RESUMEN
The two proteases beta-secretase and gamma-secretase generate the amyloid beta peptide and are drug targets for Alzheimer's disease. Here we tested the possibility of targeting the cellular environment of beta-secretase cleavage instead of the beta-secretase enzyme itself. beta-Secretase has an acidic pH optimum and cleaves the amyloid precursor protein in the acidic endosomes. We identified two drugs, bepridil and amiodarone, that are weak bases and are in clinical use as calcium antagonists. Independently of their calcium-blocking activity, both compounds mildly raised the membrane-proximal, endosomal pH and inhibited beta-secretase cleavage at therapeutically achievable concentrations in cultured cells, in primary neurons, and in vivo in guinea pigs. This shows that an alkalinization of the cellular environment could be a novel therapeutic strategy to inhibit beta-secretase. Surprisingly, bepridil and amiodarone also modulated gamma-secretase cleavage independently of endosomal alkalinization. Thus, both compounds act as dual modulators that simultaneously target beta- and gamma-secretase through distinct molecular mechanisms. In addition to Alzheimer's disease, compounds with dual properties may also be useful for drug development targeting other membrane proteins.
Asunto(s)
Amiodarona/farmacología , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Bepridil/farmacología , Inhibidores Enzimáticos/farmacología , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/enzimología , Amiodarona/química , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/sangre , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Bepridil/química , Encéfalo/efectos de los fármacos , Encéfalo/enzimología , Encéfalo/metabolismo , Línea Celular , Células Cultivadas , Inhibidores Enzimáticos/química , Femenino , Cobayas , Humanos , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Ratones , Ratones Transgénicos , Neuronas/efectos de los fármacos , Neuronas/enzimología , Neuronas/metabolismo , Nexinas de Proteasas , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismoRESUMEN
CONTEXT: Activity of beta-site APP-cleaving enzyme1 (BACE1) is required for the generation of beta-amyloid peptides, the principal constituents of plaques in the brains of patients with Alzheimer's disease. Strong BACE1 expression has also been described in pancreatic tissue. OBJECTIVE: The aim of the present study was to reveal the cell type-specific expression of BACE1 in the pancreas and to identify a substrate for BACE1 in this organ. METHODS: RT-PCR of microdissected rat pancreatic tissue was carried out in order to analyze BACE1 expression within pancreatic acini. Pancreatic juice was examined by western blot analysis and by an enzymatic activity assay in order to reveal the presence of secreted BACE1. Database analysis suggested enteropeptidase as a putative substrate for BACE1 in pancreatic juice. In vitro digestion of enteropeptidase by BACE1 was performed to demonstrate this cleavage. RESULTS: We demonstrate the expression of BACE1 in the islets of Langerhans and at the apical pole of pancreatic acinar cells. Recombinant BACE1 cleaves enteropeptidase in vitro. Furthermore, some results suggested the presence of BACE1 enzymatic activity in pancreatic juice and pancreatic tissue. DISCUSSION: We hypothesize that enteropeptidase is a BACE1 substrate in vivo. If so, BACE1 could protect the pancreas from premature trypsinogen activation due to the occasionally occurring reflux of enteropeptidase.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/fisiología , Ácido Aspártico Endopeptidasas/fisiología , Enteropeptidasa/metabolismo , Páncreas/metabolismo , Procesamiento Proteico-Postraduccional , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Ácido Aspártico Endopeptidasas/genética , Ácido Aspártico Endopeptidasas/metabolismo , Regulación Enzimológica de la Expresión Génica , Ratones , Ratas , Especificidad por Sustrato , Distribución TisularRESUMEN
The enforcement of rules for food labeling and quantitative ingredient declaration presupposes appropriate test systems. Additionally, central nervous system (CNS) tissue of ruminants is classified as specified risk material for the transmission of prion diseases, and its detection is needed to support the specified risk material ban. Existing antibody-based test systems are hampered by relatively high limits of detection and susceptibility to food processing conditions. For that reason we tested a broad panel of commercially available monoclonal antibodies to identify marker antigens appropriate for the development of a sensitive test system. Western blot analysis using organ-specific samples from cow, pig, and chicken and differently processed meat products containing defined amounts of CNS tissue revealed neurofilament light (NF-L) and protein gene product 9.5 (PGP 9.5) as suitable antigens for the organ-specific and sensitive detection of porcine and bovine CNS tissue. None of the tested PGP 9.5 antibodies displayed cross-reactivity to chicken tissues. Both antigens could be detected in moderately (F(10)121.1 = 0.84) and strongly (F(10)121.1 = 4.01) heated processed meat products containing 5% (NF-L) or 0.2% (PGP 9.5) CNS tissue, respectively. Further, two monoclonal antibodies (clones 13C4 and 31A3) directed against PGP 9.5 were used for the development of a sandwich enzyme-linked immunosorbent assay. The limits of detection of the enzyme-linked immunosorbent assay were approximately 2% added CNS tissue in fresh processed meat products and approximately 0.5% for strongly heated processed meat products (F(10)121.1 = 4.01). In conclusion this test system constitutes a valuable supplementation to existing procedures, which could improve enforcement of food safety regulations.
Asunto(s)
Contaminación de Alimentos/análisis , Productos de la Carne/análisis , Proteínas de Neurofilamentos/análisis , Ubiquitina Tiolesterasa/análisis , Animales , Anticuerpos Monoclonales/inmunología , Western Blotting , Bovinos , Pollos , Reacciones Cruzadas , Ensayo de Inmunoadsorción Enzimática , Enfermedades por Prión/prevención & control , Enfermedades por Prión/transmisión , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Especificidad de la Especie , PorcinosRESUMEN
BACKGROUND: The therapeutic capacity of human umbilical cord blood mononuclear cells (HUCB-MNC) and stem cells derived thereof is documented in animal models of focal cerebral ischemia, while mechanisms behind the reduction of lesion size and the observed improvement of behavioral skills still remain poorly understood. METHODS: A human in vitro model of neuronal hypoxia was used to address the impact of total HUCB-MNC (tMNC), a stem cell enriched fraction (CD133+, 97.38% CD133-positive cells) and a stem cell depleted fraction (CD133-, 0.06% CD133-positive cells) of HUCB-MNC by either direct or indirect co-cultivation with post-hypoxic neuronal cells (differentiated SH-SY5Y). Over three days, development of apoptosis and necrosis of neuronal cells, chemotaxis of MNC and production of chemokines (CCL2, CCL3, CCL5, CXCL8, CXCL9) and growth factors (G-CSF, GM-CSF, VEGF, bFGF) were analyzed using fluorescence microscopy, FACS and cytometric bead array. RESULTS: tMNC, CD133+ and surprisingly CD133- reduced neuronal apoptosis in direct co-cultivations significantly to levels in the range of normoxic controls (7% +/- 3%). Untreated post-hypoxic control cultures showed apoptosis rates of 85% +/- 11%. tMNC actively migrated towards injured neuronal cells. Both co-cultivation types using tMNC or CD133- reduced apoptosis comparably. CD133- produced high concentrations of CCL3 and neuroprotective G-CSF within indirect co-cultures. Soluble factors produced by CD133+ cells were not detectable in direct co-cultures. CONCLUSION: Our data show that heterogeneous tMNC and even CD133-depleted fractions have the capability not only to reduce apoptosis in neuronal cells but also to trigger the retaining of neuronal phenotypes.
Asunto(s)
Antígenos CD/análisis , Sangre Fetal/citología , Glicoproteínas/análisis , Células Madre Hematopoyéticas/citología , Neuronas/patología , Péptidos/análisis , Antígeno AC133 , Apoptosis , Diferenciación Celular , Hipoxia de la Célula , Línea Celular Tumoral , Células Cultivadas , Quimiocinas/análisis , Quimiocinas/biosíntesis , Técnicas de Cocultivo/métodos , Sangre Fetal/inmunología , Sangre Fetal/metabolismo , Citometría de Flujo , Células Madre Hematopoyéticas/inmunología , Células Madre Hematopoyéticas/metabolismo , Humanos , Recién Nacido , Péptidos y Proteínas de Señalización Intercelular/análisis , Péptidos y Proteínas de Señalización Intercelular/biosíntesis , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Microscopía Fluorescente , Neuroblastoma/inmunología , Neuroblastoma/metabolismo , Neuroblastoma/patología , Neuronas/metabolismoRESUMEN
We propose a general object counting method that does not use any prior category information. We learn from local image divisions to predict global image-level counts without using any form of local annotations. Our method separates the input image into a sets of image divisions - each fully covering the image. Each image division is composed of a set of region proposals or uniform grid cells. Our approach learns in an endto- end deep learning architecture to predict global image-level counts from local image divisions. The method incorporates a counting layer which predicts object counts in the complete image, by enforcing consistency in counts when dealing with overlapping image regions. Our counting layer is based on the inclusion-exclusion principle from set theory. We analyze the individual building blocks of our proposed approach on Pascal- VOC2007 and evaluate our method on the MS-COCO large scale generic object dataset as well as on three class-specific counting datasets: UCSD pedestrian dataset, and CARPK and PUCPR+ car datasets.
RESUMEN
In Lewis rats infected intracerebrally with the highly neurotropic Borna disease virus (BDV), the retina is one of the most severely affected central nervous system (CNS) structures. While BDV-induced damage in the brain has been previously shown to be caused by a T-cell-dependent process, the immunopathological mechanisms leading to BDV-induced retinitis remain to be elucidated. RNA samples from retinae were subjected to RNase protection assays to detect transcripts of proinflammatory cytokines and chemokines known to be involved in the recruitment of T-cells and macrophages in the CNS. The observed expression profile of proinflammatory cytokines and chemokines, as well as the immunohistochemical detection of alpha beta TCR-positive, CD4- and CD8-positive T-cells in the BDV-infected retinae, is reminiscent of the situation observed in the brains of Lewis rats during the acute phase of Borna disease (BD). This suggests that similar immunopathological mechanisms are operating in retinae and brains of infected rats.
Asunto(s)
Enfermedad de Borna/inmunología , Virus de la Enfermedad de Borna/inmunología , Retina/inmunología , Retinitis/inmunología , Enfermedad Aguda , Animales , Linfocitos B/química , Linfocitos B/inmunología , Quimiocinas/análisis , Quimiocinas/biosíntesis , Citocinas/análisis , Citocinas/biosíntesis , Inmunohistoquímica , Ratas , Ratas Endogámicas Lew , Retina/química , Retina/virología , Linfocitos T/química , Linfocitos T/inmunologíaRESUMEN
The pathophysiology of stroke is governed by immune reactions within and remote from the injured brain. Hypertension, a major cause and comorbidity of stroke, entails systemic vascular inflammation and may influence poststroke immune responses. This aspect is, however, underestimated in previous studies. Here we aimed to delineate the sequence of cellular inflammation after stroke in spontaneously hypertensive (SH) rats. Spontaneously hypertensive rats were subjected to permanent middle cerebral artery occlusion and killed after 1 or 4 days. Immune cells of the peripheral blood and those which have infiltrated the injured brain were identified and quantified by flow cytometry. The spatial distribution of myeloid cells and T lymphocytes, and the infarct volume were assessed by histology. We observed a concerted infiltration of immune cells into the ischemic brain of SH rats. At day 1, primarily neutrophils, monocytes, macrophages, and myeloid dendritic cells entered the brain, whereas the situation at day 4 was dominated by microglia, macrophages, lymphatic dendritic cells, and T cells. Postischemic inflammation did not cause secondary tissue damage during the subacute stage of experimental stroke in SH rats. Considering the intrinsic vascular pathology of SH rats, our study validates this strain for further translational research in poststroke inflammation.
Asunto(s)
Encéfalo/patología , Células Dendríticas/patología , Hipertensión/patología , Infarto de la Arteria Cerebral Media/patología , Leucocitos/patología , Microglía/patología , Animales , Encéfalo/irrigación sanguínea , Encéfalo/metabolismo , Encéfalo/fisiopatología , Células Dendríticas/metabolismo , Hipertensión/metabolismo , Hipertensión/fisiopatología , Infarto de la Arteria Cerebral Media/complicaciones , Infarto de la Arteria Cerebral Media/metabolismo , Infarto de la Arteria Cerebral Media/fisiopatología , Inflamación/etiología , Inflamación/metabolismo , Inflamación/patología , Inflamación/fisiopatología , Leucocitos/metabolismo , Microglía/metabolismo , Ratas , Ratas Endogámicas SHRRESUMEN
OBJECTIVE: Beta-site amyloid precursor protein cleaving enzyme (BACE1) is highly expressed in pancreatic ß-cells. The BACE1 gene is located in a region associated with a high diabetes risk in PIMA Indians. DESIGN AND METHODS: INS-1E cells were used to study the impact of siRNA-mediated BACE1 knockdown and glucose metabolism was characterized in Bace1(-/-) mice. BACE1 gene was sequenced in DNA samples from 48 subjects and 13 representative single nucleotide polymorphisms (SNPs) were then genotyped for association studies in 1,527 Caucasians. RESULTS: Reduction of Bace1 expression results in a significant decrease in insulin mRNA expression in INS-1E cells. Bace1(-/-) mice display significantly lower body weight, lower plasma insulin concentrations, but normal glucose tolerance and insulin sensitivity. In a case-control study including 538 healthy controls and 989 patients with type 2 diabetes (T2D), one SNP (rs535860) was significantly associated with T2D (P < 3.5 × 10(-5) , adjusted for age, sex, and BMI). CONCLUSIONS: Reduced Bace1 expression causes impaired insulin expression in pancreatic ß-cells of Bace1(-/-) mice, suggesting that BACE1 plays a role in the regulation of insulin biogenesis. The functionally relevant rs535860 SNP may decrease BACE1 expression by creating a new miR-661 binding site and could therefore contribute to T2D development.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/metabolismo , Ácido Aspártico Endopeptidasas/metabolismo , Regulación de la Expresión Génica , Insulina/sangre , ARN Mensajero/metabolismo , Adulto , Anciano , Secretasas de la Proteína Precursora del Amiloide/genética , Animales , Ácido Aspártico Endopeptidasas/genética , Índice de Masa Corporal , Estudios de Casos y Controles , Diabetes Mellitus Tipo 2/genética , Femenino , Genes Reporteros , Genotipo , Prueba de Tolerancia a la Glucosa , Humanos , Insulina/genética , Resistencia a la Insulina , Células Secretoras de Insulina/metabolismo , Masculino , Ratones , Ratones Noqueados , MicroARNs/genética , MicroARNs/metabolismo , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , ARN Mensajero/genéticaRESUMEN
The pathophysiology of sterile inflammation following focal ischemic stroke is complex and not fully understood, but there is growing evidence that it offers several therapeutic options beyond the hitherto existing treatment strategies. The identification and quantification of infiltrating inflammatory cells in animal models of stroke is crucial both for understanding post-stroke inflammation and for drug target identification. Multicolor flow cytometry plays an important role in determining subtypes and quantity of leukocytes that infiltrate the brain tissue after stroke. Until now, most investigations have been performed in mice, most likely due to a significantly broader spectrum of disposable antibodies and available knockout models. Here, we introduce a specific and reproducible method to isolate leukocytes from rat brain specimen in the context of brain ischemia to ultimately allow multi-dimensional flow cytometric characterization and further downstream methods such as cell-subtype sorting and molecular biological approaches.
RESUMEN
Experimental transplantation of human umbilical cord blood (hUCB) mononuclear cells (MNCs) in rodent stroke models revealed the therapeutic potential of these cells. However, effective cells within the heterogeneous MNC population and their modes of action are still under discussion. MNCs and MNC fractions enriched (CD34(+)) or depleted (CD34(-)) for CD34-expressing stem/progenitor cells were isolated from hUCB. Cells were transplanted intravenously following middle cerebral artery occlusion in spontaneously hypertensive rats and directly or indirectly cocultivated with hippocampal slices previously subjected to oxygen and glucose deprivation. Application of saline solution or a human T-cell line served as controls. In vivo, MNCs, CD34(+) and CD34(-) cells reduced neurofunctional deficits and diminished lesion volume as determined by magnetic resonance imaging. MNCs were superior to other fractions. However, human cells could not be identified in brain tissue 29 days after stroke induction. Following direct application on postischemic hippocampal slices, MNCs reduced neural damage throughout a 3-day observation period. CD34(+) cells provided transient protection for 2 days. The CD34(-) fraction, in contrast to in vivo results, failed to reduce neural damage. Direct cocultivation of MNCs was superior to indirect cocultivation of equal cell numbers. Indirect application of up to 10-fold MNC concentrations enhanced neuroprotection to a level comparable to direct cocultivation. After direct application, MNCs migrated into the slices. Flow cytometric analysis of migrated cells revealed that the CD34(+) cells within MNCs were preferably attracted by damaged hippocampal tissue. Our study suggests that MNCs provide the most prominent neuroprotective effect, with CD34(+) cells seeming to be particularly involved in the protective action of MNCs. CD34(+) cells preferentially home to neural tissue in vitro, but are not superior concerning the overall effect, implying that there is another, still undiscovered, protective cell population. Furthermore, MNCs did not survive in the ischemic brain for longer periods without immunosuppression.
Asunto(s)
Sangre Fetal/citología , Accidente Cerebrovascular/terapia , Animales , Antígenos CD34/metabolismo , Células Cultivadas , Humanos , Técnicas In Vitro , Imagen por Resonancia Magnética , Masculino , Factores de Crecimiento Nervioso , Ratas , Ratas Endogámicas SHRRESUMEN
Avian polyomavirus (APV) is the causative agent of an acute fatal disease in psittacine and some non-psittacine birds. In contrast to mammalian polyomaviruses, the APV genome encodes the additional capsid protein VP4 and its variant VP4Delta, truncated by an internal deletion. Both proteins induce apoptosis. Mutation of their common initiation codon prevents virus replication. Here, the generation of replication competent deletion mutants expressing either VP4 or VP4Delta is reported. In contrast to infection with wild-type virus, chicken embryo cells showed no cytopathic changes after infection with the mutants, and induction of apoptosis as well as virus release from the infected cells were delayed. Electron microscopy revealed the presence of a high proportion of small particles and tubules in preparations of the VP4 deletion mutant, indicating a scaffolding function for VP4. Wild-type and mutant viruses elicited neutralizing antibodies against APV after intramuscular and intraperitoneal infection of chicken; however, VP4-specific antibodies were only detected after infection with wild-type virus. Using the oculonasal route of infection, seroconversion was only observed in chickens infected with the wild-type virus, indicating a strongly reduced infectivity of the mutants. Based on the biological properties of the deletion mutants, they could be considered as candidates for APV marker vaccines.
Asunto(s)
Proteínas de la Cápside/fisiología , Infecciones por Polyomavirus/veterinaria , Poliomavirus/genética , Enfermedades de las Aves de Corral/virología , Eliminación de Secuencia , Ensamble de Virus , Animales , Anticuerpos Antivirales/sangre , Apoptosis , Proteínas de la Cápside/genética , Proteínas de la Cápside/inmunología , Células Cultivadas , Embrión de Pollo , Pollos , Efecto Citopatogénico Viral , Microscopía Electrónica de Transmisión , Pruebas de Neutralización , Poliomavirus/inmunología , Poliomavirus/patogenicidad , Poliomavirus/fisiología , Infecciones por Polyomavirus/virología , Virión/ultraestructura , Replicación ViralRESUMEN
Amyloid plaques, one of the neuropathological hallmarks of Alzheimer's disease, and their main constituent, the amyloid beta-peptide (Abeta), are triggers of the activation of innate inflammatory mechanisms involving the activation of microglia. To dissect the effects of a non-Abeta-specific microglial activation on the Abeta metabolism, we employed a viral infection-based model. Transgenic mice expressing a mutated form of the human amyloid precursor protein (Tg2576) were used. In preceding experiments, 2-week-old transgenic mice and non-transgenic littermates were infected intracerebrally with the neurotropic Borna disease virus and investigated at 2, 4 and 14 weeks post-infection. The Borna disease virus-inoculated mice showed a persisting, subclinical infection of cortical and limbic brain areas characterized by slight T-cell infiltrates, expression of cytokines and a massive microglial activation in the hippocampus and neocortex. Viral-induced effects reached their peak at 4 weeks post-infection. In 14-month-old Tg2576 mice, characterized by the deposition of diffuse and dense-core amyloid plaques in cortical brain regions, Borna disease virus-induced microglial activation in the vicinity of Abeta deposits was used to investigate the influence of a local inflammatory response on these deposits. At 4 weeks post-infection, histometric analyses employing Abeta immunohistochemistry revealed a decrease of the cortical and hippocampal Abeta-immunopositive area. This overall decrease was accompanied by a decrease of parenchymal thioflavin-S-positive amyloid deposits and an increase of such deposits in the walls of cerebral vessels, which indicates that the elicitation of a non-Abeta-specific microglial activation may contribute to a reduction of Abeta in the brain parenchyma.
Asunto(s)
Virus de la Enfermedad de Borna , Encéfalo/metabolismo , Encéfalo/virología , Inflamación/metabolismo , Inflamación/virología , Placa Amiloide/patología , Precursor de Proteína beta-Amiloide/genética , Animales , Animales Recién Nacidos , Benzotiazoles , Encéfalo/patología , Citocinas/genética , Citocinas/metabolismo , Electroforesis en Gel de Poliacrilamida/métodos , Ensayo de Cambio de Movilidad Electroforética/métodos , Expresión Génica/fisiología , Proteína Ácida Fibrilar de la Glía/metabolismo , Humanos , Inmunohistoquímica/métodos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuroglía/metabolismo , Neuroglía/patología , Nucleoproteínas/metabolismo , Linfocitos T/metabolismo , Linfocitos T/patología , Tiazoles , Factores de Tiempo , Proteínas Virales/metabolismoRESUMEN
The beta-site APP-cleaving enzyme (BACE1) is a prerequisite for the generation of beta-amyloid peptides, which give rise to cerebrovascular and parenchymal beta-amyloid deposits in the brain of Alzheimer's disease patients. BACE1 is neuronally expressed in the brains of humans and experimental animals such as mice and rats. In addition, we have recently shown that BACE1 protein is expressed by reactive astrocytes in close proximity to beta-amyloid plaques in the brains of aged transgenic Tg2576 mice that overexpress human amyloid precursor protein carrying the double mutation K670N-M671L. To address the question whether astrocytic BACE1 expression is an event specifically triggered by beta-amyloid plaques or whether glial cell activation by other mechanisms also induces BACE1 expression, we used six different experimental strategies to activate brain glial cells acutely or chronically. Brain sections were processed for the expression of BACE1 and glial markers by double immunofluorescence labeling and evaluated by confocal laser scanning microscopy. There was no detectable expression of BACE1 protein by activated microglial cells of the ameboid or ramified phenotype in any of the lesion paradigms studied. In contrast, BACE1 expression by reactive astrocytes was evident in chronic but not in acute models of gliosis. Additionally, we observed BACE1-immunoreactive astrocytes in proximity to beta-amyloid plaques in the brains of aged Tg2576 mice and Alzheimer's disease patients.