RESUMO
Intraneuronal aggregates of the microtubule binding protein Tau are a hallmark of different neurodegenerative diseases including Alzheimer's disease (AD). In these aggregates, Tau is modified by posttranslational modifications such as phosphorylation as well as by proteolytic cleavage. Here we identify a novel Tau cleavage site at aspartate 65 (D65) that is specific for caspase-2. In addition, we show that the previously described cleavage site at D421 is also efficiently processed by caspase-2, and both sites are cleaved in human brain samples. Caspase-2-generated Tau fragments show increased aggregation potential in vitro, but do not accumulate in vivo after AAV-mediated overexpression in mouse hippocampus. Interestingly, we observe that steady-state protein levels of caspase-2 generated Tau fragments are low in our in vivo model despite strong RNA expression, suggesting efficient clearance. Consistent with this hypothesis, we find that caspase-2 cleavage significantly improves the recognition of Tau by the ubiquitin E3 ligase CHIP, leading to increased ubiquitination and faster degradation of Tau fragments. Taken together our data thus suggest that CHIP-induced ubiquitination is of particular importance for the clearance of caspase-2 generated Tau fragments in vitro and in vivo.
Assuntos
Caspase 2 , Proteínas tau , Humanos , Masculino , Feminino , Animais , Camundongos , Modelos Animais de Doenças , Proteínas tau/química , Proteínas tau/genética , Proteínas tau/metabolismo , Caspase 2/metabolismo , Encéfalo/metabolismo , Imunoprecipitação da Cromatina , UbiquitinaçãoRESUMO
OBJECTIVES: Rapid and reliable exclusion of invasive fungal infections (IFI) by markers able to avoid unnecessary empirical antifungal treatment is still a critical unmet clinical need. We investigated the diagnostic performance of a newly available ß-d-Glucan (BDG) quantification assay, focusing on the optimisation of the BDG cut-off values for IFI exclusion. METHODS: BDG results by Wako ß-glucan assay (lower limit of detection [LLOD] = 2.16 pg/mL, positivity ≥ 11 pg/mL) on two consecutive serum samples were retrospectively analysed in 170 patients, admitted to haematological wards (N = 42), intensive care units (ICUs; N = 80), or other wards (N = 48), exhibiting clinical signs and/or symptoms suspected for IFI. Only patients with proven IFI (EORTC/MSG criteria) were considered as true positives in the assessment of BDG sensitivity, specificity and predictive values. RESULTS: Patients were diagnosed with no IFI (69.4%), proven IFI (25.3%) or probable IFI (5.3%). Two consecutive BDG values < LLOD performed within a median of 1 (interquartile range: 1-3) day were able to exclude a proven IFI with 100% sensitivity and negative predictive value (primary study goal). Test's specificity improved by using two distinct positivity and negativity cut-offs (7.7 pg/mL and LLOD, respectively), but remained suboptimal in ICU patients (50%), as compared to haematological or other patients (93% and 90%, respectively). CONCLUSIONS: The classification of Wako's results as negative when < LLOD, and positive when > 7.7 pg/mL, could be a promising diagnostic approach to confidently rule out an IFI in both ICU and non-ICU patients. The poor specificity in the ICU setting remains a concern, due to the difficulty to interpret positive results in this fragile population.
Assuntos
Testes Diagnósticos de Rotina/métodos , Infecções Fúngicas Invasivas/diagnóstico , beta-Glucanas/sangue , Idoso , Antifúngicos/uso terapêutico , Caspofungina/uso terapêutico , Feminino , Fluconazol/uso terapêutico , Humanos , Unidades de Terapia Intensiva , Infecções Fúngicas Invasivas/tratamento farmacológico , Limite de Detecção , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Estudos Retrospectivos , Sensibilidade e Especificidade , Voriconazol/uso terapêuticoRESUMO
To illustrate the effectiveness of our intensive multidisciplinary management (IMM) in the treatment of severely ill patients with necrotizing soft tissue infections (NSTIs). A retrospective observational study was conducted in a general ICU. Thirty-two consecutive patients undergoing IMM were carefully compared with 30 consecutive patients receiving a standard management (SM). IMM combined intensive care management, early surgical debridement followed by daily inspection of surgical wounds, close microbiological surveillance, and targeted high-dose antibiotics. IMM was associated with the better decrease of daily SOFA score (p = 0.04). Also, IMM caused + 12% increase in the overall number of surgical procedures (p = 0.022) and a higher number of tissue biopsies/per day (median 0.63 versus 0.32; p = 0.025), leading to a more targeted antimicrobial changes (89.6% vs 51.6%; p < 0.00001). High-dose daptomycin (75% vs 36.7%; p = 0.002) and extended/continuous infusion of beta-lactams (75% vs 43.3%; p = 0.011) were more frequently utilized. A specific efficiency score correlated with the decrease of SOFA score (efficacy) in IMM patients only (p = 0.027). Finally, IMM was associated with a significant lower ICU mortality rate (15.6% vs 40%; p = 0.032). IMM was more effective than SM as it allowed the earlier control of infection and the faster reduction of multiple organ-dysfunction.
Assuntos
Cuidados Críticos/métodos , Necrose/terapia , Infecções dos Tecidos Moles/terapia , Adulto , Idoso , Anti-Infecciosos/uso terapêutico , Cuidados Críticos/normas , Desbridamento , Feminino , Mortalidade Hospitalar , Humanos , Unidades de Terapia Intensiva/estatística & dados numéricos , Masculino , Pessoa de Meia-Idade , Necrose/patologia , Escores de Disfunção Orgânica , Avaliação de Programas e Projetos de Saúde , Estudos Retrospectivos , Infecções dos Tecidos Moles/mortalidade , Infecções dos Tecidos Moles/patologiaRESUMO
Tauopathies are neurodegenerative diseases characterized by intraneuronal inclusions of hyperphosphorylated tau protein and abnormal expression of brain-derived neurotrophic factor (BDNF), a key modulator of neuronal survival and function. The severity of both these pathological hallmarks correlate with the degree of cognitive impairment in patients. However, how tau pathology specifically modifies BDNF signaling and affects neuronal function during early prodromal stages of tauopathy remains unclear. Here, we report that the mild tauopathy developing in retinal ganglion cells (RGCs) of the P301S tau transgenic (P301S) mouse induces functional retinal changes by disrupting BDNF signaling via the TrkB receptor. In adult P301S mice, the physiological visual response of RGCs to pattern light stimuli and retinal acuity decline significantly. As a consequence, the activity-dependent secretion of BDNF in the vitreous is impaired in P301S mice. Further, in P301S retinas, TrkB receptors are selectively upregulated, but uncoupled from downstream extracellular signal-regulated kinase (ERK) 1/2 signaling. We also show that the impairment of TrkB signaling is triggered by tau pathology and mediates the tau-induced dysfunction of visual response. Overall our results identify a neurotrophin-mediated mechanism by which tau induces neuronal dysfunction during prodromal stages of tauopathy and define tau-driven pathophysiological changes of potential value to support early diagnosis and informed therapeutic decisions. SIGNIFICANCE STATEMENT: This work highlights the potential molecular mechanisms by which initial tauopathy induces neuronal dysfunction. Combining clinically used electrophysiological techniques (i.e., electroretinography) and molecular analyses, this work shows that in a relevant model of early tauopathy, the retina of the P301S mutant human tau transgenic mouse, mild tau pathology results in functional changes of neuronal activity, likely due to selective impairment of brain-derived neurotrophic factor signaling via its receptor, TrkB. These findings may have important translational implications for early diagnosis in a subset of Alzheimer's disease patients with early visual symptoms and emphasize the need to clarify the pathophysiological changes associated with distinct tauopathy stages to support informed therapeutic decisions and guide drug discovery.
Assuntos
Tauopatias/fisiopatologia , Proteínas tau , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Eletrorretinografia , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fatores de Crescimento Neural/genética , Estimulação Luminosa , Receptor trkB/genética , Células Ganglionares da Retina , Extratos de Tecidos/farmacologia , Visão Ocular , Acuidade VisualRESUMO
Adult-onset autosomal dominant leukodystrophy (ADLD) is a slowly progressive neurological disorder characterized by autonomic dysfunction, followed by cerebellar and pyramidal features. ADLD is caused by duplication of the lamin B1 gene (LMNB1), which leads to its increased expression. The molecular pathways involved in the disease are still poorly understood. Hence, we analyzed global gene expression in fibroblasts and whole blood of LMNB1 duplication carriers and used Gene Set Enrichment Analysis to explore their gene signatures. We found that LMNB1 duplication is associated with dysregulation of genes involved in the immune system, neuronal and skeletal development. Genes with an altered transcriptional profile clustered in specific genomic regions. Among the dysregulated genes, we further studied the role of RAVER2, which we found to be overexpressed at mRNA and protein level. RAVER2 encodes a putative trans regulator of the splicing repressor polypyrimidine tract binding protein (PTB) and is likely implicated in alternative splicing regulation. Functional studies demonstrated an abnormal splicing pattern of several PTB-target genes and of the myelin protein gene PLP1, previously demonstrated to be involved in ADLD. Mutant mice with different lamin B1 expression levels confirmed that Raver2 expression is dependent on lamin B1 in neural tissue and determines an altered splicing pattern of PTB-target genes and Plp1. Overall our results demonstrate that deregulation of lamin B1 expression induces modified splicing of several genes, likely driven by raver-2 overexpression, and suggest that an alteration of mRNA processing could be a pathogenic mechanism in ADLD.
Assuntos
Processamento Alternativo , Ribonucleoproteínas Nucleares Heterogêneas/genética , Lamina Tipo B/genética , Doença de Pelizaeus-Merzbacher/genética , Animais , Fibroblastos/metabolismo , Duplicação Gênica , Perfilação da Expressão Gênica , Humanos , Leucócitos/metabolismo , Camundongos , Camundongos Knockout , Doença de Pelizaeus-Merzbacher/metabolismo , Regulação para CimaRESUMO
Chromosomal rearrangements with duplication of the lamin B1 (LMNB1) gene underlie autosomal dominant adult-onset demyelinating leukodystrophy (ADLD), a rare neurological disorder in which overexpression of LMNB1 causes progressive central nervous system demyelination. However, we previously reported an ADLD family (ADLD-1-TO) without evidence of duplication or other mutation in LMNB1 despite linkage to the LMNB1 locus and lamin B1 overexpression. By custom array-CGH, we further investigated this family and report here that patients carry a large (â¼660 kb) heterozygous deletion that begins 66 kb upstream of the LMNB1 promoter. Lamin B1 overexpression was confirmed in further ADLD-1-TO tissues and in a postmortem brain sample, where lamin B1 was increased in the frontal lobe. Through parallel studies, we investigated both loss of genetic material and chromosomal rearrangement as possible causes of LMNB1 overexpression, and found that ADLD-1-TO plausibly results from an enhancer adoption mechanism. The deletion eliminates a genome topological domain boundary, allowing normally forbidden interactions between at least three forebrain-directed enhancers and the LMNB1 promoter, in line with the observed mainly cerebral localization of lamin B1 overexpression and myelin degeneration. This second route to LMNB1 overexpression and ADLD is a new example of the relevance of regulatory landscape modifications in determining Mendelian phenotypes.
Assuntos
Elementos Facilitadores Genéticos , Lamina Tipo B/genética , Doença de Pelizaeus-Merzbacher/genética , Deleção de Sequência , Animais , Sequência de Bases , Células Cultivadas , Análise Mutacional de DNA , Feminino , Expressão Gênica , Regulação da Expressão Gênica , Estudos de Associação Genética , Humanos , Lamina Tipo B/metabolismo , Masculino , Camundongos Transgênicos , Pessoa de Meia-Idade , Dados de Sequência Molecular , LinhagemRESUMO
The architecture and structural mechanics of the cell nucleus are defined by the nuclear lamina, which is formed by A- and B-type lamins. Recently, gene duplication and protein overexpression of lamin B1 (LB1) have been reported in pedigrees with autosomal dominant leukodystrophy (ADLD). However, how the overexpression of LB1 affects nuclear mechanics and function and how it may result in pathology remain unexplored. Here, we report that in primary human skin fibroblasts derived from ADLD patients, LB1, but not other lamins, is overexpressed at the nuclear lamina and specifically enhances nuclear stiffness. Transient transfection of LB1 in HEK293 and neuronal N2a cells mimics the mechanical phenotype of ADLD nuclei. Notably, in ADLD fibroblasts, reducing LB1 protein levels by shRNA knockdown restores elasticity values to those indistinguishable from control fibroblasts. Moreover, isolated nuclei from ADLD fibroblasts display a reduced nuclear ion channel open probability on voltage-step application, suggesting that biophysical changes induced by LB1 overexpression may alter nuclear signaling cascades in somatic cells. Overall, the overexpression of LB1 in ADLD cells alters nuclear mechanics and is linked to changes in nuclear signaling, which could help explain the pathogenesis of this disease.
Assuntos
Núcleo Celular/patologia , Embrião de Mamíferos/citologia , Fibroblastos/patologia , Lamina Tipo B/metabolismo , Doença de Pelizaeus-Merzbacher/patologia , Pele/citologia , Adulto , Animais , Western Blotting , Estudos de Casos e Controles , Permeabilidade da Membrana Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proliferação de Células , Células Cultivadas , Embrião de Mamíferos/metabolismo , Feminino , Fibroblastos/metabolismo , Citometria de Fluxo , Imunofluorescência , Humanos , Lamina Tipo B/antagonistas & inibidores , Lamina Tipo B/genética , Masculino , Camundongos , Pessoa de Meia-Idade , Técnicas de Patch-Clamp , Doença de Pelizaeus-Merzbacher/genética , Doença de Pelizaeus-Merzbacher/metabolismo , Fenótipo , RNA Interferente Pequeno/genética , Pele/metabolismoRESUMO
Some symmetrical and unsymmetrical thiacarbocyanines bearing NO-donor nitrooxy and furoxan moieties were synthesized and studied as candidate anti-Alzheimer's drugs. All products activated soluble guanylate cyclase (sGC) in a dose-dependent manner, depending on the presence in their structures of NO-donor groups. None displayed toxicity when tested at concentrations below 10 µM on human brain microvascular endothelial cells (hCMEC/D3). Some products were capable of inhibiting amyloid ß-protein (Aß) aggregation, with a potency in the low µM concentration range, and of inhibiting aggregation of human recombinant tau protein in amyloid fibrils when incubated with the protein at 1 µM concentration. Nitrooxy derivative 21 and furoxan derivative 22 were selected to investigate synaptic plasticity. Both products, tested at 2 µM concentration, counteracted the inhibition of long-term potentiation (LTP) induced by Aß42 in hippocampal brain slices.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Carbocianinas/uso terapêutico , Doadores de Óxido Nítrico/uso terapêutico , HumanosRESUMO
Adult-onset autosomal dominant leukodystrophy (ADLD) is a slowly progressive neurological disorder characterised by pyramidal, cerebellar, and autonomic disturbances. Duplication of the LMNB1 gene is the genetic cause of ADLD, yet the pathogenetic mechanism is not defined. In this study, we analysed cells and muscle tissue from three patients affected by ADLD, carrying an extra copy of the LMNB1 gene. Lamin B1 levels were dramatically increased in ADLD nuclei, both in skin fibroblasts and skeletal muscle fibres. Since lamin B1 is known to bind Oct-1, a transcription factor involved in the oxidative stress pathway, we investigated Oct-1 fate in ADLD. Oct-1 recruitment to the nuclear periphery was increased in ADLD cells, while nucleoplasmic localisation of the transcription factor under oxidative stress conditions was reduced. Importantly, lamin B1 degradation occurring in some, but not all ADLD cell lines, slowed down lamin B1 and Oct-1 accumulation. In skeletal muscle, focal disorganisation of sarcomeres was observed, while IIB-myosin heavy chain, an Oct-1 target gene, was under-expressed and rod-containing fibres were formed. These data show that a high degree of regulation of lamin B1 expression is implicated in the different clinical phenotypes observed in ADLD and show that altered Oct-1 nuclear localisation contributes to the disease phenotype.
Assuntos
Lamina Tipo B/metabolismo , Membrana Nuclear/metabolismo , Fator 1 de Transcrição de Octâmero/metabolismo , Doença de Pelizaeus-Merzbacher/metabolismo , Western Blotting , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Células Cultivadas , Feminino , Fibroblastos/metabolismo , Duplicação Gênica , Humanos , Lamina Tipo B/genética , Masculino , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Pessoa de Meia-Idade , Músculo Esquelético/metabolismo , Músculo Esquelético/ultraestrutura , Membrana Nuclear/ultraestrutura , Doença de Pelizaeus-Merzbacher/genéticaRESUMO
During neuronal differentiation, axonal elongation is regulated by both external and intrinsic stimuli, including neurotropic factors, cytoskeleton dynamics, second messengers such as cyclic adenosine monophosphate (cAMP), and neuronal excitability. Chloride intracellular channel 1 (CLIC1) is a cytoplasmic hydrophilic protein that, upon stimulation, dimerizes and translocates to the plasma membrane, where it contributes to increase the membrane chloride conductance. Here, we investigated the expression of CLIC1 in primary hippocampal neurons and retinal ganglion cells (RGCs) and examined how the functional expression of CLIC1 specifically modulates neurite outgrowth of neonatal murine RGCs. Using a combination of electrophysiology and immunohistochemistry, we found that CLIC1 is expressed in hippocampal neurons and RGCs and that the chloride current mediated by CLIC1 is required for maintaining growth cone morphology and sustaining cAMP-stimulated neurite elongation in dissociated immunopurified RGCs. In cultured RGCs, inhibition of CLIC1 ionic current through the pharmacological blocker IAA94 or a specific anti-CLIC1 antibody directed against its extracellular domain prevents the neurite outgrowth induced by cAMP. CLIC1-mediated chloride current, which results from an increased open probability of the channel, is detected only when cAMP is elevated. Inhibition of protein kinase A prevents such current. These results indicate that CLIC1 functional expression is regulated by cAMP via protein kinase A and is required for neurite outgrowth modulation during neuronal differentiation. Using a combination of electrophysiology and immunohistochemistry, we found that the chloride intracellular channel 1 (CLIC1) protein modulates the speed of neurite growth. The chloride current mediated by CLIC1 is essential for maintaining growth cone morphology and is required for sustaining cAMP-stimulated neurite elongation in dissociated immunopurified neurons. The presence of either the CLIC1 current blocker IAA94 or the anti-CLIC1 antibody inhibits neurite growth of Retina Ganglion Cells cultured in the presence of 10 micromolar forskolin for 24 h.
Assuntos
Canais de Cloreto/metabolismo , AMP Cíclico/farmacologia , Neuritos/efeitos dos fármacos , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/efeitos dos fármacos , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Animais , Animais Recém-Nascidos , Anticorpos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Canais de Cloreto/imunologia , Colforsina/farmacologia , Inibidores Enzimáticos/farmacologia , Glicolatos/farmacologia , Hipocampo/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Vídeo , Técnicas de Patch-Clamp , Retina/citologiaRESUMO
BACKGROUND: Gamma-hydroxybutyric acid (GHB) at low dosages has anxiolytic effects and promotes REM sleep and low-wave deep sleep. In the U.S., the legal form of GHB is prescribed to adults suffering from narcolepsy-associated cataplexy; the sodium salt of GHB is reserved for alcohol-addiction treatment. GHB is also a molecule of abuse and recreational use, it is a controlled substance in several countries, so gamma-valerolactone (GVL) has frequently been used as a legal substitute for it. GHB's abuse profile is most likely attributable to its anxiolytic, hypnotic, and euphoric properties, as well as its widespread availability and inexpensive/low cost on the illicit market. METHODS: Our study is focused on evaluating the potential effects on the mouse brain after repeated/prolonged administration of GHB and GVL at a pharmacologically active dose (100 mg/kg) through behavioral study and immunohistochemical analysis using the markers tetraspanin 17 (TSPAN17), aldehyde dehydrogenase 5 (ALDH5A1), Gamma-aminobutyric acid type A receptor (GABA-A), and Gamma-aminobutyric acid type B receptor (GABA-B). RESULTS: Our findings revealed that prolonged administration of GHB and GVL at a pharmacologically active dose (100 mg/kg) can have effects on a component of the mouse brain, the intensity of which can be assessed using immunohistochemistry. The findings revealed that long-term GHB administration causes a significant plastic alteration of the GHB signaling system, with downregulation of the putative binding site (TSPAN17) and overexpression of ALDH5A1, especially in hippocampal neurons. Our findings further revealed that GABA-A and GABA-B receptors are downregulated in these brain locations, resulting in a greater decrease in GABA-B expression. CONCLUSIONS: The goal of this study, from the point of view of forensic pathology, is to provide a new methodological strategy for better understanding the properties of this controversial substance, which could help us better grasp the unknown mechanism underlying its abuse profile.
RESUMO
Tau seed amplification assays (SAAs) directly measure the seeding activity of tau and would therefore be ideal biomarkers for clinical trials targeting seeding-competent tau in Alzheimer's disease (AD). However, the precise relationship between tau seeding measured by SAA and the levels of pathological forms of tau in the AD brain remains unknown. We developed a new tau SAA based on full-length 0N3R tau with sensitivity in the low fg/ml range and used it to characterize 103 brain samples from three independent cohorts. Tau seeding clearly discriminated between AD and control brain samples. Interestingly, seeding was absent in Progressive Supranuclear Palsy (PSP) putamen, suggesting that our tau SAA did not amplify 4R tau aggregates from PSP brain. The specificity of our tau SAA for AD brain was further supported by analysis of matched hippocampus and cerebellum samples. While seeding was detected in hippocampus from Braak stages I-II, no seeding was present in AD cerebellum that is devoid of tau inclusions. Analysis of 40 middle frontal gyrus samples encompassing all Braak stages showed that tau SAA seeding activity gradually increased with Braak stage. This relationship between seeding activity and the presence of tau inclusions in AD brain was further supported by robust correlations between tau SAA results and the levels of phosphorylated tau212/214, phosphorylated tau181, aggregated tau, and sarkosyl-insoluble tau. Strikingly, we detected tau seeding in the middle frontal gyrus already at Braak stage II-III, suggesting that tau SAA can detect tau pathology earlier than conventional immunohistochemical staining. In conclusion, our data suggest a quantitative relationship between tau seeding activity and pathological forms of tau in the human brain and provides an important basis for further development of tau SAA for accessible human samples.
Assuntos
Doença de Alzheimer , Paralisia Supranuclear Progressiva , Humanos , Doença de Alzheimer/patologia , Proteínas tau/metabolismo , Encéfalo/patologia , Paralisia Supranuclear Progressiva/patologia , Cerebelo/patologiaRESUMO
Tau protein fibrillization is implicated in the pathogenesis of several neurodegenerative diseases collectively known as Tauopathies. For decades, investigating Tau fibrillization in vitro has required the addition of polyanions or other co-factors to induce its misfolding and aggregation, with heparin being the most commonly used. However, heparin-induced Tau fibrils exhibit high morphological heterogeneity and a striking structural divergence from Tau fibrils isolated from Tauopathies patients' brains at ultra- and macro-structural levels. To address these limitations, we developed a quick, cheap, and effective method for producing completely co-factor-free fibrils from all full-length Tau isoforms and mixtures thereof. We show that Tau fibrils generated using this ClearTau method - ClearTau fibrils - exhibit amyloid-like features, possess seeding activity in biosensor cells and hiPSC-derived neurons, retain RNA-binding capacity, and have morphological properties and structures more reminiscent of the properties of the brain-derived Tau fibrils. We present the proof-of-concept implementation of the ClearTau platform for screening Tau aggregation-modifying compounds. We demonstrate that these advances open opportunities to investigate the pathophysiology of disease-relevant Tau aggregates and will facilitate the development of Tau pathology-targeting and modifying therapies and PET tracers that can distinguish between different Tauopathies.
Assuntos
Agregação Patológica de Proteínas , Proteínas tau , Proteínas tau/química , Heparina/química , Humanos , Linhagem Celular , Técnicas Biossensoriais , Células-Tronco Pluripotentes , Neurônios , Isoformas de Proteínas , Microscopia CrioeletrônicaRESUMO
Epidemiological, clinical and experimental evidence suggests a link between type 2 diabetes and Alzheimer's disease (AD). Insulin modulates metabolism of beta-amyloid precursor protein (APP) in neurons, decreasing the intracellular accumulation of beta-amyloid (Abeta) peptides, which are pivotal in AD pathogenesis. The present study investigates whether the widely prescribed insulin-sensitizing drug, metformin (Glucophage(R)), affects APP metabolism and Abeta generation in various cell models. We demonstrate that metformin, at doses that lead to activation of the AMP-activated protein kinase (AMPK), significantly increases the generation of both intracellular and extracellular Abeta species. Furthermore, the effect of metformin on Abeta generation is mediated by transcriptional up-regulation of beta-secretase (BACE1), which results in an elevated protein level and increased enzymatic activity. Unlike insulin, metformin exerts no effect on Abeta degradation. In addition, we found that glucose deprivation and various tyrphostins, known inhibitors of insulin-like growth factors/insulin receptor tyrosine kinases, do not modulate the effect of metformin on Abeta. Finally, inhibition of AMP-activated protein kinase (AMPK) by the pharmacological inhibitor Compound C largely suppresses metformin's effect on Abeta generation and BACE1 transcription, suggesting an AMPK-dependent mechanism. Although insulin and metformin display opposing effects on Abeta generation, in combined use, metformin enhances insulin's effect in reducing Abeta levels. Our findings suggest a potentially harmful consequence of this widely prescribed antidiabetic drug when used as a monotherapy in elderly diabetic patients.
Assuntos
Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/genética , Peptídeos beta-Amiloides/biossíntese , Ácido Aspártico Endopeptidases/genética , Hipoglicemiantes/farmacologia , Metformina/farmacologia , Transcrição Gênica/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Ativação Enzimática/efeitos dos fármacos , Glucose/metabolismo , Insulina/metabolismo , Insulina/farmacologia , Camundongos , Dados de Sequência Molecular , Peptídeos/metabolismo , Regiões Promotoras Genéticas , Ratos , Transdução de Sinais/efeitos dos fármacosRESUMO
In Alzheimer disease, Tau pathology is thought to propagate from cell to cell throughout interconnected brain areas. However, the forms of Tau released into the brain interstitial fluid (ISF) in vivo during the development of Tauopathy and their pathological relevance remain unclear. Combining in vivo microdialysis and biochemical analysis, we find that in Tau transgenic mice, human Tau (hTau) present in brain ISF is truncated and comprises at least 10 distinct fragments spanning the entire Tau protein. The fragmentation pattern is similar across different Tau transgenic models, pathological stages and brain areas. ISF hTau concentration decreases during Tauopathy progression, while its phosphorylation increases. ISF from mice with established Tauopathy induces Tau aggregation in HEK293-Tau biosensor cells. Notably, immunodepletion of ISF phosphorylated Tau, but not Tau fragments, significantly reduces its ability to seed Tau aggregation and only a fraction of Tau, separated by ultracentrifugation, is seeding-competent. These results indicate that ISF seeding competence is driven by a small subset of Tau, which potentially contribute to the propagation of Tau pathology.
Assuntos
Encéfalo/metabolismo , Líquido Extracelular/metabolismo , Tauopatias/metabolismo , Proteínas tau/metabolismo , Animais , Modelos Animais de Doenças , Células HEK293 , Humanos , Camundongos Transgênicos , Microdiálise , Fragmentos de Peptídeos/metabolismo , Fosforilação , Agregação Patológica de Proteínas/metabolismoRESUMO
Tau is an axonal microtubule-associated protein involved in microtubule assembly and stabilization. Mutations in Tau cause frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), and tau aggregates are present in Alzheimer's disease and other tauopathies. The mechanisms leading from tau dysfunction to neurodegeneration are still debated. The dynein-activator complex dynactin has an essential role in axonal transport and mutations in its gene are associated with lower motor neuron disease. We show here for the first time that the N-terminal projection domain of tau binds to the C-terminus of the p150 subunit of the dynactin complex. Tau and dynactin show extensive colocalization, and the attachment of the dynactin complex to microtubules is enhanced by tau. Mutations of a conserved arginine residue in the N-terminus of tau, found in patients with FTDP-17, affect its binding to dynactin, which is abnormally distributed in the retinal ganglion cell axons of transgenic mice expressing human tau with a mutation in the microtubule-binding domain. These findings, which suggest a direct involvement of tau in axonal transport, have implications for understanding the pathogenesis of tauopathies.
Assuntos
Proteínas Associadas aos Microtúbulos/química , Proteínas tau/fisiologia , Animais , Arginina/química , Axônios/metabolismo , Clonagem Molecular , Complexo Dinactina , Humanos , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Biológicos , Mutação , Neurônios/metabolismo , Transtornos Parkinsonianos/genética , Ligação Proteica , Estrutura Terciária de Proteína , Técnicas do Sistema de Duplo-Híbrido , Proteínas tau/químicaRESUMO
BACKGROUND: DNA methylation has emerged as an important epigenetic regulator of brain processes, including circadian rhythms. However, how DNA methylation intervenes between environmental signals, such as light entrainment, and the transcriptional and translational molecular mechanisms of the cellular clock is currently unknown. Here, we studied the after-hours mice, which have a point mutation in the Fbxl3 gene and a lengthened circadian period. METHODS: In this study, we used a combination of in vivo, ex vivo and in vitro approaches. We measured retinal responses in Afh animals and we have run reduced representation bisulphite sequencing (RRBS), pyrosequencing and gene expression analysis in a variety of brain tissues ex vivo. In vitro, we used primary neuronal cultures combined to micro electrode array (MEA) technology and gene expression. RESULTS: We observed functional impairments in mutant neuronal networks, and a reduction in the retinal responses to light-dependent stimuli. We detected abnormalities in the expression of photoreceptive melanopsin (OPN4). Furthermore, we identified alterations in the DNA methylation pathways throughout the retinohypothalamic tract terminals and links between the transcription factor Rev-Erbα and Fbxl3. CONCLUSIONS: The results of this study, primarily represent a contribution towards an understanding of electrophysiological and molecular phenotypic responses to external stimuli in the Afh model. Moreover, as DNA methylation has recently emerged as a new regulator of neuronal networks with important consequences for circadian behaviour, we discuss the impact of the Afh mutation on the epigenetic landscape of circadian biology.
Assuntos
Ritmo Circadiano , Metilação de DNA , Animais , Ritmo Circadiano/genética , Camundongos , Mutação , Fenótipo , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: Human tauopathies including Alzheimer's disease (AD) are characterized by alterations in the post-translational modification (PTM) pattern of Tau, which parallel the formation of insoluble Tau aggregates, neuronal dysfunction and degeneration. While PTMs on aggregated Tau have been studied in detail, much less is known about the modification patterns of soluble Tau. Furthermore, PTMs other than phosphorylation have only come into focus recently and are still understudied. Soluble Tau species are likely responsible for the spreading of pathology during disease progression and are currently being investigated as targets for immunotherapies. A better understanding of their biochemical properties is thus of high importance. METHODS: We used a mass spectrometry approach to characterize Tau PTMs on a detergent-soluble fraction of human AD and control brain tissue, which led to the discovery of novel lysine methylation events. We developed specific antibodies against Tau methylated at these sites and biochemically characterized methylated Tau species in extracts from human brain, the rTg4510 mouse model and in hiPSC-derived neurons. RESULTS: Our study demonstrates that methylated Tau levels increase with Tau pathology stage in human AD samples as well as in a mouse model of Tauopathy. Methylated Tau is enriched in soluble brain extracts and is not associated with hyperphosphorylated, high molecular weight Tau species. We also show that in hiPSC-derived neurons and mouse brain, methylated Tau preferentially localizes to the cell soma and nuclear fractions and is absent from neurites. Knock down and inhibitor studies supported by proteomics data led to the identification of SETD7 as a novel lysine methyltransferase for Tau. SETD7 specifically methylates Tau at K132, an event that facilitates subsequent methylation at K130. CONCLUSIONS: Our findings indicate that methylated Tau has a specific somatic and nuclear localization, suggesting that the methylation of soluble Tau species may provide a signal for their translocation to different subcellular compartments. Since the mislocalization and depletion of Tau from axons is associated with tauopathies, our findings may shed light onto this disease-associated phenomenon.
Assuntos
Doença de Alzheimer/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas tau/metabolismo , Animais , Humanos , Lisina/metabolismo , Metilação , Camundongos , Camundongos TransgênicosRESUMO
Intraneuronal insoluble inclusions made of Tau protein are neuropathological hallmarks of Alzheimer Disease (AD). Cleavage of Tau by legumain (LGMN) has been proposed to be crucial for aggregation of Tau into fibrils. However, it remains unclear if LGMN-cleaved Tau fragments accumulate in AD Tau inclusions.Using an in vitro enzymatic assay and non-targeted mass spectrometry, we identified four putative LGMN cleavage sites at Tau residues N167-, N255-, N296- and N368. Cleavage at N368 generates variously sized N368-Tau fragments that are aggregation prone in the Thioflavin T assay in vitro. N368-cleaved Tau is not detected in the brain of legumain knockout mice, indicating that LGMN is required for Tau cleavage in the mouse brain in vivo. Using a targeted mass spectrometry method in combination with tissue fractionation and biochemical analysis, we investigated whether N368-cleaved Tau is differentially produced and aggregated in brain of AD patients and control subjects. In brain soluble extracts, despite reduced uncleaved Tau in AD, levels of N368-cleaved Tau are comparable in AD and control hippocampus, suggesting that LGMN-mediated cleavage of Tau is not altered in AD. Consistently, levels of activated, cleaved LGMN are also similar in AD and control brain extracts. To assess the potential accumulation of N368-cleaved Tau in insoluble Tau aggregates, we analyzed sarkosyl-insoluble extracts from AD and control hippocampus. Both N368-cleaved Tau and uncleaved Tau were significantly increased in AD as a consequence of pathological Tau inclusions accumulation. However, the amount of N368-cleaved Tau represented only a very minor component (< 0.1%) of insoluble Tau.Our data indicate that LGMN physiologically cleaves Tau in the mouse and human brain generating N368-cleaved Tau fragments, which remain largely soluble and are present only in low proportion in Tau insoluble aggregates compared to uncleaved Tau. This suggests that LGMN-cleaved Tau has limited role in the progressive accumulation of Tau inclusions in AD.