Cystatin C loaded in brain-derived extracellular vesicles rescues synapses after ischemic insult in vitro and in vivo.

Synaptic loss is an early event in the penumbra area after an ischemic stroke. Promoting synaptic preservation in this area would likely improve functional neurological recovery. We aimed to detect proteins involved in endogenous protection mechanisms of synapses in the penumbra after stroke and to analyse potential beneficial effects of these candidates for a prospective stroke treatment. For this, we performed Liquid Chromatography coupled to Mass Spectrometry (LC-MS)-based proteomics of synaptosomes isolated from the ipsilateral hemispheres of mice subjected to experimental stroke at different time points (24 h, 4 and 7 days) and compared them to sham-operated mice. Proteomic analyses indicated that, among the differentially expressed proteins between the two groups, cystatin C (CysC) was significantly increased at 24 h and 4 days following stroke, before returning to steady-state levels at 7 days, thus indicating a potential transient and intrinsic rescue mechanism attempt of neurons. When CysC was applied to primary neuronal cultures subjected to an in vitro model of ischemic damage, this treatment significantly improved the preservation of synaptic structures. Notably, similar effects were observed when CysC was loaded into brain-derived extracellular vesicles (BDEVs). Finally, when CysC contained in BDEVs was administered intracerebroventricularly to stroked mice, it significantly increased the expression of synaptic markers such as SNAP25, Homer-1, and NCAM in the penumbra area compared to the group supplied with empty BDEVs. Thus, we show that CysC-loaded BDEVs promote synaptic protection after ischemic damage in vitro and in vivo, opening the possibility of a therapeutic use in stroke patients.

Retinal regions shape human and murine Müller cell proteome profile and functionality.

The human macula is a highly specialized retinal region with pit-like morphology and rich in cones. How Müller cells, the principal glial cell type in the retina, are adapted to this environment is still poorly understood. We compared proteomic data from cone- and rod-rich retinae from human and mice and identified different expression profiles of cone- and rod-associated Müller cells that converged on pathways representing extracellular matrix and cell adhesion. In particular, epiplakin (EPPK1), which is thought to play a role in intermediate filament organization, was highly expressed in macular Müller cells. Furthermore, EPPK1 knockout in a human Müller cell-derived cell line led to a decrease in traction forces as well as to changes in cell size, shape, and filopodia characteristics. We here identified EPPK1 as a central molecular player in the region-specific architecture of the human retina, which likely enables specific functions under the immense mechanical loads in vivo.

Anchorless risk or released benefit? An updated view on the ADAM10-mediated shedding of the prion protein.

The prion protein (PrP) is a broadly expressed glycoprotein linked with a multitude of (suggested) biological and pathological implications. Some of these roles seem to be due to constitutively generated proteolytic fragments of the protein. Among them is a soluble PrP form, which is released from the surface of neurons and other cell types by action of the metalloprotease ADAM10 in a process termed 'shedding'. The latter aspect is the focus of this review, which aims to provide a comprehensive overview on (i) the relevance of proteolytic processing in regulating cellular PrP functions, (ii) currently described involvement of shed PrP in neurodegenerative diseases (including prion diseases and Alzheimer's disease), (iii) shed PrP's expected roles in intercellular communication in many more (patho)physiological conditions (such as stroke, cancer or immune responses), (iv) and the need for improved research tools in respective (future) studies. Deeper mechanistic insight into roles played by PrP shedding and its resulting fragment may pave the way for improved diagnostics and future therapeutic approaches in diseases of the brain and beyond.

Multiplexed mRNA analysis of brain-derived extracellular vesicles upon experimental stroke in mice reveals increased mRNA content with potential relevance to inflammation and recovery processes.

Extracellular vesicles (EVs) are lipid bilayer-enclosed structures that represent newly discovered means for cell-to-cell communication as well as promising disease biomarkers and therapeutic tools. Apart from proteins, lipids, and metabolites, EVs can deliver genetic information such as mRNA, eliciting a response in the recipient cells. In the present study, we have analyzed the mRNA content of brain-derived EVs (BDEVs) isolated 72 h after experimental stroke in mice and compared them to controls (shams) using nCounter® Nanostring panels, with or without prior RNA isolation. We found that both panels show similar results when comparing upregulated mRNAs in stroke. Notably, the highest upregulated mRNAs were related to processes of stress and immune system responses, but also to anatomical structure development, cell differentiation, and extracellular matrix organization, thus indicating that regenerative mechanisms already take place at this time-point. The five top overrepresented mRNAs in stroke mice were confirmed by RT-qPCR and, interestingly, found to be full-length. We could reveal that the majority of the mRNA cargo in BDEVs was of microglial origin and predominantly present in small BDEVs (≤ 200 nm in diameter). However, the EV population with the highest increase in the total BDEVs pool at 72 h after stroke was of oligodendrocytic origin. Our study shows that nCounter® panels are a good tool to study mRNA content in tissue-derived EVs as they can be carried out even without previous mRNA isolation, and that the mRNA cargo of BDEVs indicates a possible participation in inflammatory but also recovery processes after stroke.

GPI-anchor signal sequence influences PrPC sorting, shedding and signalling, and impacts on different pathomechanistic aspects of prion disease in mice.

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.

A multifactorial model of pathology for age of onset heterogeneity in familial Alzheimer's disease.

Presenilin-1 (PSEN1) mutations cause familial Alzheimer's disease (FAD) characterized by early age of onset (AoO). Examination of a large kindred harboring the PSEN1-E280A mutation reveals a range of AoO spanning 30 years. The pathophysiological drivers and clinical impact of AoO variation in this population are unknown. We examined brains of 23 patients focusing on generation and deposition of beta-amyloid (Aß) and Tau pathology profile. In 14 patients distributed at the extremes of AoO, we performed whole-exome capture to identify genotype-phenotype correlations. We also studied kinome activity, proteasome activity, and protein polyubiquitination in brain tissue, associating it with Tau phosphorylation profiles. PSEN1-E280A patients showed a bimodal distribution for AoO. Besides AoO, there were no clinical differences between analyzed groups. Despite the effect of mutant PSEN1 on production of Aß, there were no relevant differences between groups in generation and deposition of Aß. However, differences were found in hyperphosphorylated Tau (pTau) pathology, where early onset patients showed severe pathology with diffuse aggregation pattern associated with increased activation of stress kinases. In contrast, late-onset patients showed lesser pTau pathology and a distinctive kinase activity. Furthermore, we identified new protective genetic variants affecting ubiquitin-proteasome function in early onset patients, resulting in higher ubiquitin-dependent degradation of differentially phosphorylated Tau. In PSEN1-E280A carriers, altered γ-secretase activity and resulting Aß accumulation are prerequisites for early AoO. However, Tau hyperphosphorylation pattern, and its degradation by the proteasome, drastically influences disease onset in individuals with otherwise similar Aß pathology, hinting toward a multifactorial model of disease for FAD. In sporadic AD (SAD), a wide range of heterogeneity, also influenced by Tau pathology, has been identified. Thus, Tau-induced heterogeneity is a common feature in both AD variants, suggesting that a multi-target therapeutic approach should be used to treat AD.

Brain-Derived Extracellular Vesicles in Health and Disease: A Methodological Perspective.

Extracellular vesicles (EVs) are double membrane structures released by presumably all cell types that transport and deliver lipids, proteins, and genetic material to near or distant recipient cells, thereby affecting their phenotype. The basic knowledge of their functions in healthy and diseased brain is still murky and many questions about their biology are unsolved. In neurological diseases, EVs are regarded as attractive biomarkers and as therapeutic tools due to their ability to cross the blood-brain barrier (BBB). EVs have been successfully isolated from conditioned media of primary brain cells and cerebrospinal fluid (CSF), but protocols allowing for the direct study of pathophysiological events mediated or influenced by EVs isolated from brain have only recently been published. This review aims to give a brief overview of the current knowledge of EVs' functions in the central nervous system (CNS) and the current protocols to isolate brain-derived EVs (BDEVs) used in different publications. By comparing the proteomic analysis of some of these publications, we also assess the influence of the isolation method on the protein content of BDEVs.

Molecular Communication of a Dying Neuron in Stroke.

When a main artery of the brain occludes, a cellular response involving multiple cell types follows. Cells directly affected by the lack of glucose and oxygen in the neuronal core die by necrosis. In the periphery surrounding the ischemic core (the so-called penumbra) neurons, astrocytes, microglia, oligodendrocytes, pericytes, and endothelial cells react to detrimental factors such as excitotoxicity, oxidative stress, and inflammation in different ways. The fate of the neurons in this area is multifactorial, and communication between all the players is important for survival. This review focuses on the latest research relating to synaptic loss and the release of apoptotic bodies and other extracellular vesicles for cellular communication in stroke. We also point out possible treatment options related to increasing neuronal survival and regeneration in the penumbra.

Inducing prion protein shedding as a neuroprotective and regenerative approach in pathological conditions of the brain: from theory to facts.

In the last decades, the role of the prion protein (PrP) in neurodegenerative diseases has been intensively investigated, initially in prion diseases of humans (e.g., Creutzfeldt-Jakob disease) and animals (e.g., scrapie in sheep, chronic wasting disease in deer and elk, or "mad cow disease" in cattle). Templated misfolding of physiological cellular prion protein (PrPC) into an aggregation-prone isoform (termed PrP "Scrapie" (PrPSc)), self-replication and spreading of the latter inside the brain and to peripheral tissues, and the associated formation of infectious proteopathic seeds (termed "prions") are among the essential pathogenic mechanisms underlying this group of fatal and transmissible spongiform encephalopathies. Later, key roles of the correctly folded PrPC were identified in more common human brain diseases (such as Alzheimer's disease or Parkinson's disease) associated with the misfolding and/or accumulation of other proteins (such as amyloid-ß, tau or α-synuclein, respectively). PrPC has also been linked with neuroprotective and regenerative functions, for instance in hypoxic/ischemic conditions such as stroke. However, despite a mixed "bouquet" of suggested functions, our understanding of pathological and, especially, physiological roles played by PrPC in the brain and beyond is certainly incomplete. Interactions with various other proteins at the cell surface or within intracellular compartments may account for the functional diversity linked with PrPC. Moreover, conserved endogenous proteolytic processing of PrPC generates several defined PrPC fragments, possibly holding intrinsic functions in physiological and pathological conditions, thus making the "true and complete biology" of this protein more complicated to be elucidated. Here, we focus on one of those released PrPC fragments, namely shed PrP (sPrP), generated by a membrane-proximate ADAM10-mediated cleavage event at the cell surface. Similar to other soluble PrPC fragments (such as the N1 fragment representing PrP's released N-terminal tail upon the major α-cleavage event) or experimentally employed recombinant PrP, sPrP is being suggested to act neuroprotective in Alzheimer's disease and other protein misfolding diseases. Several lines of evidence on extracellular PrPC (fragments) suggest that induction of PrPC release could be a future therapeutic option in various brain disorders. Our recent identification of a substrate-specific approach to stimulate the shedding by ADAM10, based on ligands binding to cell surface PrPC, may further set the stage for research into this direction.

Release of VAMP5-positive extracellular vesicles by retinal Müller glia in vivo.

Cell-cell interactions in the central nervous system are based on the release of molecules mediating signal exchange and providing structural and trophic support through vesicular exocytosis and the formation of extracellular vesicles. The specific mechanisms employed by each cell type in the brain are incompletely understood. Here, we explored the means of communication used by Müller cells, a type of radial glial cells in the retina, which forms part of the central nervous system. Using immunohistochemical, electron microscopic, and molecular analyses, we provide evidence for the release of distinct extracellular vesicles from endfeet and microvilli of retinal Müller cells in adult mice in vivo. We identify VAMP5 as a Müller cell-specific SNARE component that is part of extracellular vesicles and responsive to ischemia, and we reveal differences between the secretomes of immunoaffinity-purified Müller cells and neurons in vitro. Our findings suggest extracellular vesicle-based communication as an important mediator of cellular interactions in the retina.

SARS-CoV-2 could be spread through hospital medication dispensed to patients: A prospective observational study.

ABSTRACT: Our objective was to analyze in vitro the persistence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in the packaging material of the drugs dispensed to hospital wards. Additionally, to evaluate if the protection with a double plastic bag prevents the contamination of the medication dispensed to an intensive care unit (ICU).On the first part, different materials containing different drugs within an ICU were sampled to confirm the lack of contamination by SARS-CoV-2. The confirmation of the virus was performed using real time reverse transcription polymerase chain reaction. As a control group, in the microbiology laboratory we inoculated the virus into the different surfaces containing the same drugs included in the first part. Samples were obtained with a sterile swab at 3, 6, 8, 10, 14, 21, and 30 days after inoculation and analyzed through real time reverse transcription polymerase chain reaction.None of the studied materials containing the drugs within an ICU was contaminated by SARS-CoV-2. In the second part, SARS-CoV-2 was found in all surfaces for up to 30 days.The use of double-bag unit-dose system to deliver medication in a pandemic seems effective to prevent the potential transmission of SARS-CoV-2. A striking SARS-CoV-2 RNA stability of up to 30 days was found in the surfaces containing the drugs.

Prion protein oligomers cause neuronal cytoskeletal damage in rapidly progressive Alzheimer's disease.

BACKGROUND: High-density oligomers of the prion protein (HDPs) have previously been identified in brain tissues of patients with rapidly progressive Alzheimer's disease (rpAD). The current investigation aims at identifying interacting partners of HDPs in the rpAD brains to unravel the pathological involvement of HDPs in the rapid progression. METHODS: HDPs from the frontal cortex tissues of rpAD brains were isolated using sucrose density gradient centrifugation. Proteins interacting with HDPs were identified by co-immunoprecipitation coupled with mass spectrometry. Further verifications were carried out using proteomic tools, immunoblotting, and confocal laser scanning microscopy. RESULTS: We identified rpAD-specific HDP-interactors, including the growth arrest specific 2-like 2 protein (G2L2). Intriguingly, rpAD-specific disturbances were found in the localization of G2L2 and its associated proteins i.e., the end binding protein 1, α-tubulin, and ß-actin. DISCUSSION: The results show the involvement of HDPs in the destabilization of the neuronal actin/tubulin infrastructure. We consider this disturbance to be a contributing factor for the rapid progression in rpAD.

CD73-mediated adenosine production by CD8 T cell-derived extracellular vesicles constitutes an intrinsic mechanism of immune suppression.

Immune cells at sites of inflammation are continuously activated by local antigens and cytokines, and regulatory mechanisms must be enacted to control inflammation. The stepwise hydrolysis of extracellular ATP by ectonucleotidases CD39 and CD73 generates adenosine, a potent immune suppressor. Here we report that human effector CD8 T cells contribute to adenosine production by releasing CD73-containing extracellular vesicles upon activation. These extracellular vesicles have AMPase activity, and the resulting adenosine mediates immune suppression independently of regulatory T cells. In addition, we show that extracellular vesicles isolated from the synovial fluid of patients with juvenile idiopathic arthritis contribute to T cell suppression in a CD73-dependent manner. Our results suggest that the generation of adenosine upon T cell activation is an intrinsic mechanism of human effector T cells that complements regulatory T cell-mediated suppression in the inflamed tissue. Finally, our data underscore the role of immune cell-derived extracellular vesicles in the control of immune responses.

Ligands binding to the prion protein induce its proteolytic release with therapeutic potential in neurodegenerative proteinopathies.

The prion protein (PrPC) is a central player in neurodegenerative diseases, such as prion diseases or Alzheimer's disease. In contrast to disease-promoting cell surface PrPC, extracellular fragments act neuroprotective by blocking neurotoxic disease-associated protein conformers. Fittingly, PrPC release by the metalloprotease ADAM10 represents a protective mechanism. We used biochemical, cell biological, morphological, and structural methods to investigate mechanisms stimulating this proteolytic shedding. Shed PrP negatively correlates with prion conversion and is markedly redistributed in murine brain in the presence of prion deposits or amyloid plaques, indicating a sequestrating activity. PrP-directed ligands cause structural changes in PrPC and increased shedding in cells and organotypic brain slice cultures. As an exception, some PrP-directed antibodies targeting repetitive epitopes do not cause shedding but surface clustering, endocytosis, and degradation of PrPC. Both mechanisms may contribute to beneficial actions described for PrP-directed ligands and pave the way for new therapeutic strategies against currently incurable neurodegenerative diseases.

VDAC and ERalpha interaction in caveolae from human cortex is altered in Alzheimer's disease.

Voltage-dependent anion channel (VDAC) is a mitochondrial porin also found in the neuronal membrane (pl-VDAC), where its function may be related to redox homeostasis and apoptosis. Murine models have evidenced pl-VDAC into caveolae in a complex with estrogen receptor alpha (mERalpha), which participates in neuroprotection against amyloid beta (Abeta), and whose integration into this hydrophobic domain remains unclear. Here, we have demonstrated in caveolae of human cortex and hippocampus the presence of pl-VDAC and mERalpha, in a complex with scaffolding caveolin-1 which likely provides mERalpha stability at the plasma membrane. In Alzheimer's disease (AD) brains, VDAC was accumulated in caveolae, and it was observed in dystrophic neurites of senile plaques, whereas ERalpha was expressed in astrocytes surrounding the plaques. Together with previous data in murine neurons demonstrating the participation of pl-VDAC in Abeta-induced neurotoxicity, these data suggest that the channel may be involved in membrane dysfunctioning observed in AD neuropathology.

Show Me Your Friends and I Tell You Who You Are: The Many Facets of Prion Protein in Stroke.

Ischemic stroke belongs to the leading causes of mortality and disability worldwide. Although treatments for the acute phase of stroke are available, not all patients are eligible. There is a need to search for therapeutic options to promote neurological recovery after stroke. The cellular prion protein (PrPC) has been consistently linked to a neuroprotective role after ischemic damage: it is upregulated in the penumbra area following stroke in humans, and animal models of stroke have shown that lack of PrPC aggravates the ischemic damage and lessens the functional outcome. Mechanistically, these effects can be linked to numerous functions attributed to PrPC: (1) as a signaling partner of the PI3K/Akt and MAPK pathways, (2) as a regulator of glutamate receptors, and (3) promoting stem cell homing mechanisms, leading to angio- and neurogenesis. PrPC can be cleaved at different sites and the proteolytic fragments can account for the manifold functions. Moreover, PrPC is present on extracellular vesicles (EVs), released membrane particles originating from all types of cells that have drawn attention as potential therapeutic tools in stroke and many other diseases. Thus, identification of the many mechanisms underlying PrPC-induced neuroprotection will not only provide further understanding of the physiological functions of PrPC but also new ideas for possible treatment options after ischemic stroke.

Characterization of brain-derived extracellular vesicles reveals changes in cellular origin after stroke and enrichment of the prion protein with a potential role in cellular uptake.

Extracellular vesicles (EVs) are important means of intercellular communication and a potent tool for regenerative therapy. In ischaemic stroke, transient blockage of a brain artery leads to a lack of glucose and oxygen in the affected brain tissue, provoking neuronal death by necrosis in the core of the ischaemic region. The fate of neurons in the surrounding penumbra region depends on the stimuli, including EVs, received during the following hours. A detailed characterization of such stimuli is crucial not only for understanding stroke pathophysiology but also for new therapeutic interventions. In the present study, we characterize the EVs in mouse brain under physiological conditions and 24 h after induction of transient ischaemia in mice. We show that, in steady-state conditions, microglia are the main source of small EVs (sEVs), whereas after ischaemia the main sEV population originates from astrocytes. Brain sEVs presented high amounts of the prion protein (PrP), which were further increased after stroke. Moreover, EVs were enriched in a proteolytically truncated PrP fragment (PrP-C1). Because of similarities between PrP-C1 and certain viral surface proteins, we studied the cellular uptake of brain-derived sEVs from mice lacking (PrP-KO) or expressing PrP (WT). We show that PrP-KO-sEVs are taken up significantly faster and more efficiently than WT-EVs by primary neurons. Furthermore, microglia and astrocytes engulf PrP-KO-sEVs more readily than WT-sEVs. Our results provide novel information on the relative contribution of brain cell types to the sEV pool in murine brain and indicate that increased release of sEVs by astrocytes together with elevated levels of PrP in sEVs may play a role in intercellular communication at early stages after stroke. In addition, amounts of PrP (and probably PrP-C1) in brain sEVs seem to contribute to regulating their cellular uptake.

Transgenic Overexpression of the Disordered Prion Protein N1 Fragment in Mice Does Not Protect Against Neurodegenerative Diseases Due to Impaired ER Translocation.

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 (Aß) 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 Aß 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.

Generation and Function of Non-cell-bound CD73 in Inflammation.

Extracellular adenine nucleotides participate in cell-to-cell communication and modulate the immune response. The concerted action of ectonucleotidases CD39 and CD73 plays a major role in the local production of anti-inflammatory adenosine, but both ectonucleotidases are rarely co-expressed by human T cells. The expression of CD39 on T cells increases upon T cell activation and is high at sites of inflammation. CD73, in contrast, disappears from the cellular membrane after activation. The possibility that CD73 could act in trans would resolve the conundrum of both enzymes being co-expressed for the degradation of ATP and the generation of adenosine. An enzymatically active soluble form of CD73 has been reported, and AMPase activity has been detected in body fluids of patients with inflammation and cancer. It is not yet clear how CD73, a glycosylphosphatidylinositol (GPI)-anchored protein, is released from the cell membrane, but plausible mechanisms include cleavage by metalloproteinases and shedding mediated by cell-associated phospholipases. Importantly, like many other GPI-anchored proteins, CD73 at the cell membrane is preferentially localized in detergent-resistant domains or lipid rafts, which often contribute to extracellular vesicles (EVs). Indeed, CD73-containing vesicles of different size and origin and with immunomodulatory function have been found in the tumor microenvironment. The occurrence of CD73 as non-cell-bound molecule widens the range of action of this enzyme at sites of inflammation. In this review, we will discuss the generation of non-cell-bound CD73 and its physiological role in inflammation.

Structural and mechanistic aspects influencing the ADAM10-mediated shedding of the prion protein.

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.