RESUMO
Extracellular vesicles (EVs) are secreted, cell-derived, membrane-bound compartments implicated in various diseases for their ability to influence distant targets and as carriers of biomarkers. Here, we present a protocol for separating EVs from mammalian pancreatic cancer cells and their characterization using western blot and electron microscopy. We then demonstrate how they are utilized to affect tumor development in a murine model of metastatic pancreatic cancer including a method to quantify hepatic tumor burden in histologic samples. For complete details on the use and execution of this protocol, please refer to Dudgeon et al.1.
RESUMO
The biology of metastatic pancreatic ductal adenocarcinoma (PDAC) is distinct from that of the primary tumor due to changes in cell plasticity governed by a distinct transcriptome. Therapeutic strategies that target this distinct biology are needed. We detect an upregulation of the neuronal axon guidance molecule Netrin-1 in PDAC liver metastases that signals through its dependence receptor (DR), uncoordinated-5b (Unc5b), to facilitate metastasis in vitro and in vivo. The mechanism of Netrin-1 induction involves a feedforward loop whereby Netrin-1 on the surface of PDAC-secreted extracellular vesicles prepares the metastatic niche by inducing hepatic stellate cell activation and retinoic acid secretion that in turn upregulates Netrin-1 in disseminated tumor cells via RAR/RXR and Elf3 signaling. While this mechanism promotes PDAC liver metastasis, it also identifies a therapeutic vulnerability, as it can be targeted using anti-Netrin-1 therapy to inhibit metastasis using the Unc5b DR cell death mechanism.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Hepáticas , Neoplasias Pancreáticas , Humanos , Netrina-1 , Retinoides , Células Estreladas do Fígado/metabolismo , Linhagem Celular Tumoral , Neoplasias Pancreáticas/patologia , Carcinoma Ductal Pancreático/patologia , Neoplasias Hepáticas/metabolismo , Receptores de Netrina , Proteínas de Ligação a DNA , Fatores de Transcrição , Proteínas Proto-Oncogênicas c-etsRESUMO
Neuroinflammation, through production of proinflammatory molecules and activated glial cells, is implicated in Alzheimer's disease (AD) pathogenesis. One such proinflammatory mediator is tumor necrosis factor α (TNF-α), a multifunctional cytokine produced in excess and associated with amyloid ß-driven inflammation and cognitive decline. Long-term global inhibition of TNF receptor type I (TNF-RI) and TNF-RII signaling without cell or stage specificity in triple-transgenic AD mice exacerbates hallmark amyloid and neurofibrillary tangle pathology. These observations revealed that long-term pan anti-TNF-α inhibition accelerates disease, cautions against long-term use of anti-TNF-α therapeutics for AD, and urges more selective regulation of TNF signaling. We used adeno-associated virus vector-delivered siRNAs to selectively knock down neuronal TNF-R signaling. We demonstrate divergent roles for neuronal TNF-RI and TNF-RII where loss of opposing TNF-RII leads to TNF-RI-mediated exacerbation of amyloid ß and Tau pathology in aged triple-transgenic AD mice. Dampening of TNF-RII or TNF-RI+RII leads to a stage-independent increase in Iba-1-positive microglial staining, implying that neuronal TNF-RII may act nonautonomously on the microglial cell population. These results reveal that TNF-R signaling is complex, and it is unlikely that all cells and both receptors will respond positively to broad anti-TNF-α treatments at various stages of disease. In aggregate, these data further support the development of cell-, stage-, and/or receptor-specific anti-TNF-α therapeutics for AD.
Assuntos
Doença de Alzheimer/metabolismo , Neurônios/metabolismo , Receptores do Fator de Necrose Tumoral/biossíntese , Adenoviridae/genética , Envelhecimento/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Animais , Encéfalo/patologia , Progressão da Doença , Regulação para Baixo/fisiologia , Técnicas de Silenciamento de Genes , Vetores Genéticos , Masculino , Camundongos , Camundongos Transgênicos , Microglia/metabolismo , Placa Amiloide/metabolismo , RNA Interferente Pequeno/genética , Receptores do Fator de Necrose Tumoral/deficiência , Receptores do Fator de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/biossíntese , Receptores Tipo I de Fatores de Necrose Tumoral/deficiência , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo II do Fator de Necrose Tumoral/biossíntese , Receptores Tipo II do Fator de Necrose Tumoral/deficiência , Receptores Tipo II do Fator de Necrose Tumoral/genética , Transdução de Sinais/fisiologiaRESUMO
Effective regulation of transgene product in anatomically circumscribed brain tissue is dependent on the pharmacokinetics of the regulating agent, the kinetics of transcriptional activation and degradation of the transgene product. We evaluated rapamycin-regulated AAV2-GDNF expression in the rat brain (striatum). Regulated (a dual-component system: AAV2-FBZhGDNF + AAV2-TF1Nc) and constitutive (CMV-driven) expression vectors were compared. Constitutively active AAV2-GDNF directed stable GDNF expression in a dose-dependent manner and it increased for the first month, thereafter reaching a plateau that was maintained over a further 3 months. For the AAV2-regGDNF, rapamycin was administered in a 3-days on/4-days off cycle. Intraperitoneal, oral, and direct brain delivery (CED) of rapamycin were evaluated. Two cycles of rapamycin at an intraperitoneal dose of 10 mg/kg gave the highest GDNF level (2.75±0.01 ng/mg protein). Six cycles at 3 mg/kg resulted in lower GDNF values (1.36±0.3 ng/mg protein). Interestingly, CED of rapamycin into the brain at a very low dose (50 ng) induced GDNF levels comparable to a 6-week intraperitoneal rapamycin cycle. This study demonstrates the effectiveness of rapamycin regulation in the CNS. However, the kinetics of the transgene in brain tissue, the regulator dosing amount and schedule are critical parameters that influence the kinetics of accumulation and zenith of the encoded transgene product.
Assuntos
Dependovirus/genética , Vetores Genéticos/genética , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Sirolimo/farmacologia , Transdução Genética/métodos , Animais , Relação Dose-Resposta a Droga , Esquema de Medicação , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Humanos , Imuno-Histoquímica , Cinética , Masculino , Neostriado/efeitos dos fármacos , Neostriado/metabolismo , Ratos , Ratos Sprague-Dawley , Sirolimo/administração & dosagem , Manejo de Espécimes , Fatores de TempoRESUMO
Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by severe memory loss and cognitive impairment. Neuroinflammation, including the extensive production of pro-inflammatory molecules and the activation of microglia, has been implicated in the disease process. Tumor necrosis factor (TNF)-α, a prototypic pro-inflammatory cytokine, is elevated in AD, is neurotoxic, and colocalizes with amyloid plaques in AD animal models and human brains. We previously demonstrated that the expression of TNF-α is increased in AD mice at ages preceding the development of hallmark amyloid and tau pathological features and that long-term expression of this cytokine in these mice leads to marked neuronal death. Such observations suggest that TNF-α signaling promotes AD pathogenesis and that therapeutics suppressing this cytokine's activity may be beneficial. To dissect TNF-α receptor signaling requirements in AD, we generated triple-transgenic AD mice (3xTg-AD) lacking both TNF-α receptor 1 (TNF-RI) and 2 (TNF-RII), 3xTg-ADxTNF-RI/RII knock out, the cognate receptors of TNF-α. These mice exhibit enhanced amyloid and tau-related pathological features by the age of 15 months, in stark contrast to age-matched 3xTg-AD counterparts. Moreover, 3xTg-ADxTNF-RI/RII knock out-derived primary microglia reveal reduced amyloid-ß phagocytic marker expression and phagocytosis activity, indicating that intact TNF-α receptor signaling is critical for microglial-mediated uptake of extracellular amyloid-ß peptide pools. Overall, our results demonstrate that globally ablated TNF receptor signaling exacerbates pathogenesis and argues against long-term use of pan-anti-TNF-α inhibitors for the treatment of AD.
Assuntos
Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Receptores Tipo II do Fator de Necrose Tumoral/deficiência , Receptores Tipo I de Fatores de Necrose Tumoral/deficiência , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Envelhecimento/patologia , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/fisiopatologia , Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Região CA1 Hipocampal/patologia , Região CA1 Hipocampal/fisiopatologia , Região CA3 Hipocampal/patologia , Região CA3 Hipocampal/fisiopatologia , Proteínas de Ligação ao Cálcio/metabolismo , Cruzamentos Genéticos , Feminino , Humanos , Receptores de Lipopolissacarídeos/metabolismo , Potenciação de Longa Duração , Masculino , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/metabolismo , Microglia/metabolismo , Microglia/patologia , Fagocitose , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Sinapses/metabolismo , Transgenes/genética , Fator de Necrose Tumoral alfa/metabolismo , Proteínas tau/genéticaRESUMO
The detection of myelin disruptions in Alzheimer's disease (AD)-affected brain raises the possibility that oligodendrocytes undergo pathophysiological assault over the protracted course of this neurodegenerative disease. Oligodendrocyte compromise arising from direct toxic effects imparted by pathological amyloid-beta peptides and/or through signals derived from degenerating neurons could play an important role in the disease process. We previously demonstrated that 3xTg-AD mice, which harbor the human amyloid precursor protein Swedish mutant transgene, presenilin knock-in mutation, and tau P301L mutant transgene, exhibit significant alterations in overall myelination patterns and oligodendrocyte status at time points preceding the appearance of amyloid and tau pathology. Herein, we demonstrate that Abeta(1-42) leads to increased caspase-3 expression and apoptotic cell death of both nondifferentiated and differentiated mouse oligodendrocyte precursor (mOP) cells in vitro. Through use of a recombinant adeno-associated virus serotype-2 (rAAV2) vector expressing an Abeta(1-42)-specific intracellular antibody (intrabody), oligodendrocyte and myelin marker expression, as well as myelin integrity, were restored in the vector-infused brain regions of 3xTg-AD mice. Overall, this work provides further insights into the impact of Abeta(1-42)-mediated toxicity on the temporal and spatial progression of subtle myelin disruption during the early presymptomatic stages of AD and may help to validate new therapeutic options designed to avert these early impairments.
Assuntos
Córtex Entorrinal/patologia , Bainha de Mielina/patologia , Oligodendroglia/patologia , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Análise de Variância , Animais , Apoptose/genética , Western Blotting , Caspase 3/genética , Caspase 3/metabolismo , Linhagem Celular , Modelos Animais de Doenças , Córtex Entorrinal/metabolismo , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica , Bainha de Mielina/genética , Bainha de Mielina/metabolismo , Oligodendroglia/metabolismo , Proteínas tau/genética , Proteínas tau/metabolismoRESUMO
Alzheimer's disease (AD) is a progressive dementing disorder characterized by age-related amyloid-beta (Abeta) deposition, neurofibrillary tangles, and synapse and neuronal loss. It is widely recognized that Abeta is a principal pathogenic mediator of AD. Our goal was to develop an immunotherapeutic approach, which would specifically lead to the clearance and/or neutralization of Abeta in the triple transgenic mouse model (3xTg-AD). These mice develop the amyloid and tangle pathologies and synaptic dysfunction reminiscent of human AD. Using a human single-chain variable fragment (scFv) antibody phage display library, a novel scFv antibody specific to Abeta was isolated, its activity characterized in vitro, and its open reading frame subsequently cloned into a recombinant adeno-associated virus (rAAV) vector. Three-month-old 3xTg-AD mice were intrahippocampally infused with serotype-1 rAAV vectors encoding Abeta-scFv or a control vector using convection-enhanced delivery (CED). Mice receiving rAAV1-Abeta-scFv harbored lower levels of insoluble Abeta and hyperphosphorylated tau, and exhibited improved cognitive function as measured by the Morris Water Maze (MWM) spatial memory task. These results underscore the potential of gene-based passive vaccination for AD, and provide further rationale for the development of Abeta-targeting strategies for this debilitating disease.
Assuntos
Doença de Alzheimer/patologia , Doença de Alzheimer/terapia , Peptídeos beta-Amiloides/imunologia , Dependovirus/genética , Aprendizagem em Labirinto/fisiologia , Anticorpos de Cadeia Única/metabolismo , Doença de Alzheimer/metabolismo , Animais , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Células Cultivadas , Cricetinae , Ensaio de Imunoadsorção Enzimática , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Transgênicos , Anticorpos de Cadeia Única/genéticaRESUMO
Soluble Abeta oligomers are recognized as playing a key role in Alzheimer's disease (AD) pathophysiology. Despite their significance, many investigators encounter difficulty generating reliable preparations for in vitro and in vivo experiments. Solutions of Abeta are often unstable and soluble conformer profiles inconsistent. In this study we describe detailed methods for preparing Abeta oligomers that are stable for several weeks and are enriched for low and high molecular weight oligomeric forms, including the 56-kDa form, a conformer implicated in AD-related cognitive impairment. We characterize their structural and functional properties using Western blot, dot blot, atomic force microscopy, Thioflavine T fluorescence, and primary neuronal culture toxicity assays. These synthetic preparations should prove valuable to many studying Abeta-mediated mechanisms underlying AD.
Assuntos
Peptídeos beta-Amiloides/síntese química , Multimerização Proteica , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/ultraestrutura , Animais , Western Blotting , Morte Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Fluorescência , Immunoblotting , Camundongos , Microscopia de Força Atômica , Peso Molecular , Neurônios/citologia , Neurônios/metabolismo , Solubilidade , Fatores de TempoRESUMO
Inflammatory processes, including the episodic and/ or chronic elaboration of cytokines, have been identified as playing key roles in a number of neurological disorders. Whether these activities impart a disease-resolving and/or contributory outcome depends at least in part on the disease context, stage of pathogenesis, and cellular milieu in which these factors are released. Interferon-gamma (IFNgamma) is one such cytokine that produces pleiotropic effects in the brain. It is protective by ensuring maintenance of virus latency after infection, yet deleterious by recruiting and activating microglia that secrete potentially damaging factors at sites of brain injury. Using the triple-transgenic mouse model of Alzheimer's disease (3xTg-AD), which develops amyloid and tau pathologies in a pattern reminiscent of human Alzheimer's disease, we initiated chronic intrahippocampal expression of IFNgamma through delivery of a serotype-1 recombinant adeno-associated virus vector (rAAV1-IFNgamma). Ten months of IFNgamma expression led to an increase in microglial activation, steady-state levels of proinflammatory cytokine and chemokine transcripts, and severity of amyloid-related pathology. In contrast, these rAAV1-IFNgamma-treated 3xTg-AD mice also exhibited diminished phospho-tau pathology and evidence of increased neurogenesis. Overall, IFNgamma mediates what seem to be diametrically opposed functions in the setting of AD-related neurodegeneration. Gaining an understanding as to how these apparently divergent functions are interrelated and controlled could elucidate new therapeutic strategies designed to harness the neuroprotective activity of IFNgamma.
Assuntos
Doença de Alzheimer , Interferon gama/imunologia , Camundongos Transgênicos , Neurogênese/fisiologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Biomarcadores/metabolismo , Dependovirus/genética , Dependovirus/metabolismo , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Humanos , Interferon gama/genética , Camundongos , Microglia/citologia , Microglia/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas tau/genética , Proteínas tau/metabolismoRESUMO
Amyloid-beta (A beta) has been identified as a key component in Alzheimer's disease (AD). Significant in vitro and human pathological data suggest that intraneuronal accumulation of A beta peptides plays an early role in the neurodegenerative cascade. We hypothesized that targeting an antibody-based therapeutic to specifically abrogate intracellular A beta accumulation could prevent or slow disease onset. A beta 42-specific intracellular antibodies (intrabodies) with and without an intracellular trafficking signal were engineered from a previously characterized single-chain variable fragment (scFv) antibody. The intrabodies, one with an endoplasmic reticulum (ER) targeting signal and one devoid of a targeting sequence, were assessed in cells harboring a doxycycline (Dox)-regulated mutant human amyloid precursor protein Swedish mutant (hAPP(swe)) transcription unit for their abilities to prevent A beta peptide egress. Adeno-associated virus (AAV) vectors expressing the engineered intrabodies were administered to young adult 3xTg-AD mice, a model that develops amyloid and Tau pathologies, prior to the initial appearance of intraneuronal A beta. Chronic expression of the ER-targeted intrabody (IB) led to partial clearance of A beta 42 deposits and interestingly, in reduced staining for a pathologic phospho-Tau epitope (Thr231). This approach may provide insights into the functional relevance of intraneuronal A beta accumulation in early AD and potentially lead to the development of new therapeutics.
Assuntos
Doença de Alzheimer/prevenção & controle , Precursor de Proteína beta-Amiloide/imunologia , Anticorpos/imunologia , Dependovirus/genética , Vacinação , Doença de Alzheimer/genética , Doença de Alzheimer/imunologia , Precursor de Proteína beta-Amiloide/genética , Animais , Células Cultivadas , Modelos Animais de Doenças , Doxiciclina/farmacologia , Retículo Endoplasmático , Vetores Genéticos/administração & dosagem , Humanos , Região Variável de Imunoglobulina/imunologia , Camundongos , Camundongos Transgênicos , Proteínas tau/metabolismoRESUMO
Inflammatory mediators, such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta, appear integral in initiating and/or propagating Alzheimer's disease (AD)-associated pathogenesis. We have previously observed a significant increase in the number of mRNA transcripts encoding the pro-inflammatory cytokine TNF-alpha, which correlated to regionally enhanced microglial activation in the brains of triple transgenic mice (3xTg-AD) before the onset of overt amyloid pathology. In this study, we reveal that neurons serve as significant sources of TNF-alpha in 3xTg-AD mice. To further define the role of neuronally derived TNF-alpha during early AD-like pathology, a recombinant adeno-associated virus vector expressing TNF-alpha was stereotactically delivered to 2-month-old 3xTg-AD mice and non-transgenic control mice to produce sustained focal cytokine expression. At 6 months of age, 3xTg-AD mice exhibited evidence of enhanced intracellular levels of amyloid-beta and hyperphosphorylated tau, as well as microglial activation. At 12 months of age, both TNF receptor II and Jun-related mRNA levels were significantly enhanced, and peripheral cell infiltration and neuronal death were observed in 3xTg-AD mice, but not in non-transgenic mice. These data indicate that a pathological interaction exists between TNF-alpha and the AD-related transgene products in the brains of 3xTg-AD mice. Results presented here suggest that chronic neuronal TNF-alpha expression promotes inflammation and, ultimately, neuronal cell death in this AD mouse model, advocating the development of TNF-alpha-specific agents to subvert AD.
Assuntos
Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Inflamação/metabolismo , Neurônios/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Encéfalo/patologia , Dependovirus/genética , Dependovirus/metabolismo , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Humanos , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Camundongos Transgênicos , Microglia/citologia , Microglia/metabolismo , Neurônios/patologia , Transgenes , Fator de Necrose Tumoral alfa/genética , Proteínas tau/metabolismoRESUMO
Herpes simplex virus type-1 (HSV-1) amplicon vectors are being explored for a wide range of potential applications, including vaccine delivery and immunotherapy of cancer. While extensive effort has been directed towards the improvement of the amplicon "payload" in these vectors, relatively little attention has been paid to the effect of the packaging HSV-1 strains on the biological properties of co-packaged amplicon vectors. We therefore compared the biological properties of amplicon stocks prepared using a panel of primary HSV-1 isolates, a molecularly cloned strain used to package helper-free amplicons (designated here as F5), and two laboratory isolates (KOS and strain 17, which is the parent of the F5 clone). This analysis revealed considerable inter-strain variability in the ability of amplicon stocks packaged by different primary HSV-1 isolates to efficiently transduce established cell lines and primary human dendritic cells (DC). Amplicons packaged by both the F5 molecularly cloned virus and its laboratory-adapted parent (strain 17) were very inefficient at transducing DC, when compared to amplicons packaged by KOS or by several of the primary virus isolates. These finding have important implications for the future development of improved amplicon-based vaccine delivery systems and suggest that DC tropism may be an instrinsic property of some HSV-1 strains, independent of passage history or molecular cloning.