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Exposure to plastic nanoparticles has dramatically increased in the last 50 years, and there is evidence that plastic nanoparticles can be absorbed by organisms and cross the blood-brain-barrier (BBB). However, their toxic effects, especially on the nervous system, have not yet been extensively investigated, and most of the knowledge is based on studies using different conditions and systems, thus hard to compare. In this work, physicochemical properties of non-modified polystyrene (PS) and amine-functionalized PS (PS-NH2 ) nanoparticles are initially characterized. Advantage of a multisystemic approach is then taken to compare plastic nanoparticles effects in vitro, through cytotoxic readouts in mammalian cell culture, and in vivo, through behavioral readouts in the nematode Caenorhabditis elegans (C. elegans), a powerful 3R-complying model organism for toxicology studies. In vitro experiments in neuroblastoma cells indicate a specific cytotoxic effect of PS-NH2 particles, including a decreased neuronal differentiation and an increased Amyloid ß (Aß) secretion, a sensitive readout correlating with Alzheimer's disease pathology. In parallel, only in vivo treatments with PS-NH2 particles affect C. elegans development, decrease lifespan, and reveal higher sensitivity of animals expressing human Aß compared to wild-type animals. In summary, the multisystemic approach discloses a neurotoxic effect induced by aminated polystyrene nanoparticles.
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Nanopartículas , Poliestirenos , Animales , Humanos , Poliestirenos/toxicidad , Péptidos beta-Amiloides/toxicidad , Caenorhabditis elegans , Microplásticos/farmacología , Nanopartículas/toxicidad , Nanopartículas/química , MamíferosRESUMEN
Alzheimer's disease is the most common age-associated neurodegenerative disorder and the most frequent form of dementia in our society. Aging is a complex biological process concurrently shaped by genetic, dietary and environmental factors and natural compounds are emerging for their beneficial effects against age-related disorders. Besides their antioxidant activity often described in simple model organisms, the molecular mechanisms underlying the beneficial effects of different dietary compounds remain however largely unknown. In the present study, we exploit the nematode Caenorhabditis elegans as a widely established model for aging studies, to test the effects of different natural compounds in vivo and focused on mechanistic aspects of one of them, quercetin, using complementary systems and assays. We show that quercetin has evolutionarily conserved beneficial effects against Alzheimer's disease (AD) pathology: it prevents Amyloid beta (Aß)-induced detrimental effects in different C. elegans AD models and it reduces Aß-secretion in mammalian cells. Mechanistically, we found that the beneficial effects of quercetin are mediated by autophagy-dependent reduced expression of Abl tyrosine kinase. In turn, autophagy is required upon Abl suppression to mediate quercetin's protective effects against Aß toxicity. Our data support the power of C. elegans as an in vivo model to investigate therapeutic options for AD.
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As with toxicology in general, major challenges have emerged in its subfield neurotoxicology regarding the testing of engineered nanomaterials (ENM). This is on the one hand due to their complex physicochemical properties, like size, specific surface area, chemical composition as well as agglomeration and dissolution behavior in biological environments. On the other hand, toxicological risk assessment has faced an increasing demand for the development and implementation of non-animal alternative approaches. Regarding the investigation and interpretation of the potential adverse effects of ENM on the brain, toxicokinetic data are relatively scarce and thus hampers dose selection for in vitro neurotoxicity testing. Moreover, recent in vivo studies indicate that ENM can induce neurotoxic and behavioral effects in an indirect manner, depending on their physicochemical properties and route of exposure. Such indirect effects on the brain may proceed through the activation and spill-over of inflammatory mediators by ENM in the respiratory tract and other peripheral organs as well via ENM induced disturbance of the gut microbiome and intestinal mucus barrier. These ENM specific aspects should be incorporated into the ongoing developments of advanced in vitro neurotoxicity testing methods and strategies.
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Microbioma Gastrointestinal , Nanoestructuras , Nanoestructuras/toxicidad , Medición de RiesgoRESUMEN
The NLRP3 inflammasome plays an important role in intestinal homeostasis as well as inflammation. However, in vivo studies investigating the role of the NLRP3 inflammasome in inflammatory bowel disease (IBD) report contrasting results, leaving it unclear if the NLRP3 inflammasome augments or attenuates intestinal inflammation. To investigate the role of the NLRP3/caspase-1 pathway in a model of acute intestinal inflammation, we modified a previously established in vitro triple culture model of the healthy and inflamed intestine (Caco-2/HT29-MTX-E12/THP-1). Using THP-1 knockout cell lines, we analyzed how the NLRP3 inflammasome and its downstream enzyme caspase-1 (CASP1) affect inflammatory parameters including barrier integrity and cytotoxicity, as well as gene expression and secretion of pro-inflammatory cytokines and mucus. Furthermore, we investigated differences in inflammation-mediated cytotoxicity towards enterocyte-like (Caco-2) or goblet-like (HT29-MTX-E12) epithelial cells. As a complementary approach, inflammation-related cytotoxicity and gene expression of cytokines was analyzed in intestinal tissue explants from wildtype (WT) and Nlrp3-/- mice. Induction of intestinal inflammation impaired the barrier, caused cytotoxicity, and altered gene expression of pro-inflammatory cytokines and mucins in vitro, while the knockout of NLRP3 and CASP1 in THP 1 cells led to attenuation of these inflammatory parameters. The knockout of CASP1 tended to show a slightly stronger attenuating effect compared to the NLRP3 knockout model. We also found that the inflammation-mediated death of goblet-like cells is NLRP3/caspase-1 dependent. Furthermore, inflammation-related cytotoxicity and upregulation of pro-inflammatory cytokines was present in ileal tissue explants from WT, but not Nlrp3-/- mice. The here presented observations indicate a pro-inflammatory and adverse role of the NLRP3 inflammasome in macrophages during acute intestinal inflammation.
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Inflamasomas , Proteína con Dominio Pirina 3 de la Familia NLR , Animales , Células CACO-2 , Caspasa 1/genética , Caspasa 1/metabolismo , Citocinas/metabolismo , Humanos , Inflamasomas/metabolismo , Inflamación/genética , Inflamación/metabolismo , Intestinos/patología , Ratones , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Células THP-1RESUMEN
BACKGROUND: There is an increasing concern about the neurotoxicity of engineered nanomaterials (NMs). To investigate the effects of subchronic oral exposures to SiO2 and CeO2 NMs on Alzheimer's disease (AD)-like pathology, 5xFAD transgenic mice and their C57BL/6J littermates were fed ad libitum for 3 or 14 weeks with control food pellets, or pellets dosed with these respective NMs at 0.1% or 1% (w/w). Behaviour effects were evaluated by X-maze, string suspension, balance beam and open field tests. Brains were analysed for plaque load, beta-amyloid peptide levels, markers of oxidative stress and neuroinflammation. RESULTS: No marked behavioural impairments were observed in the mice exposed to SiO2 or CeO2 and neither treatment resulted in accelerated plaque formation, increased oxidative stress or inflammation. In contrast, the 5xFAD mice exposed to 1% CeO2 for 14 weeks showed significantly lower hippocampal Aß plaque load and improved locomotor activity compared to the corresponding controls. CONCLUSIONS: The findings from the present study suggest that long-term oral exposure to SiO2 or CeO2 NMs has no neurotoxic and AD-promoting effects. The reduced plaque burden observed in the mice following dietary CeO2 exposure warrants further investigation to establish the underlying mechanism, given the easy applicability of this administration method.
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Enfermedad de Alzheimer , Nanoestructuras , Enfermedad de Alzheimer/inducido químicamente , Enfermedad de Alzheimer/patología , Animales , Encéfalo/patología , Exposición Dietética , Modelos Animales de Enfermedad , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Nanoestructuras/toxicidad , Placa Amiloide/inducido químicamente , Dióxido de Silicio/toxicidadRESUMEN
The increasing use of engineered nanomaterials (ENM) in food has fueled the development of intestinal in vitro models for toxicity testing. However, ENM effects on intestinal mucus have barely been addressed, although its crucial role for intestinal health is evident. We investigated the effects of ENM on mucin expression and aimed to evaluate the suitability of four in vitro models of increasing complexity compared to a mouse model exposed through feed pellets. We assessed the gene expression of the mucins MUC1, MUC2, MUC5AC, MUC13 and MUC20 and the chemokine interleukin-8 in pre-confluent and confluent HT29-MTX-E12 cells, in stable and inflamed triple cultures of Caco-2, HT29-MTX-E12 and THP-1 cells, and in the ileum of mice following exposure to TiO2, Ag, CeO2 or SiO2. All ENM had shared and specific effects. CeO2 downregulated MUC1 in confluent E12 cells and in mice. Ag induced downregulation of Muc2 in mice. Overall, the in vivo data were consistent with the findings in the stable triple cultures and the confluent HT29-MTX-E12 cells but not in pre-confluent cells, indicating the higher relevance of advanced models for hazard assessment. The effects on MUC1 and MUC2 suggest that specific ENM may lead to an elevated susceptibility towards intestinal infections and inflammations.
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Rodent studies on the effects of engineered nanomaterials (ENM) on the gut microbiome have revealed contradictory results. Our aim was to assess the effects of four well-investigated model ENM using a realistic exposure scenario. Two independent ad libitum feeding studies were performed. In study 1, female mice from the local breeding facility received feed pellets containing 1% CeO2 or 1% SiO2 for three weeks. In study 2, both female and male mice were purchased and exposed to 0.2% Ag-PVP or 1% TiO2 for four weeks. A next generation 16S rDNA sequencing-based approach was applied to assess impacts on the gut microbiome. None of the ENM had an effect on the α- or ß-diversity. A decreased relative abundance of the phylum Actinobacteria was observed in SiO2 exposed mice. In female mice, the relative abundance of the genus Roseburia was increased with Ag exposure. Furthermore, in study 2, a sex-related difference in the ß-diversity was observed. A difference in the ß-diversity was also shown between the female control mice of the two studies. We did not find major effects on the gut microbiome. This contrast to other studies may be due to variations in the study design. Our investigation underlined the important role of the sex of test animals and their microbiome composition prior to ENM exposure initiation. Hence, standardization of microbiome studies is strongly required to increase comparability. The ENM-specific effects on Actinobacteria and Roseburia, two taxa pivotal for the human gut homeostasis, warrant further research on their relevance for health.
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Microbioma Gastrointestinal , Nanoestructuras , Animales , Exposición Dietética , Femenino , Masculino , Ratones , Dióxido de Silicio/toxicidad , TitanioRESUMEN
In recent years, concerns have emerged about the potential neurotoxic effects of engineered nanomaterials (NMs). Titanium dioxide and silver are among the most widely used types of metallic NMs. We have investigated the effects of these NMs on behaviour and neuropathology in male and female C57BL/6J mice following 28-day oral exposure with or without a 14-day post-exposure recovery. The mice were fed ad libitum with food pellets dosed with 10 mg/g TiO2, 2 mg/g polyvinylpyrrolidone-coated Ag or control pellets. Behaviour was evaluated by X-maze, open field, string suspension and rotarod tests. Histological alterations were analysed by immunohistochemistry and brain tissue homogenates were investigated for markers of oxidative stress, inflammation and blood-brain barrier disruption. Effects of the NMs on tyrosine and serine/threonine protein kinase activity in mouse brains were investigated by measuring kinase activity on peptide microarrays. Markers of inflammation, oxidative stress and blood-brain barrier integrity were not significantly affected in the male and female mice following exposure to Ag or TiO2. Both types of NMs also revealed no consistent significant treatment-related effects on anxiety and cognition. However, in the Ag NM exposed mice altered motor performance effects were observed by the rotarod test that differed between sexes. At 1-week post-exposure, a diminished performance in this test was observed exclusively in the female animals. Cortex tissues of female mice also showed a pronounced increase in tyrosine kinase activity following 28 days oral exposure to Ag NM. A subsequent Inductively Coupled Plasma - Mass Spectrometry (ICP-MS) based toxicokinetic study in female mice revealed a rapid and persistent accumulation of Ag in various internal organs including liver, kidney, spleen and the brain up to 4 weeks post-exposure. In conclusion, our study demonstrated that subacute exposure to foodborne TiO2 and Ag NMs does not cause substantial neuropathological changes in mice. However, the toxicokinetic and specific toxicodynamic findings indicate that long-term exposures to Ag NM can cause neurotoxicity, possibly in a sex-dependent manner.
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Encéfalo/efectos de los fármacos , Ingeniería Química/métodos , Nanopartículas del Metal/química , Nanopartículas del Metal/toxicidad , Nanoestructuras/química , Nanoestructuras/toxicidad , Animales , Encéfalo/metabolismo , Evaluación Preclínica de Medicamentos/métodos , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Plata/química , Plata/metabolismo , Plata/toxicidad , Titanio/química , Titanio/metabolismo , Titanio/toxicidadRESUMEN
Increasing evidence from toxicological and epidemiological studies indicates that the brain is an important target for ambient (ultrafine) particles. Disturbance of redox-homeostasis and inflammation in the brain are proposed as possible mechanisms that can contribute to neurotoxic and neurodegenerative effects. Whether and how engineered nanoparticles (NPs) may cause neurotoxicity and promote neurodegenerative diseases such as Alzheimer's disease (AD) is largely unstudied. We have assessed the neurological effects of subacute inhalation exposures (4 mg/m3 for 3 h/day, 5 days/week for 4 weeks) to cerium dioxide (CeO2) NPs doped with different amounts of zirconium (Zr, 0%, 27% and 78%), to address the influence of particle redox-activity in the 5xFAD transgenic mouse model of AD. Four weeks post-exposure, effects on behaviour were evaluated and brain tissues were analysed for amyloid-ß plaque formation and reactive microglia (Iba-1 staining). Behaviour was also evaluated in concurrently exposed non-transgenic C57BL/6J littermates, as well as in Western diet-fed apolipoprotein E-deficient (ApoE-/-) mice as a model of vascular disease. Markers of inflammation and oxidative stress were evaluated in brain cortex. The brains of the NP-exposed 5xFAD mice revealed no accelerated amyloid-ß plaque formation. No significant treatment-related behaviour impairments were observed in the healthy C57BL/6J mice. In the 5xFAD and ApoE-/- models, the NP inhalation exposures did not affect the alternation score in the X-maze indicating absence of spatial working memory deficits. However, following inhalation exposure to the 78% Zr-doped CeO2 NPs changes in forced motor performance (string suspension) and exploratory motor activity (X-maze) were observed in ApoE-/- and 5xFAD mice, respectively. Exposure to the 78% doped NPs also caused increased cortical expression of glial fibrillary acidic protein (GFAP) in the C57BL/6J mice. No significant treatment-related changes neuroinflammation and oxidative stress were observed in the 5xFAD and ApoE-/- mice. Our study findings reveal that subacute inhalation exposure to CeO2 NPs does not accelerate the AD-like phenotype of the 5xFAD model. Further investigation is warranted to unravel whether the redox-activity dependent effects on motor activity as observed in the mouse models of AD and vascular disease result from specific neurotoxic effects of these NPs.
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Enfermedad de Alzheimer/patología , Cerio/administración & dosificación , Exposición por Inhalación , Actividad Motora/efectos de los fármacos , Nanopartículas/administración & dosificación , Enfermedades Vasculares/patología , Circonio/administración & dosificación , Enfermedad de Alzheimer/inducido químicamente , Enfermedad de Alzheimer/psicología , Animales , Cerio/efectos adversos , Modelos Animales de Enfermedad , Femenino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Actividad Motora/fisiología , Nanopartículas/efectos adversos , Enfermedades Vasculares/inducido químicamente , Enfermedades Vasculares/psicología , Circonio/efectos adversosRESUMEN
Altered processing of the Amyloid Precursor Protein (APP) is a well-recognized central pathogenic mechanism in Alzheimer's Disease (AD), and regulation of APP processing is a major focus of research in the AD field. However, how age-associated cellular and molecular changes contribute to changes in the amyloidogenic processing of APP have not been extensively clarified so far. We here provide evidence that the processing of APP is influenced by the e3 ubiquitin ligase Mahogunin (MGRN1), a neuroprotective molecule whose levels decrease with aging. Specifically, the expression of MGRN1 inhibits the maturation of APP by sequestering it in the secretory pathway. This sequestration significantly delayed the proteolytic processing of APP, resulting in a reduced ß-amyloid (Aß) peptide release into the extracellular environment. Accordingly, a reduction of MGRN1 levels in hippocampal neurons, as it occurs during physiological aging, leads to an increased Aß40 and Aß42 release. We therefore propose that age contributes to the amyloidogenic processing of APP by altering its intracellular trafficking along the secretory pathway due in part to the down-regulation of MGRN1.
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BACKGROUND: Increasing evidence from toxicological and epidemiological studies indicates that the central nervous system is an important target for ambient air pollutants. We have investigated whether long-term inhalation exposure to diesel engine exhaust (DEE), a dominant contributor to particulate air pollution in urban environments, can aggravate Alzheimer's Disease (AD)-like effects in female 5X Familial AD (5XFAD) mice and their wild-type female littermates. Following 3 and 13 weeks exposures to diluted DEE (0.95 mg/m3, 6 h/day, 5 days/week) or clean air (controls) behaviour tests were performed and amyloid-ß (Aß) plaque formation, pulmonary histopathology and systemic inflammation were evaluated. RESULTS: In a string suspension task, assessing for grip strength and motor coordination, 13 weeks exposed 5XFAD mice performed significantly less than the 5XFAD controls. Spatial working memory deficits, assessed by Y-maze and X-maze tasks, were not observed in association with the DEE exposures. Brains of the 3 weeks DEE-exposed 5XFAD mice showed significantly higher cortical Aß plaque load and higher whole brain homogenate Aß42 levels than the clean air-exposed 5XFAD littermate controls. After the 13 weeks exposures, with increasing age and progression of the AD-phenotype of the 5XFAD mice, DEE-related differences in amyloid pathology were no longer present. Immunohistochemical evaluation of lungs of the mice revealed no obvious genetic background-related differences in tissue structure, and the DEE exposure did not cause histopathological changes in the mice of both backgrounds. Luminex analysis of plasma cytokines demonstrated absence of sustained systemic inflammation upon DEE exposure. CONCLUSIONS: Inhalation exposure to DEE causes accelerated plaque formation and motor function impairment in 5XFAD transgenic mice. Our study provides further support that the brain is a relevant target for the effects of inhaled DEE and suggests that long-term exposure to this ubiquitous air pollution mixture may promote the development of Alzheimer's disease.
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Contaminantes Atmosféricos/toxicidad , Enfermedad de Alzheimer/patología , Exposición por Inhalación/efectos adversos , Material Particulado/toxicidad , Placa Amiloide/patología , Emisiones de Vehículos/toxicidad , Animales , Conducta Animal/efectos de los fármacos , Modelos Animales de Enfermedad , Femenino , Exposición por Inhalación/análisis , Memoria a Corto Plazo/efectos de los fármacos , Ratones EndogámicosRESUMEN
The world's population is steadily ageing and as a result, health conditions related to ageing, such as dementia, have become a major public health concern. In 2001, it was estimated that there were almost 5 million Europeans suffering from Alzheimer's disease (AD) and this figure has been projected to almost double by 2040. About 40% of people over 85 suffer from AD, and another 10% from Parkinson's disease (PD). The majority of AD and PD cases are of sporadic origin and environmental factors play an important role in the aetiology. Epidemiological research identified airborne particulate matter (PM) as one of the environmental factors potentially involved in AD and PD pathogenesis. Also, cumulating evidence demonstrates that the smallest sizes of the inhalable fraction of ambient particulate matter, also referred to as ultrafine particulate matter or nano-sized particles, are capable of inducing effects beyond the respiratory system. Translocation of very small particles via the olfactory epithelium in the nose or via uptake into the circulation has been demonstrated through experimental rodent studies with engineered nanoparticles. Outdoor air pollution has been linked to several health effects including oxidative stress and neuroinflammation that may ultimately result in neurodegeneration and cognitive impairment. This review aims to evaluate the relationship between exposure to inhaled ambient particles and neurodegeneration.
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Exposición por Inhalación , Enfermedades del Sistema Nervioso/inducido químicamente , Enfermedades Neurodegenerativas/inducido químicamente , Material Particulado/efectos adversos , Animales , Humanos , Exposición por Inhalación/estadística & datos numéricos , Enfermedades del Sistema Nervioso/epidemiología , Enfermedades Neurodegenerativas/epidemiologíaRESUMEN
Dyshomeostasis of amyloid-ß peptide (Aß) is responsible for synaptic malfunctions leading to cognitive deficits ranging from mild impairment to full-blown dementia in Alzheimer's disease. Aß appears to skew synaptic plasticity events toward depression. We found that inhibition of PTEN, a lipid phosphatase that is essential to long-term depression, rescued normal synaptic function and cognition in cellular and animal models of Alzheimer's disease. Conversely, transgenic mice that overexpressed PTEN displayed synaptic depression that mimicked and occluded Aß-induced depression. Mechanistically, Aß triggers a PDZ-dependent recruitment of PTEN into the postsynaptic compartment. Using a PTEN knock-in mouse lacking the PDZ motif, and a cell-permeable interfering peptide, we found that this mechanism is crucial for Aß-induced synaptic toxicity and cognitive dysfunction. Our results provide fundamental information on the molecular mechanisms of Aß-induced synaptic malfunction and may offer new mechanism-based therapeutic targets to counteract downstream Aß signaling.
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Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/fisiopatología , Trastornos del Conocimiento/fisiopatología , Fosfohidrolasa PTEN/fisiología , Transmisión Sináptica/fisiología , Enfermedad de Alzheimer/complicaciones , Péptidos beta-Amiloides/toxicidad , Animales , Trastornos del Conocimiento/complicaciones , Modelos Animales de Enfermedad , Técnicas de Sustitución del Gen , Ratones , Ratones Transgénicos , Dominios PDZ/genética , Dominios PDZ/fisiología , Fosfohidrolasa PTEN/antagonistas & inhibidores , Fosfohidrolasa PTEN/genética , Cultivo Primario de Células , Ratas , Transmisión Sináptica/efectos de los fármacosRESUMEN
The Fragile X mental retardation protein (FMRP) regulates neuronal RNA metabolism, and its absence or mutations leads to the Fragile X syndrome (FXS). The ß-amyloid precursor protein (APP) is involved in Alzheimer's disease, plays a role in synapse formation, and is upregulated in intellectual disabilities. Here, we show that during mouse synaptogenesis and in human FXS fibroblasts, a dual dysregulation of APP and the α-secretase ADAM10 leads to the production of an excess of soluble APPα (sAPPα). In FXS, sAPPα signals through the metabotropic receptor that, activating the MAP kinase pathway, leads to synaptic and behavioral deficits. Modulation of ADAM10 activity in FXS reduces sAPPα levels, restoring translational control, synaptic morphology, and behavioral plasticity. Thus, proper control of ADAM10-mediated APP processing during a specific developmental postnatal stage is crucial for healthy spine formation and function(s). Downregulation of ADAM10 activity at synapses may be an effective strategy for ameliorating FXS phenotypes.
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Proteínas ADAM/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Síndrome del Cromosoma X Frágil/patología , Regulación de la Expresión Génica/genética , Proteínas de la Membrana/metabolismo , Sinapsis/patología , Proteínas ADAM/genética , Proteína ADAM10 , Adolescente , Adulto , Factores de Edad , Secretasas de la Proteína Precursora del Amiloide/genética , Animales , Animales Recién Nacidos , Células Cultivadas , Corteza Cerebral/citología , Niño , Femenino , Síndrome del Cromosoma X Frágil/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/ultraestructura , Factores de Tiempo , Adulto JovenRESUMEN
Inherited familial Alzheimer's disease (AD) is characterized by small increases in the ratio of Aß42 versus Aß40 peptide which is thought to drive the amyloid plaque formation in the brain of these patients. Little is known however whether ageing, the major risk factor for sporadic AD, affects amyloid beta-peptide (Aß) generation as well. Here we demonstrate that the secretion of Aß is enhanced in an in vitro model of neuronal ageing, correlating with an increase in γ-secretase complex formation. Moreover we found that peroxynitrite (ONOO(-)), produced by the reaction of superoxide anion with nitric oxide, promoted the nitrotyrosination of presenilin 1 (PS1), the catalytic subunit of γ-secretase. This was associated with an increased association of the two PS1 fragments, PS1-CTF and PS1-NTF, which constitute the active catalytic centre. Furthermore, we found that peroxynitrite shifted the production of Aß towards Aß(42) and increased the Aß(42) /Aß(40) ratio. Our work identifies nitrosative stress as a potential mechanistic link between ageing and AD.
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Enfermedad de Alzheimer/enzimología , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Senescencia Celular/efectos de los fármacos , Neuronas/metabolismo , Ácido Peroxinitroso/farmacología , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Animales , Encéfalo/metabolismo , Dominio Catalítico , Células Cultivadas , Humanos , Ratones , Neuronas/citología , Fragmentos de Péptidos/metabolismo , Presenilina-1/química , Presenilina-1/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Factores de Riesgo , Superóxido Dismutasa/antagonistas & inhibidores , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismoRESUMEN
Several studies suggest that the generation of Aß is highly dependent on the levels of cholesterol within membranes' detergent-resistant microdomains (DRM). Indeed, the ß-amyloid precursor protein (APP) cleaving machinery, namely ß- and γ-secretases, has been shown to be present in DRM and its activity depends on membrane cholesterol levels. Counterintuitive to the localization of the cleavage machinery, the substrate, APP, localizes to membranes' detergent-soluble microdomains enriched in phospholipids (PL), indicating that Aß generation is highly dependent on the capacity of enzyme and substrate to diffuse along the lateral plane of the membrane and therefore on the internal equilibrium of the different lipids of DRM and non-DRM domains. Here, we studied to which extent changes in the content of a main non-DRM lipid might affect the proteolytic processing of APP. As phosphatidylethanolamine (PE) accounts for the majority of PL, we focused on its impact on the regulation of APP proteolysis. In mammalian cells, siRNA-mediated knock-down of PE synthesis resulted in decreased Aß owing to a dual effect: promoted α-secretase cleavage and decreased γ-secretase processing of APP. In vivo, in Drosophila melanogaster, genetic reduction in PL synthesis results in decreased γ-secretase-dependent cleavage of APP. These results suggest that modulation of the membrane-soluble domains could be a valuable alternative to reduce excessive Aß generation.
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Enfermedad de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/biosíntesis , Drosophila melanogaster/metabolismo , Neuronas/metabolismo , Fosfatidiletanolaminas/metabolismo , Enfermedad de Alzheimer/genética , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Membrana Celular/genética , Membrana Celular/metabolismo , Colesterol/metabolismo , Detergentes/farmacología , Modelos Animales de Enfermedad , Drosophila melanogaster/genética , Femenino , Células HEK293 , Hipocampo/citología , Hipocampo/metabolismo , Humanos , Masculino , Microdominios de Membrana/química , Microdominios de Membrana/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Neuronas/citología , Fosfatidiletanolaminas/genética , Proteolisis , ARN Interferente Pequeño/genética , RatasRESUMEN
Clinical, pharmacological, biochemical, and genetic evidence support the notion that alteration of cholesterol homeostasis strongly predisposes to Alzheimer disease (AD). The ATP-binding cassette transporter-2 (Abca2), which plays a role in intracellular sterol trafficking, has been genetically linked to AD. It is unclear how these two processes are related. Here we demonstrate that down-regulation of Abca2 in mammalian cells leads to decreased amyloid-ß (Aß) generation. In vitro studies revealed altered γ-secretase complex formation in Abca2 knock-out cells due to the altered levels, post-translational modification, and subcellular localization of Nicastrin. Reduced Abca2 levels in mammalian cells in vitro, in Drosophila melanogaster and in mice resulted in altered γ-secretase processing of APP, and thus Aß generation, without affecting Notch cleavage.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/biosíntesis , Transportadoras de Casetes de Unión a ATP/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Proteínas de Drosophila/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Secretasas de la Proteína Precursora del Amiloide/genética , Precursor de Proteína beta-Amiloide/genética , Animales , Regulación hacia Abajo/genética , Proteínas de Drosophila/genética , Drosophila melanogaster , Células HEK293 , Humanos , Glicoproteínas de Membrana/genética , Ratones , Proteínas del Tejido Nervioso/genética , RatasRESUMEN
Amyloid-ß (Aß) oligomer toxicity is a crucial factor in the development of Alzheimer's disease. Therefore, the aim of therapeutic research is to target the modification of secretase activity, increase Aß degradation, reduce Aß formation, and modulate Aß-induced neuroinflammation. Recently, the p38 MAP kinase inhibitor CNI-1493 has been shown to reduce plaque load and has led to an improvement in memory performance in a transgenic mouse model. We examined the role of CNI-1493 in the microglial inflammatory response to Aß using both a microglia cell line as well as primary microglia isolated from mesocortices. MTT assays were performed to quantify cell viability. FACS analysis was used to measure phagocytosis. We used ELISA to analyse cytokine concentrations in response to CNI-1493 treatment. Western-blot/Dot-blot techniques were used to show the interaction of CNI-1493 with Aß-oligomers as well as to measure apoptosis in microglia cells. RT-PCR was used to analyze secretase expression, and secretase function was determined using fluorimetric assays. CNI-1493 is able to prevent oligomer formation as well as apoptosis in microglia. A significant reduction was found in the Aß-induced release of IL-6 and TNF-α in the presence of CNI-1493. Phagocytosis is an essential Aß removal mechanism and was enhanced by CNI-1493 in primary microglia. CNI-1493 also influenced the α-secretase product C83 with an increase in the treated cells, while a simultaneous reduction in Aß secretion was also observed. We hypothesize that CNI-1493 not only reduces neuroinflammation and consequent neurodegeneration, but also leads to a shift in AßPP-processing towards the non-amyloidogenic pathway. Therefore, CNI-1493 is a promising candidate for the treatment of AD.
Asunto(s)
Péptidos beta-Amiloides/toxicidad , Hidrazonas/farmacología , Inmunosupresores/farmacología , Microglía/efectos de los fármacos , Fragmentos de Péptidos/toxicidad , Animales , Apoptosis/efectos de los fármacos , Caspasa 12/metabolismo , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Embrión de Mamíferos , Ratones , Fagocitosis/efectos de los fármacos , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismoRESUMEN
The beta-amyloid peptide (Abeta) is a major component of Alzheimer disease (AD)-associated senile plaques and is generated by sequential cleavage of the beta-amyloid precursor protein (APP) by beta-secretase (BACE1) and gamma-secretase. BACE1 cleaves APP at the N terminus of the Abeta domain, generating a membrane-bound C-terminal fragment (CTF-beta) that can be subsequently cleaved by gamma-secretase within the transmembrane domain to release Abeta. Because BACE1 initiates Abeta generation, it represents a potential target molecule to interfere with Abeta production in therapeutic strategies for AD. BACE1 interacts with Golgi-localized, gamma-ear-containing, ADP ribosylation factor-binding (GGA) proteins that are involved in the subcellular trafficking of BACE1. Here, we show that GGA1 is preferentially expressed in neurons of the human brain. GGA1 was also detected in activated microglia surrounding amyloid plaques in AD brains. Functional analyses with cultured cells demonstrate that GGA1 is implicated in the proteolytic processing of APP. Overexpression of GGA1 or a dominant-negative variant reduced cleavage of APP by BACE1 as indicated by a decrease in CTF-beta generation. Importantly, overexpression of GGA1 reduced, whereas RNAi-mediated suppression of GGA1 increased the secretion of Abeta. The modulation of APP processing by GGA1 is independent of a direct interaction of both proteins. Because total cellular activity of BACE1 was not affected by GGA1 expression, our data indicate that changes in the subcellular trafficking of BACE1 or other GGA1-dependent proteins contribute to changes in APP processing and Abeta generation. Thus, GGA proteins might be involved in the pathogenesis of AD.
Asunto(s)
Factores de Ribosilacion-ADP/fisiología , Proteínas Adaptadoras del Transporte Vesicular/fisiología , Péptidos beta-Amiloides/biosíntesis , Encéfalo/metabolismo , Regulación de la Expresión Génica/fisiología , Factores de Ribosilacion-ADP/biosíntesis , Factores de Ribosilacion-ADP/genética , Proteínas Adaptadoras del Transporte Vesicular/biosíntesis , Proteínas Adaptadoras del Transporte Vesicular/genética , Anciano , Anciano de 80 o más Años , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/metabolismo , Encéfalo/patología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Procesamiento Proteico-Postraduccional/fisiologíaRESUMEN
The beta-amyloid peptide (Abeta) is a major component of the Alzheimer's disease (AD)-associated senile plaques and is generated by sequential cleavage of the beta-amyloid precursor protein (APP) by beta-secretase and gamma-secretase. Since BACE1 initiates Abeta generation it represents a valuable target to interfere with Abeta production and treatment of AD. While the enzymatic activity of BACE1 resides in the extracellular domain, the protein also contains a short cytoplasmic tail (B1-CT). This domain serves as a binding site for at least two proteins, the copper chaperone for superoxide dismutase-1 (CCS), and the Golgi-localized, gamma-ear-containing, ADP ribosylation factor-binding (GGA1) protein, and contains a single phosphorylation site. However, the precise role of the B1-CT for the overall biological function of this protein is largely unknown. Functional studies focusing on the activity of this domain would strongly benefit from the availability of domain-specific inhibitors. Here we describe the isolation and characterization of RNA aptamers that selectively target the B1-CT. We show that these RNAs bind to authentic BACE1 and provide evidence that the binding site is restricted to the membrane-proximal half of the C terminus. Aptamer-binding specifically interferes with the recruitment of CCS, but still permits GGA1 association and casein kinase-dependent phosphorylation, consistent with selective binding site targeting within this short peptide. Because phosphorylation and GGA1 binding to B1-CT regulate BACE1 transport, these RNA inhibitors could be applied to investigate B1-CT activity without affecting the subcellular localization of BACE1.