RESUMEN
Abnormal accumulation of misfolded and hyperphosphorylated tau protein in brain is the defining feature of several neurodegenerative diseases called tauopathies, including Alzheimer's disease (AD). In AD, this pathological change is reflected by highly specific cerebrospinal fluid (CSF) tau biomarkers, including both phosphorylated and non-phosphorylated variants. Interestingly, despite tau pathology being at the core of all tauopathies, CSF tau biomarkers remain unchanged in certain tauopathies, e.g., progressive supranuclear palsy (PSP), Pick's disease (PiD), and corticobasal neurodegeneration (CBD). To better understand commonalities and differences between tauopathies, we report a multiplex assay combining immunoprecipitation and high-resolution mass spectrometry capable of detecting and quantifying peptides from different tau protein isoforms as well as non-phosphorylated and phosphorylated peptides, including those carrying multiple phosphorylations. We investigated the tau proteoforms in soluble and insoluble fractions of brain tissue from subjects with autopsy-confirmed tauopathies, including sporadic AD (n = 10), PSP (n = 11), PiD (n = 10), and CBD (n = 10), and controls (n = 10). Our results demonstrate that non-phosphorylated tau profiles differ across tauopathies, generally showing high abundance of microtubule-binding region (MTBR)-containing peptides in insoluble protein fractions compared with controls; the AD group showed 12-72 times higher levels of MTBR-containing aggregates. Quantification of tau isoforms showed the 3R being more abundant in PiD and the 4R isoform being more abundant in CBD and PSP in the insoluble fraction. Twenty-three different phosphorylated peptides were quantified. Most phosphorylated peptides were measurable in all investigated tauopathies. All phosphorylated peptides were significantly increased in AD insoluble fraction. However, doubly and triply phosphorylated peptides were significantly increased in AD even in the soluble fraction. Results were replicated using a validation cohort comprising AD (n = 10), CBD (n = 10), and controls (n = 10). Our study demonstrates that abnormal levels of phosphorylation and aggregation do indeed occur in non-AD tauopathies, however, both appear pronouncedly increased in AD, becoming a distinctive characteristic of AD pathology.
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Encéfalo , Tauopatías , Proteínas tau , Humanos , Proteínas tau/metabolismo , Tauopatías/metabolismo , Anciano , Encéfalo/metabolismo , Encéfalo/patología , Masculino , Femenino , Persona de Mediana Edad , Fosforilación , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Anciano de 80 o más Años , Parálisis Supranuclear Progresiva/metabolismo , Isoformas de Proteínas/metabolismoRESUMEN
Defective DNA damage signalling and repair is a hallmark of age-related and genetic neurodegenerative disease. One mechanism implicated in disease progression is DNA damage-driven neuroinflammation, which is largely mediated by tissue-resident immune cells, microglia. Here, we utilise human microglia-like cell models of persistent DNA damage and ATM kinase deficiency to investigate how genome instability shapes microglial function. We demonstrate that upon DNA damage the cytosolic DNA sensing cGAS-STING axis drives chronic inflammation and a robust chemokine response, exemplified by production of CCL5 and CXCL10. Transcriptomic analyses revealed that cell migratory pathways were highly enriched upon IFN-ß treatment of human iPSC-derived microglia, indicating that the chemokine response to DNA damage mirrors type I interferon signalling. Furthermore, we find that STING deletion leads to a defect in microglial chemotaxis under basal conditions and upon ATM kinase loss. Overall, this work provides mechanistic insights into cGAS-STING-dependent neuroinflammatory mechanisms and consequences of genome instability in the central nervous system.
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Microglía , Enfermedades Neurodegenerativas , Transducción de Señal , Humanos , Quimiocinas , Quimiotaxis/genética , Microglía/metabolismo , Enfermedades Neurodegenerativas/genética , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismoRESUMEN
INTRODUCTION: Recombinant adeno-associated viruses(rAAVs) are an attractive tool to ensure long-term expression monoclonal antibody(mAb) in the central nervous system(CNS). It is still unclear whether systemic injection or local CNS administration of AAV9 is more beneficial for the exposure of the expressed mAb in the brain. Hence, we compared the biodistribution and transgene expression following AAV9-Trastuzumab administration through different routes. METHODS AND RESULT: In-house generated AAV9-Trastuzumab vectors were administered at 5E+11 Vgs/rat through intravenous(IV), intracerebroventricular(ICV), intra-cisterna magna(ICM) and intrastriatal(IST) routes. Vector and trastuzumab blood/plasma concentrations were assessed at different time points up to the terminal time point of 21 days. Different brain regions in addition to the spinal cord, cerebrospinal fluid(CSF) and interstitial fluid(ISF), were also analyzed at the terminal time point. Our results show that vector biodistribution and Trastuzumab expression in the brain could the ranked as follows: IST>ICM>ICV>IV. Rapid clearance of vector was observed after administration via the ICM and ICV routes. The ICV route produced similar expression levels across different brain regions, while the ICM route had better expression in the hindbrain and spinal cord region. The IST route had higher expression in the forebrain region compared to the hindbrain region. A sharp decline in trastuzumab plasma concentration was observed across all routes of administration due to anti-trastuzumab antibody response. CONCLUSION: In this study we have characterized vector biodistribution and transgene mAb expression after AAV9 vector administration through different routes in rats. IST and ICM represent the best administration routes to deliver antibody genes to the brain.
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Encéfalo , Terapia Genética , Ratas , Animales , Transducción Genética , Terapia Genética/métodos , Distribución Tisular , Trastuzumab , Encéfalo/metabolismo , Vectores GenéticosRESUMEN
The purinoceptor P2X7R is a promising therapeutic target for tauopathies, including Alzheimer's disease (AD). Pharmacological inhibition or genetic knockdown of P2X7R ameliorates cognitive deficits and reduces pathological tau burden in mice that model aspects of tauopathy, including mice expressing mutant human frontotemporal dementia (FTD)-causing forms of tau. However, disagreements remain over which glial cell types express P2X7R and therefore the mechanism of action is unresolved. Here, we show that P2X7R protein levels increase in human AD post-mortem brain, in agreement with an upregulation of P2RX7 mRNA observed in transcriptome profiles from the AMP-AD consortium. P2X7R protein increases mirror advancing Braak stage and coincide with synapse loss. Using RNAScope we detect P2RX7 mRNA in microglia and astrocytes in human AD brain, including in the vicinity of senile plaques. In cultured microglia, P2X7R activation modulates the NLRP3 inflammasome pathway by promoting the formation of active complexes and release of IL-1ß. In astrocytes, P2X7R activates NFκB signalling and increases production of the cytokines CCL2, CXCL1 and IL-6 together with the acute phase protein Lcn2. To further explore the role of P2X7R in a disease-relevant context, we expressed wild-type or FTD-causing mutant forms of tau in mouse organotypic brain slice cultures. Inhibition of P2X7R reduces insoluble tau levels without altering soluble tau phosphorylation or synaptic localisation, suggesting a non-cell autonomous role of glial P2X7R on pathological tau aggregation. These findings support further investigations into the cell-type specific effects of P2X7R-targeting therapies in tauopathies.
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Enfermedad de Alzheimer , Demencia Frontotemporal , Tauopatías , Animales , Humanos , Ratones , Enfermedad de Alzheimer/metabolismo , Astrocitos/metabolismo , Encéfalo/metabolismo , Demencia Frontotemporal/genética , Demencia Frontotemporal/metabolismo , Demencia Frontotemporal/patología , Microglía/metabolismo , ARN Mensajero/metabolismo , Proteínas tau/genética , Proteínas tau/metabolismo , Tauopatías/metabolismoRESUMEN
Amyloid ß protein (Aß) and tau, the two main proteins implicated in causing Alzheimer's disease (AD), are posited to trigger synaptic dysfunction long before significant synaptic loss occurs in vulnerable circuits. Whereas soluble Aß aggregates from AD brain are well recognized potent synaptotoxins, less is known about the synaptotoxicity of soluble tau from AD or other tauopathy patient brains. Minimally manipulated patient-derived aqueous brain extracts contain the more diffusible native forms of these proteins. Here, we explore how intracerebral injection of Aß and tau present in such aqueous extracts of patient brain contribute to disruption of synaptic plasticity in the CA1 area of the male rat hippocampus. Aqueous extracts of certain AD brains acutely inhibited long-term potentiation (LTP) of synaptic transmission in a manner that required both Aß and tau. Tau-containing aqueous extracts of a brain from a patient with Pick's disease (PiD) also impaired LTP, and diffusible tau from either AD or PiD brain lowered the threshold for AD brain Aß to inhibit LTP. Remarkably, the disruption of LTP persisted for at least 2 weeks after a single injection. These findings support a critical role for diffusible tau in causing rapid onset, persistent synaptic plasticity deficits, and promoting Aß-mediated synaptic dysfunction.SIGNIFICANCE STATEMENT The microtubule-associated protein tau forms relatively insoluble fibrillar deposits in the brains of people with neurodegenerative diseases including Alzheimer's and Pick's diseases. More soluble aggregates of disease-associated tau may diffuse between cells and could cause damage to synapses in vulnerable circuits. We prepared aqueous extracts of diseased cerebral cortex and tested their ability to interfere with synaptic function in the brains of live rats. Tau in these extracts rapidly and persistently disrupted synaptic plasticity and facilitated impairments caused by amyloid ß protein, the other major pathologic protein in Alzheimer's disease. These findings show that certain diffusible forms of tau can mediate synaptic dysfunction and may be a target for therapy.
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Enfermedad de Alzheimer , Péptidos beta-Amiloides , Masculino , Ratas , Animales , Péptidos beta-Amiloides/metabolismo , Potenciación a Largo Plazo , Enfermedad de Alzheimer/metabolismo , Proteínas tau/metabolismo , Plasticidad Neuronal , Sinapsis/metabolismo , Hipocampo/metabolismo , Encéfalo/metabolismoRESUMEN
Challenges in obtaining efficient transduction of brain and spinal cord following systemic AAV delivery have led to alternative administration routes being used in clinical trials that directly infuse the virus into the CNS. However, data comparing different direct AAV injections into the brain remain limited making it difficult to choose optimal routes. Here we tested both AAV9-egfp and AAV9-fLuc delivery via intrastriatal (IST), intracisterna magna (ICM) and lumbar intrathecal (LIT) routes in adult rats and assessed vector distribution and transduction in brain, spinal cord and peripheral tissues. We find that IST infusion leads to robust transgene expression in the striatum, thalamus and cortex with lower peripheral tissue transduction and anti-AAV9 capsid titers compared to ICM or LIT. ICM delivery provided strong GFP and luciferase expression across more brain regions than the other routes and similar expression in the spinal cord to LIT injections, which itself largely failed to transduce the rat brain. Our data highlight the strengths and weaknesses of each direct CNS delivery route which will help with future clinical targeting.
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Técnicas de Transferencia de Gen , Médula Espinal , Ratas , Animales , Transducción Genética , Médula Espinal/metabolismo , Encéfalo/metabolismo , Transgenes , Vectores Genéticos/genética , Dependovirus/genética , Dependovirus/metabolismoRESUMEN
INTRODUCTION: With the pressing need to develop treatments that slow or stop the progression of Alzheimer's disease, new tools are needed to reduce clinical trial duration and validate new targets for human therapeutics. Such tools could be derived from neurophysiological measurements of disease. METHODS AND ANALYSIS: The New Therapeutics in Alzheimer's Disease study (NTAD) aims to identify a biomarker set from magneto/electroencephalography that is sensitive to disease and progression over 1 year. The study will recruit 100 people with amyloid-positive mild cognitive impairment or early-stage Alzheimer's disease and 30 healthy controls aged between 50 and 85 years. Measurements of the clinical, cognitive and imaging data (magnetoencephalography, electroencephalography and MRI) of all participants will be taken at baseline. These measurements will be repeated after approximately 1 year on participants with Alzheimer's disease or mild cognitive impairment, and clinical and cognitive assessment of these participants will be repeated again after approximately 2 years. To assess reliability of magneto/electroencephalographic changes, a subset of 30 participants with mild cognitive impairment or early-stage Alzheimer's disease will also undergo repeat magneto/electroencephalography 2 weeks after baseline. Baseline and longitudinal changes in neurophysiology are the primary analyses of interest. Additional outputs will include atrophy and cognitive change and estimated numbers needed to treat each arm of simulated clinical trials of a future disease-modifying therapy. ETHICS AND DATA STATEMENT: The study has received a favourable opinion from the East of England Cambridge Central Research Ethics Committee (REC reference 18/EE/0042). Results will be disseminated through internal reports, peer-reviewed scientific journals, conference presentations, website publication, submission to regulatory authorities and other publications. Data will be made available via the Dementias Platform UK Data Portal on completion of initial analyses by the NTAD study group.
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Enfermedad de Alzheimer , Disfunción Cognitiva , Humanos , Persona de Mediana Edad , Anciano , Anciano de 80 o más Años , Estudios Longitudinales , Reproducibilidad de los Resultados , Progresión de la Enfermedad , Estudios de CohortesRESUMEN
Neurodegenerative diseases are an enormous public health problem, affecting tens of millions of people worldwide. Nearly all of these diseases are characterized by oligomerization and fibrillization of neuronal proteins, and there is great interest in therapeutic targeting of these aggregates. Here, we show that soluble aggregates of α-synuclein and tau bind to plate-immobilized PrP in vitro and on mouse cortical neurons, and that this binding requires at least one of the same N-terminal sites at which soluble Aß aggregates bind. Moreover, soluble aggregates of tau, α-synuclein and Aß cause both functional (impairment of LTP) and structural (neuritic dystrophy) compromise and these deficits are absent when PrP is ablated, knocked-down, or when neurons are pre-treated with anti-PrP blocking antibodies. Using an all-human experimental paradigm involving: (1) isogenic iPSC-derived neurons expressing or lacking PRNP, and (2) aqueous extracts from brains of individuals who died with Alzheimer's disease, dementia with Lewy bodies, and Pick's disease, we demonstrate that Aß, α-synuclein and tau are toxic to neurons in a manner that requires PrPC. These results indicate that PrP is likely to play an important role in a variety of late-life neurodegenerative diseases and that therapeutic targeting of PrP, rather than individual disease proteins, may have more benefit for conditions which involve the aggregation of more than one protein.
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Péptidos beta-Amiloides/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Neuronas/metabolismo , Priones/metabolismo , alfa-Sinucleína/metabolismo , Proteínas tau/metabolismo , Animales , Encéfalo/metabolismo , Humanos , Ratones , Unión ProteicaRESUMEN
There are no approved drug therapies that can prevent or slow the progression of Parkinson's disease (PD). Accumulation and aggregation of α-synuclein protein is observed throughout the nervous system in PD. α-Synuclein is a core component of Lewy bodies and neurites that neuropathologically define PD, suggesting that α-synuclein may be a key causative agent in PD. Recent experimental data suggest that PD progression may arise due to spreading of pathological forms of extracellular α-synuclein throughout the brain via a cellular release, uptake and seeding mechanism. We have developed a high affinity α-synuclein antibody, MEDI1341, that can enter the brain, sequester extracellular α-synuclein and attenuate α-synuclein spreading in vivo. MEDI1341 binds both monomeric and aggregated forms of α-synuclein. In vitro, MEDI1341 blocks cell-to-cell transmission of pathologically relevant α-synuclein preformed fibrils (pffs). After intravenous injection into rats and cynomolgus monkeys, MEDI1341 rapidly enters the central nervous system and lowers free extracellular α-synuclein levels in the interstitial fluid (ISF) and cerebrospinal fluid (CSF) compartments. Using a novel lentiviral-based in vivo mouse model of α-synuclein spreading in the brain, we show that treatment with MEDI1341 significantly reduces α-synuclein accumulation and propagation along axons. In this same model, we demonstrate that an effector-null version of the antibody was equally as effective as one with effector function. MEDI1341 is now in Phase 1 human clinical trial testing as a novel treatment for α-synucleinopathies including PD with the aim to slow or halt disease progression.
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Anticuerpos Monoclonales/farmacología , Encéfalo/efectos de los fármacos , alfa-Sinucleína/antagonistas & inhibidores , Animales , Especificidad de Anticuerpos , Humanos , Macaca fascicularis , Ratones , RatasRESUMEN
Soluble synaptotoxic aggregates of the main pathological proteins of Alzheimer's disease, amyloid ß-protein (Aß) and tau, have rapid and potent inhibitory effects on long-term potentiation (LTP). Although the promotion of synaptic weakening mechanisms, including long-term depression (LTD), is posited to mediate LTP inhibition by Aß, little is known regarding the action of exogenous tau on LTD. The present study examined the ability of different assemblies of full-length human tau to affect LTD in the dorsal hippocampus of the anaesthetized rat. Unlike Aß, intracerebroventricular injection of soluble aggregates of tau (SτAs), but not monomers or fibrils, potently increased the threshold for LTD induction in a manner that required cellular prion protein. However, MTEP, an antagonist of the putative prion protein coreceptor metabotropic glutamate receptor 5, did not prevent the disruption of synaptic plasticity by SτAs. In contrast, systemic treatment with Ro 25-6981, a selective antagonist at GluN2B subunit-containing NMDA receptors, reduced SτA-mediated inhibition of LTD, but not LTP. Intriguingly, SτAs completely blocked Aß-facilitated LTD, whereas a subthreshold dose of SτAs facilitated Aß-mediated inhibition of LTP. Overall, these findings support the importance of cellular prion protein in mediating a range of, sometimes opposing, actions of soluble Aß and tau aggregates with different effector mechanisms on synaptic plasticity.
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Péptidos beta-Amiloides/farmacología , Hipocampo/efectos de los fármacos , Depresión Sináptica a Largo Plazo/efectos de los fármacos , Agregado de Proteínas/fisiología , Proteínas tau/metabolismo , Animales , Hipocampo/metabolismo , Depresión Sináptica a Largo Plazo/fisiología , Masculino , Piridinas/farmacología , Ratas , Receptor del Glutamato Metabotropico 5/agonistas , Sinapsis/efectos de los fármacos , Sinapsis/fisiología , Tiazoles/farmacologíaRESUMEN
INTRODUCTION: The tau protein plays a central role in Alzheimer's disease (AD), and there is huge interest in measuring tau in blood and cerebrospinal fluid (CSF). METHODS: We developed a set of immunoassays to measure tau in specimens from humans diagnosed based on current best clinical and CSF biomarker criteria. RESULTS: In CSF, mid-region- and N-terminal-detected tau predominated and rose in disease. In plasma, an N-terminal assay (NT1) detected elevated levels of tau in AD and AD-mild cognitive impairment (MCI). Plasma NT1 measurements separated controls from AD-MCI (area under the curve [AUC] = 0.88) and AD (AUC = 0.96) in a discovery cohort and in a Validation Cohort (with AUCs = 0.79 and 0.75, respectively). DISCUSSION: The forms of tau in CSF and plasma are distinct, but in each specimen type, the levels of certain fragments are increased in AD. Measurement of plasma NT1 tau should be aggressively pursued as a potential blood-based screening test for AD/AD-MCI.
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Enfermedad de Alzheimer/sangre , Disfunción Cognitiva/sangre , Inmunoensayo , Proteínas tau/sangre , Anciano , Enfermedad de Alzheimer/líquido cefalorraquídeo , Biomarcadores/sangre , Biomarcadores/líquido cefalorraquídeo , Disfunción Cognitiva/líquido cefalorraquídeo , Estudios de Cohortes , Diagnóstico Diferencial , Espacio Extracelular , Femenino , Humanos , Inmunoensayo/métodos , Masculino , Persona de Mediana Edad , Sensibilidad y Especificidad , Proteínas tau/líquido cefalorraquídeoRESUMEN
Intracellular neurofibrillary tangles (NFTs) composed of tau protein are a neuropathological hallmark of several neurodegenerative diseases, the most common of which is Alzheimer's disease (AD). For some time NFTs were considered the primary cause of synaptic dysfunction and neuronal death, however, more recent evidence suggests that soluble aggregates of tau are key drivers of disease. Here we investigated the effect of different tau species on synaptic plasticity in the male rat hippocampus in vivo Intracerebroventricular injection of soluble aggregates formed from either wild-type or P301S human recombinant tau potently inhibited hippocampal long-term potentiation (LTP) at CA3-to-CA1 synapses. In contrast, tau monomers and fibrils appeared inactive. Neither baseline synaptic transmission, paired-pulse facilitation nor burst response during high-frequency conditioning stimulation was affected by the soluble tau aggregates. Similarly, certain AD brain soluble extracts inhibited LTP in a tau-dependent manner that was abrogated by either immunodepletion with, or coinjection of, a mid-region anti-tau monoclonal antibody (mAb), Tau5. Importantly, this tau-mediated block of LTP was prevented by administration of mAbs selective for the prion protein (PrP). Specifically, mAbs to both the mid-region (6D11) and N-terminus (MI-0131) of PrP prevented inhibition of LTP by both recombinant and brain-derived tau. These findings indicate that PrP is a mediator of tau-induced synaptic dysfunction.SIGNIFICANCE STATEMENT Here we report that certain soluble forms of tau selectively disrupt synaptic plasticity in the live rat hippocampus. Further, we show that monoclonal antibodies to cellular prion protein abrogate the impairment of long-term potentiation caused both by recombinant and Alzheimer's disease brain-derived soluble tau. These findings support a critical role for cellular prion protein in the deleterious synaptic actions of extracellular soluble tau in tauopathies, including Alzheimer's disease. Thus, approaches targeting cellular prion protein, or downstream pathways, might provide an effective strategy for developing therapeutics.
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Hipocampo/metabolismo , Hipocampo/patología , Plasticidad Neuronal/fisiología , Proteínas PrPC/metabolismo , Proteínas tau/metabolismo , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Inhibidores de la Angiogénesis/farmacología , Animales , Femenino , Hipocampo/efectos de los fármacos , Humanos , Masculino , Plasticidad Neuronal/efectos de los fármacos , Proteínas Priónicas/metabolismo , RatasRESUMEN
Tau is normally a highly soluble phosphoprotein found predominantly in neurons. Six different isoforms of tau are expressed in the adult human CNS. Under pathological conditions, phosphorylated tau aggregates are a defining feature of neurodegenerative disorders called tauopathies. Recent findings have suggested a potential role of the gut-brain axis in CNS homeostasis, and therefore we set out to examine the isoform profile and phosphorylation state of tau in the enteric nervous system (ENS) under physiological conditions and in tauopathies. Surgical specimens of human colon from controls, Parkinson's disease (PD) and progressive supranuclear palsy (PSP) patients were analyzed by Western Blot and immunohistochemistry using a panel of anti-tau antibodies. We found that adult human ENS primarily expresses two tau isoforms, localized in the cell bodies and neuronal processes. We did not observe any difference in the enteric tau isoform profile and phosphorylation state between PSP, PD and control subjects. The htau mouse model of tauopathy also expressed two main isoforms of human tau in the ENS, and there were no apparent differences in ENS tau localization or phosphorylation between wild-type and htau mice. Tau in both human and mouse ENS was found to be phosphorylated but poorly susceptible to dephosphorylation with lambda phosphatase. To investigate ENS tau phosphorylation further, primary cultures from rat enteric neurons, which express four isoforms of tau, were pharmacologically manipulated to show that ENS tau phosphorylation state can be regulated, at least in vitro. Our study is the first to characterize tau in the rodent and human ENS. As a whole, our findings provide a basis to unravel the functions of tau in the ENS and to further investigate the possibility of pathological changes in enteric neuropathies and tauopathies.
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Sistema Nervioso Entérico/metabolismo , Enfermedad de Parkinson/patología , Parálisis Supranuclear Progresiva/patología , Proteínas tau/metabolismo , Anciano , Animales , Antiinfecciosos/farmacología , Benzofenantridinas/farmacología , Encéfalo/metabolismo , Encéfalo/patología , Células Cultivadas , Colon/metabolismo , Colon/patología , Embrión de Mamíferos , Sistema Nervioso Entérico/efectos de los fármacos , Femenino , Humanos , Isoquinolinas/farmacología , Masculino , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Plexo Mientérico/metabolismo , Embarazo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratas , Ratas Sprague-Dawley , Plexo Submucoso/metabolismo , Tubulina (Proteína)/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Adulto Joven , Proteínas tau/genéticaRESUMEN
In Alzheimer's disease, neurofibrillary tangle pathology appears to spread along neuronal connections, proposed to be mediated by the release and uptake of abnormal, disease-specific forms of microtubule-binding protein tau MAPT. It is currently unclear whether transfer of tau between neurons is a toxic gain-of-function process in dementia or reflects a constitutive biological process. We report two entry mechanisms for monomeric tau to human neurons: a rapid dynamin-dependent phase typical of endocytosis and a second, slower actin-dependent phase of macropinocytosis. Aggregated tau entry is independent of actin polymerization and largely dynamin dependent, consistent with endocytosis and distinct from macropinocytosis, the major route for aggregated tau entry reported for non-neuronal cells. Anti-tau antibodies abrogate monomeric tau entry into neurons, but less efficiently in the case of aggregated tau, where internalized tau carries antibody with it into neurons. These data suggest that tau entry to human neurons is a physiological process and not a disease-specific phenomenon.
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Neuronas/metabolismo , Proteínas tau/metabolismo , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Dinaminas/antagonistas & inhibidores , Dinaminas/metabolismo , Endocitosis , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Fosforilación , Agregación Patológica de ProteínasRESUMEN
Sequential cleavage of the amyloid-ß protein precursor (AßPP) by BACE1 (ß-secretase) followed by theγ-secretase complex, is strongly implicated in Alzheimer's disease (AD) but the initial cellular responses to these cleavage events are not fully defined. ß-secretase-mediated AßPP processing yields an extracellular domain (sAßPPß) and a C-terminal fragment of AßPP of 99 amino acids (C99). Subsequent cleavage by γ-secretase produces amyloid-ß (Aß) and an AßPP intracellular domain (AICD). A cellular screen based on the generation of AICD from an AßPP-Gal4 fusion protein was adapted by introducing familial AD (FAD) mutations into the AßPP sequence and linking the assay to Gal4-UAS driven luciferase and GFP expression, to identify responses immediately downstream of AßPP processing in neurons with a focus on the transcription factor Foxo3a which has been implicated in neurodegeneration. The K670N/M671L, E682K, E693G, and V717I FAD mutations and the A673T protective mutation, were introduced into the AßPP sequence by site directed mutagenesis. When expressed in mouse cortical neurons, AßPP-Gal4-UAS driven luciferase and GFP expression was substantially reduced by γ-secretase inhibitors, lowered by ß-secretase inhibitors, and enhanced by α-secretase inhibitors suggesting that AICD is a product of the ßγ-secretase pathway. AßPP-Gal4-UAS driven GFP expression was exploited to identify individual neurons undergoing amyloidogenic AßPP processing, revealing increased nuclear localization of Foxo3a and enhanced Foxo3a-mediated transcription downstream of AICD production. Foxo3a translocation was not driven by AICD directly but correlated with reduced Akt phosphorylation. Collectively this suggests that ßγ-secretase-mediated AßPP processing couples to Foxo3a which could be an early neuronal signaling response in AD.
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Enfermedad de Alzheimer/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Proteína Forkhead Box O3/metabolismo , Neuronas/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animales , Modelos Animales de Enfermedad , Proteína Forkhead Box O3/genética , Ratones , Mutagénesis Sitio-Dirigida , Mutación , Fosforilación , Transporte de Proteínas , Transducción de SeñalRESUMEN
The spatiotemporal transmission of pathological tau in the brain is characteristic of Alzheimer's disease. Release of both soluble and abnormal tau species from healthy neurons is increased upon stimulation of neuronal activity. It is not yet understood whether the mechanisms controlling soluble tau release from healthy neurons is the same as those involved in the spread of pathological tau species. To begin to understand these events, we have studied tau distribution and release using organotypic brain slice cultures. The slices were cultured from postnatal wild-type and 3xTg-AD mice for up to 1 month. Tau distribution in subcellular compartments was examined by western blotting, and tau release into culture medium was determined using a sensitive sandwich ELISA. We show here that 3xTg-AD cultures have an accelerated development of pathological tau abnormalities including the redistribution of tau to synaptic and membrane compartments. The 3xTg-AD slice cultures show elevated basal tau release relative to total tau when compared with wild-type cultures. However, tau release from 3xTg-AD slices cannot be further stimulated when neuronal activity is increased with potassium chloride. Moreover, we report that there is an increased pool of dephosphorylated membrane-associated tau in conditions where tau release is increased. These data suggest that there may be differential patterns of tau release when using integrated slice culture models of wild-type and transgenic mouse brain, although it will be important to determine the effect of tau overexpression for these findings. These results further increase our knowledge of the molecular mechanisms underlying tau release and propagation in neurodegenerative tauopathies.
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Encéfalo/metabolismo , Membrana Celular/metabolismo , Proteínas tau/metabolismo , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Animales , Emparejamiento Cromosómico/fisiología , Modelos Animales de Enfermedad , Ratones , Ratones Transgénicos/metabolismo , Neuronas/metabolismo , Fosforilación/fisiologíaRESUMEN
Flavonoids, a group of dietary polyphenols have been shown to possess cognitive health benefits. Epidemiologic evidence suggests that they could play a role in risk reduction in dementia. Amyloid precursor protein processing and the subsequent generation of amyloid beta (Aß) are central to the pathogenesis of Alzheimer's disease, as soluble, oligomeric Aß is thought to be the toxic species driving disease progression. We undertook an in vitro screen to identify flavonoids with bioactivity at ßγ-mediated amyloid precursor protein processing, which lead to identification of a number of flavonoids bioactive at 100 nM. Because of known bioavailability, we investigated the catechin family further and identified epigallocatechin and (-)-epicatechin as potent (nanomolar) inhibitors of amyloidogenic processing. Supporting this finding, we have shown reduced Aß pathology and Aß levels following short term, a 21-day oral delivery of (-)-epicatechin in 7-month-old TASTPM mice. Further, in vitro mechanistic studies suggest this is likely because of indirect BACE1 inhibition. Taken together, our results suggest that orally delivered (-)-epicatechin may be a potential prophylactic for Alzheimer's disease.
Asunto(s)
Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/prevención & control , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Catequina/administración & dosificación , Catequina/farmacología , Administración Oral , Animales , Encéfalo/metabolismo , Catequina/análogos & derivados , Células Cultivadas , Progresión de la Enfermedad , Masculino , Ratones TransgénicosRESUMEN
Soluble oligomeric amyloid ß peptide (Aß) generated from processing of the amyloid precursor protein (APP) plays a central role in the pathogenesis of Alzheimer's Disease (AD) and through actions at glutamatergic synapses affects excitability and plasticity. The physiological control of APP processing is not fully understood but stimulation of synaptic NMDA receptors (NMDAR) can suppress Aß levels through an ERK-dependent increase in α-secretase activity. AMPA-type glutamate receptors (AMPAR) couple to ERK phosphorylation independently of NMDAR activation raising the possibility that stimulation of AMPAR might similarly promote non-amyloidogenic APP processing. We have tested this hypothesis by investigating whether AMPAR directly regulate APP processing in cultured mouse cortical neurons, by analyzing APP C-terminal fragments (CTFs), soluble APP (sAPP), Aß levels, and cleavage of an APP-GAL4 reporter protein. We report that direct stimulation of AMPAR increases non-amyloidogenic α-secretase-mediated APP processing and inhibits Aß production. Processing was blocked by the matrix metalloproteinase inhibitor TAPI-1 but was only partially dependent on Ca(2+) influx and ERK activity. AMPAR can therefore, be added to the repertoire of receptors that couple to non-amyloidogenic APP processing at glutamatergic synapses and thus pharmacological targeting of AMPAR could potentially influence the development and progression of Aß pathology in AD.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Receptores AMPA/metabolismo , Péptidos beta-Amiloides/genética , Precursor de Proteína beta-Amiloide/genética , Animales , Células Cultivadas , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Ratones , Fosforilación , Receptores AMPA/genética , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismoRESUMEN
Disruption to axonal transport is an early pathological feature in Alzheimer's disease. The amyloid precursor protein (APP) is a key axonal transport cargo in Alzheimer's disease since perturbation of its transport increases APP processing and production of amyloid-ß peptide (Aß) that is deposited in the brains of Alzheimer's disease patients. APP is transported anterogradely through axons on kinesin-1 motors. One favoured route for attachment of APP to kinesin-1 involves the scaffolding protein c-Jun N-terminal kinase-interacting protein-1 (JIP1), which has been shown to bind both APP and kinesin-1 light chain (KLC). However, direct experimental evidence to support a role of JIP1 in APP transport is lacking. Notably, the effect of loss of JIP1 on movement of APP through axons of living neurons, and the impact of such loss on APP processing and Aß production has not been reported. To address these issues, we monitored how siRNA mediated loss of JIP1 influenced transport of enhanced green fluorescent protein (EGFP)-tagged APP through axons and production of endogenous Aß in living neurons. Surprisingly, we found that knockdown of JIP1 did not affect either APP transport or Aß production. These results have important implications for our understanding of APP trafficking in Alzheimer's disease.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Transporte Axonal , Neuronas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/genética , Animales , Axones/metabolismo , Encéfalo/metabolismo , Embrión de Mamíferos/metabolismo , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Masculino , Persona de Mediana Edad , RatasRESUMEN
Understanding the mechanisms that control processing of the amyloid precursor protein (APP) to produce amyloid-ß (Aß) peptide represents a key area of Alzheimer's disease research. Here, we show that siRNA-mediated loss of calsyntenin-1 in cultured neurons alters APP processing to increase production of Aß. We also show that calsyntenin-1 is reduced in Alzheimer's disease brains and that the extent of this reduction correlates with increased Aß levels. Calsyntenin-1 is a ligand for kinesin-1 light chains and APP is transported through axons on kinesin-1 molecular motors. Defects in axonal transport are an early pathological feature in Alzheimer's disease and defective APP transport is known to increase Aß production. We show that calsyntenin-1 and APP are co-transported through axons and that siRNA-induced loss of calsyntenin-1 markedly disrupts axonal transport of APP. Thus, perturbation to axonal transport of APP on calsyntenin-1 containing carriers induces alterations to APP processing that increase production of Aß. Together, our findings suggest that disruption of calsyntenin-1-associated axonal transport of APP is a pathogenic mechanism in Alzheimer's disease.