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The misfolding of the cellular prion protein (PrP C ) causes fatal neurodegenerative diseases. Yet PrP C is highly conserved in mammals, suggesting that it exerts beneficial functions preventing its evolutionary elimination. Ablation of PrP C in mice results in well-defined structural and functional alterations in the peripheral nervous system. Many additional phenotypes were ascribed to the lack of PrP C , but some of these were found to arise from genetic artifacts of the underlying mouse models. Here, we revisit the proposed physiological roles of PrP C in the central and peripheral nervous systems and highlight the need for their critical reassessment using new, rigorously controlled animal models.

The cellular prion protein PrP C promotes peripheral myelin homeostasis by acting on a G protein-coupled receptor to increase levels of cyclic AMP in Schwann cells. What does prion protein do? The cellular prion protein PrP C is known chiefly in the context of prion diseases. The prion gene Prnp was cloned in 1985, and Prnp -knockout mice have been available since 1992, but the role of PrP C in normal physiology remains unknown. Adriano Aguzzi and colleagues now show that PrP C is expressed in neurons where it promotes myelin homeostasis by acting on a promyelinating G protein-coupled receptor, Adgrg6 (also known as Gpr126), to increase levels of cyclic AMP in Schwann cells. Ablation of the cellular prion protein PrP C leads to a chronic demyelinating polyneuropathy affecting Schwann cells. Neuron-restricted expression of PrP C prevents the disease 1 , suggesting that PrP C acts in trans through an unidentified Schwann cell receptor. Here we show that the cAMP concentration in sciatic nerves from PrP C -deficient mice is reduced, suggesting that PrP C acts via a G protein-coupled receptor (GPCR). The amino-terminal flexible tail (residues 23–120) of PrP C triggered a concentration-dependent increase in cAMP in primary Schwann cells, in the Schwann cell line SW10, and in HEK293T cells overexpressing the GPCR Adgrg6 (also known as Gpr126). By contrast, naive HEK293T cells and HEK293T cells expressing several other GPCRs did not react to the flexible tail, and ablation of Gpr126 from SW10 cells abolished the flexible tail-induced cAMP response. The flexible tail contains a polycationic cluster (KKRPKPG) similar to the GPRGKPG motif of the Gpr126 agonist type-IV collagen 2 . A KKRPKPG-containing PrP C -derived peptide (FT 23–50 ) sufficed to induce a Gpr126-dependent cAMP response in cells and mice, and improved myelination in hypomorphic gpr126 mutant zebrafish ( Danio rerio ). Substitution of the cationic residues with alanines abolished the biological activity of both FT 23–50 and the equivalent type-IV collagen peptide. We conclude that PrP C promotes myelin homeostasis through flexible tail-mediated Gpr126 agonism. As well as clarifying the physiological role of PrP C , these observations are relevant to the pathogenesis of demyelinating polyneuropathies—common debilitating diseases for which there are limited therapeutic options.

Prion infections cause inexorable, progressive neurological dysfunction and neurodegeneration. Expression of the cellular prion protein PrPC is required for toxicity, suggesting the existence of deleterious PrPC-dependent signaling cascades. Because group-I metabotropic glutamate receptors (mGluR1 and mGluR5) can form complexes with the cellular prion protein (PrPC), we investigated the impact of mGluR1 and mGluR5 inhibition on prion toxicity ex vivo and in vivo. We found that pharmacological inhibition of mGluR1 and mGluR5 antagonized dose-dependently the neurotoxicity triggered by prion infection and by prion-mimetic anti-PrPC antibodies in organotypic brain slices. Prion-mimetic antibodies increased mGluR5 clustering around dendritic spines, mimicking the toxicity of Aβ oligomers. Oral treatment with the mGluR5 inhibitor, MPEP, delayed the onset of motor deficits and moderately prolonged survival of prion-infected mice. Although group-I mGluR inhibition was not curative, these results suggest that it may alleviate the neurological dysfunctions induced by prion diseases.

The adhesion G-protein coupled receptor Adgrg6 (formerly Gpr126) is instrumental in the development, maintenance and repair of peripheral nervous system myelin. The prion protein (PrP) is a potent activator of Adgrg6 and could be used as a potential therapeutic agent in treating peripheral demyelinating and dysmyelinating diseases. We designed a dimeric Fc-fusion protein comprising the myelinotrophic domain of PrP (FT 2 Fc), which activated Adgrg6 in vitro and exhibited favorable pharmacokinetic properties for in vivo treatment of peripheral neuropathies. While chronic FT 2 Fc treatment elicited specific transcriptomic changes in the sciatic nerves of PrP knockout mice, no amelioration of the early molecular signs demyelination was detected. Instead, RNA sequencing of sciatic nerves revealed downregulation of cytoskeletal and sarcomere genes, akin to the gene expression changes seen in myopathic skeletal muscle of PrP overexpressing mice. These results call for caution when devising myelinotrophic therapies based on PrP-derived Adgrg6 ligands. While our treatment approach was not successful, Adgrg6 remains an attractive therapeutic target to be addressed in other disease models or by using different biologically active Adgrg6 ligands.

Bovine spongiform encephalopathy (BSE) is a prion disease of cattle that is caused by the misfolding of the cellular prion protein (PrP C ) into an infectious conformation (PrP Sc ). PrP C is a predominantly α-helical membrane protein that misfolds into a β-sheet rich, infectious state, which has a high propensity to self-assemble into amyloid fibrils. Three strains of BSE prions can cause prion disease in cattle, including classical BSE (C-type) and two atypical strains, named L-type and H-type BSE. To date, there is no detailed information available about the structure of any of the infectious BSE prion strains. In this study, we purified L-type BSE prions from transgenic mouse brains and investigated their biochemical and ultrastructural characteristics using electron microscopy, image processing, and immunogold labeling techniques. By using phosphotungstate anions (PTA) to precipitate PrP Sc combined with sucrose gradient centrifugation, a high yield of proteinase K-resistant BSE amyloid fibrils was obtained. A morphological examination using electron microscopy, two-dimensional class averages, and three-dimensional reconstructions revealed two structural classes of L-type BSE amyloid fibrils; fibrils that consisted of two protofilaments with a central gap and an average width of 22.5 nm and one-protofilament fibrils that were 10.6 nm wide. The one-protofilament fibrils were found to be more abundant compared to the thicker two-protofilament fibrils. Both fibrillar assemblies were successfully decorated with monoclonal antibodies against N- and C-terminal epitopes of PrP using immunogold-labeling techniques, confirming the presence of polypeptides that span residues 100–110 to 227–237. The fact that the one-protofilament fibrils contain both N- and C-terminal PrP epitopes constrains molecular models for the structure of the infectious conformer in favour of a compact four-rung β-solenoid fold.

Prion immunotherapy may hold great potential, but antibodies against certain PrP epitopes can be neurotoxic. Here, we identified > 6,000 PrP‐binding antibodies in a synthetic human Fab phage display library, 49 of which we characterized in detail. Antibodies directed against the flexible tail of PrP conferred neuroprotection against infectious prions. We then mined published repertoires of circulating B cells from healthy humans and found antibodies similar to the protective phage‐derived antibodies. When expressed recombinantly, these antibodies exhibited anti‐PrP reactivity. Furthermore, we surveyed 48,718 samples from 37,894 hospital patients for the presence of anti‐PrP IgGs and found 21 high‐titer individuals. The clinical files of these individuals did not reveal any enrichment of specific pathologies, suggesting that anti‐PrP autoimmunity is innocuous. The existence of anti‐prion antibodies in unbiased human immunological repertoires suggests that they might clear nascent prions early in life. Combined with the reported lack of such antibodies in carriers of disease‐associated PRNP mutations, this suggests a link to the low incidence of spontaneous prion diseases in human populations. Synopsis This study assessed the anti‐prion protein (PrP) immunoreactivity in human immunoglobulin repertoires and patient samples. Anti‐PrP autoimmunity can exist in human communities, appears to be innocuous, and may protect against prion infections. >6,000 antibodies against various PrP epitopes were recovered from a synthetic human antibody (Fab) phage library. Of the phage display derived Fabs, 49 Fabs were characterized in detail. Fabs directed against the N‐terminal flexible tail of PrP conferred neuroprotection against infectious prions. HCDR3 sequences similar to a protective phage‐derived Fab were identified in published repertoires of B cells from healthy humans. When expressed recombinantly, these antibodies reacted to human PrP. By interrogating a large cohort of 37,894 hospital patients, twenty‐one individuals with high‐titer anti‐PrP autoreactivity in the plasma were found. The presence of anti‐PrP autoantibodies did not correlate to any neurological condition or any other diseases. Graphical Abstract This study assessed the anti‐prion protein (PrP) immunoreactivity in human immunoglobulin repertoires and patient samples. Anti‐PrP autoimmunity can exist in human communities, appears to be innocuous, and may protect against prion infections.

Prions are the infectious agents causing transmissible spongiform encephalopathies (TSE), progressive, inexorably lethal neurological diseases. Antibodies targeting the globular domain (GD) of the cellular prion protein PrPC trigger a neurotoxic syndrome morphologically and molecularly similar to prion disease. This phenomenon raises the question whether such antibodies induce infectious prions de novo. Here we exposed cerebellar organotypic cultured slices (COCS) to the neurotoxic antibody, POM1. We then inoculated COCS homogenates into tga20 mice, which overexpress PrPC and are commonly utilized as sensitive indicators of prion infectivity. None of the mice inoculated with COCS-derived lysates developed any signs of disease, and all mice survived for at least 200 days post-inoculation. In contrast, all mice inoculated with bona fide prions succumbed to TSE after 55-95 days. Post-mortem analyses did not reveal any signs of prion pathology in mice inoculated with POM1-COCS lysates. Also, lysates from POM1-exposed COCS were unable to convert PrP by quaking. Hence, anti-GD antibodies do not catalyze the generation of prion infectivity. These data indicate that prion replication can be separated from prion toxicity, and suggest that anti-GD antibodies exert toxicity by acting downstream of prion replication.

Recently, several genome-wide association studies identified PHACTR1 as key locus for five diverse vascular disorders: coronary artery disease, migraine, fibromuscular dysplasia, cervical artery dissection and hypertension. Although these represent significant risk factors or comorbidities for ischemic stroke, PHACTR1 role in brain small vessel ischemic disease and ischemic stroke most important survival mechanism, such as the recruitment of brain collateral arteries like posterior communicating arteries (PcomAs), remains unknown. Therefore, we applied exome and genome sequencing in a multi-ethnic cohort of 180 early-onset independent familial and apparently sporadic brain small vessel ischemic disease and CADASIL-like Caucasian patients from US, Portugal, Finland, Serbia and Turkey and in 2 C57BL/6J stroke mouse models (bilateral common carotid artery stenosis [BCCAS] and middle cerebral artery occlusion [MCAO]), characterized by different degrees of PcomAs patency. We report 3 very rare coding variants in the small vessel ischemic disease-CADASIL-like cohort (p.Glu198Gln, p.Arg204Gly, p.Val251Leu) and a stop-gain mutation (p.Gln273*) in one MCAO mouse. These coding variants do not cluster in PHACTR1 known pathogenic domains and are not likely to play a critical role in small vessel ischemic disease or brain collateral circulation. We also exclude the possibility that copy number variants (CNVs) or a variant enrichment in Phactr1 may be associated with PcomA recruitment in BCCAS mice or linked to diverse vascular traits (cerebral blood flow pre-surgery, PcomA size, leptomeningeal microcollateral length and junction density during brain hypoperfusion) in C57BL/6J mice, respectively. Genetic variability in PHACTR1 is not likely to be a common susceptibility factor influencing small vessel ischemic disease in patients and PcomA recruitment in C57BL/6J mice. Nonetheless, rare variants in PHACTR1 RPEL domains may influence the stroke outcome and are worth investigating in a larger cohort of small vessel ischemic disease patients, different ischemic stroke subtypes and with functional studies.

Mammalian models are essential for brain aging research. However, the long lifespan and poor amenability to genetic and pharmacological perturbations have hindered the use of mammals for dissecting aging-regulatory molecular networks and discovering new anti-aging interventions. To circumvent these limitations, we developed an ex vivo model system that faithfully mimics the aging process of the mammalian brain using cultured mouse brain slices. Genome-wide gene expression analyses showed that cultured brain slices spontaneously upregulated senescence-associated genes over time and reproduced many of the transcriptional characteristics of aged brains. Treatment with rapamycin, a classical anti-aging compound, largely abolished the time-dependent transcriptional changes in naturally aged brain slice cultures. Using this model system, we discovered that prions drastically accelerated the development of age-related molecular signatures and the pace of brain aging. We confirmed this finding in mouse models and human victims of Creutzfeldt-Jakob disease. These data establish an innovative, eminently tractable mammalian model of brain aging, and uncover a surprising acceleration of brain aging in prion diseases. Cultured brain slices from mice spontaneously upregulate senescence-associated genes over time and reproduce the transcriptional characteristics of aged brains; prions accelerate brain aging in brain slice cultures, animal models and human patients.