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Internalization of molecules can occur throughout many different pathways: the classical, clathrin-mediated pathway (1); the clathrin-independent raft-dependent pathway (2); the raft-mediated clathrin-dependent route (3) and caveolae (4). At the cell surface of FRT cells PrPC is localized in DRMs. As PrPC remains in these domains during its internalization a classical clathrin-dependent pathway (1) seems to be excluded. Conversely PrPC can undertake a rafts dependent pathway (2) and/or rafts might promote its recruitment to clathrin domains inducing its internalization (3). PrPC resides in caveolae at the plasma membrane but is not internalized via the caveolar pathway (4).
Source publication
The cellular prion protein (PrP(C)) plays a key role in the pathogenesis of Transmissible Spongiform Encephalopathies in which the protein undergoes post-translational conversion to the infectious form (PrP(Sc)). Although endocytosis appears to be required for this conversion, the mechanism of PrP(C) internalization is still debated, as caveolae/ra...
Citations
... However, it can be involved in other uptake mechanisms, like clathrin-and caveolae-independent endocytosis, macropinocytosis, and phagocytosis. [173,[175][176][177][178][179][180] Below, we address lipid raft-mediated uptake of liposomes and EVs, as well as the impact of the entry pathway for transfection. Furthermore, methods and strategies are described to enhance delivery by exploiting this pathway. ...
A key aspect for successful drug delivery via lipid‐based nanoparticles is their internalization in target cells. Two prominent examples of such drug delivery systems are artificial phospholipid‐based carriers, such as liposomes, and their biological counterparts, the extracellular vesicles (EVs). Despite a wealth of literature, it remains unclear which mechanisms precisely orchestrate nanoparticle‐mediated cargo delivery to recipient cells and the subsequent intracellular fate of therapeutic cargo. In this review, internalization mechanisms involved in the uptake of liposomes and EVs by recipient cells are evaluated, also exploring their intracellular fate after intracellular trafficking. Opportunities are highlighted to tweak these internalization mechanisms and intracellular fates to enhance the therapeutic efficacy of these drug delivery systems. Overall, literature to date shows that both liposomes and EVs are predominantly internalized through classical endocytosis mechanisms, sharing a common fate: accumulation inside lysosomes. Studies tackling the differences between liposomes and EVs, with respect to cellular uptake, intracellular delivery and therapy efficacy, remain scarce, despite its importance for the selection of an appropriate drug delivery system. In addition, further exploration of functionalization strategies of both liposomes and EVs represents an important avenue to pursue in order to control internalization and fate, thereby improving therapeutic efficacy.
... 148,150 This endocytosis then occurs via a clathrin-mediated process which also necessitates the presence of the N-terminal of the prion protein. 148,149 Further, the role of clathrin has also been reported for the uptake of PrP C in Fischer rat thyroid (FRT) cells, 151 in vesiclemediated intracellular transport of PrP Sc in prion-infected N2a-3 cells 152 and copper-mediated endocytosis of PrP Sc in neuroblastoma cells. 153 In addition, lipid rafts have been shown to play a role in both clathrin-mediated and clathrin-independent endocytic uptake of prion proteins. ...
... 153 In addition, lipid rafts have been shown to play a role in both clathrin-mediated and clathrin-independent endocytic uptake of prion proteins. 147,151 Apart from these conventional endocytic mechanisms, an Arf6-mediated mechanism for the uptake of PrP C has also been demonstrated in N2a neuroblastoma cells. 154 ...
Endocytosis is the fundamental uptake process through which cells internalize extracellular materials and species. Neurodegenerative diseases (NDs) are characterized by a progressive accumulation of intrinsically disordered protein species, leading to neuronal death. Misfolding in many proteins leads to various NDs such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS) and other disorders. Despite the significance of disordered protein species in neurodegeneration, their spread between cells and the cellular uptake of extracellular species is not entirely understood. This review discusses the major internalization mechanisms of the different conformer species of these proteins and their endocytic mechanisms. We briefly introduce the broad types of endocytic mechanisms found in cells and then summarize what is known about the endocytosis of monomeric, oligomeric and aggregated conformations of tau, Aβ, α-Syn, Huntingtin, Prions, SOD1, TDP-43 and other proteins associated with neurodegeneration. We also highlight the key players involved in internalizing these disordered proteins and the several techniques and approaches to identify their endocytic mechanisms. Finally, we discuss the obstacles involved in studying the endocytosis of these protein species and the need to develop better techniques to elucidate the uptake mechanisms of a particular disordered protein species.
... Similarly, endocytosis of many lipid-anchored proteins such as glycosylphosphatidylinositol-anchored proteins (GPI-APs) also does not appear to require any of the well-characterized coat proteins [38]. However, the GPI-anchored prion protein (PrP) might be internalized through both CIE and CME endocytosis, depending on the expression of the LRP-1 receptor, which drives PrP to CME [39,40]. The two main features that distinguish these pathways are the dependency on dynamin and the main protein mediators involved. ...
Endocytosis is a critical process for cell growth and viability. It mediates nutrient uptake, guarantees plasma membrane homeostasis, and generates intracellular signaling cascades. Moreover, it plays an important role in dead cell clearance and defense against external microbes. Finally, endocytosis is an important cellular route for the delivery of nanomedicines for therapeutic treatments. Thus, it is not surprising that both environmental and genetic perturbation of endocy-tosis have been associated with several human conditions, such as cancer, neurological disorders, and virus infections, among others. Over the last decades, a lot of research has been focused on developing advanced imaging methods to monitor endocytosis events with high resolution in living cells and tissues. These include fluorescence imaging, electron microscopy, and correlative and super-resolution microscopy. In this review, we outline the major endocytic pathways and briefly discuss how defects in the molecular machinery of these pathways lead to disease. We then discuss the current imaging methodologies used to study endocytosis in different contexts, high-lighting strengths and weaknesses.
... How cells internalize PrP C is an important consideration in prion disease as endocytic vesicles that contain PrP C may be central sites of prion conversion if cells take up PrP Sc via the same route that PrP C is taken up. Cell surface associated PrP C is predominately internalized via clathrin-coated pits and caveolae before trafficking to the early endosome (Fig. 1A, B, and D) with uptake of PrP C by these mechanisms being modulated by association of PrP C with lipid rafts and metal ions (Hooper et al. 2008;Peters et al. 2003;Sarnataro et al. 2009). The exact uptake pathway for PrP C can also be cell type dependent. ...
The properties of infectious prions and the pathology of the diseases they cause are dependent upon the unique conformation of each prion strain. How the pathology of prion disease correlates with different strains and genetic backgrounds has been investigated via in vivo assays, but how interactions between specific prion strains and cell types contribute to the pathology of prion disease has been dissected more effectively using in vitro cell lines. Observations made through in vivo and in vitro assays have informed each other with regard to not only how genetic variation influences prion properties, but also how infectious prions are taken up by cells, modified by cellular processes and propagated, and the cellular components they rely on for persistent infection. These studies suggest that persistent cellular infection results from a balance between prion propagation and degradation. This balance may be shifted depending upon how different cell lines process infectious prions, potentially altering prion stability, and how fast they can be transported to the lysosome. Thus, in vitro studies have given us a deeper understanding of the interactions between different prions and cell types and how they may influence prion disease phenotypes in vivo.
... We observed a reduction in S100B uptake in response to nystatin, which provide evidence for the involvement of lipid-raft mediated mechanisms. In addition, Sarnataro et al. (2009), reported that both lipid rafts and clathrin contribute to the efficiency of PrPC protein uptake, and there is evidence that EGFR is capable of endocytosis of both clathrin dependent and non-clathrin mechanisms (Sigismund et al., 2008), which support our findings. Interestingly, none of the inhibitors of either CME or lipid rafts resulted in complete inhibition of S100B-Alexa488 uptake, indicating a contribution from both pathways, further experiments are required to clarify whether inhibition of both pathways had an additive effect on S100B internalization. ...
Mesenchymal stem cells (MSCs) are promising tools for cancer therapy, but there is a risk of malignant transformation in their clinical application. Our previous work revealed that the paracrine protein S100B in the glioma microenvironment induces malignant transformation of MSCs and upregulates intracellular S100B, which could affect cell homeostasis by interfering with p53. The purpose of this study was to investigate whether extracellular S100B can be internalized by MSCs and the specific endocytic pathway involved in S100B internalization. By using real-time confocal microscopy and structured illumination microscopy (SIM), we visualized the uptake of fluorescently labeled S100B protein (S100B-Alexa488) and monitored the intracellular trafficking of internalized vesicles. The results showed that S100B-Alexa488 was efficiently internalized into MSCs in a time-dependent manner and transported through endolysosomal pathways. After that, we used chemical inhibitors and RNA interference approaches to investigate possible mechanisms involved in S100B-Alexa488 uptake. The internalization of S100B-Alexa488 was inhibited by pitstop-2 or dyngo-4a treatment or RNA-mediated silencing of clathrin or dynamin, and the lipid raft-mediated endocytosis inhibitors nystatin and MβCD. In conclusion, our findings show that clathrin and lipid rafts contribute to the internalization of S100B-Alexa488, which provides promising interventions for the safe application of MSCs in glioma therapy.
... In addition, it has been previously shown to cross the blood-brain barrier in the setting of neurological injury (14,15). Cellular release of PrPC is primarily achieved through ectodomain shedding by a member of the disintegrin and metalloproteinase (ADAM) family, ADAM10 (16)(17)(18)(19). ADAM10 is a transmembrane protein located exclusively in the non-raft regions of the plasma membrane, and acts as the major regulator of PrPC (16). ...
Background
Cellular prion protein (PrPC) is a lipid raft protein abundant within CNS. It is regulated by a disintegrin and metalloproteinase domain containing protein 10 (ADAM10). PrPC has previously been implicated as a biomarker for TBI. ADAM10 has not been investigated as a TBI biomarker.
Objective
We evaluated PrPC and ADAM10 as candidate biomarkers for TBI.
Methods
We performed ELISA for ADAM10 and PrPC on plasma samples of patients with TBI admitted to Brigham and Women’s Hospital. Plasma samples from 20 patients admitted for isolated TBI were acquired from a biobank with clinical information. Control plasma (37 samples) was acquired from a commercial source. GraphPad was used to conduct statistical analysis.
Results
37 controls and 20 TBI samples were collected. Of the patients with TBI, eight were mild, three were moderate, and nine were severe. Both PrPC and ADAM10 were elevated in patients with TBI compared with control (p < .001). ADAM10 exhibited greater expression in patients with worse clinical grade. There was no significant association of either PrPC or ADAM10 with time after injury.
Conclusions
Our results indicate that PrPC and ADAM10 appear to be useful potential tools for screening of TBI. ADAM10 is closely associated with clinical grade.
... Some studies suggested the absence of rafts in CCPs (Nichols, 2003b), and there were general observations that rafts and raft-associated proteins internalize through CIE (Sato et al., 2004). However, membrane rafts can cooperate with clathrin in the internalization of some molecules, such as the B cell antigen receptor (BCR) (Stoddart et al., 2002) or certain GPI-anchored proteins (Rollason et al., 2007;Sarnataro et al., 2009). ...
Membrane rafts are dynamic, small (10–200 nm) domains enriched with cholesterol and sphingolipids that compartmentalize cellular processes. Rafts participate in roles essential to the lifecycle of different viral families including virus entry, assembly and/or budding events. Rafts seem to participate in virus attachment and recruitment to the cell surface, as well as the endocytic and non-endocytic mechanisms some viruses use to enter host cells. In this review, we will introduce the specific role of rafts in viral entry and define cellular factors implied in the choice of one entry pathway over the others. Finally, we will summarize the most relevant information about raft participation in the entry process of enveloped and non-enveloped viruses.
... GPI-anchored PrP C , preferentially located in lipid rafts on the cell membrane, functions as a cell surface receptor or co-receptor in concert with numerous ligands. PrP C is transported by specific vesicular trafficking events through endocytic/secretory membrane systems to find a correct functional destination, and conversely towards recycling or degradation [48][49][50]. In different cell types, through distinct modes of internalization, and interacting with specific ligands, PrP C triggers particular signaling cascades [9,51]. ...
... The co-participation of the laminin receptor precursor (LRP) and the low-density lipoprotein receptor-related protein 1 (LRP1) has been reported, and both proteins are highly active in internalization processes of clathrin-coated pits, including in CME of PrP C [59][60][61][62]. As such, the lack of clathrin and disruption of lipid rafts-by drugs such as filipin and nystatin [63] or inactivation of Cdc42, an actin-remodeling GTPase usually recruited to lipid rafts-blocks PrP C internalization [49]. Association of PrP C with lipid rafts remains during the whole CME process, suggesting that PrP C and clathrin engage specifically at this location on cell membrane [49]. ...
... As such, the lack of clathrin and disruption of lipid rafts-by drugs such as filipin and nystatin [63] or inactivation of Cdc42, an actin-remodeling GTPase usually recruited to lipid rafts-blocks PrP C internalization [49]. Association of PrP C with lipid rafts remains during the whole CME process, suggesting that PrP C and clathrin engage specifically at this location on cell membrane [49]. As briefly mentioned before, PrP C internalization is dependent upon dynamin, and transits in endocytic vesicles positive for Rab5, prior to be accumulated in the perinuclear region [64]. ...
The mobility of cellular prion protein (PrPC) in specific cell membrane domains and among distinct cell compartments dictates its molecular interactions and directs its cell function. PrPC works in concert with several partners to organize signaling platforms implicated in various cellular processes. The scaffold property of PrPC is able to gather a molecular repertoire to create heterogeneous membrane domains that favor endocytic events. Dynamic trafficking of PrPC through multiple pathways, in a well-orchestrated mechanism of intra and extracellular vesicular transport, defines its functional plasticity, and also assists the conversion and spreading of its infectious isoform associated with neurodegenerative diseases. In this review, we highlight how PrPC traffics across intra- and extracellular compartments and the consequences of this dynamic transport in governing cell functions and contributing to prion disease pathogenesis.
... 139,140 Evidence for a cooperation between clathrin 138,141,142 and rafts [143][144][145] in the internalization of PrP C was found. 146 Clathrin is a large, oligomeric protein assembling into lattice structures on the inner surface of the plasma membrane. Thereby, it causes the membrane to invaginate and pinch off to form clathrincoated vesicles (CCVs), which can then fuse with other intracellular organelles. ...
Deciphering the pathophysiologic events in prion diseases is challenging, and the role of posttranslational modifications (PTMs) such as glypidation and glycosylation remains elusive due to the lack of homogeneous protein preparations. So far, experimental studies have been limited in directly analyzing the earliest events of the conformational change of cellular prion protein (PrPC) into scrapie prion protein (PrPSc) that further propagates PrPC misfolding and aggregation at the cellular membrane, the initial site of prion infection, and PrP misfolding, by a lack of suitably modified PrP variants. PTMs of PrP, especially attachment of the glycosylphosphatidylinositol (GPI) anchor, have been shown to be crucially involved in the PrPSc formation. To this end, semisynthesis offers a unique possibility to understand PrP behavior invitro and invivo as it provides access to defined site‐selectively modified PrP variants. This approach relies on the production and chemoselective linkage of peptide segments, amenable to chemical modifications, with recombinantly produced protein segments. In this article, advances in understanding PrP conversion using semisynthesis as a tool to obtain homogeneous posttranslationally modified PrP will be discussed. The key pathophysiologic event in prion diseases is based on a conformational change of cellular (PrPC) into scrapie prion protein (PrPSc) and is closely linked to posttranslational modifications (PTMs). Semisynthesis offers a unique opportunity to study the impact of PTMs on prion conversion, transmission, and pathogenicity, which is the major focus of this review.
... PrP intracellular localization and transport are of fundamental importance for the generation of the pathological PrP Sc [49][50][51][52]. For some PrP mutants, it has been hypothesized that the mutations are able to lower the energy barrier for the conversion of the normal cellular PrP into the scrapie, pathological PrP Sc isoform [2], and/or to induce an aberrant trafficking and accumulation inside the cells, triggering abnormal interaction with other unknown cofactors [52]. ...
... PrP intracellular localization and transport are of fundamental importance for the generation of the pathological PrP Sc [49][50][51][52]. ...
Misfolded and abnormal β-sheets forms of wild-type proteins, such as cellular prion protein (PrPC) and amyloid beta (Aβ), are believed to be the vectors of neurodegenerative diseases, prion and Alzheimer’s disease (AD), respectively. Increasing evidence highlights the “prion-like” seeding of protein aggregates as a mechanism for pathological spread in AD, tauopathy, as well as in other neurodegenerative diseases, such as Parkinson’s. Mutations in both PrPC and Aβ precursor protein (APP), have been associated with the pathogenesis of these fatal disorders with clear evidence for their pathogenic significance. In addition, a critical role for the gut microbiota is emerging; indeed, as a consequence of gut–brain axis alterations, the gut microbiota has been involved in the regulation of Aβ production in AD and, through the microglial inflammation, in the amyloid fibril formation, in prion diseases. Here, we aim to review the role of microbiome (“the other human genome”) alterations in AD and prion disease pathogenesis.
