RESUMEN
[18 F]FDG-PET/CT is a high sensitive functional diagnostic imaging modality to monitor tumor but also immune cell activation by determination of the glucose metabolism. Our results show that the anti-inflammatory effects of immunotherapeutics like DMF can be assessed non invasively in vivo during Th1/Th17 cell-mediated encephalomyelitis (EAE) by [18 F]FDG-PET/CT imaging of the draining lymph nodes.
Asunto(s)
Dimetilfumarato/inmunología , Monitoreo de Drogas/métodos , Encefalomielitis Autoinmune Experimental/inmunología , Glucosa/metabolismo , Ganglios Linfáticos/inmunología , Tomografía Computarizada por Tomografía de Emisión de Positrones/métodos , Animales , Dimetilfumarato/uso terapéutico , Encefalomielitis Autoinmune Experimental/diagnóstico por imagen , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Fluorodesoxiglucosa F18/metabolismo , Humanos , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/metabolismo , Ratones , Células TH1/efectos de los fármacos , Células TH1/inmunología , Células TH1/metabolismo , Células Th17/efectos de los fármacos , Células Th17/inmunología , Células Th17/metabolismoRESUMEN
The prostate-specific membrane antigen (PSMA) has been demonstrated in numerous studies to be expressed specifically on prostate carcinoma cells and on the neovasculature of several other cancer entities. However, the simultaneous expression of PSMA on both, tumor cells as well as tumor vessels remains unclear, even if such "dual" expression would constitute an important asset to facilitate sufficient influx of effector cells to a given tumor site. We report here on the generation of a PSMA antibody, termed 10B3, which exerts superior dual reactivity on sections of prostate carcinoma and squamous cell carcinoma of the lung. 10B3 was used for the construction of T-cell recruiting bispecific PSMAxCD3 antibodies in Fab- and IgG-based formats, designated Fabsc and IgGsc, respectively. In vitro, both molecules exhibited comparable activity. In contrast, only the larger IgGsc molecule induced complete and durable elimination of established tumors in humanized mice due to favorable pharmacokinetic properties. Upon treatment of three patients with metastasized prostate carcinoma with the IgGsc reagent, marked activation of T cells and rapid reduction of elevated PSA levels were observed.
Asunto(s)
Anticuerpos Biespecíficos , Neoplasias de la Próstata , Animales , Antígenos de Superficie , Humanos , Inmunoglobulina G , Masculino , Ratones , Neoplasias de la Próstata/tratamiento farmacológico , Linfocitos TRESUMEN
BACKGROUND: Limited knowledge of stem cell therapies` mechanisms of action hampers their sustainable implementation into the clinic. Specifically, the interactions of transplanted stem cells with the host vasculature and its implications for their therapeutic efficacy are not elucidated. We tested whether adhesion receptors and chemokine receptors on stem cells can be functionally modulated, and consequently if such modulation may substantially affect therapeutically relevant stem cell interactions with the host endothelium. METHODS: We investigated the effects of cationic molecule polyethylenimine (PEI) treatment with or without nanoparticles on the functions of adhesion receptors and chemokine receptors of human bone marrow-derived Mesenchymal Stem Cells (MSC). Analyses included MSC functions in vitro, as well as homing and therapeutic efficacy in rodent models of central nervous system´s pathologies in vivo. FINDINGS: PEI treatment did not affect viability, immunomodulation or differentiation potential of MSC, but increased the CCR4 expression and functionally blocked their adhesion receptors, thus decreasing their adhesion capacity in vitro. Intravenously applied in a rat model of brain injury, the homing rate of PEI-MSC in the brain was highly increased with decreased numbers of adherent PEI-MSC in the lung vasculature. Moreover, in comparison to untreated MSC, PEI-MSC featured increased tumour directed migration in a mouse glioblastoma model, and superior therapeutic efficacy in a murine model of stroke. INTERPRETATION: Balanced stem cell adhesion and migration in different parts of the vasculature and tissues together with the local microenvironment impacts their therapeutic efficacy. FUNDING: Robert Bosch Stiftung, IZEPHA grant, EU grant 7 FP Health.
Asunto(s)
Adhesión Celular , Movimiento Celular , Endotelio/metabolismo , Células Madre/metabolismo , Animales , Biomarcadores , Diferenciación Celular , Línea Celular , Tratamiento Basado en Trasplante de Células y Tejidos , Células Cultivadas , Microambiente Celular , Modelos Animales de Enfermedad , Glioma/diagnóstico , Glioma/patología , Glioma/terapia , Humanos , Inmunofenotipificación , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratas , Trasplante de Células Madre , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Stem cells` (SC) functional heterogeneity and its poorly understood aetiology impedes clinical development of cell-based therapies in regenerative medicine and oncology. Recent studies suggest a strong correlation between the SC migration potential and their therapeutic efficacy in humans. Designating SC migration as a denominator of functional SC heterogeneity, we sought to identify highly migrating subpopulations within different SC classes and evaluate their therapeutic properties in comparison to the parental non-selected cells. METHODS: We selected highly migrating subpopulations from mesenchymal and neural SC (sMSC and sNSC), characterized their features including but not limited to migratory potential, trophic factor release and transcriptomic signature. To assess lesion-targeted migration and therapeutic properties of isolated subpopulations in vivo, surgical transplantation and intranasal administration of MSCs in mouse models of glioblastoma and Alzheimer's disease respectively were performed. FINDINGS: Comparison of parental non-selected cells with isolated subpopulations revealed superior motility and migratory potential of sMSC and sNSC in vitro. We identified podoplanin as a major regulator of migratory features of sMSC/sNSC. Podoplanin engineering improved oncovirolytic activity of virus-loaded NSC on distantly located glioblastoma cells. Finally, sMSC displayed more targeted migration to the tumour site in a mouse glioblastoma model and remarkably higher potency to reduce pathological hallmarks and memory deficits in transgenic Alzheimer's disease mice. INTERPRETATION: Functional heterogeneity of SC is associated with their motility and migration potential which can serve as predictors of SC therapeutic efficacy. FUNDING: This work was supported in part by the Robert Bosch Stiftung (Stuttgart, Germany) and by the IZEPHA grant.
Asunto(s)
Movimiento Celular , Células Madre/fisiología , Enfermedad de Alzheimer/terapia , Animales , Biomarcadores , Supervivencia Celular , Rastreo Celular/métodos , Células Cultivadas , Modelos Animales de Enfermedad , Expresión Génica , Perfilación de la Expresión Génica , Humanos , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/fisiología , Ratones , Ratones Transgénicos , Células-Madre Neurales/citología , Células-Madre Neurales/fisiología , Viroterapia Oncolítica , Trasplante de Células Madre , Células Madre/citología , Resultado del TratamientoRESUMEN
The inner clock of biological organisms plays a pivotal role and has strong effects on metabolic processes such as glucose consumption. Since the commonly used positron emission tomography (PET) tracer 18F-flourodeoxygucose (FDG) is a glucose analogue, it is not surprising that the FDG distribution in mice and humans has been shown to succumb to daily rhythms. In preclinical studies, the circadian rhythm of animals is often not considered, and studies are performed at different times of day. Only a few studies have analyzed the effect of the circadian rhythm on FDG uptake in mice, and none of these studies included human tumor xenografts. Therefore, it is not known how strongly a preclinical tumor study is influenced by the time of day. In this work, the effect of the circadian rhythm on FDG uptake in human tumor xenografts and other organs was analyzed. CD1 nu/nu mice were kept for three weeks under a 12 h light/12 h dark rhythm and then injected s.c. with PC3 or A431 tumor cells. When the tumors had reached an appropriate volume, FDG-PET scans were performed on different animal groups (n = 4-5) every 4 h over a time period from 8 A.M. to 8 P.M. Tracer uptake in the tumors and in other organs was determined based on the PET scans and biodistribution studies. The standardized uptake value and %injected dose/cc of the tumors remained constant over the whole observed time period, and no statistically significant differences were determined according to the PET analysis. In the brain, we found a small but statistically significant increase from noon to 4 P.M., which led to a decrease in the tumor-to-brain ratio. No evidence for an effect of the circadian rhythm on FDG uptake could be found in subcutaneous tumors, however, in brain studies the circadian rhythm needs to be considered.
Asunto(s)
Química Encefálica , Ritmo Circadiano , Xenoinjertos/metabolismo , Tomografía de Emisión de Positrones , Animales , Glucemia/análisis , Glucemia/fisiología , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Química Encefálica/fisiología , Femenino , Fluorodesoxiglucosa F18 , Xenoinjertos/diagnóstico por imagen , Humanos , Ratones , Músculo Esquelético/diagnóstico por imagen , Trasplante de Neoplasias/diagnóstico por imagen , Neuroimagen , Células PC-3RESUMEN
Pertussis toxin (PTX) is a potent virulence factor in patients suffering from whooping cough, but in its detoxified version, it is applied for vaccination. It is thought to contribute to the pathology of the disease including various CNS malfunctions. Based on its enzymatic activity, PTX disrupts GPCR-dependent signaling by modifying the α-subunit of heterotrimeric Gi/o-proteins. It is also extensively used as a research tool to study neuronal functions in vivo and in vitro. However, data demonstrating the penetration of PTX from the blood into the brain are missing. Here, we examined the Gαi/o-modifying activity of PTX in murine brains after its parenteral application. Ex vivo biodistribution analysis of [124I]-PTX displayed poor distribution to the brain while relatively high concentrations were visible in the pancreas. PTX affected CNS and endocrine functions of the pancreas as shown by open-field and glucose tolerance tests, respectively. However, while pancreatic islet Gαi/o-proteins were modified, their neuronal counterparts in brain tissue were resistant towards PTX as indicated by different autoradiographic and immunoblot SDS-PAGE analyses. In contrast, PTX easily modified brain Gαi/o-proteins ex vivo. An attempt to increase BBB permeability by application of hypertonic mannitol did not show PTX activity on neuronal G proteins. Consistent with these findings, in vivo MRI analysis did not point to an increased blood-brain barrier (BBB) permeability following PTX treatment. Our data demonstrate that the CNS is protected from PTX. Thus, we hypothesize that the BBB hinders PTX to penetrate into the CNS and to deliver its enzymatic activity to brain Gαi/o-proteins. KEY MESSAGES: i.p. applied PTX is poorly retained in the brain while reaches high concentration in the pancreas. Pancreatic islet Gαi/o- but not cerebral Gαi/o-proteins are modified by i.p. administered PTX. Gαi/o-proteins from isolated cerebral cell membranes were easily modified by PTX ex vivo. CNS is protected from i.p. administered PTX. PTX does not permeabilize the BBB.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Inyecciones/métodos , Neuroprotección , Toxina del Pertussis/administración & dosificación , Toxina del Pertussis/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Barrera Hematoencefálica/diagnóstico por imagen , Barrera Hematoencefálica/metabolismo , Permeabilidad Capilar/efectos de los fármacos , Membrana Celular/metabolismo , Femenino , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/antagonistas & inhibidores , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/deficiencia , Radioisótopos de Yodo , Islotes Pancreáticos/diagnóstico por imagen , Islotes Pancreáticos/metabolismo , Imagen por Resonancia Magnética , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas/metabolismo , Distribución TisularRESUMEN
Doxorubicin-induced nephropathy in mice is a model for studying experimental nephrotic syndrome. It corresponds to puromycin aminonucleoside nephrosis in rats. In this model, susceptible 129 S1/SvImJ mice are administered a rapid intravenous injection that can be accomplished via either the lateral tail vein or the retrobulbar sinus. Because doxorubicin is a highly toxic substance, extravasation must be avoided during the administration of the intravenous injection to prevent the development of large necrotizing lesions and exacerbation of the animals' stress. In the present study, we compared the safety and stress of these two injection routes by using histopathological analyses of the animals' orbital cavities or tails, respectively. The injection of 14.5 µg/g body weight doxorubicin into the mice's lateral tail veins (n = 9) or retrobulbar sinuses (n = 19) caused no clinically detectable stress or impairment. Histopathologies of the specimens five days after doxorubicin injection revealed inflammatory lesions at the injection sites in both groups. In the orbital sinus specimens from the retrobulbar-injected group, fibrosis was evident 25 days after injection. Moreover, while all of the retrobulbar-injected mice (100%) developed nephrotic syndrome, tail vein-injected mice had a significantly lower response rate (66%, p = 0.047, Fisher's exact test) and exhibited only attenuated features of nephrotic syndrome. It was therefore concluded that doxorubicin administration via either lateral tail vein or retrobulbar sinus injections led to a similar induction of histopathological changes with no effects on the clinical well-being of the mice. However, retrobulbar sinus injections were more efficient for inducing experimental nephrotic syndrome.
Asunto(s)
Administración Intranasal , Doxorrubicina/efectos adversos , Inyecciones Intravenosas , Síndrome Nefrótico/inducido químicamente , Animales , Femenino , Masculino , Ratones , Proyectos Piloto , Cola (estructura animal)RESUMEN
The goal of this study was to validate the use of an MR-compatible blood sampler (BS) with a detector system based on a lutetium oxyorthosilicate scintillator and avalanche photodiodes for small-animal PET. Methods: Five rats underwent a 60-min 18F-FDG study. For each animal, the arterial input function (AIF) was derived from the BS recording, from manual sampling (MS), and from the PET image. These AIFs were applied for kinetic modeling of the striatum using the irreversible 2-tissue-compartment model. The MS-based technique with a dispersion correction served as a reference approach, and the kinetic parameters that were estimated with the BS- and the image-derived AIFs were compared with the reference values. Additionally, the effect of applying a population-based activity ratio for plasma to whole blood (p/wb) and the dispersion correction was assessed. Results: The K1, k2, and k3 values estimated with the reference approach were 0.174 ± 0.037 mL/min/cm3, 0.342 ± 0.080 1/min, and 0.048 ± 0.009 1/min, respectively. The corresponding parameters obtained with the BS- and image-derived AIFs deviated from these values by 0.6%-18.8% and 16.7%-47.9%, respectively. To compensate for the error in the BS-based technique, data from one MS collected at the end of the experiment were combined with the data from the first 10 min of the BS recording. This approach reduced the deviation in the kinetic parameters to 1.8%-6.3%. Using p/wb led to a 1.7%-8.3% difference from the reference parameters. The sensitivity of the BS was 23%, the energy resolution for the 511-keV photopeak was 19%, and the timing resolution was 11.2 ns. Conclusion: Online recording of the blood activity level with the BS allows precise measurement of AIF, without loss of blood volume. Combining the BS data with one MS is the most accurate approach for the data analysis. The high sensitivity of the device may allow application of lower radioactivity doses.
Asunto(s)
Arterias/fisiología , Tomografía de Emisión de Positrones , Animales , Fluorodesoxiglucosa F18 , Cinética , Masculino , Modelos Biológicos , Ratas , Conteo por Cintilación/instrumentaciónRESUMEN
Manganese in its divalent state (Mn2+) has features that make it a unique tool for tracing neuronal pathways. It is taken up and transported by neurons in an activity-dependent manner and it can cross synapses. It also acts as a contrast agent for magnetic resonance imaging (MRI) enabling visualization of neuronal tracts. However, due to the limited sensitivity of MRI systems relatively high Mn2+ doses are required. This is undesirable, especially in long-term studies, because of the known toxicity of the metal. In order to overcome this limitation, we propose 52Mn as a positron emission tomography (PET) neuronal tract tracer. We used 52Mn for imaging dopaminergic pathways after a unilateral injection into the ventral tegmental area (VTA), as well as the striatonigral pathway after an injection into the dorsal striatum (STR) in rats. Furthermore, we tested potentially noxious effects of the radioactivity dose with a behavioral test and histological staining. 24 h after 52Mn administration, the neuronal tracts were clearly visible in PET images and statistical analysis confirmed the observed distribution of the tracer. We noticed a behavioral impairment in some animals treated with 170 kBq of 52Mn, most likely caused by dysfunction of dopaminergic cells. Moreover, there was a substantial DNA damage in the brain tissue after applying 150 kBq of the tracer. However, all those effects were completely eliminated by reducing the 52Mn dose to 20-30 kBq. Crucially, the reduced dose was still sufficient for PET imaging.
Asunto(s)
Mapeo Encefálico/métodos , Encéfalo/efectos de los fármacos , Manganeso/toxicidad , Tomografía de Emisión de Positrones/métodos , Radiofármacos/toxicidad , Animales , Masculino , Radioisótopos/toxicidad , RatasRESUMEN
The clinical use of Magnetic Resonance Imaging (MRI) and Positron Emission Tomography (PET) has proven to be a strong diagnostic tool in the field of neurology. The reliability of these methods to confirm clinical diagnoses has guided preclinical research to utilize these techniques for the characterization of animal disease models. Previously, we demonstrated that an endothelial cell-specific ablation of the murine Serum Response Factor (SrfiECKO) results in blood brain barrier (BBB) breakdown and hemorrhagic stroke. Taking advantage of this mouse model we here perform a comprehensive longitudinal, multiparametric and in vivo imaging approach to reveal pathophysiological processes occurring before and during the appearance of cerebral microbleeds using combined PET and MRI. We complement our imaging results with data regarding animal behavior and immunohistochemistry. Our results demonstrate diffusion abnormalities in the cortical brain tissue prior to the onset of cerebral microbleeds. Diffusion reductions were accompanied by significant increments of [18F]FAZA uptake before the onset of the lesions in T2WI. The Open Field behavioral tests revealed reduced activity of SrfiECKO animals, whereas histology confirmed the presence of hemorrhages in cortical regions of the mouse brain and iron deposition at lesion sites with increased hypoxia inducible factor 1α, CD31 and glial fibrillary acidic protein expression. For the first time, we performed a thorough evaluation of the prodromal period before the occurrence of spontaneous cerebral microbleeds. Using in vivo PET and MRI, we show the pathological tissue changes that occur previous to gross blood brain barrier (BBB) disruption and breakage. In addition, our results show that apparent diffusion coefficient (ADC) reduction may be an early biomarker of BBB disruption proposing an alternate clinical interpretation. Furthermore, our findings remark the usefulness of this novel SrfiECKO mouse model to study underlying mechanisms of hemorrhagic stroke.
Asunto(s)
Barrera Hematoencefálica/diagnóstico por imagen , Corteza Cerebral/diagnóstico por imagen , Hemorragias Intracraneales/diagnóstico por imagen , Imagen por Resonancia Magnética/métodos , Tomografía de Emisión de Positrones/métodos , Síntomas Prodrómicos , Accidente Cerebrovascular/diagnóstico , Animales , Modelos Animales de Enfermedad , Masculino , Ratones , Ratones TransgénicosRESUMEN
PURPOSE: Great advances have recently been made in treating patients with metastatic melanoma. However, existing therapies are less effective on cerebral than extracerebral metastases. This highlights the potential role of the brain environment on tumor progression and drug resistance and underlines the need for "brain-specific" therapies. We previously showed that the PI3K-AKT survival pathway is hyperactivated in brain but not extracerebral melanoma metastases and that astrocyte-conditioned medium activates AKT in melanoma cells in vitro We therefore tested the PI3K inhibitor buparlisib as an antitumor agent for melanoma brain metastases. EXPERIMENTAL DESIGN AND RESULTS: Buparlisib inhibited AKT activity, decreased proliferation, and induced apoptosis in metastatic melanoma cell lines and short-term brain melanoma cells, irrespective of their BRAF and NRAS mutation status. In addition, buparlisib inhibited hyperactivated AKT and induced apoptosis in melanoma cells that were stimulated with astrocyte-conditioned medium. The growth of tumors induced by injecting human BRAF- and NRAS-mutant metastatic melanoma cells into the brain of mice was significantly inhibited by buparlisib. CONCLUSIONS: These results emphasize the value of targeting the PI3K pathway as a strategy to develop drugs for melanoma brain metastases. Clin Cancer Res; 22(23); 5818-28. ©2016 AACR.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Melanoma/tratamiento farmacológico , Metástasis de la Neoplasia/patología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Humanos , Melanoma/metabolismo , Melanoma/patología , Ratones , Mutación/efectos de los fármacos , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
Metabolic syndrome is characterized by insulin resistance, obesity, and dyslipidemia. It is the consequence of an imbalance between caloric intake and energy consumption. Adiponectin protects against metabolic syndrome. Insulin-induced signaling includes activation of PI3 kinase and protein kinase B (PKB)/Akt. PKB/Akt in turn inactivates glycogen synthase kinase (GSK) 3, a major regulator of metabolism. Here, we studied the significance of PI3K-dependent GSK3 inactivation for adiponectin formation in diet-induced metabolic syndrome. Mice expressing PI3K-insensitive GSK3 (gsk3(KI)) and wild-type mice (gsk3(WT)) were fed a high-fat diet. Compared with gsk3(WT) mice, gsk3(KI) mice were protected against the development of metabolic syndrome as evident from a markedly lower weight gain, lower total body and liver fat accumulation, better glucose tolerance, stronger hepatic insulin-dependent PKB/Akt phosphorylation, lower serum insulin, cholesterol, and triglyceride levels, as well as higher energy expenditure. Serum adiponectin concentration and the activity of transcription factor C/EBPα controlling the expression of adiponectin in adipose tissue was significantly higher in gsk3(KI) mice than in gsk3(WT) mice. Treatment with GSK3 inhibitor lithium significantly decreased the serum adiponectin concentration of gsk3(KI) mice and abrogated the difference in C/EBPα activity between the genotypes. Taken together, our data demonstrate that the expression of PI3K-insensitive GSK3 stimulates the production of adiponectin and protects from diet-induced metabolic syndrome.
Asunto(s)
Adiponectina/biosíntesis , Glucógeno Sintasa Quinasa 3/fisiología , Síndrome Metabólico/enzimología , Tejido Adiposo/metabolismo , Animales , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Dieta Alta en Grasa/efectos adversos , Intolerancia a la Glucosa/enzimología , Resistencia a la Insulina , Hígado/enzimología , Masculino , Síndrome Metabólico/etiología , Ratones Transgénicos , Fosfohidrolasa PTEN/metabolismo , Fosfatidilinositol 3-Quinasas/fisiologíaRESUMEN
White matter (WM) atrophy is a significant feature of Huntington disease (HD), although its aetiology and early pathological manifestations remain poorly defined. In this study, we aimed to characterize WM-related features in the transgenic YAC128 and BACHD models of HD. Using diffusion tensor magnetic resonance imaging (DT-MRI), we demonstrate that microstructural WM abnormalities occur from an early age in YAC128 mice. Similarly, electron microscopy analysis of myelinated fibres of the corpus callosum indicated that myelin sheaths are thinner in YAC128 mice as early as 1.5 months of age, well before any neuronal loss can be detected. Transcript levels of myelin-related genes in striatal and cortical tissues were significantly lower in YAC128 mice from 2 weeks of age, and these findings were replicated in differentiated primary oligodendrocytes from YAC128 mice, suggesting a possible mechanistic explanation for the observed structural deficits. Concordant with these observations, we demonstrate reduced expression of myelin-related genes at 3 months of age and WM microstructural abnormalities using DT-MRI at 12 months of age in the BACHD rats. These findings indicate that WM deficits in HD are an early phenotype associated with cell-intrinsic effects of mutant huntingtin on myelin-related transcripts in oligodendrocytes, and raise the possibility that WM abnormalities may be an early contributing factor to the pathogenesis of HD.
Asunto(s)
Enfermedad de Huntington/genética , Vaina de Mielina/fisiología , Sustancia Blanca/fisiopatología , Animales , Atrofia/patología , Encéfalo/metabolismo , Cuerpo Calloso/metabolismo , Cuerpo Estriado/metabolismo , Imagen de Difusión Tensora/métodos , Modelos Animales de Enfermedad , Expresión Génica , Humanos , Enfermedad de Huntington/etiología , Ratones , Ratones Transgénicos , Vaina de Mielina/genética , Vaina de Mielina/metabolismo , Neostriado/metabolismo , Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Oligodendroglía/metabolismo , RatasRESUMEN
Huntington's disease is a fatal human neurodegenerative disorder caused by a CAG repeat expansion in the HTT gene, which translates into a mutant huntingtin protein. A key event in the molecular pathogenesis of Huntington's disease is the proteolytic cleavage of mutant huntingtin, leading to the accumulation of toxic protein fragments. Mutant huntingtin cleavage has been linked to the overactivation of proteases due to mitochondrial dysfunction and calcium derangements. Here, we investigated the therapeutic potential of olesoxime, a mitochondria-targeting, neuroprotective compound, in the BACHD rat model of Huntington's disease. BACHD rats were treated with olesoxime via the food for 12 months. In vivo analysis covered motor impairments, cognitive deficits, mood disturbances and brain atrophy. Ex vivo analyses addressed olesoxime's effect on mutant huntingtin aggregation and cleavage, as well as brain mitochondria function. Olesoxime improved cognitive and psychiatric phenotypes, and ameliorated cortical thinning in the BACHD rat. The treatment reduced cerebral mutant huntingtin aggregates and nuclear accumulation. Further analysis revealed a cortex-specific overactivation of calpain in untreated BACHD rats. Treated BACHD rats instead showed significantly reduced levels of mutant huntingtin fragments due to the suppression of calpain-mediated cleavage. In addition, olesoxime reduced the amount of mutant huntingtin fragments associated with mitochondria, restored a respiration deficit, and enhanced the expression of fusion and outer-membrane transport proteins. In conclusion, we discovered the calpain proteolytic system, a key player in Huntington's disease and other neurodegenerative disorders, as a target of olesoxime. Our findings suggest that olesoxime exerts its beneficial effects by improving mitochondrial function, which results in reduced calpain activation. The observed alleviation of behavioural and neuropathological phenotypes encourages further investigations on the use of olesoxime as a therapeutic for Huntington's disease.
Asunto(s)
Calpaína/metabolismo , Colestenonas/farmacología , Colestenonas/uso terapéutico , Enfermedad de Huntington/tratamiento farmacológico , Enfermedad de Huntington/metabolismo , Proteínas Mutantes/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Proteolisis/efectos de los fármacos , Animales , Conducta Animal/efectos de los fármacos , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Calpaína/antagonistas & inhibidores , Colestenonas/sangre , Colestenonas/metabolismo , Modelos Animales de Enfermedad , Activación Enzimática/efectos de los fármacos , Proteína Huntingtina , Enfermedad de Huntington/enzimología , Enfermedad de Huntington/genética , Masculino , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/genética , Mutación , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Ratas , Ratas TransgénicasRESUMEN
Intracerebral hemorrhagic stroke and vascular dementia are age- and hypertension-associated manifestations of human cerebral small vessel disease (SVD). Cerebral microvessels are formed by endothelial cells (ECs), which are connected through tight junctions, adherens junctions, and stabilizing basement membrane structures. These endothelial connections ensure both vessel stability and blood-brain barrier (BBB) functions, the latter enabling selective exchange of ions, bioactive molecules, and cells between the bloodstream and brain tissue. Srf(iECKO) mice, permitting conditional EC-specific depletion of the transcription factor Serum Response Factor (SRF), suffer from loss of BBB integrity and intracerebral hemorrhaging. Cerebral microbleeds and larger hemorrhages developed upon postnatal and adult depletion of either SRF or its cofactors Myocardin Related Transcription Factor (MRTF-A/-B), revealing essential requirements of ongoing SRF/MRTF activity for maintenance of cerebral small vessel integrity. In vivo magnetic resonance imaging allowed detection, localization, and time-resolved quantification of BBB permeability and hemorrhage formation in Srf(iECKO) brains. At the molecular level, direct and indirect SRF/MRTF target genes, encoding structural components of tight junctions (Claudins and ZO proteins), adherens junctions (VE-cadherin, α-Actinin), and the basement membrane (Collagen IV), were down-regulated upon SRF depletion. These results identify SRF and its MRTF cofactors as major transcriptional regulators of EC junctional stability, guaranteeing physiological functions of the cerebral microvasculature. We hypothesize that impairments in SRF/MRTF activity contribute to human SVD pathology.
Asunto(s)
Hemorragia Cerebral/complicaciones , Células Endoteliales/metabolismo , Factor de Respuesta Sérica/metabolismo , Accidente Cerebrovascular/etiología , Accidente Cerebrovascular/metabolismo , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Animales , Animales Recién Nacidos , Astrocitos/metabolismo , Astrocitos/patología , Membrana Basal/metabolismo , Membrana Basal/patología , Barrera Hematoencefálica/metabolismo , Encéfalo/irrigación sanguínea , Encéfalo/metabolismo , Encéfalo/patología , Encéfalo/fisiopatología , Cadherinas/metabolismo , Hemorragia Cerebral/metabolismo , Hemorragia Cerebral/patología , Hemorragia Cerebral/fisiopatología , Colágeno Tipo IV/metabolismo , Regulación hacia Abajo , Azul de Evans/metabolismo , Conducta Exploratoria , Extravasación de Materiales Terapéuticos y Diagnósticos , Eliminación de Gen , Imagen por Resonancia Magnética , Ratones Noqueados , Microvasos/metabolismo , Microvasos/patología , Actividad Motora , Permeabilidad , Factor de Respuesta Sérica/genética , Accidente Cerebrovascular/patología , Accidente Cerebrovascular/fisiopatología , Uniones Estrechas/metabolismo , Factores de TiempoRESUMEN
The dynamics of ß-amyloid deposition and related second-order physiological effects, such as regional cerebral blood flow (rCBF), are key factors for a deeper understanding of Alzheimer's disease (AD). We present longitudinal in vivo data on the dynamics of ß-amyloid deposition and the decline of rCBF in two different amyloid precursor protein (APP) transgenic mouse models of AD. Using a multiparametric positron emission tomography and magnetic resonance imaging approach, we demonstrate that in the presence of cerebral ß-amyloid angiopathy (CAA), ß-amyloid deposition is accompanied by a decline of rCBF. Loss of perfusion correlates with the growth of ß-amyloid plaque burden but is not related to the number of CAA-induced microhemorrhages. However, in a mouse model of parenchymal ß-amyloidosis and negligible CAA, rCBF is unchanged. Because synaptically driven spontaneous network activity is similar in both transgenic mouse strains, we conclude that the disease-related decline of rCBF is caused by CAA.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Encéfalo/patología , Angiopatía Amiloide Cerebral/patología , Hemorragia Cerebral/patología , Circulación Cerebrovascular , Placa Amiloide/patología , Precursor de Proteína beta-Amiloide/genética , Compuestos de Anilina , Animales , Benzotiazoles , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Angiopatía Amiloide Cerebral/diagnóstico por imagen , Angiopatía Amiloide Cerebral/metabolismo , Hemorragia Cerebral/diagnóstico por imagen , Modelos Animales de Enfermedad , Femenino , Estudios Longitudinales , Imagen por Resonancia Magnética , Ratones , Ratones Transgénicos , Imagen Multimodal , Imagen de Perfusión , Placa Amiloide/diagnóstico por imagen , Placa Amiloide/metabolismo , Tomografía de Emisión de Positrones , Radiofármacos , TiazolesRESUMEN
Spinocerebellar ataxia 17 (SCA17) is an autosomal-dominant, late-onset neurodegenerative disorder caused by an expanded polyglutamine (polyQ) repeat in the TATA-box-binding protein (TBP). To further investigate this devastating disease, we sought to create a first transgenic rat model for SCA17 that carries a full human cDNA fragment of the TBP gene with 64 CAA/CAG repeats (TBPQ64). In line with previous observations in mouse models for SCA17, TBPQ64 rats show a severe neurological phenotype including ataxia, impairment of postural reflexes, and hyperactivity in early stages followed by reduced activity, loss of body weight, and early death. Neuropathologically, the severe phenotype of SCA17 rats was associated with neuronal loss, particularly in the cerebellum. Degeneration of Purkinje, basket, and stellate cells, changes in the morphology of the dendrites, nuclear TBP-positive immunoreactivity, and axonal torpedos were readily found by light and electron microscopy. While some of these changes are well recapitulated in existing mouse models for SCA17, we provide evidence that some crucial characteristics of SCA17 are better mirrored in TBPQ64 rats. Thus, this SCA17 model represents a valuable tool to pursue experimentation and therapeutic approaches that may be difficult or impossible to perform with SCA17 transgenic mice. We show for the first time positron emission tomography (PET) and diffusion tensor imaging (DTI) data of a SCA animal model that replicate recent PET studies in human SCA17 patients. Our results also confirm that DTI are potentially useful correlates of neuropathological changes in TBPQ64 rats and raise hope that DTI imaging could provide a biomarker for SCA17 patients.
Asunto(s)
Imagen de Difusión Tensora , Modelos Animales de Enfermedad , Ataxias Espinocerebelosas , Proteína de Unión a TATA-Box/genética , Expansión de Repetición de Trinucleótido/genética , Animales , Ansiedad/etiología , Ansiedad/genética , Peso Corporal/genética , Encéfalo/metabolismo , Encéfalo/patología , Encéfalo/ultraestructura , Procesamiento Automatizado de Datos , Femenino , Genotipo , Humanos , Masculino , Aprendizaje por Laberinto , Actividad Motora , Examen Neurológico , Tomografía de Emisión de Positrones , Desempeño Psicomotor/fisiología , Racloprida/farmacocinética , Ratas , Ratas Transgénicas , Prueba de Desempeño de Rotación con Aceleración Constante , Índice de Severidad de la Enfermedad , Ataxias Espinocerebelosas/diagnóstico , Ataxias Espinocerebelosas/genética , Ataxias Espinocerebelosas/fisiopatología , Tubulina (Proteína)/metabolismoRESUMEN
Conversion of soluble α-synuclein into insoluble and fibrillar inclusions is a hallmark of Parkinson's disease and other synucleinopathies. Accumulating evidence points towards a relationship between its generation at nerve terminals and structural synaptic pathology. Little is known about the pathogenic impact of α-synuclein conversion and deposition at nigrostriatal dopaminergic synapses in transgenic mice, mainly owing to expression limitations of the α-synuclein construct. Here, we explore whether both the rat as a model and expression of the bacterial artificial chromosome construct consisting of human full-length wild-type α-synuclein could exert dopaminergic neuropathological effects. We found that the human promoter induced a pan-neuronal expression, matching the rodent α-synuclein expression pattern, however, with prominent C-terminally truncated fragments. Ageing promoted conversion of both full-length and C-terminally truncated α-synuclein species into insolube and proteinase K-resistant fibres, with strongest accumulation in the striatum, resembling biochemical changes seen in human Parkinson's disease. Transgenic rats develop early changes in novelty-seeking, avoidance and smell before the progressive motor deficit. Importantly, the observed pathological changes were associated with severe loss of the dopaminergic integrity, thus resembling more closely the human pathology.
Asunto(s)
Cromosomas Artificiales Bacterianos/genética , Neuronas Dopaminérgicas/patología , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patología , Fenotipo , alfa-Sinucleína/genética , Animales , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Neuronas Dopaminérgicas/metabolismo , Humanos , Enfermedad de Parkinson/metabolismo , Ratas , Ratas Sprague-Dawley , Ratas Transgénicas , alfa-Sinucleína/biosíntesis , alfa-Sinucleína/toxicidadRESUMEN
Huntington disease (HD) is an inherited progressive neurodegenerative disorder, characterized by motor, cognitive, and psychiatric deficits as well as neurodegeneration and brain atrophy beginning in the striatum and the cortex and extending to other subcortical brain regions. The genetic cause is an expansion of the CAG repeat stretch in the HTT gene encoding huntingtin protein (htt). Here, we generated an HD transgenic rat model using a human bacterial artificial chromosome (BAC), which contains the full-length HTT genomic sequence with 97 CAG/CAA repeats and all regulatory elements. BACHD transgenic rats display a robust, early onset and progressive HD-like phenotype including motor deficits and anxiety-related symptoms. In contrast to BAC and yeast artificial chromosome HD mouse models that express full-length mutant huntingtin, BACHD rats do not exhibit an increased body weight. Neuropathologically, the distribution of neuropil aggregates and nuclear accumulation of N-terminal mutant huntingtin in BACHD rats is similar to the observations in human HD brains. Aggregates occur more frequently in the cortex than in the striatum and neuropil aggregates appear earlier than mutant htt accumulation in the nucleus. Furthermore, we found an imbalance in the striatal striosome and matrix compartments in early stages of the disease. In addition, reduced dopamine receptor binding was detectable by in vivo imaging. Our data demonstrate that this transgenic BACHD rat line may be a valuable model for further understanding the disease mechanisms and for preclinical pharmacological studies.
Asunto(s)
Cromosomas Artificiales Bacterianos/genética , Enfermedad de Huntington/genética , Enfermedad de Huntington/patología , Proteínas del Tejido Nervioso/genética , Empalme Alternativo , Animales , Ansiedad/genética , Ansiedad/psicología , Conducta Animal/fisiología , Western Blotting , Peso Corporal/fisiología , Ingestión de Alimentos/fisiología , Trastornos Neurológicos de la Marcha/psicología , Dosificación de Gen , Humanos , Proteína Huntingtina , Enfermedad de Huntington/psicología , Inmunohistoquímica , Actividad Motora/fisiología , Tomografía de Emisión de Positrones , Equilibrio Postural/fisiología , Regiones Promotoras Genéticas , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Ratas Transgénicas , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Conflicts in spatial stimulus-response tasks occur when the task-relevant feature of a stimulus implies a response toward a certain location which does not match the location of stimulus presentation. This conflict leads to increased error rates and longer reaction times, which has been termed Simon effect. A model of dual route processing (automatic and intentional) of stimulus features has been proposed, predicting response conflicts if the two routes are incongruent. Although there is evidence that the prefrontal cortex, notably the anterior cingulate cortex (ACC), plays a crucial role in conflict processing, the neuronal basis of dual route architecture is still unknown. In this study, we pursue a novel approach using positron emission tomography (PET) to identify relevant brain areas in a rat model of an auditory Simon task, a neuropsychological interference task, which is commonly used to study conflict processing in humans. For combination with PET we used the metabolic tracer [(18)F]fluorodeoxyglucose, which accumulates in metabolically active brain cells during the behavioral task. Brain areas involved in conflict processing are supposed to be activated when automatic and intentional route processing lead to different responses (dual route model). Analysis of PET data revealed specific activation patterns for different task settings applicable to the dual route model as established for response conflict processing. The rat motor cortex (M1) may be part of the automatic route or involved in its facilitation, while premotor (M2), prelimbic, and ACC seemed to be essential for inhibiting the incorrect, automatic response, indicating conflict monitoring functions. Our findings and the remarkable similarities to the pattern of activated regions reported during conflict processing in humans demonstrate that our rodent model opens novel opportunities to investigate the anatomical basis of conflict processing and dual route architecture.