RESUMEN
Soluble oligomers arising from the aggregation of the amyloid beta peptide (Aß) have been identified as the main pathogenic agents in Alzheimer's disease (AD). Prefibrillar oligomers of the 42-residue form of Aß (Aß42 O) show membrane-binding capacity and trigger the disruption of Ca2+ homeostasis, a causative event in neuron degeneration. Since bioactive lipids have been recently proposed as potent protective agents against Aß toxicity, we investigated the involvement of sphingosine 1-phosphate (S1P) signalling pathway in Ca2+ homeostasis in living neurons exposed to Aß42 O. We show that both exogenous and endogenous S1P rescued neuronal Ca2+ dyshomeostasis induced by toxic Aß42 O in primary rat cortical neurons and human neuroblastoma SH-SY5Y cells. Further analysis revealed a strong neuroprotective effect of S1P1 and S1P4 receptors, and to a lower extent of S1P3 and S1P5 receptors, which activate the Gi -dependent signalling pathways, thus resulting in the endocytic internalization of the extrasynaptic GluN2B-containing N-methyl-D-aspartate receptors (NMDARs). Notably, the S1P beneficial effect can be sustained over time by sphingosine kinase-1 overexpression, thus counteracting the down-regulation of the S1P signalling induced by Aß42 O. Our findings disclose underlying mechanisms of S1P neuronal protection against harmful Aß42 O, suggesting that S1P and its signalling axis can be considered promising targets for therapeutic approaches for AD.
Asunto(s)
Enfermedad de Alzheimer , Neuroblastoma , Ratas , Humanos , Animales , Receptores de N-Metil-D-Aspartato/genética , Péptidos beta-Amiloides/metabolismo , Neuroblastoma/metabolismo , Neuronas/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismoRESUMEN
Trodusquemine is an aminosterol with a variety of biological and pharmacological functions, such as acting as an antimicrobial, stimulating body weight loss and interfering with the toxicity of proteins involved in the development of Alzheimer's and Parkinson's diseases. The mechanisms of interaction of aminosterols with cells are, however, still largely uncharacterized. Here, by using fluorescently labeled trodusquemine (TRO-A594 and TRO-ATTO565), we show that trodusquemine binds initially to the plasma membrane of living cells, that the binding affinity is dependent on cholesterol, and that trodusquemine is then internalized and mainly targeted to lysosomes after internalization. We also found that TRO-A594 is able to strongly and selectively bind to myelinated fibers in fixed mouse brain slices, and that it is a marker compatible with tissue clearing and light-sheet fluorescence microscopy or expansion microscopy. In conclusion, this work contributes to further characterize the biology of aminosterols and provides a new tool for nerve labeling suitable for the most advanced microscopy techniques.
Asunto(s)
Colestanos , Animales , Ratones , Colestanos/farmacología , Espermina/farmacología , Microscopía Fluorescente/métodos , ColesterolRESUMEN
Alzheimer's disease is characterized by the accumulation in the brain of the amyloid ß (Aß) peptide in the form of senile plaques. According to the amyloid hypothesis, the aggregation process of Aß also generates smaller soluble misfolded oligomers that contribute to disease progression. One of the mechanisms of Aß oligomer cytotoxicity is the aberrant interaction of these species with the phospholipid bilayer of cell membranes, with a consequent increase in cytosolic Ca2+ levels, flowing from the extracellular space, and production of reactive oxygen species (ROS). Here we investigated the relationship between the increase in Ca2+ and ROS levels immediately after the exposure to misfolded protein oligomers, asking whether they are simultaneous or instead one precedes the other. Using Aß42-derived diffusible ligands (ADDLs) and type A HypF-N model oligomers (OAs), we followed the kinetics of ROS production and Ca2+ influx in human neuroblastoma SH-SY5Y cells and rat primary cortical neurons in a variety of conditions. In all cases we found a faster increase of intracellular Ca2+ than ROS levels, and a lag phase in the latter process. A Ca2+-deprived cell medium prevented the increase of intracellular Ca2+ ions and abolished ROS production. By contrast, treatment with antioxidant agents prevented ROS formation, did not prevent the initial Ca2+ flux, but allowed the cells to react to the initial calcium dyshomeostasis, restoring later the normal levels of the ions. These results reveal a mechanism in which the entry of Ca2+ causes the production of ROS in cells challenged by aberrant protein oligomers.
Asunto(s)
Enfermedad de Alzheimer , Neuroblastoma , Péptidos beta-Amiloides , Animales , Humanos , Estrés Oxidativo , Ratas , Especies Reactivas de OxígenoRESUMEN
Alzheimer's disease, which is the most common form of dementia, is characterized by the aggregation of the amyloid ß peptide (Aß) and by an impairment of calcium homeostasis caused by excessive activation of glutamatergic receptors (excitotoxicity). Here, we studied the effects on calcium homeostasis caused by the formation of Aß oligomeric assemblies. We found that Aß oligomers cause a rapid influx of calcium ions (Ca2+) across the cell membrane by rapidly activating extrasynaptic N-methyl-d-aspartate (NMDA) receptors and, to a lower extent, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. We also observed, however, that misfolded oligomers do not interact directly with these receptors. Further experiments with lysophosphatidylcholine and arachidonic acid, which cause membrane compression and stretch, respectively, indicated that these receptors are activated through a change in membrane tension induced by the oligomers and transmitted mechanically to the receptors via the lipid bilayer. Indeed, lysophosphatidylcholine is able to neutralize the oligomer-induced activation of the NMDA receptors, whereas arachidonic acid activates the receptors similarly to the oligomers with no additive effects. An increased rotational freedom observed for a fluorescent probe embedded within the membrane in the presence of the oligomers also indicates a membrane stretch. These results reveal a mechanism of toxicity of Aß oligomers in Alzheimer's disease through the perturbation of the mechanical properties of lipid membranes sensed by NMDA and AMPA receptors.
Asunto(s)
Enfermedad de Alzheimer , Receptores de N-Metil-D-Aspartato , Péptidos beta-Amiloides/metabolismo , Calcio/metabolismo , Homeostasis , Humanos , Neuronas/metabolismo , Fragmentos de Péptidos/metabolismo , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol PropiónicoRESUMEN
Accumulation of ubiquitin-positive, tau- and α-synuclein-negative intracellular inclusions of TDP-43 in the central nervous system represents the major hallmark correlated to amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U). Such inclusions have variably been described as amorphous aggregates or more structured deposits having amyloid properties. Here we have purified full-length TDP-43 (FL TDP-43) and its C-terminal domain (Ct TDP-43) to investigate the morphological, structural and tinctorial features of aggregates formed in vitro by them at pH 7.4 and 37 °C. AFM images indicate that both protein variants show a tendency to form filaments. Moreover, we show that both FL TDP-43 and Ct TDP-43 filaments possess a largely disordered secondary structure, as ascertained by far-UV circular dichroism and Fourier transform infra-red spectroscopy, do not bind Congo red and induce a very weak increase of thioflavin T fluorescence, indicating the absence of a clear amyloid-like signature.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Encéfalo/metabolismo , Proteínas de Unión al ADN/genética , Demencia Frontotemporal/genética , Amiloide/genética , Amiloide/ultraestructura , Proteínas Amiloidogénicas/genética , Proteínas Amiloidogénicas/ultraestructura , Esclerosis Amiotrófica Lateral/patología , Encéfalo/patología , Encéfalo/ultraestructura , Proteínas de Unión al ADN/ultraestructura , Escherichia coli/genética , Demencia Frontotemporal/patología , Humanos , Cuerpos de Inclusión/genética , Cuerpos de Inclusión/patología , Cuerpos de Inclusión/ultraestructura , Agregación Patológica de Proteínas/genética , Agregación Patológica de Proteínas/patología , Conformación Proteica , Dominios Proteicos/genética , Estructura Secundaria de ProteínaRESUMEN
Alzheimer's disease (AD) is the most prevalent form of dementia and soluble amyloid ß (Aß) oligomers are thought to play a critical role in AD pathogenesis. Cellular prion protein (PrPC) is a high-affinity receptor for Aß oligomers and mediates some of their toxic effects. The N-terminal region of PrPC can interact with Aß, particularly the region encompassing residues 95-110. In this study, we identified a soluble and unstructured prion-derived peptide (PrP107-120) that is external to this region of the sequence and was found to successfully reduce the mitochondrial impairment, intracellular ROS generation and cytosolic Ca2+ uptake induced by oligomeric Aß42 ADDLs in neuroblastoma SH-SY5Y cells. PrP107-120 was also found to rescue SH-SY5Y cells from Aß42 ADDL internalization. The peptide did not change the structure and aggregation pathway of Aß42 ADDLs, did not show co-localization with Aß42 ADDLs in the cells and showed a partial colocalization with the endogenous cellular PrPC. As a sequence region that is not involved in Aß binding but in PrP self-recognition, the peptide was suggested to protect against the toxicity of Aß42 oligomers by interfering with cellular PrPC and/or activating a signaling that protected the cells. These results strongly suggest that PrP107-120 has therapeutic potential for AD.
Asunto(s)
Péptidos beta-Amiloides/antagonistas & inhibidores , Calcio/metabolismo , Neuronas/efectos de los fármacos , Fragmentos de Péptidos/antagonistas & inhibidores , Péptidos/farmacología , Proteínas PrPC/química , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/toxicidad , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Humanos , Transporte Iónico , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/patología , Modelos Biológicos , Neuronas/metabolismo , Neuronas/patología , Fragmentos de Péptidos/toxicidad , Péptidos/química , Proteínas PrPC/metabolismo , Unión Proteica , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Especies Reactivas de Oxígeno/metabolismo , SolubilidadRESUMEN
Frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) are progressive and fatal neurodegenerative disorders showing mislocalization and cytosolic accumulation of TDP-43 inclusions in the central nervous system. The decrease in the efficiency of the clearance systems in aging, as well as the presence of genetic mutations of proteins associated with cellular proteostasis in the familial forms of TDP-43 proteinopathies, suggest that a failure of these protein degradation systems is a key factor in the aetiology of TDP-43 associated disorders. Here we show that the internalization of human pre-formed TDP-43 aggregates in the murine neuroblastoma N2a cells promptly resulted in their ubiquitination and hyperphosphorylation by endogenous machineries, mimicking the post-translational modifications observed in patients. Moreover, our data identify mitochondria as the main responsible sites for the alteration of calcium homeostasis induced by TDP-43 aggregates, which, in turn, stimulates an increase in reactive oxygen species and, finally, caspase activation. The inhibition of TDP-43 proteostasis in the presence of selective inhibitors against the proteasome and macroautophagy systems revealed that these two systems are both severely involved in TDP-43 accumulation and have a strong influence on each other in neurodegenerative disorders associated with TDP-43.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Enfermedades Neurodegenerativas/etiología , Enfermedades Neurodegenerativas/metabolismo , Agregado de Proteínas , Agregación Patológica de Proteínas , Proteostasis , Animales , Autofagia , Calcio/metabolismo , Caspasa 3/metabolismo , Supervivencia Celular , Humanos , Mitocondrias/metabolismo , Enfermedades Neurodegenerativas/patología , Neuronas/metabolismo , Neuronas/patología , Fosforilación , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Proteolisis , Especies Reactivas de Oxígeno/metabolismo , UbiquitinaciónRESUMEN
The formation of misfolded protein oligomers during early stages of amyloid aggregation and the activation of neuroinflammatory responses are two key events associated with neurodegenerative diseases. Although it has been established that misfolded oligomers are involved in the neuroinflammatory process, the links between their structural features and their functional effects on the immune response remain unknown. To explore such links, we took advantage of two structurally distinct soluble oligomers (type A and B) of protein HypF-N and compared the elicited microglial inflammatory responses. By using confocal microscopy, protein pull-down, and high-throughput mass spectrometry, we found that, even though both types bound to a common pool of microglial proteins, type B oligomers-with a lower solvent-exposed hydrophobicity-showed enhanced protein binding, correlating with the observed inflammatory response. Furthermore, the interactome associated with inflammatory-mediated neurodegeneration revealed previously unidentified receptors and signaling molecules likely to be involved in the oligomer-elicited innate immune response.
Asunto(s)
Transferasas de Carboxilo y Carbamoilo/inmunología , Proteínas de Escherichia coli/inmunología , Inmunidad Innata/inmunología , Microglía/inmunología , Agregación Patológica de Proteínas/inmunología , Animales , Línea Celular , Cricetinae , Humanos , Ratones , Microglía/patología , Agregación Patológica de Proteínas/patología , Unión ProteicaRESUMEN
The involvement of transactivation response (TAR) DNA-binding protein 43 (TDP-43) in neurodegenerative diseases was revealed in 2006, when it was first reported to be the main component of the intracellular inclusions in patients with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration. After 12 yr it is not yet possible to purify to a reasonable yield and in a reproducible manner a stable full-length protein, which has limited so far the characterization of its structure, function, molecular interactors, and pathobiology. Using a novel protocol we have achieved the purification of the full-length TDP-43, with both a short pectate lyase B tag and a glutathione S-transferase tag, which consisted in its expression in bacteria, solubilization from inclusion bodies, purification under denaturing conditions, refolding, and a final size exclusion chromatography (SEC) step. Differential scanning fluorimetry was used to find the best buffers and combination of additives to increase both its solubility and its stability. The protein is pure, as determined with electrophoresis, Western blotting, and mass spectrometry; properly refolded, as revealed by circular dichroism and fluorescence spectroscopies; functional, because it binds to DNA and protein partners; and stable to degradation and aggregation in a physiologic solution. Analyses with dynamic light scattering and SEC revealed that the protein is a dimer.-Vivoli Vega, M., Nigro, A., Luti, S., Capitini, C., Fani, G., Gonnelli, L., Boscaro, F., Chiti, F. Isolation and characterization of soluble human full-length TDP-43 associated with neurodegeneration.
Asunto(s)
Proteínas de Unión al ADN/aislamiento & purificación , Enfermedades Neurodegenerativas/metabolismo , Secuencia de Aminoácidos , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Cromatografía en Gel , Dicroismo Circular , Clonación Molecular , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Dispersión Dinámica de Luz , Degeneración Lobar Frontotemporal/genética , Degeneración Lobar Frontotemporal/metabolismo , Humanos , Espectrometría de Masas , Enfermedades Neurodegenerativas/genética , Pliegue de Proteína , Estabilidad Proteica , Estructura Cuaternaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , SolubilidadRESUMEN
Amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitin-positive inclusions are neurodegenerative disorders that share the cytosolic deposition of TDP-43 (TAR DNA-binding protein 43) in the CNS. TDP-43 is well known as being actively degraded by both the proteasome and macroautophagy. The well-documented decrease in the efficiency of these clearance systems in aging and neurodegeneration, as well as the genetic evidence that many of the familial forms of TDP-43 proteinopathies involve genes that are associated with them, suggest that a failure of these protein degradation systems is a major factor that contributes to the onset of TDP-43-associated disorders. Here, we inserted preformed human TDP-43 aggregates in the cytosol of murine NSC34 and N2a cells in diffuse form and observed their degradation under conditions in which exogenous TDP-43 is not expressed and endogenous nuclear TDP-43 is not recruited, thereby allowing a time zero to be established in TDP-43 degradation and to observe its disposal kinetically and analytically. TDP-43 degradation was observed in the absence and presence of selective inhibitors and small interfering RNAs against the proteasome and autophagy. We found that cytosolic diffuse aggregates of TDP-43 can be distinguished in 3 different classes on the basis of their vulnerability to degradation, which contributed to the definition-with previous reports-of a total of 6 distinct classes of misfolded TDP-43 species that range from soluble monomer to undegradable macroaggregates. We also found that the proteasome and macroautophagy-degradable pools of TDP-43 are fully distinguishable, rather than in equilibrium between them on the time scale required for degradation, and that a significant crosstalk exists between the 2 degradation processes.-Cascella, R., Fani, G., Capitini, C., Rusmini, P., Poletti, A., Cecchi, C., Chiti, F. Quantitative assessment of the degradation of aggregated TDP-43 mediated by the ubiquitin proteasome system and macroautophagy.
Asunto(s)
Autofagia/fisiología , Proteínas de Unión al ADN/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina/metabolismo , Animales , Autofagia/genética , Línea Celular , Proteínas de Unión al ADN/genética , Humanos , Cuerpos de Inclusión/metabolismo , Ratones , Enfermedad de la Neurona Motora/genética , Enfermedad de la Neurona Motora/metabolismo , Agregación Patológica de Proteínas/genética , Agregación Patológica de Proteínas/metabolismo , Proteolisis , Interferencia de ARN , Ubiquitina/genéticaRESUMEN
Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin positive inclusions (FTLD-U) are two clinically distinct neurodegenerative conditions sharing a similar histopathology characterized by the nuclear clearance of TDP-43 and its associated deposition into cytoplasmic inclusions in different areas of the central nervous system. Given the concomitant occurrence of TDP-43 nuclear depletion and cytoplasmic accumulation, it has been proposed that TDP-43 proteinopathies originate from either a loss-of-function (LOF) mechanism, a gain-of-function (GOF) process, or both. We have addressed this issue by transfecting murine NSC34 and N2a cells with siRNA for endogenous murine TDP-43 and with human recombinant TDP-43 inclusion bodies (IBs). These two strategies allowed the depletion of nuclear TDP-43 and the accumulation of cytoplasmic TDP-43 aggregates to occur separately and independently. Endogenous and exogenous TDP-43 were monitored and quantified using both immunofluorescence and Western blotting analysis, and nuclear functional TDP-43 was measured by monitoring the sortilin 1 mRNA splicing activity. Various degrees of TDP-43 cytoplasmic accumulation and nuclear TDP-43 depletion were achieved and the resulting cellular viability was evaluated, leading to a quantitative global analysis on the relative effects of LOF and GOF on the overall cytotoxicity. These were found to be â¼55% and 45%, respectively, in both cell lines and using both readouts of cell toxicity, showing that these two mechanisms are likely to contribute apparently equally to the pathologies of ALS and FTLD-U.