transmissible spongiform encephalopathy affecting the central nervous system in sheep. The key event in such neurodegeneration is the conversion of the normal prion protein (PrP^C) into the pathological isoform (PrP^Sc). Misfolded prion proteins are normally degraded by the proteasome. This work, analyzing models of scrapie disease, describes the in vivo relationship between the proteasome and prions. We report that the disease is associated with an increase of proteasome functionality, most likely as a means of counteracting the increased levels of oxidative stress. Here, we show that prions coprecipitate with the 20S proteasome and that they colocalize within the same neuron, thus raising the possibility that PrP interacts with the proteasome in both normal and diseased brain, affecting substrate trafficking and proteasome functionality. This interaction, inducing proteasome activation, leads to different neuronal alterations and triggers apoptosis. Furthermore, testing the effects of isolated PrP^C on purified 20S proteasomes, we obtain a concentration‐ and proteasome composition‐dependent decrease in the complex activity. © 2009 Wiley‐Liss, Inc.     

Prion protein amyloidosis with divergent phenotype associated with two novel nonsense mutations in PRNP

Stop codon mutations in the gene encoding the prion protein (PRNP) are very rare and have thus far only been described in two patients with prion protein cerebral amyloid angiopathy (PrP-CAA). In this report, we describe the clinical, histopathological and pathological prion protein (PrP^Sc) characteristics of two Dutch patients carrying novel adjacent stop codon mutations in the C-terminal part of PRNP, resulting in either case in hereditary prion protein amyloidoses, but with strikingly different clinicopathological phenotypes. The patient with the shortest disease duration (27 months) carried a Y226X mutation and showed PrP-CAA without any neurofibrillary lesions, whereas the patient with the longest disease duration (72 months) had a Q227X mutation and showed an unusual Gerstmann-Sträussler-Scheinker disease phenotype with numerous cerebral multicentric amyloid plaques and severe neurofibrillary lesions without PrP-CAA. Western blot analysis in the patient with the Q227X mutation demonstrated the presence of a 7 kDa unglycosylated PrP^Sc fragment truncated at both the N- and C-terminal ends. Our observations expand the spectrum of clinicopathological phenotypes associated with PRNP mutations and show that a single tyrosine residue difference in the PrP C-terminus may significantly affect the site of amyloid deposition and the overall phenotypic expression of the prion disease. Furthermore, it confirms that the absence of the glycosylphosphatidylinositol anchor in PrP predisposes to amyloid plaque formation.     

Accumulation of prion protein in the vagus nerve in creutzfeldt–jakob disease

Disease‐associated proteins are thought to propagate along neuronal processes in neurodegenerative diseases. To detect disease‐associated prion protein (PrP^Sc) in the vagus nerve in different forms and molecular subtypes of Creutzfeldt–Jakob disease (CJD), we applied 3 different anti‐PrP antibodies. We screened the vagus nerve in 162 sporadic and 30 genetic CJD cases. Four of 31 VV‐2 type sporadic CJD and 7 of 30 genetic CJD cases showed vagal PrP^Sc immunodeposits with distinct morphology. Thus, PrP^Sc in CJD affects the vagus nerve analogously to α‐synuclein in Parkinson disease. The morphologically diverse deposition of PrP^Sc in genetic and sporadic CJD argues against uniform mechanisms of propagation of PrP^Sc. Ann Neurol 2019;85:782–787     

A novel seven-octapeptide repeat insertion in the prion protein gene (PRNP) in a Dutch pedigree with Gerstmann�CStr?ussler�CScheinker disease phenotype: comparison with similar cases from the literature

Human prion diseases can be sporadic, inherited or acquired by infection and show considerable phenotypic heterogeneity. We describe the clinical, histopathological and pathological prion protein (PrP^Sc) characteristics of a Dutch family with a novel 7-octapeptide repeat insertion (7-OPRI) in PRNP, the gene encoding the prion protein (PrP). Clinical features were available in four, neuropathological features in three and biochemical characteristics in two members of this family. The clinical phenotype was characterized by slowly progressive cognitive decline, personality change, lethargy, depression with anxiety and panic attacks, apraxia and a hypokinetic-rigid syndrome. Neuropathological findings consisted of numerous multi- and unicentric amyloid plaques throughout the cerebrum and cerebellum with varying degrees of spongiform degeneration. Genetic and molecular studies were performed in two male family members. One of them was homozygous for valine and the other heterozygous for methionine and valine at codon 129 of PRNP. Sequence analysis identified a novel 168?bp insertion [R2?CR2?CR2?CR2?CR3g?CR2?CR2] in the octapeptide repeat region of PRNP. Both patients carried the mutation on the allele with valine at codon 129. Western blot analysis showed type 1 PrP^Sc in both patients and detected a smaller ~8?kDa PrP^Sc fragment in the cerebellum in one patient. The features of this Dutch kindred define an unusual neuropathological phenotype and a novel PRNP haplotype among the previously documented 7-OPRI mutations, further expanding the spectrum of genotype?Cphenotype correlations in inherited prion diseases.     

Excitotoxicity Through NMDA Receptors Mediates Cerebellar Granule Neuron Apoptosis Induced by Prion Protein 90-231 Fragment

Prion diseases recognize, as a unique molecular trait, the misfolding of CNS-enriched prion protein (PrP^C) into an aberrant isoform (PrP^Sc). In this work, we characterize the in vitro toxicity of amino-terminally truncated recombinant PrP fragment (amino acids 90-231, PrP90-231), on rat cerebellar granule neurons (CGN), focusing on glutamatergic receptor activation and Ca²⁺ homeostasis impairment. This recombinant fragment assumes a toxic conformation (PrP90-231^TOX) after controlled thermal denaturation (1 h at 53 °C) acquiring structural characteristics identified in PrP^Sc (enrichment in β-structures, increased hydrophobicity, partial resistance to proteinase K, and aggregation in amyloid fibrils). By annexin-V binding assay, and evaluation of the percentage of fragmented and condensed nuclei, we show that treatment with PrP90-231^TOX, used in pre-fibrillar aggregation state, induces CGN apoptosis. This effect was associated with a delayed, but sustained elevation of [Ca²⁺]_i. Both CGN apoptosis and [Ca²⁺]_i increase were not observed using PrP90-231 in PrP^C-like conformation. PrP90-231^TOX effects were significantly reduced in the presence of ionotropic glutamate receptor antagonists. In particular, CGN apoptosis and [Ca²⁺]_i increase were largely reduced, although not fully abolished, by pre-treatment with the NMDA antagonists APV and memantine, while the AMPA antagonist CNQX produced a lower, although still significant, effect. In conclusion, we report that CGN apoptosis induced by PrP90-231^TOX correlates with a sustained elevation of [Ca²⁺]_i mediated by the activation of NMDA and AMPA receptors.     

Mutant prion protein D202N associated with familial prion disease is retained in the endoplasmic reticulum and forms ‘curly’ intracellular aggregates

Transmissible Spongiform Encephalopathies are fatal neurodegenerative disorders of humans and animals that are familial, sporadic, and infectious in nature. Familial disorders of humans include Gerstmann–Straussler–Scheinker disease (GSS), familial Creutzfeldt–Jakob disease (CJD), and fatal familial insomnia, and result from point mutations in the prion protein gene. Although neurotoxicity in familial cases is believed to result from a spontaneous change in conformation of mutant prion protein (PrP) to the pathogenic PrP-scrapie (PrP^Sc) form, emerging evidence indicates otherwise. We have investigated the processing and metabolism of mutant PrP D202N (PrP^202N) in cell models to elucidate possible mechanisms of cytotoxicity. In this report, we demonstrate that PrP^202N expressed in human neuroblastoma cells fails to achieve a mature conformation following synthesis and accumulates in the endoplasmic reticulum as ‘curly’ aggregates. In addition, PrP^202N cells show increased sensitivity to free radicals, indicating that neuronal susceptibility to oxidative damage may account for the neurotoxicity observed in cases of GSS resulting from PrP D202N mutation. Yaping Gu and Susamma Verghese contributed equally.