RESUMEN
Corruption of cellular prion protein (PrPC) function(s) at the plasma membrane of neurons is at the root of prion diseases, such as Creutzfeldt-Jakob disease and its variant in humans, and Bovine Spongiform Encephalopathies, better known as mad cow disease, in cattle. The roles exerted by PrPC, however, remain poorly elucidated. With the perspective to grasp the molecular pathways of neurodegeneration occurring in prion diseases, and to identify therapeutic targets, achieving a better understanding of PrPC roles is a priority. Based on global approaches that compare the proteome and metabolome of the PrPC expressing 1C11 neuronal stem cell line to those of PrPnull-1C11 cells stably repressed for PrPC expression, we here unravel that PrPC contributes to the regulation of the energetic metabolism by orienting cells towards mitochondrial oxidative degradation of glucose. Through its coupling to cAMP/protein kinase A signaling, PrPC tones down the expression of the pyruvate dehydrogenase kinase 4 (PDK4). Such an event favors the transfer of pyruvate into mitochondria and its conversion into acetyl-CoA by the pyruvate dehydrogenase complex and, thereby, limits fatty acids ß-oxidation and subsequent onset of oxidative stress conditions. The corruption of PrPC metabolic role by pathogenic prions PrPSc causes in the mouse hippocampus an imbalance between glucose oxidative degradation and fatty acids ß-oxidation in a PDK4-dependent manner. The inhibition of PDK4 extends the survival of prion-infected mice, supporting that PrPSc-induced deregulation of PDK4 activity and subsequent metabolic derangements contribute to prion diseases. Our study posits PDK4 as a potential therapeutic target to fight against prion diseases.
Asunto(s)
Glucosa/metabolismo , Degeneración Nerviosa/metabolismo , Proteínas PrPSc/metabolismo , Enfermedades por Prión/metabolismo , Enfermedades por Prión/patología , Animales , Modelos Animales de Enfermedad , Masculino , Ratones , Ratones Endogámicos C57BL , Degeneración Nerviosa/patología , Estrés Oxidativo/fisiología , Proteínas Quinasas/metabolismoRESUMEN
BACKGROUND: Epidemiological emerging evidence shows that human exposure to some nanosized materials present in the environment would contribute to the onset and/or progression of Alzheimer's disease (AD). The cellular and molecular mechanisms whereby nanoparticles would exert some adverse effects towards neurons and take part in AD pathology are nevertheless unknown. RESULTS: Here, we provide the prime evidence that titanium dioxide (TiO2) and carbon black (CB) nanoparticles (NPs) bind the cellular form of the prion protein (PrPC), a plasma membrane protein well known for its implication in prion diseases and prion-like diseases, such as AD. The interaction between TiO2- or CB-NPs and PrPC at the surface of neuronal cells grown in culture corrupts PrPC signaling function. This triggers PrPC-dependent activation of NADPH oxidase and subsequent production of reactive oxygen species (ROS) that alters redox equilibrium. Through PrPC interaction, NPs also promote the activation of 3-phosphoinositide-dependent kinase 1 (PDK1), which in turn provokes the internalization of the neuroprotective TACE α-secretase. This diverts TACE cleavage activity away from (i) TNFα receptors (TNFR), whose accumulation at the plasma membrane augments the vulnerability of NP-exposed neuronal cells to TNFα -associated inflammation, and (ii) the amyloid precursor protein APP, leading to overproduction of neurotoxic amyloid Aß40/42 peptides. The silencing of PrPC or the pharmacological inhibition of PDK1 protects neuronal cells from TiO2- and CB-NPs effects regarding ROS production, TNFα hypersensitivity, and Aß rise. Finally, we show that dysregulation of the PrPC-PDK1-TACE pathway likely occurs in the brain of mice injected with TiO2-NPs by the intra-cerebro-ventricular route as we monitor a rise of TNFR at the cell surface of several groups of neurons located in distinct brain areas. CONCLUSION: Our in vitro and in vivo study thus posits for the first time normal cellular prion protein PrPC as being a neuronal receptor of TiO2- and CB-NPs and identifies PrPC-coupled signaling pathways by which those nanoparticles alter redox equilibrium, augment the intrinsic sensitivity of neurons to neuroinflammation, and provoke a rise of Aß peptides. By identifying signaling cascades dysregulated by TiO2- and CB-NPs in neurons, our data shed light on how human exposure to some NPs might be related to AD.
Asunto(s)
Enfermedad de Alzheimer , Nanopartículas , Priones , Enfermedad de Alzheimer/inducido químicamente , Enfermedad de Alzheimer/patología , Animales , Homeostasis , Humanos , Ratones , Nanopartículas/toxicidad , Neuronas/patología , Proteínas Priónicas/metabolismo , Priones/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Hollín/toxicidad , Titanio , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
In prion diseases, synapse dysfunction, axon retraction and loss of neuronal polarity precede neuronal death. The mechanisms driving such polarization defects, however, remain unclear. Here, we examined the contribution of RhoA-associated coiled-coil containing kinases (ROCK), key players in neuritogenesis, to prion diseases. We found that overactivation of ROCK signaling occurred in neuronal stem cells infected by pathogenic prions (PrPSc) and impaired the sprouting of neurites. In reconstructed networks of mature neurons, PrPSc-induced ROCK overactivation provoked synapse disconnection and dendrite/axon degeneration. This overactivation of ROCK also disturbed overall neurotransmitter-associated functions. Importantly, we demonstrated that beyond its impact on neuronal polarity ROCK overactivity favored the production of PrPSc through a ROCK-dependent control of 3-phosphoinositide-dependent kinase 1 (PDK1) activity. In non-infectious conditions, ROCK and PDK1 associated within a complex and ROCK phosphorylated PDK1, conferring basal activity to PDK1. In prion-infected neurons, exacerbated ROCK activity increased the pool of PDK1 molecules physically interacting with and phosphorylated by ROCK. ROCK-induced PDK1 overstimulation then canceled the neuroprotective α-cleavage of normal cellular prion protein PrPC by TACE α-secretase, which physiologically precludes PrPSc production. In prion-infected cells, inhibition of ROCK rescued neurite sprouting, preserved neuronal architecture, restored neuronal functions and reduced the amount of PrPSc. In mice challenged with prions, inhibition of ROCK also lowered brain PrPSc accumulation, reduced motor impairment and extended survival. We conclude that ROCK overactivation exerts a double detrimental effect in prion diseases by altering neuronal polarity and triggering PrPSc accumulation. Eventually ROCK emerges as therapeutic target to combat prion diseases.
Asunto(s)
Proteínas PrPSc/metabolismo , Enfermedades por Prión/metabolismo , Enfermedades por Prión/patología , Quinasas Asociadas a rho/metabolismo , Animales , Western Blotting , Modelos Animales de Enfermedad , Técnica del Anticuerpo Fluorescente , Inmunoprecipitación , Dispositivos Laboratorio en un Chip , Ratones , Ratones Endogámicos C57BL , Neuritas/metabolismo , Neurogénesis , Proteínas PrPC/metabolismoRESUMEN
More than 650 reversible and irreversible post-translational modifications (PTMs) of proteins have been listed so far. Canonical PTMs of proteins consist of the covalent addition of functional or chemical groups on target backbone amino-acids or the cleavage of the protein itself, giving rise to modified proteins with specific properties in terms of stability, solubility, cell distribution, activity, or interactions with other biomolecules. PTMs of protein contribute to cell homeostatic processes, enabling basal cell functions, allowing the cell to respond and adapt to variations of its environment, and globally maintaining the constancy of the milieu interieur (the body's inner environment) to sustain human health. Abnormal protein PTMs are, however, associated with several disease states, such as cancers, metabolic disorders, or neurodegenerative diseases. Abnormal PTMs alter the functional properties of the protein or even cause a loss of protein function. One example of dramatic PTMs concerns the cellular prion protein (PrPC), a GPI-anchored signaling molecule at the plasma membrane, whose irreversible post-translational conformational conversion (PTCC) into pathogenic prions (PrPSc) provokes neurodegeneration. PrPC PTCC into PrPSc is an additional type of PTM that affects the tridimensional structure and physiological function of PrPC and generates a protein conformer with neurotoxic properties. PrPC PTCC into PrPSc in neurons is the first step of a deleterious sequence of events at the root of a group of neurodegenerative disorders affecting both humans (Creutzfeldt-Jakob diseases for the most representative diseases) and animals (scrapie in sheep, bovine spongiform encephalopathy in cow, and chronic wasting disease in elk and deer). There are currently no therapies to block PrPC PTCC into PrPSc and stop neurodegeneration in prion diseases. Here, we review known PrPC PTMs that influence PrPC conversion into PrPSc. We summarized how PrPC PTCC into PrPSc impacts the PrPC interactome at the plasma membrane and the downstream intracellular controlled protein effectors, whose abnormal activation or trafficking caused by altered PTMs promotes neurodegeneration. We discussed these effectors as candidate drug targets for prion diseases and possibly other neurodegenerative diseases.
RESUMEN
Cytoskeleton modifications are required for neuronal stem cells to acquire neuronal polarization. Little is known, however, about mechanisms that orchestrate cytoskeleton remodeling along neuritogenesis. Here, we show that the silencing of the cellular prion protein (PrP(C)) impairs the initial sprouting of neurites upon induction of differentiation of the 1C11 neuroectodermal cell line, indicating that PrP(C) is necessary to neuritogenesis. Such PrP(C) function relies on its capacity to negatively regulate the clustering, activation, and signaling activity of ß1 integrins at the plasma membrane. ß1 Integrin aggregation caused by PrP(C) depletion triggers overactivation of the RhoA-Rho kinase-LIMK-cofilin pathway, which, in turn, alters the turnover of focal adhesions, increases the stability of actin microfilaments, and in fine impairs neurite formation. Inhibition of Rho kinases is sufficient to compensate for the lack of PrP(C) and to restore neurite sprouting. We also observe an increased secretion of fibronectin in the surrounding milieu of PrP(C)-depleted 1C11 cells, which likely self-sustains ß1 integrin signaling overactivation and contributes to neuritogenesis defect. Our overall data reveal that PrP(C) contributes to the acquisition of neuronal polarization by modulating ß1 integrin activity, cell interaction with fibronectin, and cytoskeleton dynamics.
Asunto(s)
Integrina beta1/metabolismo , Neuritas/metabolismo , Neurogénesis/fisiología , Proteínas PrPC/metabolismo , Factores Despolimerizantes de la Actina/metabolismo , Actinas/metabolismo , Animales , Secuencia de Bases , Diferenciación Celular , Línea Celular , Polaridad Celular , Citoesqueleto/metabolismo , Fibronectinas/metabolismo , Quinasas Lim/metabolismo , Ratones , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Neuronas/citología , Neuronas/metabolismo , Proteínas PrPC/antagonistas & inhibidores , Proteínas PrPC/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , Transducción de Señal , Proteínas de Unión al GTP rho/metabolismo , Quinasas Asociadas a rho/metabolismo , Proteína de Unión al GTP rhoARESUMEN
Amyloid-based neurodegenerative diseases such as prion, Alzheimer's, and Parkinson's diseases have distinct etiologies and clinical manifestations, but they share common pathological events. These diseases are caused by abnormally folded proteins (pathogenic prions PrPSc in prion diseases, ß-amyloids/Aß and Tau in Alzheimer's disease, α-synuclein in Parkinson's disease) that display ß-sheet-enriched structures, propagate and accumulate in the nervous central system, and trigger neuronal death. In prion diseases, PrPSc-induced corruption of the physiological functions exerted by normal cellular prion proteins (PrPC) present at the cell surface of neurons is at the root of neuronal death. For a decade, PrPC emerges as a common cell surface receptor for other amyloids such as Aß and α-synuclein, which relays, at least in part, their toxicity. In lipid-rafts of the plasma membrane, PrPC exerts a signaling function and controls a set of effectors involved in neuronal homeostasis, among which are the RhoA-associated coiled-coil containing kinases (ROCKs). Here we review (i) how PrPC controls ROCKs, (ii) how PrPC-ROCK coupling contributes to neuronal homeostasis, and (iii) how the deregulation of the PrPC-ROCK connection in amyloid-based neurodegenerative diseases triggers a loss of neuronal polarity, affects neurotransmitter-associated functions, contributes to the endoplasmic reticulum stress cascade, renders diseased neurons highly sensitive to neuroinflammation, and amplifies the production of neurotoxic amyloids.
Asunto(s)
Enfermedad de Alzheimer/enzimología , Enfermedades por Prión/enzimología , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas ADAM/metabolismo , Proteína ADAM17 , Inhibidores Enzimáticos/uso terapéutico , Humanos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Piruvato Deshidrogenasa Quinasa Acetil-TransferidoraRESUMEN
Serotonin transporter, SERT (SLC64A for solute carrier family 6, member A4), is a twelve transmembrane domain (TMDs) protein that assumes the uptake of serotonin (5-HT) through dissipation of the Na+ gradient established by the electrogenic pump Na/K ATPase. Abnormalities in 5-HT level and signaling have been associated with various disorders of the central nervous system (CNS) such as depression, obsessive-compulsive disorder, anxiety disorders, and autism spectrum disorder. Since the 50s, SERT has raised a lot of interest as being the target of a class of antidepressants, the Serotonin Selective Reuptake Inhibitors (SSRIs), used in clinics to combat depressive states. Because of the refractoriness of two-third of patients to SSRI treatment, a better understanding of the mechanisms regulating SERT functions is of priority. Here, we review how genetic and epigenetic regulations, post-translational modifications of SERT, and specific interactions between SERT and a set of diverse partners influence SERT expression, trafficking to and away from the plasma membrane and activity, in connection with the neuronal adaptive cell response to SSRI antidepressants.
RESUMEN
The presence of amyloid beta (Aß) plaques in the brain of some individuals with Creutzfeldt-Jakob or Gertsmann-Straussler-Scheinker diseases suggests that pathogenic prions (PrPSc) would have stimulated the production and deposition of Aß peptides. We here show in prion-infected neurons and mice that deregulation of the PDK1-TACE α-secretase pathway reduces the Amyloid Precursor Protein (APP) α-cleavage in favor of APP ß-processing, leading to Aß40/42 accumulation. Aß predominates as monomers, but is also found as trimers and tetramers. Prion-induced Aß peptides do not affect prion replication and infectivity, but display seedable properties as they can deposit in the mouse brain only when seeds of Aß trimers are co-transmitted with PrPSc. Importantly, brain Aß deposition accelerates death of prion-infected mice. Our data stress that PrPSc, through deregulation of the PDK1-TACE-APP pathway, provokes the accumulation of Aß, a prerequisite for the onset of an Aß seeds-induced Aß pathology within a prion-infectious context.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Enfermedades por Prión/metabolismo , Priones/metabolismo , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora/metabolismo , Proteína ADAM17/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/líquido cefalorraquídeo , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Conducta Animal , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Enfermedades Neurodegenerativas/metabolismo , Neuronas/metabolismo , Fragmentos de Péptidos/líquido cefalorraquídeo , Placa Amiloide/metabolismo , Enfermedades por Prión/líquido cefalorraquídeo , Enfermedades por Prión/patología , Células MadreRESUMEN
The cellular prion protein PrP(C) is the normal counterpart of the scrapie prion protein PrP(Sc), the main component of the infectious agent of transmissible spongiform encephalopathies (TSEs). It is a ubiquitous cell-surface glycoprotein, abundantly expressed in neurons, which constitute the targets of TSE pathogenesis. Taking advantage of the 1C11 neuroectodermal cell line, endowed with the capacity to convert into 1C11(5-HT) serotonergic or 1C11(NE) noradrenergic neuronal cells, allowed us to ascribe a signaling function to PrP(C). Antibody-mediated ligation of PrP(C) recruits transduction pathways, which involve nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-dependent reactive oxygen species production and target the extracellular-regulated kinases ERK1/2. In fully differentiated cells only, these effectors are under the control of a PrP(C)-caveolin-Fyn platform, located on neuritic extensions. In addition to its proper signaling activity, PrP(C) modulates the agonist-induced response of the three serotonergic G protein-coupled receptors present on the 1C11(5-HT) differentiated cells. The impact of PrP(C) ligation on the receptor couplings depends on the receptor subtype and the pathway considered. The implementation of the PrP(C)-caveolin complex again is mandatory for PrP(C) to exert its action on 5-HT receptor signaling. Our current data argue that PrP(C) interferes with the intensities and/or dynamics of G protein activation by agonist-bound 5-HT receptors. By mobilizing transduction cascades controlling the cellular redox state and the ERK1/2 kinases and by altering 5-HT receptor-mediated intracellular response, PrP(C) takes part in the homeostasis of serotonergic neuronal cells. These findings may have implications for future research aiming at understanding the fate of serotonergic neurons in prion diseases.
Asunto(s)
Neuronas/metabolismo , Proteínas PrPC/metabolismo , Transducción de Señal , Animales , Caveolinas/metabolismo , Diferenciación Celular , Línea Celular , Ectodermo/metabolismo , Proteínas de Unión al GTP/metabolismo , Regulación de la Expresión Génica , Glicoproteínas/metabolismo , Ratones , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Modelos BiológicosRESUMEN
Although cellular prion protein PrPC is well known for its implication in Transmissible Spongiform Encephalopathies, its functions remain elusive. Combining in vitro and in vivo approaches, we here show that PrPC displays the intrinsic capacity to protect neuronal cells from a pro-inflammatory TNFα noxious insult. Mechanistically, PrPC coupling to the NADPH oxidase-TACE α-secretase signaling pathway promotes TACE-mediated cleavage of transmembrane TNFα receptors (TNFRs) and the release of soluble TNFR, which limits the sensitivity of recipient cells to TNFα. We further show that PrPC expression is necessary for TACE α-secretase to stay at the plasma membrane in an active state for TNFR shedding. Such PrPC control of TACE localization depends on PrPC modulation of ß1 integrin signaling and downstream activation of ROCK-I and PDK1 kinases. Loss of PrPC provokes TACE internalization, which in turn cancels TACE-mediated cleavage of TNFR and renders PrPC-depleted neuronal cells as well as PrPC knockout mice highly vulnerable to pro-inflammatory TNFα insult. Our work provides the prime evidence that in an inflammatory context PrPC adjusts the response of neuronal cells targeted by TNFα through TACE α-secretase. Our data also support the view that abnormal TACE trafficking and activity in prion diseases originate from a-loss-of-PrPC cytoprotective function.
Asunto(s)
Proteína ADAM17/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Mediadores de Inflamación/metabolismo , Proteínas Priónicas/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Línea Celular , Ratones , NADPH Oxidasas/metabolismo , Neuronas/metabolismo , Proteínas PrPC/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Transducción de Señal , Quinasas Asociadas a rho/metabolismoRESUMEN
A major determinant of neuronal homeostasis is the proper integration of cell signaling pathways recruited by a variety of neuronal and non-neuronal factors. By taking advantage of a neuroectodermal cell line (1C11) endowed with the capacity to differentiate into serotonergic (1C115-HT) or noradrenergic (1C11NE) neurons, we identified serotonin (5-hydroxytryptamine, 5-HT)- and norepinephrine (NE)-dependent signaling cascades possibly involved in neuronal functions. First, we establish that 5-HT2B receptors and 1D adrenoceptors are functionally coupled to reactive oxygen species (ROS) synthesis through NADPH oxidase activation in 1C115-HT and 1C11NE cells. This observation constitutes the prime evidence that bioaminergic autoreceptors take part in the control of the cellular redox equilibrium in a neuronal context. Second, our data identify TACE (TNF- Converting Enzyme), a member of a disintegrin and metalloproteinase (ADAM) family, as a downstream target of the 5-HT2B and 1D receptor-NADPH oxidase signaling pathways. Upon 5-HT2B or 1D receptor stimulation, ROS fully govern TNF- shedding in the surrounding milieu of 1C115-HT or 1C11NE cells. Third, 5-HT2B and 1Dreceptor couplings to the NADPH oxidase-TACE cascade are strictly restricted to 1C11-derived progenies that have implemented a complete serotonergic or noradrenergic phenotype. Overall, these observations suggest that 5-HT2B and 1D autoreceptors may play a role in the maintenance of neuron- and neurotransmitter-associated functions. Eventually, our study may have implications regarding the origin of oxidative stress as well as up-regulated expression of proinflammatory cytokines in neurodegenerative disorders, which may relate to the deviation of normal signaling pathways.
Asunto(s)
Proteínas ADAM/metabolismo , Autorreceptores/fisiología , Neuronas/enzimología , Especies Reactivas de Oxígeno/metabolismo , Receptor de Serotonina 5-HT1D/fisiología , Receptor de Serotonina 5-HT2B/fisiología , Proteína ADAM17 , Animales , Diferenciación Celular , Línea Celular , Activación Enzimática , Homeostasis , Ratones , NADPH Oxidasas/fisiología , Enfermedades Neurodegenerativas/etiología , Transducción de SeñalRESUMEN
Homeostasis of the central nervous system relies on the proper integration of cell-signaling pathways recruited by a variety of neuronal and non-neuronal factors, with the aim of tightly controlling neurotransmitter metabolism, storage, and transport. We took advantage of the 1C11 neuroectodermal cell line, endowed with the capacity to selectively differentiate into serotonergic (1C11(5-HT)) or noradrenergic (1C11(NE)) neurons, to identify functional targets of serotonin (5-hydroxytryptamine [5-HT]) and norepinephrine (NE) autoreceptors possibly involved in the control of neuronal functions. We demonstrate that 5-HT(2B) and adreno alpha(1D) receptors are coupled to reactive oxygen species (ROS) production through NADPH oxidase activation in 1C11(5-HT) and 1C11(NE) neuronal cells, respectively. In the signaling cascade linking 5-HT(2B) receptors to NADPH oxidase, phospholipase A2-mediated arachidonic acid production is required for ROS synthesis. ROS, in turn, act as second message signals and control the activation of TACE (TNF-alpha converting enzyme), a member of a disintegrin and metalloproteinase family. 5-HT(2B) and alpha(1D) receptor stimulation triggers TACE-dependent TNF-alpha shedding in the surrounding milieu of 1C11(5-HT) and 1C11(NE) cells. In these cells, shed TNF-alpha triggers degradation of 5-HT and NE into 5-HIAA and MHPG, respectively. Finally, we observe that 5-HT(2B) and alpha(1D) receptor couplings to the NADPH oxidase-TACE cascade are strictly restricted to 1C11-derived progenies that have implemented a complete neuronal phenotype. Altogether, our data indicate that couplings of 5-HT(2B) and alpha(1D) autoreceptors to ROS and TNF-alpha signaling control neurotransmitter metabolism in 1C11-derived neuronal cells. Eventually, we might explain the origin of oxidative stress and high level of TNF-alpha in neurodegenerative diseases as a consequence of deviation of normal signaling pathways coupled to neurotransmitters.
Asunto(s)
Aminas Biogénicas/metabolismo , Neuronas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Receptor de Serotonina 5-HT2B/fisiología , Receptores Adrenérgicos alfa 1/fisiología , Transducción de Señal/fisiología , Factor de Necrosis Tumoral alfa/fisiología , Animales , Línea Celular , Metoxihidroxifenilglicol/metabolismo , RatonesRESUMEN
α-secretase-mediated cleavage of amyloid precursor protein (APP) precludes formation of neurotoxic amyloid-ß (Aß) peptides, and α-cleavage of cellular prion protein (PrP(C)) prevents its conversion into misfolded, pathogenic prions (PrP(Sc)). The mechanisms leading to decreased α-secretase activity in Alzheimer's and prion disease remain unclear. Here, we find that tumor necrosis factor-α-converting enzyme (TACE)-mediated α-secretase activity is impaired at the surface of neurons infected with PrP(Sc) or isolated from APP-transgenic mice with amyloid pathology. 3-phosphoinositide-dependent kinase-1 (PDK1) activity is increased in neurons infected with prions or affected by Aß deposition and in the brains of individuals with Alzheimer's disease. PDK1 induces phosphorylation and caveolin-1-mediated internalization of TACE. This dysregulation of TACE increases PrP(Sc) and Aß accumulation and reduces shedding of TNF-α receptor type 1 (TNFR1). Inhibition of PDK1 promotes localization of TACE to the plasma membrane, restores TACE-dependent α-secretase activity and cleavage of APP, PrP(C) and TNFR1, and attenuates PrP(Sc)- and Aß-induced neurotoxicity. In mice, inhibition or siRNA-mediated silencing of PDK1 extends survival and reduces motor impairment following PrP(Sc) infection and in APP-transgenic mice reduces Alzheimer's disease-like pathology and memory impairment.
Asunto(s)
Proteínas ADAM/metabolismo , Enfermedad de Alzheimer/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Enfermedades por Prión/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína ADAM17 , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Encéfalo/metabolismo , Encéfalo/patología , Caveolina 1/metabolismo , Supervivencia Celular , Células Cultivadas , Progresión de la Enfermedad , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fosforilación , Priones/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora , Interferencia de ARN , ARN Interferente Pequeño , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The cellular prion protein PrP(C) is the normal counterpart of the scrapie prion protein PrP(Sc), the main component of the infectious agent of transmissible spongiform encephalopathies (TSEs). It is a ubiquitous cell-surface glycoprotein, abundantly expressed in neurons, which constitute the targets of TSE pathogenesis. The presence of PrP(C) at the surface of neurons is an absolute requirement for the development of prion diseases and corruption of PrP(C) function(s) within an infectious context emerges as a proximal cause for PrP(Sc)-induced neurodegeneration. Experimental evidence gained over the past decade indicates that PrP(C) has the capacity to mobilize promiscuous signal transduction cascades that, notably, contribute to cell homeostasis. Beyond ubiquitous effectors, much data converge onto a neurospecificity of PrP(C) signaling, which may be the clue to neuronal cell demise in prion disorders. In this article, we highlight the requirement of PrP(C) for TSEs-associated neurodegeneration and review the current knowledge of PrP(C)-dependent signal transduction in neuronal cells and its implications for PrP(Sc)-mediated neurotoxicity.
Asunto(s)
Neuronas/metabolismo , Proteínas PrPC/fisiología , Enfermedades por Prión/fisiopatología , Proteínas ADAM/metabolismo , Proteína ADAM17 , Fosfatasa Alcalina/metabolismo , Animales , Calcio/metabolismo , Cobre/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Activación Enzimática , Humanos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , NADPH Oxidasas/metabolismo , Oxidación-Reducción , Fragmentos de Péptidos/fisiología , Proteínas PrPC/metabolismo , Proteínas PrPSc/metabolismo , Enfermedades por Prión/metabolismo , Priones/metabolismo , Priones/fisiología , Especies Reactivas de Oxígeno/metabolismo , Receptores Acoplados a Proteínas G/fisiología , Transducción de Señal , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Various experimental models are used to study brain development and degeneration. They range from whole animal models, which preserve anatomical structures but strongly limit investigations at the cellular level, to dissociated cell culture systems that allow detailed observation of cell phenotypes but lack the highly ordered physiological neuron connection architecture. We describe here a platform comprising independent cell culture chambers separated by an array of "axonal diodes". This array involves asymmetric micro-channels, imposing unidirectional axon connectivity with 97% selectivity. It allows the construction of complex, oriented neuronal networks not feasible with earlier platforms. Different neuronal subtypes could be co-cultivated for weeks, and sequential seeding of different cell populations reproduced physiological network development. To illustrate possible applications, we created and characterized a cortico-striatal oriented network. Functional synaptic connections were established. The activation of striatal differentiation by cortical axons, and the synchronization of neural activity were demonstrated. Each neuronal population and subcompartment could be chemically addressed individually. The directionality of neural pathways being a key feature of the nervous system organization, the axon diode concept brings in a paradigmatic change in neuronal culture platforms, with potential applications for studying neuronal development, synaptic transmission and neurodegenerative disorder such as Alzheimer and Parkinson diseases at the sub-cellular, cellular and network levels.
Asunto(s)
Axones/fisiología , Técnicas Analíticas Microfluídicas , Red Nerviosa/citología , Neuronas/citología , Compuestos de Anilina/química , Animales , Calcio/metabolismo , Diferenciación Celular , Células Cultivadas , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Ratones , Ratones Transgénicos , Red Nerviosa/metabolismo , Red Nerviosa/fisiología , Neuronas/metabolismo , Xantenos/químicaRESUMEN
BACKGROUND: The cellular prion protein, PrP(C), is GPI anchored and abundant in lipid rafts. The absolute requirement of PrP(C) in neurodegeneration associated to prion diseases is well established. However, the function of this ubiquitous protein is still puzzling. Our previous work using the 1C11 neuronal model, provided evidence that PrP(C) acts as a cell surface receptor. Besides a ubiquitous signaling function of PrP(C), we have described a neuronal specificity pointing to a role of PrP(C) in neuronal homeostasis. 1C11 cells, upon appropriate induction, engage into neuronal differentiation programs, giving rise either to serotonergic (1C11(5-HT)) or noradrenergic (1C11(NE)) derivatives. METHODOLOGY/PRINCIPAL FINDINGS: The neuronal specificity of PrP(C) signaling prompted us to search for PrP(C) partners in 1C11-derived bioaminergic neuronal cells. We show here by immunoprecipitation an association of PrP(C) with an 80 kDa protein identified by mass spectrometry as the tissue non-specific alkaline phosphatase (TNAP). This interaction occurs in lipid rafts and is restricted to 1C11-derived neuronal progenies. Our data indicate that TNAP is implemented during the differentiation programs of 1C11(5-HT) and 1C11(NE) cells and is active at their cell surface. Noteworthy, TNAP may contribute to the regulation of serotonin or catecholamine synthesis in 1C11(5-HT) and 1C11(NE) bioaminergic cells by controlling pyridoxal phosphate levels. Finally, TNAP activity is shown to modulate the phosphorylation status of laminin and thereby its interaction with PrP. CONCLUSION/SIGNIFICANCE: The identification of a novel PrP(C) partner in lipid rafts of neuronal cells favors the idea of a role of PrP in multiple functions. Because PrP(C) and laminin functionally interact to support neuronal differentiation and memory consolidation, our findings introduce TNAP as a functional protagonist in the PrP(C)-laminin interplay. The partnership between TNAP and PrP(C) in neuronal cells may provide new clues as to the neurospecificity of PrP(C) function.
Asunto(s)
Fosfatasa Alcalina/metabolismo , Aminas Biogénicas/metabolismo , Neuronas/metabolismo , Proteínas PrPC/metabolismo , Secuencia de Aminoácidos , Animales , Western Blotting , Catecolaminas/biosíntesis , Diferenciación Celular , Membrana Celular/metabolismo , Homeostasis , Espectrometría de Masas , Datos de Secuencia Molecular , Neuronas/citología , Fosforilación , Proteínas PrPC/química , Unión Proteica , Serotonina/biosíntesisRESUMEN
Transmissible spongiform encephalopathies, also called prion diseases, are characterized by neuronal loss linked to the accumulation of PrP(Sc), a pathologic variant of the cellular prion protein (PrP(C)). Although the molecular and cellular bases of PrP(Sc)-induced neuropathogenesis are not yet fully understood, increasing evidence supports the view that PrP(Sc) accumulation interferes with PrP(C) normal function(s) in neurons. In the present work, we exploit the properties of PrP-(106-126), a synthetic peptide encompassing residues 106-126 of PrP, to investigate into the mechanisms sustaining prion-associated neuronal damage. This peptide shares many physicochemical properties with PrP(Sc) and is neurotoxic in vitro and in vivo. We examined the impact of PrP-(106-126) exposure on 1C11 neuroepithelial cells, their neuronal progenies, and GT1-7 hypothalamic cells. This peptide triggers reactive oxygen species overflow, mitogen-activated protein kinase (ERK1/2), and SAPK (p38 and JNK1/2) sustained activation, and apoptotic signals in 1C11-derived serotonergic and noradrenergic neuronal cells, while having no effect on 1C11 precursor and GT1-7 cells. The neurotoxic action of PrP-(106-126) relies on cell surface expression of PrP(C), recruitment of a PrP(C)-Caveolin-Fyn signaling platform, and overstimulation of NADPH-oxidase activity. Altogether, these findings provide actual evidence that PrP-(106-126)-induced neuronal injury is caused by an amplification of PrP(C)-associated signaling responses, which notably promotes oxidative stress conditions. Distorsion of PrP(C) signaling in neuronal cells could hence represent a causal event in transmissible spongiform encephalopathy pathogenesis.