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The cellular prion protein (PrPC) is a central player in neurodegenerative diseases caused by protein misfolding, such as prion diseases or Alzheimer’s disease (AD). Expression levels of this GPI-anchored glycoprotein, especially at the neuronal cell surface, critically correlate with various pathomechanistic aspects underlying these diseases, such as templated misfolding (in prion diseases) and neurotoxicity and, hence, with disease progression and severity. In stark contrast to cell-associated PrPC, soluble extracellular forms or fragments of PrP are linked with neuroprotective effects, which is likely due to their ability to interfere with neurotoxic disease-associated protein conformers in the interstitial fluid. Fittingly, the endogenous proteolytic release of PrPCby the metalloprotease ADAM10 (‘shedding’) was characterized as a protective mechanism. Here, using a recently generated cleavage-site specific antibody, we shed new light on earlier studies by demonstrating that shed PrP (sPrP) negatively correlates with conformational conversion (in prion disease) and is markedly redistributed in murine brain in the presence of prion deposits or AD-associated amyloid plaques indicating a blocking and sequestrating activity. Importantly, we reveal that administration of certain PrP-directed antibodies and other ligands results in increased PrP shedding in cells and organotypic brain slice cultures. We also provide mechanistic and structural insight into this shedding-stimulating effect. In addition, we identified a striking exception to this, as one particular neuroprotective antibody, due to its special binding characteristics, did not cause increased shedding but rather strong surface clustering followed by fast endocytosis and degradation of PrPC. Both mechanisms may contribute to the beneficial action described for some PrP-directed antibodies/ligands and pave the way for new therapeutic strategies against devastating and currently incurable neurodegenerative diseases.

Recent computational advancements in the simulation of biochemical processes allow investigating the mechanisms involved in protein regulation with realistic physics-based models, at an atomistic level of resolution. Using these techniques to study the negative regulation of the androgen receptor (AR), we discovered a key functional role played by non-native metastable states appearing along the folding pathway of this protein. This unexpected observation inspired us to design a completely novel drug discovery approach, named Pharmacological Protein Inactivation by Folding Intermediate Targeting (PPI-FIT), based on the rationale of negatively regulating protein expression by targeting folding intermediates. Here, PPI-FIT was tested for the first time on the cellular prion protein (PrP), a cell surface glycoprotein playing a key role in fatal and transmissible neurodegenerative pathologies known as prion diseases. We predicted the all-atom structure of an intermediate appearing along the folding pathway of PrP, and identified four different small molecule ligands for this conformer, all capable of selectively lowering the expression of the protein by promoting its degradation. Our data support the notion that the level of target proteins could be modulated by acting on their folding pathways, implying a previously unappreciated role for folding intermediates in the biological regulation of protein expression.

Cofactors are essential for driving recombinant prion protein into pathogenic conformers. Polyanions promote prion aggregation in vitro, yet the cofactors that modulate prion assembly in vivo remain largely unknown. Here we report that the endogenous glycosaminoglycan, heparan sulfate (HS), impacts prion propagation kinetics and deposition sites in the brain. Exostosin-1 haploinsufficient (Ext1+/-) mice, which produce short HS chains, show a prolonged survival and a redistribution of plaques from the parenchyma to vessels when infected with fibrillar prions, and a modest delay when infected with subfibrillar prions. Notably, the fibrillar, plaque-forming prions are composed of ADAM10-cleaved prion protein lacking a glycosylphosphatidylinositol anchor, indicating that these prions are mobile and assemble extracellularly. By analyzing the prion-bound HS using liquid chromatography-mass spectrometry (LC-MS), we identified the disaccharide signature of HS differentially bound to fibrillar compared to subfibrillar prions, and found approximately 20-fold more HS bound to the fibrils. Finally, LC-MS of prion-bound HS from human patients with familial and sporadic prion disease also showed distinct HS signatures and higher HS levels associated with fibrillar prions. This study provides the first in vivo evidence of an endogenous cofactor that accelerates prion disease progression and enhances parenchymal deposition of ADAM10-cleaved, mobile prions.

The structurally disordered N-terminal half of the prion protein (PrPC) is constitutively released into the extracellular space by an endogenous proteolytic cleavage event. Once liberated, this N1 fragment acts neuroprotective in ischemic conditions and interferes with toxic peptides associated with neurodegenerative diseases, such as amyloid-beta (Aβ) in Alzheimer’s disease. Since analog protective effects of N1 in prion diseases, such as Creutzfeldt-Jakob disease, have not been studied, and given that the protease releasing N1 has not been identified to date, we have generated and characterized transgenic mice overexpressing N1 (TgN1). Upon intracerebral inoculation of TgN1 mice with prions, no protective effects were observed at the levels of survival, clinical course, neuropathological, or molecular assessment. Likewise, primary neurons of these mice did not show protection against Aβ toxicity. Our biochemical and morphological analyses revealed that this lack of protective effects is seemingly due to an impaired ER translocation of the disordered N1 resulting in its cytosolic retention with an uncleaved signal peptide. Thus, TgN1 mice represent the first animal model to prove the inefficient ER translocation of intrinsically disordered domains (IDD). In contrast to earlier studies, our data challenge roles of cytoplasmic N1 as a cell penetrating peptide or as a potent “anti-prion” agent. Lastly, our study highlights both the importance of structured domains in the nascent chain for proteins to be translocated and aspects to be considered when devising novel N1-based therapeutic approaches against neurodegenerative diseases. Electronic supplementary material The online version of this article (10.1007/s12035-020-01917-2) contains supplementary material, which is available to authorized users.

The cellular prion protein (PrPC) is a cell surface glycoprotein attached to the membrane by a glycosylphosphatidylinositol (GPI)-anchor and plays a critical role in transmissible, neurodegenerative and fatal prion diseases. Alterations in membrane attachment influence PrPC-associated signaling, and the development of prion disease, yet our knowledge of the role of the GPI-anchor in localization, processing, and function of PrPC in vivo is limited We exchanged the PrPC GPI-anchor signal sequence of for that of Thy-1 (PrPCGPIThy-1) in cells and mice. We show that this modifies the GPI-anchor composition, which then lacks sialic acid, and that PrPCGPIThy-1 is preferentially localized in axons and is less prone to proteolytic shedding when compared to PrPC. Interestingly, after prion infection, mice expressing PrPCGPIThy-1 show a significant delay to terminal disease, a decrease of microglia/astrocyte activation, and altered MAPK signaling when compared to wild-type mice. Our results are the first to demonstrate in vivo, that the GPI-anchor signal sequence plays a fundamental role in the GPI-anchor composition, dictating the subcellular localization of a given protein and, in the case of PrPC, influencing the development of prion disease., Author summary The prion protein (PrPC) is a glycoprotein attached to the neuronal surface via a GPI-anchor. When misfolded to PrPSc, it leads to fatal neurodegenerative diseases which propagates from host to host. PrPSc is the principal component of the infectious agent of prion diseases, the “prion”. Misfolding occurs at the plasma membrane, and when PrPC lacks the GPI-anchor, neuropathology and incubation time of prion disease are strongly modified. Moreover, the composition of the PrPC GPI-anchor impacts on the conversion process. To study the role of the GPI-anchor in the pathophysiology of prion diseases in vivo, we have generated transgenic mice where the PrPC GPI-signal sequence (GPI-SS) is replaced for the one of Thy-1, a neuronal protein with a distinct GPI-anchor and membrane localization. We found that the resulting protein, PrPCGPIThy-1, shows a different GPI-anchor composition, increased axonal localization, and reduced enzymatic shedding. After prion infection, disease progression is significantly delayed, and the neuropathology and cellular signaling are changed. The present work demonstrates that the GPI-SS per se determines the GPI-anchor composition and localization of a given protein and it stresses the importance of PrPC membrane anchorage in prion disease.

Background: Proteolytic processing of the prion protein (PrPC) by endogenous proteases generates bioactive membrane-bound and soluble fragments which may help to explain the pleiotropic roles of this protein in the nervous system and in brain diseases. Shedding of almost full-length PrPC into the extracellular space by the metalloprotease ADAM10 is of peculiar relevance since soluble PrP stimulates axonal outgrowth and is protective in neurodegenerative conditions such as Alzheimer’s and prion disease. However, molecular determinates and mechanisms regulating the shedding of PrP are entirely unknown. Methods: We produced an antibody recognizing the neo-epitope of shed PrP generated by ADAM10 in biological samples and used it to study structural and mechanistic aspects affecting the shedding. For this, we investigated genetically modified cellular and murine models by biochemical and morphological approaches. Results: We show that the novel antibody specifically detects shed PrP in cell culture supernatants and murine brain. We demonstrate that ADAM10 is the exclusive sheddase of PrPC in the nervous system and reveal that the glycosylation state and type of membrane-anchorage of PrPC severely affect its shedding. Furthermore, we provide evidence that PrP shedding can be modulated by pharmacological inhibition and stimulation and present data suggesting that shedding is a relevant part of a compensatory network ensuring PrPC homeostasis of the cell. Conclusions: With the new antibody, our study introduces a new tool to reliably investigate PrP-shedding. In addition, this study provides novel and important insight into the regulation of this cleavage event, which is likely to be relevant for diagnostic and therapeutic approaches even beyond neurodegeneration.

Proteolytic processing of the cellular and disease-associated form of the prion protein leads to generation of bioactive soluble prion protein fragments and modifies the structure and function of its cell-bound form. The nature of proteases responsible for shedding, α-, β-, and γ-cleavage of the prion protein are only partially identified and their regulation is largely unknown. Here, we provide an overview of the increasingly multifaceted picture of prion protein proteolysis and shed light on physiological and pathological roles associated with these cleavages. This article is part of a Special Issue entitled: Proteolysis as a Regulatory Event in Pathophysiology edited by Stefan Rose-John.

Misfolding of proteins in the biosynthetic pathway in neurons may cause disturbed protein homeostasis and neurodegeneration. The prion protein (PrPC) is a GPI-anchored protein that resides at the plasma membrane and may be misfolded to PrPSc leading to prion diseases. We show that a deletion in the C-terminal domain of PrPC (PrPΔ214–229) leads to partial retention in the secretory pathway causing a fatal neurodegenerative disease in mice that is partially rescued by co-expression of PrPC. Transgenic (Tg(PrPΔ214–229)) mice show extensive neuronal loss in hippocampus and cerebellum and activation of p38-MAPK. In cell culture under stress conditions, PrPΔ214–229 accumulates in the Golgi apparatus possibly representing transit to the Rapid ER Stress-induced ExporT (RESET) pathway together with p38-MAPK activation. Here we describe a novel pathway linking retention of a GPI-anchored protein in the early secretory pathway to p38-MAPK activation and a neurodegenerative phenotype in transgenic mice.

Proteolytic processing regulates key processes in health and disease. The cellular prion protein (PrPC) is subject to at least 3 cleavage events, α-cleavage, β-cleavage and shedding. In contrast to α- and β-cleavage where there is an ongoing controversy on the identity of relevant proteases, the metalloprotease ADAM10 represents the only relevant PrP sheddase. Here we focus on the roles that ADAM10-mediated shedding of PrPC and its pathogenic isoform (PrPSc) might play in regulating their physiological and pathogenic functions, respectively. As revealed by our recent study using conditional ADAM10 knockout mice (Altmeppen et al., 2015), shedding of PrP seems to be involved in key processes of prion diseases. These aspects and several open questions arising from them are discussed. Increased knowledge on this topic can shed new light on prion diseases and other neurodegenerative conditions as well.

The prion protein (PrPC) is highly expressed in the nervous system and critically involved in prion diseases where it misfolds into pathogenic PrPSc. Moreover, it has been suggested as a receptor mediating neurotoxicity in common neurodegenerative proteinopathies such as Alzheimer's disease. PrPC is shed at the plasma membrane by the metalloprotease ADAM10, yet the impact of this on prion disease remains enigmatic. Employing conditional knockout mice, we show that depletion of ADAM10 in forebrain neurons leads to posttranslational increase of PrPC levels. Upon prion infection of these mice, clinical, biochemical, and morphological data reveal that lack of ADAM10 significantly reduces incubation times and increases PrPSc formation. In contrast, spatiotemporal analysis indicates that absence of shedding impairs spread of prion pathology. Our data support a dual role for ADAM10-mediated shedding and highlight the role of proteolytic processing in prion disease. DOI: http://dx.doi.org/10.7554/eLife.04260.001, eLife digest Prion proteins are anchored to the surface of brain cells called neurons. Normally, prion proteins are folded into a specific three-dimensional shape that enables them to carry out their normal roles in the brain. However, they can be misfolded into a different shape known as PrPSc, which can cause Creutzfeldt-Jakob disease and other serious conditions that affect brain function and ultimately lead to death. The PrPSc proteins can force normal prion proteins to change into the PrPSc form, so that over time this form accumulates in the brain. They are essential components of infectious particles termed ‘prions’ and this is why prion diseases are infectious: if prions from one individual enter the brain of another individual they can cause disease in the recipient. The UK outbreak of variant Creutzfeldt-Jakob disease in humans in the 1990s is thought to be due to the consumption of meat from cattle with a prion disease known as mad cow disease. An enzyme called ADAM10 can cut normal prion proteins from the surface of neurons. However, it is not clear whether ADAM10 can also target the PrPSc proteins and what impact this may have on the development of prion diseases. Here, Altmeppen et al. studied mutant mice that were missing ADAM10 in neurons in the front portion of their brain. These mice had a higher number of normal prion proteins on the surface of their neurons than normal mice did. When mice missing ADAM10 were infected with prions, more PrPSc accumulated in their brain and disease symptoms developed sooner than when normal mice were infected. This supports the view that mice with higher numbers of prion proteins are more vulnerable to prion disease. However, disease symptoms did not spread as quickly to other parts of the brain in the mice missing ADAM10. This suggests that by releasing prion proteins from the surface of neurons, ADAM10 helps PrPSc proteins to spread around the brain. Recently, it has been suggested that prion proteins may also play a role in Alzheimer's disease and other neurodegenerative conditions. Therefore, Altmeppen et al.'s findings may help to develop new therapies for other forms of dementia. The next challenge is to understand the precise details of how ADAM10 works. DOI: http://dx.doi.org/10.7554/eLife.04260.002