RESUMO
The transition between soluble intrinsically disordered tau protein and aggregated tau in neurofibrillary tangles in Alzheimer's disease is unknown. Here, we propose that soluble tau species can undergo liquid-liquid phase separation (LLPS) under cellular conditions and that phase-separated tau droplets can serve as an intermediate toward tau aggregate formation. We demonstrate that phosphorylated or mutant aggregation prone recombinant tau undergoes LLPS, as does high molecular weight soluble phospho-tau isolated from human Alzheimer brain. Droplet-like tau can also be observed in neurons and other cells. We found that tau droplets become gel-like in minutes, and over days start to spontaneously form thioflavin-S-positive tau aggregates that are competent of seeding cellular tau aggregation. Since analogous LLPS observations have been made for FUS, hnRNPA1, and TDP43, which aggregate in the context of amyotrophic lateral sclerosis, we suggest that LLPS represents a biophysical process with a role in multiple different neurodegenerative diseases.
Assuntos
Doença de Alzheimer/metabolismo , Esclerose Lateral Amiotrófica/metabolismo , Encéfalo/metabolismo , Neurônios/metabolismo , Agregação Patológica de Proteínas/metabolismo , Proteínas tau/química , Proteínas tau/isolamento & purificação , Proteínas tau/metabolismo , Idoso de 80 Anos ou mais , Sequência de Aminoácidos , Animais , Benzotiazóis/metabolismo , Fenômenos Biofísicos , Clonagem Molecular , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Feminino , Células HEK293 , Ribonucleoproteína Nuclear Heterogênea A1/metabolismo , Humanos , Extração Líquido-Líquido , Camundongos , Camundongos Transgênicos , Peso Molecular , Neuroblastoma/metabolismo , Doenças Neurodegenerativas/metabolismo , Emaranhados Neurofibrilares/metabolismo , Fosforilação , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análise de Sequência de Proteína , Células Sf9RESUMO
Apolipoprotein E (ApoE) is a secreted apolipoprotein with three isoforms, E2, E3, and E4, that binds to lipids and facilitates their transport in the extracellular environment of the brain and the periphery. The E4 allele is a major genetic risk factor for the sporadic form of Alzheimer's disease (AD), and studies of human brain and mouse models have revealed that E4 significantly exacerbates the deposition of amyloid beta (Aß). It has been suggested that this deposition could be attributed to the formation of soluble ApoE isoform-specific ApoE-Aß complexes. However, previous studies have reported conflicting results regarding the directionality and strength of those interactions. In this study, using a series of flow cytometry assays that maintain the physiological integrity of ApoE-Aß complexes, we systematically assessed the association of Aß with ApoE2, E3, or E4. We used ApoE secreted from HEK cells or astrocytes overexpressing ApoE fused with a GFP tag. As a source of soluble Aß peptide, we used synthetic Aß40 or Aß42 or physiological Aß secreted from CHO cell lines overexpressing WT or V717F variant amyloid precursor protein (APP). We observed significant interactions between the different ApoE isoforms and Aß, with E4 interacting with Aß more strongly than the E2 and E3 isoforms. We also found subtle differences depending on the Aß type and the ApoE-producing cell type. In conclusion, these results indicate that the strength of the ApoE-Aß association depends on the source of Aß or ApoE.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Apolipoproteína E2/metabolismo , Apolipoproteína E3/metabolismo , Apolipoproteína E4/metabolismo , Astrócitos/metabolismo , Citometria de Fluxo/métodos , Neurônios/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Astrócitos/citologia , Bioensaio , Linhagem da Célula , Células HEK293 , Humanos , Técnicas In Vitro , Masculino , Camundongos , Camundongos Transgênicos , Neurônios/citologia , Isoformas de ProteínasRESUMO
Several studies have now supported the use of a tau lowering agent as a possible therapy in the treatment of tauopathy disorders, including Alzheimer's disease. In human Alzheimer's disease, however, concurrent amyloid-ß deposition appears to synergize and accelerate tau pathological changes. Thus far, tau reduction strategies that have been tested in vivo have been examined in the setting of tau pathology without confounding amyloid-ß deposition. To determine whether reducing total human tau expression in a transgenic model where there is concurrent amyloid-ß plaque formation can still reduce tau pathology and protect against neuronal loss, we have taken advantage of the regulatable tau transgene in APP/PS1 × rTg4510 mice. These mice develop both neurofibrillary tangles as well as amyloid-ß plaques throughout the cortex and hippocampus. By suppressing human tau expression for 6 months in the APP/PS1 × rTg4510 mice using doxycycline, AT8 tau pathology, bioactivity, and astrogliosis were reduced, though importantly to a lesser extent than lowering tau in the rTg4510 alone mice. Based on non-denaturing gels and proteinase K digestions, the remaining tau aggregates in the presence of amyloid-ß exhibit a longer-lived aggregate conformation. Nonetheless, lowering the expression of the human tau transgene was sufficient to equally ameliorate thioflavin-S positive tangles and prevent neuronal loss equally well in both the APP/PS1 × rTg4510 mice and the rTg4510 cohort. Together, these results suggest that, although amyloid-ß stabilizes tau aggregates, lowering total tau levels is still an effective strategy for the treatment of tau pathology and neuronal loss even in the presence of amyloid-ß deposition.
Assuntos
Placa Amiloide/patologia , Tauopatias/metabolismo , Proteínas tau/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Modelos Animais de Doenças , Hipocampo/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Emaranhados Neurofibrilares/patologia , Neurônios/metabolismo , Fosforilação , Placa Amiloide/metabolismo , Presenilina-1/metabolismoRESUMO
Apolipoprotein E (apoE) has an important role in the pathogenesis of Alzheimer's disease with its three isoforms having distinct effects on disease risk. Here, we assessed the conformational differences between those isoforms using a novel flow cytometry-Forster resonance energy transfer (FRET) assay. We showed that the conformation of intracellular apoE within HEK cells and astrocytes adopts a directional pattern; in other words, E4 adopts the most closed conformation, E2 adopts the most open conformation, and E3 adopts an intermediate conformation. However, this pattern was not maintained upon secretion of apoE from astrocytes. Intermolecular interactions between apoE molecules were isoform-specific, indicating a great diversity in the structure of apoE lipoparticles. Finally, we showed that secreted E4 is the most lipidated isoform in astrocytes, suggesting that increased lipidation acts as a folding chaperone enabling E4 to adopt a closed conformation. In conclusion, this study gives insights into apoE biology and establishes a robust screening system to monitor apoE conformation.
Assuntos
Apolipoproteínas E/química , Astrócitos/química , Transferência Ressonante de Energia de Fluorescência , Apolipoproteínas E/metabolismo , Citometria de Fluxo , Células HEK293 , Humanos , Conformação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismoRESUMO
BACKGROUND: Misfolding of microtubule-associated protein tau (MAPT) within neurons into neurofibrillary tangles is an important pathological feature of Alzheimer's disease (AD). Tau pathology correlates with cognitive decline in AD and follows a stereotypical anatomical course; several recent studies indicate that tau pathology spreads inter-neuronally via misfolded tau "seeds." Previous research has focused on neurons as the source of these tau seeds. However, recent studies as well as the data contained herein suggest that microglia, the resident immune cells of the central nervous system, play a direct role in the spread of tau pathology. METHODS: Primary adult microglia were isolated from human AD cases and the rTg4510 tauopathy mouse model and used for analysis of gene expression, tau protein by Simoa technology, and quantification of tau seeding using a highly sensitive fluorescence resonance energy transfer (FRET) biosensing cell line for tau seeding and aggregation. RESULTS: Here, we show that microglia isolated from both human tauopathy and AD cases and the rTg4510 tauopathy mouse model stably contain tau seeds, despite not synthesizing any tau. Microglia releases these tau seeds in vitro into their conditioned media (CM). This suggests that microglia have taken up tau but are incapable of entirely neutralizing its seeding activity. Indeed, when in vitro microglia are given media containing tau seeds, they reduce (but do not eliminate) tau seeding. When microglia are treated with inflammagens such as lipopolysaccharide (LPS), interleukin-1ß (IL1ß), tumor necrosis factor α (TNFα), or amyloid-ß, their ability to reduce tau seeding is unchanged and these factors do not induce seeding activity on their own. CONCLUSIONS: Overall, these data suggest that microglia have a complex role: they are capable of taking up and breaking down seed competent tau, but do so inefficiently and could therefore potentially play a role in the spread of tau pathology.
Assuntos
Doença de Alzheimer/patologia , Encéfalo/patologia , Regulação da Expressão Gênica/genética , Microglia/metabolismo , Proteínas tau/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Citocinas/metabolismo , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas dos Microfilamentos/metabolismo , Mutação/genética , Emaranhados Neurofibrilares , Neurônios/metabolismo , Proteínas tau/genéticaRESUMO
Amyloid plaques and neurofibrillary tangles co-occur in Alzheimer disease, but with different topological and temporal patterns. Whether these two lesions are independent or pathobiologically related is uncertain. For example, amyloid deposition in the neocortex precedes the spread of tau neurofibrillary tangles from the limbic areas to the cortex. We examined the aggregation properties of tau isolated from human cases with early tau pathology (Braak II) with and without plaques. Using a well-established HEK cell biosensor assay, we show that tau from cases with plaques has an enhanced ability to induce tau aggregates compared to tau from cases without plaques. To further explore this effect, we combined mice carrying the APP/PS1 transgene array that develop plaques with rTg4510 mice carrying the P301L mutant human tau transgene that develop extensive tau pathology with age. The resulting APP/PS1-rTg4510 mice had a threefold increase in tau seeding activity over the rTg4510 strain, without change in tau production or extracellular release. Surprisingly, this effect was observed before overt amyloid deposition. The enhancement of tau aggregation was also apparent by an increase in histological measures of tau pathology in young APP/PS1-rTg4510 mice and an increase in high-molecular-weight tau. Overall, these data provide evidence that amyloid ß acts to enhance tau pathology by increasing the formation of tau species capable of seeding new aggregates.
Assuntos
Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Emaranhados Neurofibrilares/patologia , Placa Amiloide/patologia , Proteínas tau/metabolismo , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/genética , Animais , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Neocórtex/metabolismo , Neocórtex/patologia , Emaranhados Neurofibrilares/metabolismo , Fosforilação , Placa Amiloide/metabolismo , Agregação Patológica de Proteínas , Proteínas tau/genéticaRESUMO
The clinical progression of Alzheimer disease (AD) is associated with the accumulation of tau neurofibrillary tangles, which may spread throughout the cortex by interneuronal tau transfer. If so, targeting extracellular tau species may slow the spreading of tau pathology and possibly cognitive decline. To identify suitable target epitopes, we tested the effects of a panel of tau antibodies on neuronal uptake and aggregation in vitro. Immunodepletion was performed on brain extract from tau-transgenic mice and postmortem AD brain and added to a sensitive fluorescence resonance energy transfer-based tau uptake assay to assess blocking efficacy. The antibodies reduced tau uptake in an epitope-dependent manner: N-terminal (Tau13) and middomain (6C5 and HT7) antibodies successfully prevented uptake of tau species, whereas the distal C-terminal-specific antibody (Tau46) had little effect. Phosphorylation-dependent (40E8 and p396) and C-terminal half (4E4) tau antibodies also reduced tau uptake despite removing less total tau by immunodepletion, suggesting specific interactions with species involved in uptake. Among the seven antibodies evaluated, 6C5 most efficiently blocked uptake and subsequent aggregation. More important, 6C5 also blocked neuron-to-neuron spreading of tau in a unique three-chamber microfluidic device. Furthermore, 6C5 slowed down the progression of tau aggregation even after uptake had begun. Our results imply that not all antibodies/epitopes are equally robust in terms of blocking tau uptake of human AD-derived tau species.
Assuntos
Doença de Alzheimer/metabolismo , Neurônios/metabolismo , Proteínas tau/metabolismo , Idoso de 80 Anos ou mais , Doença de Alzheimer/patologia , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/imunologia , Encéfalo/metabolismo , Encéfalo/patologia , Células Cultivadas , Epitopos/imunologia , Feminino , Humanos , Interneurônios/metabolismo , Masculino , Camundongos Transgênicos , Técnicas Analíticas Microfluídicas , Terapia de Alvo Molecular/métodos , Emaranhados Neurofibrilares/metabolismo , Emaranhados Neurofibrilares/patologia , Fosforilação , Proteínas tau/antagonistas & inibidores , Proteínas tau/imunologiaRESUMO
OBJECTIVE: Cerebrospinal fluid (CSF) tau is an excellent surrogate marker for assessing neuropathological changes that occur in Alzheimer's disease (AD) patients. However, whether the elevated tau in AD CSF is just a marker of neurodegeneration or, in fact, a part of the disease process is uncertain. Moreover, it is unknown how CSF tau relates to the recently described soluble high-molecular-weight (HMW) species that is found in the postmortem AD brain and can be taken up by neurons and seed aggregates. METHODS: We have examined seeding and uptake properties of brain extracellular tau from various sources, including interstitial fluid (ISF) and CSF from an AD transgenic mouse model and postmortem ventricular and antemortem lumbar CSF from AD patients. RESULTS: We found that brain ISF and CSF tau from the AD mouse model can be taken up by cells and induce intracellular aggregates. Ventricular CSF from AD patients contained a rare HMW tau species that exerted a higher seeding activity. Notably, the HMW tau species was also detected in lumbar CSF from AD patients, and its levels were significantly elevated compared to control subjects. HMW tau derived from CSF of AD patients was seed competent in vitro. INTERPRETATION: These findings suggest that CSF from an AD brain contains potentially bioactive HMW tau species, giving new insights into the role of CSF tau and biomarker development for AD. Ann Neurol 2016;80:355-367.
Assuntos
Doença de Alzheimer/líquido cefalorraquidiano , Encéfalo/metabolismo , Proteínas tau/líquido cefalorraquidiano , Idoso , Animais , Biomarcadores/líquido cefalorraquidiano , Líquido Extracelular/metabolismo , Feminino , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-IdadeRESUMO
INTRODUCTION: Little is known about the utility of plasma amyloid beta (Aß) in clinical trials of Alzheimer's disease (AD). METHODS: We analyzed longitudinal plasma samples from two large multicenter clinical trials: (1) donezepil and vitamin E in mild cognitive impairment (n = 405, 24 months) and (2) simvastatin in mild to moderate AD (n = 225, 18 months). RESULTS: Baseline plasma Aß was not related to cognitive or clinical progression. We observed a decrease in plasma Aß40 and 42 among apolipoprotein E epsilon 4 (APOE ε4) carriers relative to noncarriers in the mild cognitive impairment trial. Patients treated with simvastatin showed a significant increase in Aß compared with placebo. We found significant storage time effects and considerable plate-to-plate variation. DISCUSSION: We found no support for the utility of plasma Aß as a prognostic factor or correlate of cognitive change. Analysis of stored specimens requires careful standardization and experimental design, but plasma Aß may prove useful in pharmacodynamic studies of antiamyloid drugs.
Assuntos
Doença de Alzheimer/sangue , Peptídeos beta-Amiloides/sangue , Disfunção Cognitiva/sangue , Fragmentos de Peptídeos/sangue , Idoso , Doença de Alzheimer/tratamento farmacológico , Apolipoproteína E4/genética , Biomarcadores/sangue , Análise Química do Sangue , Disfunção Cognitiva/tratamento farmacológico , Disfunção Cognitiva/genética , Progressão da Doença , Donepezila , Feminino , Heterozigoto , Humanos , Indanos/uso terapêutico , Estudos Longitudinais , Masculino , Nootrópicos/uso terapêutico , Piperidinas/uso terapêutico , Índice de Gravidade de Doença , Sinvastatina/uso terapêutico , Vitamina E/uso terapêuticoRESUMO
Neurofibrillary tangles (NFTs), a marker of neuronal alterations in Alzheimer's disease (AD) and other tauopathies, are comprised of aggregates of hyperphosphorylated tau protein. We recently studied the formation of NFTs in the entorhinal cortex (EC) and their subsequent propagation through neural circuits in the rTgTauEC mouse model (de Calignon et al., 2012). We now examine the consequences of suppressing transgene expression with doxycycline on the NFT-associated pathological features of neuronal system deafferentation, NFT progression and propagation, and neuronal loss. At 21 months of age we observe that EC axonal lesions are associated with an abnormal sprouting response of acetylcholinesterase (AChE)-positive fibers, a phenotype reminiscent of human AD. At 24 months, NFTs progress, tau inclusions propagate to the dentate gyrus, and neuronal loss is evident. Suppression of the transgene expression from 18 to 24 months led to reversal of AChE sprouting, resolution of Gallyas-positive and Alz50-positive NFTs, and abrogation of progressive neuronal loss. These data suggest that propagation of NFTs, as well as some of the neural system consequences of NFTs, can be reversed in an animal model of NFT-associated toxicity, providing proof in principle that these lesions can be halted, even in established disease.
Assuntos
Doença de Alzheimer/patologia , Córtex Entorrinal/patologia , Emaranhados Neurofibrilares/patologia , Proteínas tau/metabolismo , Acetilcolinesterase , Doença de Alzheimer/metabolismo , Animais , Western Blotting , Modelos Animais de Doenças , Córtex Entorrinal/metabolismo , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Fenótipo , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise Serial de Tecidos , Proteínas tau/genéticaRESUMO
There is a growing body of evidence that soluble oligomeric forms of amyloid ß (Aß) play a critical role in Alzheimer's disease (AD). Despite the importance of soluble Aß oligomers as a therapeutic target for AD, the dynamic metabolism of these Aß species in vivo has not been elucidated because of the difficulty in monitoring brain Aß oligomers in living animals. Here, using a unique large pore-sized membrane microdialysis, we characterized soluble Aß oligomers in brain interstitial fluid (ISF) of awake, freely moving APP/PS1 transgenic and control WT mice. We could detect high-molecular-weight (HMW) and low-molecular-weight (LMW) Aß oligomers in the brain ISF of living animals, which increased dramatically in an age-dependent manner (5- to 8-fold increase, 4 vs. 17-18 mo). Notably, HMW Aß decreased more slowly than other forms of Aß after acute γ-secretase inhibition [% decrease from the baseline (HMW vs. LMW) was 36.9 vs. 74.1% (Aß40, P<0.05) and 25.4 vs. 88.0% (Aß42, P<0.01)], suggesting that HMW Aß oligomers clear more slowly than other forms from the brain. These data reveal the dynamic metabolism of neurotoxic Aß oligomers in AD brain and could provide new insights into Aß-targeted therapies for AD.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Modelos Animais de Doenças , Fatores Etários , Doença de Alzheimer/genética , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/química , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Benzodiazepinonas/farmacologia , Encéfalo/efeitos dos fármacos , Líquido Extracelular/efeitos dos fármacos , Líquido Extracelular/metabolismo , Humanos , Immunoblotting , Camundongos , Camundongos Transgênicos , Microdiálise/métodos , Peso Molecular , Presenilina-1/genética , Presenilina-1/metabolismo , Multimerização ProteicaRESUMO
Clinical studies suggest that exposure to stress can increase risk for Alzheimer's disease (AD). Although the precise links between stress and vulnerability to develop AD remain uncertain, recent animal work suggests that stress may promote susceptibility to AD pathology by activating tau kinases and inducing tau phosphorylation (tau-P). Our previous findings indicate the differential involvement of corticotropin-releasing factor receptor (CRFR) types 1 and 2 in regulating tau-P in the hippocampus induced by acute restraint, an emotional stressor. To assess the generality of CRFR involvement in stress-induced tau-P and tau kinase activity, the present study extends our investigation to a well-characterized physiological stressor, i.e. immune challenge induced by bacterial lipopolysaccharide (LPS). Acute systemic administration of LPS (100 µg/kg) robustly increased hippocampal (but not isocortical or cerebellar) tau-P, peaking at 40-120 min postinjection and abating thereafter. Assessments of the genotype dependence of this effect yielded results that were distinct from the restraint model. Treatment with LPS increased phosphorylation in wild-type, single and double CRFR knockouts with only subtle variation, which included a reliable exaggeration of tau-P responses in CRFR1-deficient mice. Parallel analyses implicated glycogen synthase kinase-3 and cyclin-dependent kinase-5 as likely cellular mediators of LPS-induced tau-P. Conversely, our data suggest that temperature-dependent fluctuations in tau protein phosphatase 2A (PP2A) may not play a role in this context. Thus, neither the strict CRFR1 dependence of restraint-induced tau-P nor the exaggeration of these responses in CRFR2 null mice generalize to the LPS model. CRFR mediation of stress-induced hippocampal tau-P may be limited to emotional stressors.
Assuntos
Quinase 3 da Glicogênio Sintase/metabolismo , Hipocampo/efeitos dos fármacos , Hipocampo/fisiologia , Lipopolissacarídeos/farmacologia , Receptores de Hormônio Liberador da Corticotropina/metabolismo , Proteínas tau/metabolismo , Animais , Temperatura Corporal/efeitos dos fármacos , Ativação Enzimática , Feminino , Hipocampo/citologia , Inflamação/induzido quimicamente , Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Receptores de Hormônio Liberador da Corticotropina/genética , Estresse Fisiológico , Estresse PsicológicoRESUMO
Apolipoprotein E (APOE) effects on brain function remain controversial. Removal of APOE not only impairs cognitive functions but also reduces neuritic amyloid plaques in mouse models of Alzheimer's disease (AD). Can APOE simultaneously protect and impair neural circuits? Here, we dissociated the role of APOE in AD versus aging to determine its effects on neuronal function and synaptic integrity. Using two-photon calcium imaging in awake mice to record visually evoked responses, we found that genetic removal of APOE improved neuronal responses in adult APP/PSEN1 mice (8-10 mo). These animals also exhibited fewer neuritic plaques with less surrounding synapse loss, fewer neuritic dystrophies, and reactive glia. Surprisingly, the lack of APOE in aged mice (18-20 mo), even in the absence of amyloid, disrupted visually evoked responses. These results suggest a dissociation in APOE's role in AD versus aging: APOE may be neurotoxic during early stages of amyloid deposition, although being neuroprotective in latter stages of aging.
Assuntos
Envelhecimento/fisiologia , Apolipoproteínas E/genética , Regeneração/fisiologia , Córtex Visual/fisiologia , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Amiloidose , Animais , Modelos Animais de Doenças , Potenciais Evocados Visuais/genética , Humanos , Mutação com Perda de Função/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neuroglia/metabolismo , Neurônios/metabolismo , Placa Amiloide/patologia , Presenilina-1/genética , Sinapses/metabolismoRESUMO
A synergy between ß-amyloid (Aß) and tau appears to occur in Alzheimer disease (AD), but the mechanisms of interaction, and potential locations, are little understood. This study investigates the possibility of such interactions within the cortical synaptic compartments of APP/PS1 mice. We used label-free quantitative mass spectrometry to study the phosphoproteome of synaptosomes, covering 2400 phosphopeptides and providing an unbiased survey of phosphorylation changes associated with amyloid pathology. Hyperphosphorylation was detected on 36 synaptic proteins, many of which are associated with the cytoskeleton. Importantly, tau is one of the most hyperphosphorylated proteins at the synapse, upregulated at both proline-directed kinase (PDK) sites (S199/S202, S396/S404) and nonPDK sites (S400). These PDK sites correspond to well-known pathological tau epitopes in AD patients, recognized by AT8 and PHF-1 antibodies, respectively. Hyperphosphorylation at S199/S202, a rarely examined combination, was further validated in patient-derived human synaptosomes by immunoblotting. Global surveys of upregulated phosphosites revealed 2 potential kinase motifs, which resemble those of cyclin-dependent kinase 5 (CDK5, a PDK) and casein kinase II (CK2, a nonPDK). Our data demonstrate that, within synaptic compartments, amyloid pathology is associated with tau hyperphosphorylation at disease-relevant epitopes. This provides a plausible mechanism by which Aß promotes the spreading of tauopathy.
Assuntos
Doença de Alzheimer/patologia , Córtex Cerebral/patologia , Hipocampo/patologia , Sinapses/metabolismo , Sinaptossomos/patologia , Proteínas tau/metabolismo , Doença de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Animais , Cromatografia de Afinidade , Modelos Animais de Doenças , Regulação da Expressão Gênica/genética , Humanos , Camundongos , Camundongos Transgênicos , Mutação/genética , Fosforilação , Presenilina-1/genética , Sinapses/patologia , Sinaptossomos/metabolismoRESUMO
The spread of neurofibrillary tangle (NFT) pathology through the human brain is a hallmark of Alzheimer's disease (AD), which is thought to be caused by the propagation of "seeding" competent soluble misfolded tau. "TauC3", a C-terminally truncated form of tau that is generated by caspase-3 cleavage at D421, has previously been observed in NFTs and has been implicated in tau toxicity. Here we show that TauC3 is found in the seeding competent high molecular weight (HMW) protein fraction of human AD brain. Using a specific TauC3 antibody, we were able to substantially block the HMW tau seeding activity of human AD brain extracts in an in vitro tau seeding FRET assay. We propose that TauC3 could contribute to the templated tau misfolding that leads to NFT spread in AD brains.
Assuntos
Doença de Alzheimer/metabolismo , Anticorpos/metabolismo , Encéfalo/metabolismo , Proteínas tau/imunologia , Idoso de 80 Anos ou mais , Especificidade de Anticorpos , Caspase 3/metabolismo , Linhagem Celular , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Emaranhados Neurofibrilares/metabolismo , Dobramento de Proteína , Proteínas tau/químicaRESUMO
OBJECTIVE: To better understand cross-sectional relationships between CSF and PET measures of tau pathology, we compared regional and global measures of (18)F-T807 (AV-1451) PET to CSF protein levels of total tau (t-tau), phosphorylated tau (p-tau), and ß-amyloid 1-42 (Aß42). METHODS: T-tau, p-tau, and Aß42 levels were assessed using INNOTEST xMAP immunoassays. Linear regression was used to compare regional and global measures of (18)F-T807 standardized uptake value ratios (SUVR) to CSF protein levels using data from 31 cognitively unimpaired elderly participants in the Harvard Aging Brain study. RESULTS: After controlling for sex and age, total cortical (18)F-T807 binding was significantly correlated with p-tau (partial r = 0.48; p < 0.01) and at trend level with t-tau (partial r = 0.30; p = 0.12). Regional (18)F-T807 measures were more strongly correlated with CSF protein levels than the global measure, with both t-tau and p-tau significantly correlated with (18)F-T807 SUVR in entorhinal, parahippocampal, and inferior temporal cortical regions (partial r = 0.53-0.73). Peak correlations between CSF and PET measures of tau were similar to those between CSF and PET measures of amyloid burden. Finally, we observed significantly higher temporal T807 SUVR in individuals with high amyloid burden. CONCLUSIONS: These data support the link between (18)F-T807 PET and CSF measures of tau pathology. In these cognitively normal individuals with (18)F-T807 binding largely restricted to the temporal lobe, (18)F-T807 SUVR in temporal regions appeared more reflective of CSF t-tau and p-tau than a total cortical measure.
Assuntos
Encéfalo/diagnóstico por imagem , Carbolinas/farmacocinética , Lobo Temporal/diagnóstico por imagem , Proteínas tau/líquido cefalorraquidiano , Idoso , Idoso de 80 Anos ou mais , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Apolipoproteína E4/genética , Estudos Transversais , Feminino , Avaliação Geriátrica , Humanos , Imageamento por Ressonância Magnética , Masculino , Entrevista Psiquiátrica Padronizada , Fosforilação , Tomografia por Emissão de Pósitrons , Lobo Temporal/efeitos dos fármacosRESUMO
Stress exposure and the corticotropin-releasing factor (CRF) system have been implicated as mechanistically involved in both Alzheimer's disease (AD) and associated rodent models. In particular, the major stress receptor, CRF receptor type 1 (CRFR1), modulates cellular activity in many AD-relevant brain areas, and has been demonstrated to impact both tau phosphorylation and amyloid-ß (Aß) pathways. The overarching goal of our laboratory is to develop and characterize agents that impact the CRF signaling system as disease-modifying treatments for AD. In the present study, we developed a novel transgenic mouse to determine whether partial or complete ablation of CRFR1 was feasible in an AD transgenic model and whether this type of treatment could impact Aß pathology. Double transgenic AD mice (PSAPP) were crossed to mice null for CRFR1; resultant CRFR1 heterozygous (PSAPP-R1(+/-)) and homozygous (PSAPP-R1(-/-)) female offspring were used at 12 months of age to examine the impact of CRFR1 disruption on the severity of AD Aß levels and pathology. We found that both PSAPP-R1(+/-) and PSAPP-R1(-/-) had significantly reduced Aß burden in the hippocampus, insular, rhinal, and retrosplenial cortices. Accordingly, we observed dramatic reductions in Aß peptides and AßPP-CTFs, providing support for a direct relationship between CRFR1 and Aß production pathways. In summary, our results suggest that interference of CRFR1 in an AD model is tolerable and is efficacious in impacting Aß neuropathology.
Assuntos
Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Receptores de Hormônio Liberador da Corticotropina/deficiência , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Placa Amiloide/metabolismo , Placa Amiloide/patologia , Presenilina-1/genética , Presenilina-1/metabolismo , Receptores de Hormônio Liberador da Corticotropina/genéticaRESUMO
Clinical and basic science research suggests that stress and/or changes in central stress signaling intermediates may be involved in Alzheimer's disease (AD) pathogenesis. Although the links between stress and AD remain unsettled, data from our group and others have established that stress exposure in rodents may confer susceptibility to AD pathology by inducing hippocampal tau phosphorylation (tau-P). Work in our laboratory has shown that stress-induced tau-P requires activation of the type-1 corticotropin-releasing factor receptor (CRFR1). CRF overexpressing (CRF-OE) mice are a model of chronic stress that display cognitive impairment at 9-10 month of age. In this study we used 6-7 month old CRF-OE mice to examine whether sustained exposure to CRF and stress steroids would impact hippocampal tau-P and kinase activity in the presence or absence of the CRFR1-specific antagonist, R121919, given daily for 30 days. CRF-OE mice had significantly elevated tau-P compared to wild type (WT) mice at the AT8 (S202/T204), PHF-1 (S396/404), S262, and S422 sites. Treating CRF-OE mice with R121919 blocked phosphorylation at the AT8 (S202/T204) and PHF-1 (S396/404) sites, but not at the S262 and S422 sites and reduced phosphorylation of c-Jun N Terminal Kinase (JNK). Examination of hippocampal extracts from CRF-OE mice at the ultrastructural level revealed negatively stained round/globular aggregates that were positively labeled by PHF-1. These data suggest critical roles for CRF and CRFR1 in tau-P and aggregation and may have implications for the development of AD cognitive decline.
Assuntos
Hormônio Liberador da Corticotropina/metabolismo , Hipocampo/metabolismo , Agregação Patológica de Proteínas/metabolismo , Receptores de Hormônio Liberador da Corticotropina/metabolismo , Proteínas tau/metabolismo , Animais , Hormônio Liberador da Corticotropina/genética , Hipocampo/patologia , Camundongos , Camundongos Transgênicos , Fosforilação , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/patologia , Receptores de Hormônio Liberador da Corticotropina/genéticaRESUMO
Tau pathology is known to spread in a hierarchical pattern in Alzheimer's disease (AD) brain during disease progression, likely by trans-synaptic tau transfer between neurons. However, the tau species involved in inter-neuron propagation remains unclear. To identify tau species responsible for propagation, we examined uptake and propagation properties of different tau species derived from postmortem cortical extracts and brain interstitial fluid of tau-transgenic mice, as well as human AD cortices. Here we show that PBS-soluble phosphorylated high-molecular-weight (HMW) tau, though very low in abundance, is taken up, axonally transported, and passed on to synaptically connected neurons. Our findings suggest that a rare species of soluble phosphorylated HMW tau is the endogenous form of tau involved in propagation and could be a target for therapeutic intervention and biomarker development.