RESUMO
Clinical relevance of miRNAs as biomarkers is growing due to their stability and detection in biofluids. In this, diagnosis at asymptomatic stages of Alzheimer's disease (AD) remains a challenge since it can only be made at autopsy according to Braak NFT staging. Achieving the objective of detecting AD at early stages would allow possible therapies to be addressed before the onset of cognitive impairment. Many studies have determined that the expression pattern of some miRNAs is dysregulated in AD patients, but to date, none has been correlated with downregulated expression of cellular prion protein (PrPC) during disease progression. That is why, by means of cross studies of miRNAs up-regulated in AD with in silico identification of potential miRNAs-binding to 3'UTR of human PRNP gene, we selected miR-519a-3p for our study. Then, in vitro experiments were carried out in two ways. First, we validated miR-519a-3p target on 3'UTR-PRNP, and second, we analyzed the levels of PrPC expression after using of mimic technology on cell culture. In addition, RT-qPCR was performed to analyzed miR-519a-3p expression in human cerebral samples of AD at different stages of disease evolution. Additionally, samples of other neurodegenerative diseases such as other non-AD tauopathies and several synucleinopathies were included in the study. Our results showed that miR-519a-3p overlaps with PRNP 3'UTR in vitro and promotes downregulation of PrPC. Moreover, miR-519a-3p was found to be up-regulated exclusively in AD samples from stage I to VI, suggesting its potential use as a novel label of preclinical stages of the disease.
Assuntos
Regiões 3' não Traduzidas , Doença de Alzheimer , Biomarcadores , MicroRNAs , Proteínas Priônicas , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Doença de Alzheimer/diagnóstico , Proteínas Priônicas/genética , Proteínas Priônicas/metabolismo , Biomarcadores/metabolismo , Regiões 3' não Traduzidas/genética , Feminino , Idoso , Masculino , Idoso de 80 Anos ou mais , Proteínas PrPC/metabolismo , Proteínas PrPC/genéticaRESUMO
The cellular prion protein (PrPC) is required for skeletal muscle function. Here, we report that a higher level of PrPC accumulates in the cytoplasm of the skeletal muscle of six myopathy patients compared to controls. PrPC inhibits skeletal muscle cell autophagy, and blocks myoblast differentiation. PrPC selectively binds to a subset of miRNAs during myoblast differentiation, and the colocalization of PrPC and miR-214-3p was observed in the skeletal muscle of six myopathy patients with excessive PrPC. We demonstrate that PrPC is overexpressed in skeletal muscle cells under pathological conditions, inhibits muscle cell differentiation by physically interacting with a subset of miRNAs, and selectively recruits these miRNAs into its phase-separated condensate in living myoblasts, which in turn enhances liquid-liquid phase separation of PrPC, promotes pathological aggregation of PrP, and results in the inhibition of autophagy-related protein 5-dependent autophagy and muscle bundle formation in myopathy patients characterized by incomplete muscle regeneration.
Assuntos
MicroRNAs , Doenças Musculares , Proteínas PrPC , Humanos , Diferenciação Celular/genética , Proliferação de Células , MicroRNAs/genética , MicroRNAs/metabolismo , Desenvolvimento Muscular/fisiologia , Músculo Esquelético/metabolismo , Doenças Musculares/metabolismo , Proteínas PrPC/metabolismoRESUMO
Oligomers formed from monomers of the amyloid ß-protein (Aß) are thought to be central to the pathogenesis of Alzheimer's disease (AD). Unsurprisingly for a complex disease, current mouse models of AD fail to fully mimic the clinical disease in humans. Moreover, results obtained in a given mouse model are not always reproduced in a different model. Cellular prion protein (PrPC) is now an established receptor for Aß oligomers. However, studies of the Aß-PrPC interaction in different mouse models have yielded contradictory results. Here we performed a longitudinal study assessing a range of biochemical and histological features in the commonly used J20 and APP-PS1 mouse models. Our analysis demonstrated that PrPC ablation had no effect on amyloid accumulation or oligomer production. However, we found that APP-PS1 mice had higher levels of oligomers, that these could bind to recombinant PrPC, and were recognised by the OC antibody which distinguishes parallel, in register fibrils. On the other hand, J20 mice had a lower level of Aß oligomers, which did not interact with PrPC when tested in vitro and were OC-negative. These results suggest the two mouse models produce diverse Aß assemblies that could interact with different targets, highlighting the necessity to characterise the conformation of the Aß oligomers concomitantly with the toxic cascade elicited by them. Our results provide an explanation for the apparent contradictory results found in APP-PS1 mice and the J20 mouse line in regards to Aß toxicity mediated by PrPC.
Assuntos
Doença de Alzheimer , Proteínas PrPC , Príons , Humanos , Camundongos , Animais , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Proteínas Priônicas/genética , Estudos Longitudinais , Proteínas PrPC/genética , Proteínas PrPC/metabolismo , Camundongos TransgênicosRESUMO
The cellular prion protein (PrPC) is well-known for its involvement, under its pathogenic protease-resistant form (PrPSc), in a group of neurodegenerative diseases, known as prion diseases. PrPC is expressed in nervous system, as well as in other peripheral organs, and has been found overexpressed in several types of solid tumors. Notwithstanding, studies in recent years have disclosed an emerging role for PrPC in various cancer associated processes. PrPC has high binding affinity for 37/67 kDa laminin receptor (RPSA), a molecule that acts as a key player in tumorigenesis, affecting cell growth, adhesion, migration, invasion and cell death processes. Recently, we have characterized at cellular level, small molecules able to antagonize the direct PrPC binding to RPSA and their intracellular trafficking. These findings are very crucial considering that the main function of RPSA is to modulate key events in the metastasis cascade. Elucidation of the role played by PrPC/RPSA interaction in regulating tumor development, progression and response to treatment, represents a very promising challenge to gain pathogenetic information and discover novel specific biomarkers and/or therapeutic targets to be exploited in clinical settings. This review attempts to convey a detailed description of the complexity surrounding these multifaceted proteins from the perspective of cancer hallmarks, but with a specific focus on the role of their interaction in the control of proliferation, migration and invasion, genome instability and mutation, as well as resistance to cell death controlled by autophagic pathway.
Assuntos
Neoplasias , Proteínas PrPC , Doenças Priônicas , Príons , Humanos , Proteínas Priônicas/genética , Proteínas Priônicas/metabolismo , Doenças Priônicas/metabolismo , Receptores de Laminina/genética , Receptores de Laminina/metabolismo , Neoplasias/genética , Biologia , Proteínas PrPC/genética , Proteínas PrPC/metabolismoRESUMO
Currently, no effective therapeutics exist for the treatment of incurable neurodegenerative diseases such as Alzheimer's disease (AD). The cellular prion protein (PrPC) acts as a high-affinity receptor for amyloid beta oligomers (AßO), a main neurotoxic species mediating AD pathology. The interaction of AßO with PrPC subsequently activates Fyn tyrosine kinase and neuroinflammation. Herein, we used our previously developed peptide aptamer 8 (PA8) binding to PrPC as a therapeutic to target the AßO-PrP-Fyn axis and prevent its associated pathologies. Our in vitro results indicated that PA8 prevents the binding of AßO with PrPC and reduces AßO-induced neurotoxicity in mouse neuroblastoma N2a cells and primary hippocampal neurons. Next, we performed in vivo experiments using the transgenic 5XFAD mouse model of AD. The 5XFAD mice were treated with PA8 and its scaffold protein thioredoxin A (Trx) at a 14.4 µg/day dosage for 12 weeks by intraventricular infusion through Alzet® osmotic pumps. We observed that treatment with PA8 improves learning and memory functions of 5XFAD mice as compared to Trx-treated 5XFAD mice. We found that PA8 treatment significantly reduces AßO levels and Aß plaques in the brain tissue of 5XFAD mice. Interestingly, PA8 significantly reduces AßO-PrP interaction and its downstream signaling such as phosphorylation of Fyn kinase, reactive gliosis as well as apoptotic neurodegeneration in the 5XFAD mice compared to Trx-treated 5XFAD mice. Collectively, our results demonstrate that treatment with PA8 targeting the AßO-PrP-Fyn axis is a promising and novel approach to prevent and treat AD.
Assuntos
Doença de Alzheimer , Aptâmeros de Peptídeos , Proteínas PrPC , Camundongos , Animais , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Camundongos Transgênicos , Peptídeos beta-Amiloides/metabolismo , Proteínas PrPC/metabolismo , Modelos Animais de DoençasRESUMO
The carcinogenesis of glial tumors appears complex because of the many genetic and epigenetic phenomena involved. Among these, cellular prion protein (PrPC) is considered a key factor in cell-death resistance and important aspect implicated in tumorigenesis. Autophagy also plays an important role in cell death in various pathological conditions. These two cellular phenomena are related and share the same activation by specific alterations in the cellular microenvironment. Furthermore, there is an interdependence between autophagy and prion activity in glioma tumorigenesis. Glioma is one of the most aggressive known cancers, and the fact that such poorly studied processes as autophagy and PrPC activity are so strongly involved in its carcinogenesis suggests that by better understanding their interaction, more can be understood about its origin and treatment. Few studies in the literature relate these two cellular phenomena, much less try to explain their combined activity and role in glioma carcinogenesis. In this study, we explored the recent findings on the molecular mechanism and regulation pathways of autophagy, examining the role of PrPC in autophagy processes and how they may play a central role in glioma tumorigenesis. Among the many molecular interactions that PrP physiologically performs, it appears that processes shared with autophagy activity are those most implicated in glial tumor carcinogeneses such as activity on MAP kinases, PI3K, and mTOR. This work can be supportive and valuable as a basis for further future studies on this topic.
Assuntos
Glioma , Proteínas PrPC , Príons , Humanos , Proteínas Priônicas , Príons/metabolismo , Glioma/genética , Autofagia , Carcinogênese/genética , Transformação Celular Neoplásica/genética , Proteínas PrPC/metabolismo , Microambiente TumoralRESUMO
The prion protein (PrPC) is subjected to several conserved endoproteolytic events producing bioactive fragments that are of increasing interest for their physiological functions and their implication in the pathogenesis of prion diseases and other neurodegenerative diseases. However, systematic and comprehensive investigations on the full spectrum of PrPC proteoforms have been hampered by the lack of methods able to identify all PrPC-derived proteoforms. Building on previous knowledge of PrPC endoproteolytic processing, we thus developed an optimized Western blot assay able to obtain the maximum information about PrPC constitutive processing and the relative abundance of PrPC proteoforms in a complex biological sample. This approach led to the concurrent identification of the whole spectrum of known endoproteolytic-derived PrPC proteoforms in brain homogenates, including C-terminal, N-terminal and, most importantly, shed PrPC-derived fragments. Endoproteolytic processing of PrPC was remarkably similar in the brain of widely used wild type and transgenic rodent models, with α-cleavage-derived C1 representing the most abundant proteoform and ADAM10-mediated shedding being an unexpectedly prominent proteolytic event. Interestingly, the relative amount of shed PrPC was higher in WT mice than in most other models. Our results indicate that constitutive endoproteolytic processing of PrPC is not affected by PrPC overexpression or host factors other than PrPC but can be impacted by PrPC primary structure. Finally, this method represents a crucial step in gaining insight into pathophysiological roles, biomarker suitability, and therapeutic potential of shed PrPC and for a comprehensive appraisal of PrPC proteoforms in therapies, drug screening, or in the progression of neurodegenerative diseases.
Assuntos
Western Blotting , Fragmentos de Peptídeos , Proteínas PrPC , Proteólise , Animais , Camundongos , Western Blotting/métodos , Doenças Priônicas/metabolismo , Doenças Priônicas/patologia , Doenças Priônicas/fisiopatologia , Proteínas PrPC/química , Proteínas PrPC/genética , Proteínas PrPC/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Encéfalo/metabolismoRESUMO
Amyloid ß (Aß) oligomers play a key role in the progression of Alzheimer's disease (AD). Multiple forms of Aß assemblies have been identified by in vitro and in vivo analyses; however, it is uncertain which oligomer is highly neurotoxic. Thus, understanding the pathogenesis of AD by detecting toxic Aß oligomers is crucial. In this study, we report a fusion protein of cellular prion protein (PrPc) and alkaline phosphatase (ALP) from Escherichia coli as a sensing element for toxic Aß oligomers. Since the N-terminus domain of PrPc (residue 23-111) derived from mice is known to bind to toxic Aß oligomers in vitro, we genetically fused PrPc23-111 to ALP. The developed fusion protein, PrP-ALP, retained both the binding ability of PrPc and enzymatic activity of ALP. We showed that PrP-ALP strongly bound to high molecular weight (HMW) oligomers but showed little or no affinity toward monomers. The observation that PrP-ALP neutralized the toxic effect of Aß oligomers indicated an interaction between PrP-ALP and toxic HMW oligomers. Based on ALP activity, we succeeded in detecting Aß oligomers. PrP-ALP may serve as a powerful tool for detecting toxic Aß oligomers that may be related to AD progression.
Assuntos
Doença de Alzheimer , Proteínas PrPC , Príons , Camundongos , Animais , Peptídeos beta-Amiloides/metabolismo , Proteínas Priônicas/genética , Proteínas Priônicas/química , Proteínas PrPC/metabolismo , Fosfatase Alcalina/genética , Doença de Alzheimer/metabolismoRESUMO
Cellular prion protein (PrPC) is a highly conserved glycoprotein, present both anchored in the cell membrane and soluble in the extracellular medium. It has a diversity of ligands and is variably expressed in numerous tissues and cell subtypes, most notably in the central nervous system (CNS). Its importance has been brought to light over the years both under physiological conditions, such as embryogenesis and immune system homeostasis, and in pathologies, such as cancer and neurodegenerative diseases. During development, PrPC plays an important role in CNS, participating in axonal growth and guidance and differentiation of glial cells, but also in other organs such as the heart, lung, and digestive system. In diseases, PrPC has been related to several types of tumors, modulating cancer stem cells, enhancing malignant properties, and inducing drug resistance. Also, in non-neoplastic diseases, such as Alzheimer's and Parkinson's diseases, PrPC seems to alter the dynamics of neurotoxic aggregate formation and, consequently, the progression of the disease. In this review, we explore in detail the multiple functions of this protein, which proved to be relevant for understanding the dynamics of organism homeostasis, as well as a promising target in the treatment of both neoplastic and degenerative diseases.
Assuntos
Neoplasias , Doenças Neurodegenerativas , Proteínas PrPC , Sistema Nervoso Central/metabolismo , Humanos , Células-Tronco Neoplásicas/metabolismo , Proteínas PrPC/genética , Proteínas PrPC/metabolismoRESUMO
Previous studies have shown that amyloid-ß oligomers (AßO) bind with high affinity to cellular prion protein (PrPC ). The AßO-PrPC complex binds to cell-surface co-receptors, including the laminin receptor (67LR). Our current studies revealed that in Neuroscreen-1 cells, 67LR is the major co-receptor involved in the cellular uptake of AßO and AßΟ-induced cell death. Both pharmacological (dibutyryl-cAMP, forskolin and rolipram) and physiological (pituitary adenylate cyclase-activating polypeptide) cAMP-elevating agents decreased cell-surface PrPC and 67LR, thereby attenuating the uptake of AßO and the resultant neuronal cell death. These cAMP protective effects are dependent on protein kinase A, but not dependent on the exchange protein directly activated by cAMP. Conceivably, cAMP protects neuronal cells from AßO-induced cytotoxicity by decreasing cell-surface-associated PrPC and 67LR.
Assuntos
Peptídeos beta-Amiloides , Proteínas PrPC , Peptídeos beta-Amiloides/metabolismo , Proteínas Priônicas , Proteínas PrPC/metabolismo , Laminina/metabolismo , Morte Celular , Receptores de Laminina/genética , Polipeptídeo Hipofisário Ativador de Adenilato CiclaseRESUMO
We propose a model to explain the pathogenesis of Alzheimer's disease (AD) based on the theory that any disease affecting a healthy organism originates from a bistable feedback loop that shifts the system from a physiological to a pathological condition. We focused on the known double inhibitory loop involving the cellular prion protein (PrPC) and the enzyme BACE1 that produces amyloid-beta (Aß) peptides. BACE1 is inhibited by PrPC, but its inhibitory activity is lost when PrPC binds to Aß oligomers (Aßo). Excessive Aßo formation would switch the loop to a pathogenic condition involving the Aßo-PrPC-mGluR5 complex, Fyn kinase activation, tau, and NMDAR phosphorylation, ultimately leading to neurodegeneration. Based on the emerging role of cyclic nucleotides in Aß production, and thereby in synaptic plasticity and cognitive processes, cAMP and cGMP can be considered as modulatory factors capable of inducing the transition from a physiological steady state to a pathogenic one. This would imply that critical pharmacological targets for AD treatment lie within pathways that lead to an imbalance of cyclic nucleotides in neurons. If this hypothesis is confirmed, it will provide precise indications for the development of preventive or therapeutic treatments for the disease.
Assuntos
Doença de Alzheimer , Proteínas PrPC , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide , Peptídeos beta-Amiloides/metabolismo , Ácido Aspártico Endopeptidases , Humanos , Nucleotídeos Cíclicos , Proteínas PrPC/metabolismo , Proteínas Priônicas/metabolismoRESUMO
Current therapeutic strategies for Alzheimer's disease (AD) mainly focus on amyloid ß oligomer (AßO) formation or clearance. However, most of them have failed to yield good clinical results. There is an urgent need to explore an alternative therapeutic target for AD treatments. Recent studies have indicated that the cellular prion protein (PrPC) is one of the cell-surface receptors of AßO that mediates related neurotoxicity. Besides, as a neuroprotective protein, the dimerization of PrPC seems to be critical for its trophic activity. We presume that modulating PrPC receptor activity could be another potential approach to abrogate AßO toxicity. In the present work, using an aptamer-induced dimerization (AID) strategy, we enforce PrPC dimerization and modulate its neurotrophic signaling. The AID strategy can attenuate AßO toxic action by (i) interfering with AßO-PrPC interaction and promoting neuroprotective shedding of PrPC; (ii) preventing the AßO-induced mitochondrial dysfunction and the caspase-3-induced apoptosis; and (iii) reducing the secretion of inflammatory cytokines and relieving the neuroinflammation microenvironment. Our findings suggest that the strategy targeting PrPC signaling may shed light on validating new therapeutic strategies in AD.
Assuntos
Doença de Alzheimer , Proteínas PrPC , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/toxicidade , Dimerização , Humanos , Proteínas PrPC/metabolismo , Proteínas PriônicasRESUMO
The cellular isoform of prion protein, designated PrPC, is a membrane glycoprotein expressed most abundantly in the brain, particularly by neurons, and its conformational conversion into the abnormally folded, amyloidogenic isoform, PrPSc, is an underlying mechanism in the pathogenesis of prion diseases, a group of neurodegenerative disorders in humans and animals. Most cases of these diseases are sporadic and their aetiologies are unknown. We recently found that a neurotropic strain of influenza A virus (IAV/WSN) caused the conversion of PrPC into PrPSc and the subsequent formation of infectious prions in mouse neuroblastoma cells after infection. These results show that IAV/WSN is the first non-prion pathogen capable of inducing the conversion of PrPC into PrPSc and propagating infectious prions in cultured neuronal cells, and also provide the intriguing possibility that IAV infection in neurons might be a cause of or be associated with sporadic prion diseases. Here, we present our findings of the IAV/WSN-induced conversion of PrPC into PrPSc and subsequent propagation of infectious prions, and also discuss the biological significance of the conversion of PrPC into PrPSc in virus infections.
Assuntos
Orthomyxoviridae , Proteínas PrPC , Doenças Priônicas , Príons , Animais , Encéfalo/patologia , Camundongos , Proteínas PrPC/metabolismo , Doenças Priônicas/metabolismo , Proteínas Priônicas/metabolismo , Príons/metabolismoRESUMO
Nonpathogenic cellular prion protein (PrPC) demonstrates anti-inflammatory activity; however, the responsible mechanisms are incompletely defined. PrPC exists as a GPI-anchored membrane protein in diverse cells; however, PrPC may be released from cells by ADAM proteases or when packaged into extracellular vesicles (EVs). In this study, we show that a soluble derivative of PrPC (S-PrP) counteracts inflammatory responses triggered by pattern recognition receptors in macrophages, including TLR2, TLR4, TLR7, TLR9, NOD1, and NOD2. S-PrP also significantly attenuates the toxicity of LPS in mice. The response of macrophages to S-PrP is mediated by a receptor assembly that includes the N-methyl-d-aspartate receptor (NMDA-R) and low-density lipoprotein receptor-related protein-1 (LRP1). PrPC was identified in EVs isolated from human plasma. These EVs replicated the activity of S-PrP, inhibiting cytokine expression and IκBα phosphorylation in LPS-treated macrophages. The effects of plasma EVs on LPS-treated macrophages were blocked by PrPC-specific Ab, by antagonists of LRP1 and the NMDA-R, by deleting Lrp1 in macrophages, and by inhibiting Src family kinases. Phosphatidylinositol-specific phospholipase C dissociated the LPS-regulatory activity from EVs, rendering the EVs inactive as LPS inhibitors. The LPS-regulatory activity that was lost from phosphatidylinositol-specific phospholipase C-treated EVs was recovered in solution. Collectively, these results demonstrate that GPI-anchored PrPC is the essential EV component required for the observed immune regulatory activity of human plasma EVs. S-PrP and EV-associated PrPC regulate innate immunity by engaging the NMDA-R/LRP1 receptor system in macrophages. The scope of pattern recognition receptors antagonized by S-PrP suggests that released forms of PrPC may have broad anti-inflammatory activity.
Assuntos
Membrana Celular/metabolismo , Vesículas Extracelulares/metabolismo , Inflamação/metabolismo , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas PrPC/metabolismo , Receptores de N-Metil-D-Aspartato/imunologia , Animais , Células Cultivadas , Humanos , Imunidade Inata , Lipopolissacarídeos/imunologia , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Macrófagos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas PrPC/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores de Reconhecimento de Padrão/metabolismoRESUMO
Conformational conversion of the cellular isoform of prion protein, PrPC, into the abnormally folded, amyloidogenic isoform, PrPSc, is an underlying pathogenic mechanism in prion diseases. The diseases manifest as sporadic, hereditary, and acquired disorders. Etiological mechanisms driving the conversion of PrPC into PrPSc are unknown in sporadic prion diseases, while prion infection and specific mutations in the PrP gene are known to cause the conversion of PrPC into PrPSc in acquired and hereditary prion diseases, respectively. We recently reported that a neurotropic strain of influenza A virus (IAV) induced the conversion of PrPC into PrPSc as well as formation of infectious prions in mouse neuroblastoma cells after infection, suggesting the causative role of the neuronal infection of IAV in sporadic prion diseases. Here, we discuss the conversion mechanism of PrPC into PrPSc in different types of prion diseases, by presenting our findings of the IAV infection-induced conversion of PrPC into PrPSc and by reviewing the so far reported transgenic animal models of hereditary prion diseases and the reverse genetic studies, which have revealed the structure-function relationship for PrPC to convert into PrPSc after prion infection.
Assuntos
Síndrome de Creutzfeldt-Jakob/genética , Doença de Gerstmann-Straussler-Scheinker/genética , Influenza Humana/genética , Insônia Familiar Fatal/genética , Proteínas PrPC/genética , Proteínas PrPSc/genética , Proteínas Priônicas/genética , Animais , Linhagem Celular Tumoral , Síndrome de Creutzfeldt-Jakob/metabolismo , Síndrome de Creutzfeldt-Jakob/patologia , Síndrome de Creutzfeldt-Jakob/virologia , Doença de Gerstmann-Straussler-Scheinker/metabolismo , Doença de Gerstmann-Straussler-Scheinker/patologia , Doença de Gerstmann-Straussler-Scheinker/virologia , Humanos , Vírus da Influenza A/genética , Vírus da Influenza A/crescimento & desenvolvimento , Vírus da Influenza A/patogenicidade , Influenza Humana/metabolismo , Influenza Humana/patologia , Influenza Humana/virologia , Insônia Familiar Fatal/metabolismo , Insônia Familiar Fatal/patologia , Insônia Familiar Fatal/virologia , Camundongos , Camundongos Transgênicos , Mutação , Neurônios/metabolismo , Neurônios/patologia , Neurônios/virologia , Proteínas PrPC/química , Proteínas PrPC/metabolismo , Proteínas PrPSc/química , Proteínas PrPSc/metabolismo , Proteínas Priônicas/química , Proteínas Priônicas/metabolismo , Conformação Proteica , Genética Reversa/métodosRESUMO
Recently, we reported ß-cleavage of the prion protein (PrPC) in human ocular tissues. Here, we explored whether this is unique to the human eye, and its functional implications. A comparison of the cleavage pattern of PrPC in human ocular tissues with common nocturnal and diurnal animals revealed mainly ß-cleavage in humans, and mostly full-length PrPC in animal retinas. Soluble FL PrPC and N-terminal fragment (N2) released from ß-cleavage was observed in the aqueous and vitreous humor (AH & VH). Expression of human PrPC in ARPE-19 cells, a human retinal pigmented epithelial cell line, also showed ß-cleaved PrPC. Surprisingly, ß-cleavage was not altered by a variety of insults, including oxidative stress, suggesting a unique role of this cleavage in the human eye. It is likely that ß-cleaved C- or N-terminal fragments of PrPC protect from various insults unique to the human eye. On the contrary, ß-cleaved C-terminus of PrPC is susceptible to conversion to the pathological PrP-scrapie form, and includes the binding sites for ß1-integrin and amyloid-ß, molecules implicated in several ocular disorders. Considering the species and tissue-specific cleavage of PrPC, our data suggest re-evaluation of prion infectivity and other ocular disorders of the human eye conducted in mouse models.
Assuntos
Oftalmopatias/metabolismo , Proteínas PrPC/metabolismo , Clivagem do RNA/fisiologia , Epitélio Pigmentado da Retina/metabolismo , Animais , Linhagem Celular , Modelos Animais de Doenças , Humanos , Camundongos , Epitélio Pigmentado da Retina/patologiaRESUMO
Despite having therapeutic potential, anti-PrP antibodies caused a major controversy due to their neurotoxic effects. For instance, treating mice with ICSM antibodies delayed prion disease onset, but both were found to be either toxic or innocuous to neurons by researchers following cross-linking PrPC. In order to elucidate and understand the reasons that led to these contradictory outcomes, we conducted a comprehensive in silico study to assess the antibody-specific toxicity. Since most therapeutic anti-PrP antibodies were generated against human truncated recombinant PrP91-231 or full-length mouse PrP23-231, we reasoned that host specificity (human vs murine) of PrPC might influence the nature of the specific epitopes recognized by these antibodies at the structural level possibly explaining the 'toxicity' discrepancies reported previously. Initially, molecular dynamics simulation and pro-motif analysis of full-length human (hu)PrP and mouse (mo)PrP 3D structure displayed conspicuous structural differences between huPrP and moPrP. We identified 10 huPrP and 6 moPrP linear B-cell epitopes from the prion protein 3D structure where 5 out of 10 huPrP and 3 out of 6 moPrP B-cell epitopes were predicted to be potentially toxic in immunoinformatics approaches. Herein, we demonstrate that some of the predicted potentially 'toxic' epitopes identified by the in silico analysis were similar to the epitopes recognized by the toxic antibodies such as ICSM18 (146-159), POM1 (138-147), D18 (133-157), ICSM35 (91-110), D13 (95-103) and POM3 (95-100). This in silico study reveals the role of host specificity of PrPC in epitope-specific anti-PrP antibody toxicity.
Assuntos
Proteínas PrPC , Doenças Priônicas , Príons , Animais , Epitopos , Humanos , Camundongos , Proteínas PrPC/metabolismo , Proteínas PriônicasRESUMO
Conversion of normal prion protein (PrPC) to the pathogenic PrPSc conformer is central to prion diseases such as Creutzfeldt-Jakob disease and scrapie; however, the detailed mechanism of this conversion remains obscure. To investigate how the N-terminal polybasic region of PrP (NPR) influences the PrPC-to-PrPSc conversion, we analyzed two PrP mutants: ΔN6 (deletion of all six amino acids in NPR) and Met4-1 (replacement of four positively charged amino acids in NPR with methionine). We found that ΔN6 and Met4-1 differentially impacted the binding of recombinant PrP (recPrP) to the negatively charged phospholipid 1-palmitoyl-2-oleoylphosphatidylglycerol, a nonprotein cofactor that facilitates PrP conversion. Both mutant recPrPs were able to form recombinant prion (recPrPSc) in vitro, but the convertibility was greatly reduced, with ΔN6 displaying the lowest convertibility. Prion infection assays in mammalian RK13 cells expressing WT or NPR-mutant PrPs confirmed these differences in convertibility, indicating that the NPR affects the conversion of both bacterially expressed recPrP and post-translationally modified PrP in eukaryotic cells. We also found that both WT and mutant recPrPSc conformers caused prion disease in WT mice with a 100% attack rate, but the incubation times and neuropathological changes caused by two recPrPSc mutants were significantly different from each other and from that of WT recPrPSc. Together, our results support that the NPR greatly influences PrPC-to-PrPSc conversion, but it is not essential for the generation of PrPSc. Moreover, the significant differences between ΔN6 and Met4-1 suggest that not only charge but also the identity of amino acids in NPR is important to PrP conversion.
Assuntos
Encéfalo/metabolismo , Mutação , Proteínas PrPC/metabolismo , Proteínas PrPSc/metabolismo , Doenças Priônicas/metabolismo , Animais , Linhagem Celular , Camundongos , Proteínas PrPC/genética , Proteínas PrPSc/genética , Doenças Priônicas/genética , CoelhosRESUMO
Fyn is a non-receptor tyrosine kinase belonging to the Src family of kinases (SFKs) which has been implicated in several integral functions throughout the central nervous system (CNS), including myelination and synaptic transmission. More recently, Fyn dysfunction has been associated with pathological processes observed in neurodegenerative diseases, such as multiple sclerosis (MS), Alzheimer's disease (AD) and Parkinson's disease (PD). Neurodegenerative diseases are amongst the leading cause of death and disability worldwide and, due to the ageing population, prevalence is predicted to rise in the coming years. Symptoms across neurodegenerative diseases are both debilitating and degenerative in nature and, concerningly, there are currently no disease-modifying therapies to prevent their progression. As such, it is important to identify potential new therapeutic targets. This review will outline the role of Fyn in normal/homeostatic processes, as well as degenerative/pathological mechanisms associated with neurodegenerative diseases, such as demyelination, pathological protein aggregation, neuroinflammation and cognitive dysfunction.
Assuntos
Proteínas do Tecido Nervoso/fisiologia , Doenças Neurodegenerativas/enzimologia , Proteínas Proto-Oncogênicas c-fyn/fisiologia , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/enzimologia , Doença de Alzheimer/fisiopatologia , Peptídeos beta-Amiloides/metabolismo , Benzamidas/farmacologia , Benzamidas/uso terapêutico , Sistema Nervoso Central/enzimologia , Dasatinibe/farmacologia , Dasatinibe/uso terapêutico , Humanos , Terapia de Alvo Molecular , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/enzimologia , Bainha de Mielina/fisiologia , Proteínas do Tecido Nervoso/efeitos dos fármacos , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/fisiopatologia , Oligodendroglia/metabolismo , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/enzimologia , Doença de Parkinson/fisiopatologia , Piperidinas/farmacologia , Piperidinas/uso terapêutico , Proteínas PrPC/metabolismo , Proteínas Proto-Oncogênicas c-fyn/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-fyn/efeitos dos fármacos , Piridinas/farmacologia , Piridinas/uso terapêutico , Receptores de N-Metil-D-Aspartato/metabolismo , Especificidade do Receptor de Antígeno de Linfócitos T , Subpopulações de Linfócitos T/enzimologia , Subpopulações de Linfócitos T/imunologia , Tiazóis/farmacologia , Tiazóis/uso terapêutico , Proteínas tau/metabolismoRESUMO
Cytoplasmic aggregation of the primarily nuclear TAR DNA-binding protein 43 (TDP-43) affects neurons in most amyotrophic lateral sclerosis (ALS) and approximately half of frontotemporal lobar degeneration (FTLD) cases. The cellular prion protein, PrPC, has been recognized as a common receptor and downstream effector of circulating neurotoxic species of several proteins involved in neurodegeneration. Here, capitalizing on our recently adapted TDP-43 real time quaking induced reaction, we set reproducible protocols to obtain standardized preparations of recombinant TDP-43 fibrils. We then exploited two different cellular systems (human SH-SY5Y and mouse N2a neuroblastoma cells) engineered to express low or high PrPC levels to investigate the link between PrPC expression on the cell surface and the internalization of TDP-43 fibrils. Fibril uptake was increased in cells overexpressing either human or mouse prion protein. Increased internalization was associated with detrimental consequences in all PrP-overexpressing cell lines but was milder in cells expressing the human form of the prion protein. As described for other amyloids, treatment with TDP-43 fibrils induced a reduction in the accumulation of the misfolded form of PrPC, PrPSc, in cells chronically infected with prions. Our results expand the list of misfolded proteins whose uptake and detrimental effects are mediated by PrPC, which encompass almost all pathological amyloids involved in neurodegeneration.