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Mistargeting of secretory proteins in the cytosol can trigger their aggregation and subsequent proteostasis decline. We have identified a VCP/p97-dependent pathway that directs non-ER-imported prion protein (PrP) into the nucleus to prevent the formation of toxic aggregates in the cytosol. Upon impaired translocation into the ER, PrP interacts with VCP/p97, which facilitates nuclear import mediated by importin-ß. Notably, the cytosolic interaction of PrP with VCP/p97 and its nuclear import are independent of ubiquitination. In vitro experiments revealed that VCP/p97 binds non-ubiquitinated PrP and prevents its aggregation. Inhibiting binding of PrP to VCP/p97, or transient proteotoxic stress, promotes the formation of self-perpetuating and partially proteinase resistant PrP aggregates in the cytosol, which compromised cellular proteostasis and disrupted further nuclear targeting of PrP. In the nucleus, RNAs keep PrP in a soluble and non-toxic conformation. Our study revealed a novel ubiquitin-independent role of VCP/p97 in the nuclear targeting of non-imported secretory proteins and highlights the impact of the chemical milieu in triggering protein misfolding., (© 2024 Banik et al.)

A common pathological denominator of various neurodegenerative diseases is the accumulation of protein aggregates. Neurotoxic effects are caused by a loss of the physiological activity of the aggregating protein and/or a gain of toxic function of the misfolded protein conformers. In transmissible spongiform encephalopathies or prion diseases, neurodegeneration is caused by aberrantly folded isoforms of the prion protein (PrP). However, it is poorly understood how pathogenic PrP conformers interfere with neuronal viability. Employing in vitro approaches, cell culture, animal models and patients' brain samples, we show that misfolded PrP can induce aggregation and inactivation of TAR DNA-binding protein-43 (TDP-43). Purified PrP aggregates interact with TDP-43 in vitro and in cells and induce the conversion of soluble TDP-43 into non-dynamic protein assemblies. Similarly, mislocalized PrP conformers in the cytosol bind to and sequester TDP-43 in cytosolic aggregates. As a consequence, TDP-43-dependent splicing activity in the nucleus is significantly decreased, leading to altered protein expression in cells with cytosolic PrP aggregates. Finally, we present evidence for cytosolic TDP-43 aggregates in neurons of transgenic flies expressing mammalian PrP and Creutzfeldt-Jakob disease patients. Our study identified a novel mechanism of how aberrant PrP conformers impair physiological pathways by cross-seeding., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Formation of biomolecular condensates through liquid-liquid phase separation (LLPS) has been described for several pathogenic proteins linked to neurodegenerative diseases and is discussed as an early step in the formation of protein aggregates with neurotoxic properties. In prion diseases, neurodegeneration and formation of infectious prions is caused by aberrant folding of the cellular prion protein (PrP C ). PrP C is characterized by a large intrinsically disordered N-terminal domain and a structured C-terminal globular domain. A significant fraction of mature PrP C is proteolytically processed in vivo into an entirely unstructured fragment, designated N1, and the corresponding C-terminal fragment C1 harboring the globular domain. Notably, N1 contains a polybasic motif that serves as a binding site for neurotoxic Aβ oligomers. PrP can undergo LLPS; however, nothing is known how phase separation of PrP is triggered on a molecular scale. Here, we show that the intrinsically disordered N1 domain is necessary and sufficient for LLPS of PrP. Similar to full-length PrP, the N1 fragment formed highly dynamic liquid-like droplets. Remarkably, a slightly shorter unstructured fragment, designated N2, which lacks the Aβ-binding domain and is generated under stress conditions, failed to form liquid-like droplets and instead formed amorphous assemblies of irregular structures. Through a mutational analysis, we identified three positively charged lysines in the postoctarepeat region as essential drivers of condensate formation, presumably largely via cation-π interactions. These findings provide insights into the molecular basis of LLPS of the mammalian prion protein and reveal a crucial role of the Aβ-binding domain in this process., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Despite their devastating impact, no effective therapeutic yet exists for prion diseases at the symptomatic stage in humans or animals. Progress is hampered by the difficulty in identifying compounds that affect PrP (Sc) and the necessity of any potential therapeutic to gain access to the CNS. Synthetic polymers known as dendrimers are a particularly promising candidate in this area. Studies with cell culture models of prion disease and prion infected brain homogenate have demonstrated that numerous species of dendrimers eliminate PrP (Sc) in a dose and time dependent fashion and specific glycodendrimers are capable of crossing the CNS. However, despite their potential a number of important questions remained unanswered such as what makes an effective dendrimer and how dendrimers eliminate prions intracellularly. In a number of recent studies we have tackled these questions and revealed for the first time that a specific dendrimer can inhibit the intracellular conversion of PrP (C) to PrP (Sc) and that a high density of surface reactive groups is a necessity for dendrimers in vitro anti-prion activity. Understanding how a therapeutic works is a vital component in maximising its activity and these studies therefore represent a significant development in the race to find effective treatments for prion diseases.

Prion diseases in humans and animals comprise a group of invariably fatal neurodegenerative diseases characterized by the formation of a pathogenic protein conformer designated PrP(Sc) and infectious particles denoted prions. The cellular prion protein (PrP(C)) has a central role in the pathogenesis of prion disease. First, it is the precursor of PrP(Sc) and infectious prions and second, its expression on neuronal cells is required to mediate toxic effects of prions. To specifically study the role of PrP(C) as a mediator of toxic signaling, we have developed novel cell culture models, including primary neurons prepared from PrP-deficient mice. Using these approaches we have been able to show that PrP(C) can interact with and mediate toxic signaling of various β-sheet-rich conformers of different origins, including amyloid β, suggesting a pathophysiological role of the prion protein beyond prion diseases., (Copyright © 2011 S. Karger AG, Basel.)

Eukaryotic cell surface proteins are often modified by a glycosylphosphatidylinositol (GPI) anchor. More than 200 of these post-translationally altered proteins are presently known, a prominent example being the prion protein (PrP). Although the significance of the GPI anchor is well recognized, efforts to study its function are hampered due to its complex chemical nature, which combines hydrophilic glycosyl chains with hydrophobic lipid moieties. Here we describe a general method for the synthesis of a GPI-anchored peptide containing an N-terminal Cys. This module can be employed for the production of proteins containing a natural GPI anchor using expressed protein ligation., (Copyright (c) 2010 Wiley Periodicals, Inc.)

Prion diseases are characterized by the conformational transition of the cellular prion protein (PrP(C)) into an aberrant protein conformer, designated scrapie-prion protein (PrP(Sc)). A causal link between protein misfolding and neurodegeneration has been established for a variety of neurodegenerative disease, such as Alzheimer's disease, Parkinson's disease and polyglutamine diseases, but there is an ongoing debate about the nature of the neurotoxic species and how non-native conformers can damage neuronal populations. PrP is normally imported into the endoplasmic reticulum (ER) and targeted to the outer leaflet of the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor. However, several conditions, such as ER stress or some pathogenic mutations in the PrP gene, can induce the mislocalization of PrP in the cytosol, where it has a neurotoxic potential as demonstrated in cell culture and transgenic mouse models. In this review we focus on intrinsic factors and cellular pathways implicated in the import of PrP into the ER and its mistargeting to the cytosol. The findings summarized here not only reveal a complex regulation of the biogenesis of PrP, but also provide interesting new insight into toxic activities of pathogenic protein conformers and quality control pathways of ER-targeted proteins.

Prion diseases are caused by conversion of a normally folded, non-pathogenic isoform of the prion protein (PrP(C)) to a misfolded, pathogenic isoform (PrP(Sc)). Prion inoculation experiments in mice expressing homologous PrP(C) molecules on different genetic backgrounds displayed different incubation times, indicating that the conversion reaction may be influenced by other gene products. To identify genes that contribute to prion pathogenesis, we analysed incubation times of prions in mice in which the gene product was inactivated, knocked out or overexpressed. We tested 20 candidate genes, because their products either colocalize with PrP, are associated with Alzheimer's disease, are elevated during prion disease, or function in PrP-mediated signalling, PrP glycosylation, or protein maintenance. Whereas some of the candidates tested may have a role in the normal function of PrP(C), our data show that many genes previously implicated in prion replication have no discernible effect on the pathogenesis of prion disease. While most genes tested did not significantly affect survival times, ablation of the amyloid beta (A4) precursor protein (App) or interleukin-1 receptor, type I (Il1r1), and transgenic overexpression of human superoxide dismutase 1 (SOD1) prolonged incubation times by 13, 16 and 19 %, respectively.

The infectious agent in prion diseases is an aberrant-folded isoform of the cellular prion protein (PrPC). This scrapie-related prion protein (PrPSc) has an increased beta-sheet content, is detergent insoluble and proteinase K resistant, and accumulates in prion-infected organisms and cells. In vitro, PrPSc self-aggregates into amyloid fibrils. However, there is no direct experimental proof for the occurrence of PrPSc-containing fibrils in vivo or in cell cultures. Applying atomic force microscopy (AFM) to scrapie-infected mouse neuroblastoma (ScN2a) cells, we discovered growing patch-like assemblies of amyloid-like fibrillar structures on the cell surfaces. Immunofluorescence and AFM images showed heterogeneous accumulation and aggregation of PrPSc in ScN2a cell cultures. The percentage of cells having characteristic fibrils on their surface increased with time after scrapie infection. These endogeneous fibrils had lengths from 0.5 to 3 microm and protruded from the cell surface by 108 +/- 30 nm, and thus resembled the heterogeneous shapes and networks of in vitro prepared amyloid fibrils.

The biochemical nature and the replication of infectious prions have been intensively studied in recent years. Much less is known about the cellular events underlying neuronal dysfunction and cell death. As the cellular function of the normal cellular isoform of prion protein is not exactly known, the impact of gain of toxic function or loss of function, or a combination of both, in prion pathology is still controversial. There is increasing evidence that the normal cellular isoform of the prion protein is a key mediator in prion pathology. Transgenic models were instrumental in dissecting propagation of prions, disease-associated isoforms of prion protein and amyloid production, and induction of neurodegeneration. Four experimental avenues will be discussed here which address scenarios of inappropriate trafficking, folding, or targeting of the prion protein.

A hallmark of prion diseases in mammals is a conformational transition of the cellular prion protein (PrP(C)) into a pathogenic isoform termed PrP(Sc). PrP(C) is highly conserved in mammals, moreover, genes of PrP-related proteins have been recently identified in fish. While there is only little sequence homology to mammalian PrP, PrP-related fish proteins were predicted to be modified with N-linked glycans and a C-terminal glycosylphosphatidylinositol (GPI) anchor. We biochemically characterized two PrP-related proteins from zebrafish in cultured cells and show that both zePrP1 and zeSho2 are imported into the endoplasmic reticulum and are post-translationally modified with complex glycans and a C-terminal GPI anchor.

An inert polysaccharide scaffold identified as a 5-15% component of prion rods (PrP 27-30) is unambiguously distinguishable from the N-glycosyl groups and the GPI anchor of PrP, and consists predominantly of 1,4-linked glucose with some branching via 1,4,6-linked glucose. We show that this polysaccharide scaffold is a common secondary component of prions found in hamster full-length PrP(Sc), prion rods and in mouse ScN2a prions from cell culture. The preparation from prion rods was improved, resulting in a polysaccharide scaffold free of remaining infectivity. Furthermore, we determined the stereochemistry of the glycoside linkages as pre-dominantly if not entirely alpha-glycosidic. The origin of the polysaccharide, its interaction with PrP and its potential relation to glycogen and corpora amylacea are discussed.

Conformational changes and aggregation of specific proteins are hallmarks of a number of diseases, like Alzheimer's disease, Parkinson's disease, and prion diseases. In the case of prion diseases, the prion protein (PrP), a neuronal glycoprotein, undergoes a conformational change from the normal, mainly alpha-helical conformation to a disease-associated, mainly beta-sheeted scrapie isoform (PrP(Sc)), which forms amyloid aggregates. This conversion, which is crucial for disease progression, depends on direct PrP(C)/PrP(Sc) interaction. We developed a high-throughput assay based on scanning for intensely fluorescent targets (SIFT) for the identification of drugs which interfere with this interaction at the molecular level. Screening of a library of 10,000 drug-like compounds yielded 256 primary hits, 80 of which were confirmed by dose response curves with half-maximal inhibitory effects ranging from 0.3 to 60 microM. Among these, six compounds displayed an inhibitory effect on PrP(Sc) propagation in scrapie-infected N2a cells. Four of these candidate drugs share an N'-benzylidene-benzohydrazide core structure. Thus, the combination of high-throughput in vitro assay with the established cell culture system provides a rapid and efficient method to identify new antiprion drugs, which corroborates that interaction of PrP(C) and PrP(Sc) is a crucial molecular step in the propagation of prions. Moreover, SIFT-based screening may facilitate the search for drugs against other diseases linked to protein aggregation.

Several proteins linked to neurodegenerative diseases, such as the beta-amyloid precursor protein, amyloid beta-peptide, beta-secretase, and tau, undergo selective polarized sorting. We investigated polarized sorting of the mammalian prion protein (PrP(C)) and its homologue doppel (Dpl). In contrast to Dpl, which accumulates on the apical surface, PrP(C) is targeted selectively to the basolateral side in Madin-Darby canine kidney cells. An extensive deletion and domain swapping analysis revealed that the internal hydrophobic domain (HD) of PrP (amino acids 113-133) confers basolateral sorting in a dominant manner. PrP mutants lacking the HD are sorted apically, while Dpl chimeras containing the HD of PrP are directed to the basolateral membrane. Furthermore, a pathogenic PrP missense mutation within the HD leads to aberrant apical sorting of PrP as well.

Suramin induces misfolding of the cellular prion protein (PrP(C)) and interferes with the propagation of infectious scrapie prions. A mechanistic analysis of this effect revealed that suramin-induced misfolding occurs at the plasma membrane and is dependent on the proximal region of the C-terminal domain (aa 90-158) of PrP(C). The conformational transition induces rapid internalization, mediated by the unstructured N-terminal domain, and subsequent intracellular degradation of PrP(C). As a consequence, PrP Delta N adopts a misfolded conformation at the plasma membrane; however, internalization is significantly delayed. We also found that misfolding and intracellular retention of PrP(C) can be induced by copper and that, moreover, copper interferes with the propagation of the pathogenic prion protein (PrP(Sc)) in scrapie-infected N2a cells. Our study revealed a quality control pathway for aberrant PrP conformers present at the plasma membrane and identified distinct PrP domains involved.

The mammalian prion protein (PrP) is composed of an unstructured flexible N-terminal region and a C-terminal globular domain. We examined the import of PrP into the endoplasmic reticulum (ER) of neuronal cells and show that information present in the C-terminal globular domain is required for ER import of the N terminus. N-terminal fragments of PrP, devoid of structural domains located in the C terminus, remained in the cytosol with an uncleaved signal peptide and were rapidly degraded by the proteasome. Conversely, the separate C-terminal domain of PrP, comprising the highly ordered helix 2-loop-helix 3 motif, was entirely imported into the ER. As a consequence, two PrP mutants linked to inherited prion disease in humans, PrP-W145Stop and PrP-Q160Stop, were partially retained in the cytosol. The cytosolic fraction was characterized by an uncleaved N-terminal signal peptide and was degraded by the proteasome. Our study identified a new regulatory element in the C-terminal globular domain of PrP necessary and sufficient to promote import of PrP into the ER.

Aberrant folding of the mammalian prion protein (PrP) is linked to prion diseases in humans and animals. We show that during post-translational targeting of PrP to the endoplasmic reticulum (ER) the putative transmembrane domain induces misfolding of PrP in the cytosol and interferes with its import into the ER. Unglycosylated and misfolded PrP with an uncleaved N-terminal signal sequence associates with ER membranes, and, moreover, decreases cell viability. PrP expressed in the cytosol, lacking the N-terminal ER targeting sequence, also adopts a misfolded conformation; however, this has no adverse effect on cell growth. PrP processing, productive ER import, and cellular viability can be restored either by deleting the putative transmembrane domain or by using a N-terminal signal sequence specific for co-translational ER import. Our study reveals that the putative transmembrane domain features in the formation of misfolded PrP conformers and indicates that post-translational targeting of PrP to the ER can decrease cell viability.

Prion diseases are fatal and transmissible neurodegenerative disorders linked to an aberrant conformation of the cellular prion protein (PrP(c)). We show that the chemical compound Suramin induced aggregation of PrP in a post-ER/Golgi compartment and prevented further trafficking of PrP(c) to the outer leaflet of the plasma membrane. Instead, misfolded PrP was efficiently re-routed to acidic compartments for intracellular degradation. In contrast to PrP(Sc) in prion-infected cells, PrP aggregates formed in the presence of Suramin did not accumulate, were entirely sensitive to proteolytic digestion, had distinct biophysical properties, and were not infectious. The prophylactic potential of Suramin-induced intracellular re-routing was tested in mice. After intraperitoneal infection with scrapie prions, peripheral application of Suramin around the time of inoculation significantly delayed onset of prion disease. Our data reveal a novel quality control mechanism for misfolded PrP isoforms and introduce a new molecular mechanism for anti-prion compounds.

The kinetics of PrP(Sc) and insoluble PrP accumulation in the spleens and brains of CD-1 mice were studied. The mice were inoculated intracerebrally with RML prions and euthanized at various times between inoculation and the onset of illness at approximately 130 days. Protease-resistant PrP(Sc), PrP 27-30, was first detected in brain by histoblotting 49 days after inoculation and by Western immunoblotting at 70 days. In spleen, PrP 27-30 was first detected by Western immunoblotting at 28 days after inoculation. Like PrP 27-30, substantial increases in detergent-insoluble PrP were first detected at 70 days after inoculation in brain and 28 days in spleen. In addition, a progressive increase in detergent-soluble PrP was detected beginning 70 days after inoculation. Further characterization of detergent soluble and insoluble PrP with respect to protease-sensitive PrP(Sc) and prion infectivity will be of considerable interest.

1. Prion diseases include kuru, Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker disease (GSS), and fatal familia insomnia (FFI) of humans, as well as scrapie and bovine spongiform encephalopathy (BSE) of animals. 2. All these disorders involve conversion of the normal, cellular prion protein (PrPC) into the corresponding scrapie isoform (PrPSc). PrPC adopts a structure rich in alpha-helices and devoid of beta-sheet, in contrast to PrPSc, which has a high beta-sheet content and is resistant to limited digestion by proteases. That a conformational transition features in the conversion of PrPC into PrPSc implies that prion diseases are disorders of protein conformation. 3. This concept has been extended by our studies with heat shock proteins (Hsp), many of which are thought to function as molecular chaperones. We found that the induction of some Hsps but not others was profoundly altered in scrapie-infected cells and that the distribution of Hsp73 is unusual in these cells. 4. Whether the conversion of PrPC into PrPSc is assisted by molecular chaperones, or if the accumulation of the abnormally folded PrPSc is complexed with Hsps remains to be established.

Syrian hamster prion protein (PrPC) and a truncated Syrian hamster prion protein lacking the glycosylphosphatidylinositol (GPI) anchor C-terminal signal sequence (GPI-) were expressed in Chinese hamster ovary cells using a glutamine synthetase selection and amplification system. The CHO cell clones expressing the GPI- PrP secreted the majority of the protein into the media, whereas most of the PrP produced by clones expressing the full-length protein with the GPI anchor was located on the cell surface, as demonstrated by its release upon treatment with phosphatidylinositol-specific phospholipase C (PIPLC). A cell clone that expressed the highest levels of full length PrP was subcloned to obtain clone 30C3-1. PrP from clone 30C3-1 was shown to be sensitive to proteolysis by proteinase K and to react with monoclonal and polyclonal antibodies that recognize native PrPC. The recombinant PrP migrated as a diffuse band of 19-40 kDa but removal of the N-linked oligosaccharides with peptide N-glycosidase F (PNGase F) revealed three protein species of 19, 17 and 15 kDa. The 19 kDa band corresponding to deglycosylated full-length PrP was quantified and found to be expressed at a level approximately 14-fold higher than that of PrPC found in Syrian hamster brain.

Two prion strains with identical incubation periods in mice exhibited distinct incubation periods and different neuropathological profiles upon serial transmission to transgenic mice expressing chimeric Syrian hamster/mouse (MH2M) prion protein (PrP) genes [Tg(MH2M) mice] and subsequent transmission to Syrian hamsters. After transmission to Syrian hamsters, the Me7 strain was indistinguishable from the previously established Syrian hamster strain Sc237, despite having been derived from an independent ancestral source. This apparent convergence suggests that prion diversity may be limited. The Me7 mouse strain could also be transmitted directly to Syrian hamsters, but when derived in this way, its properties were distinct from those of Me7 passaged through Tg(MH2M) mice. The Me7 strain did not appear permanently altered in either case, since the original incubation period could be restored by effectively reversing the series of passages. Prion diversity enciphered in the conformation of the scrapie isoform of PrP (PrP(Sc)) (G. C. Telling et al., Science 274:2079-2082, 1996) seems to be limited by the sequence of the PrP substrates serially converted into PrP(Sc), while prions are propagated through interactions between the cellular and scrapie isoforms of PrP.

The fundamental event underlying scrapie infection seems to be a conformational change in the prion protein. To investigate proteins that might feature in the conversion of the cellular prion protein (PrPC) into the scrapie isoform (PrPSc), we examined mouse neuroblastoma N2a cells for the expression and cellular distribution of heat shock proteins (Hsps), some of which function as molecular chaperones. In scrapie-infected N2a (ScN2a) cells, Hsp72 and Hsp28 were not induced by heat shock, sodium arsenite, or an amino acid analog, in contrast to uninfected control N2a cells, while other inducible Hsps were increased by these treatments. Following heat shock of the N2a cells, constitutively expressed Hsp73 was translocated from the cytoplasm into the nucleus and nucleolus. In contrast, the distribution of Hsp73 in ScN2a cells was not altered by heat shock; the discrete cytoplasmic structures containing Hsp73 were largely resistant to detergent extraction. These alterations in the expression and subcellular translocation of specific Hsps in ScN2a cells may reflect the cellular response to the accumulation of PrPSc. Whether any of these Hsps feature in the conversion of PrPC into PrPSc or the pathogenesis of prion diseases remains to be established.