RESUMEN
Elucidation of the mutational landscape of human cancer has progressed rapidly and been accompanied by the development of therapeutics targeting mutant oncogenes. However, a comprehensive mapping of cancer dependencies has lagged behind and the discovery of therapeutic targets for counteracting tumor suppressor gene loss is needed. To identify vulnerabilities relevant to specific cancer subtypes, we conducted a large-scale RNAi screen in which viability effects of mRNA knockdown were assessed for 7,837 genes using an average of 20 shRNAs per gene in 398 cancer cell lines. We describe findings of this screen, outlining the classes of cancer dependency genes and their relationships to genetic, expression, and lineage features. In addition, we describe robust gene-interaction networks recapitulating both protein complexes and functional cooperation among complexes and pathways. This dataset along with a web portal is provided to the community to assist in the discovery and translation of new therapeutic approaches for cancer.
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Neoplasias/genética , Neoplasias/patología , Interferencia de ARN , Línea Celular Tumoral , Biblioteca de Genes , Redes Reguladoras de Genes , Humanos , Complejos Multiproteicos/metabolismo , Neoplasias/metabolismo , Oncogenes , ARN Interferente Pequeño , Transducción de Señal , Factores de Transcripción/metabolismoRESUMEN
AIM: Tau becomes hyperphosphorylated in Alzheimer's disease (AD) and frontotemporal lobar degeneration (FTLD-tau), resulting in functional deficits of neurones, neurofibrillary tangle (NFT) formation and eventually dementia. Expression of mutant human tau in the brains of transgenic mice has produced different lines that recapitulate various aspects of FTLD-tau and AD. In this study, we characterized the novel P301S mutant tau transgenic mouse line, TAU58/2. METHODS: Both young and aged TAU58/2 mice underwent extensive motor testing, after which brain tissue was analysed with immunohistochemistry, silver staining, electron microscopy and Western blotting. Tissue from various FTLD subtypes and AD patients was also analysed for comparison. RESULTS: TAU58/2 mice presented with early-onset motor deficits, which became more pronounced with age. Throughout the brains of these mice, tau was progressively hyperphosphorylated resulting in increased NFT formation with age. In addition, frequent axonal swellings that stained intensively for neurofilament (NF) were present in young TAU58/2 mice prior to NFT formation. Similar axonal pathology was also observed in human FTLD-tau and AD. Interestingly, activated microglia were found in close proximity to neurones harbouring transgenic tau, but were not associated with NF-positive axonal swellings. CONCLUSIONS: In TAU58/2 mice, early tau pathology induces functional deficits of neurones associated with NF pathology. This appears to be specific to tau, as similar changes are observed in FTLD-tau, but not in FTLD with TDP-43 inclusions. Therefore, TAU58/2 mice recapitulate aspects of human FTLD-tau and AD pathology, and will become instrumental in studying disease mechanisms and therapeutics in the future.
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Axones/patología , Encéfalo/patología , Degeneración Lobar Frontotemporal/patología , Neuronas/patología , Animales , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Degeneración Lobar Frontotemporal/genética , Degeneración Lobar Frontotemporal/metabolismo , Ratones , Ratones Transgénicos , Neuronas/metabolismo , Proteínas tau/genética , Proteínas tau/metabolismoRESUMEN
An early role of amyloid-ß peptide (Aß) aggregation in Alzheimer's disease pathogenesis is well established. However, the contribution of intracellular or extracellular forms of Aß to the neurodegenerative process is a subject of considerable debate. We here describe transgenic mice expressing Aß1-40 (APP47) and Aß1-42 (APP48) with a cleaved signal sequence to insert both peptides during synthesis into the endoplasmic reticulum. Although lower in transgene mRNA, APP48 mice reach a higher brain Aß concentration. The reduced solubility and increased aggregation of Aß1-42 may impair its degradation. APP48 mice develop intracellular Aß lesions in dendrites and lysosomes. The hippocampal neuron number is reduced already at young age. The brain weight decreases during aging in conjunction with severe white matter atrophy. The mice show a motor impairment. Only very few Aß1-40 lesions are found in APP47 mice. Neither APP47 nor APP48 nor the bigenic mice develop extracellular amyloid plaques. While intracellular membrane expression of Aß1-42 in APP48 mice does not lead to the AD-typical lesions, Aß aggregates develop within cells accompanied by considerable neurodegeneration.
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Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/biosíntesis , Péptidos beta-Amiloides/genética , Regulación de la Expresión Génica , Degeneración Nerviosa/genética , Neuronas/metabolismo , Fragmentos de Péptidos/biosíntesis , Fragmentos de Péptidos/genética , Factores de Edad , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Animales , Humanos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/patología , Neuronas/patología , RatasRESUMEN
Cerebral amyloid angiopathy (CAA) is a common feature of Alzheimer's disease (AD). More advanced stages are accompanied by microhemorrhages and vasculitis. Peripheral blood-borne macrophages are intimately linked to cerebrovascular pathology coincident with AD. Magnetic resonance imaging (MRI) was used to noninvasively study microvascular lesions in amyloid precursor protein transgenic mouse AD models. Foci of signal attenuation were detected in cortical and thalamic brain regions of aged APP23 mice. Their strength and number was considerably enhanced by intravenous administration of iron oxide nanoparticles, which are taken up by macrophages through absorptive endocytosis, 24 h before image acquisition. The number of cortical sites displaying signal attenuation increased with age. Histology at these sites demonstrated the presence of iron-containing macrophages in the vicinity of CAA-affected blood vessels. A fraction of the sites additionally showed thickened vessel walls and vasculitis. Consistent with the visualization of CAA-associated lesions, MRI detected a much smaller number of attenuated signal sites in APP23xPS45 mice, for which a strong presenilin mutation caused a shift toward amyloid ß(42), thus reducing vascular amyloid. Similar results were obtained with APP24 and APP51 mice, which develop significantly less CAA and microvascular pathology than APP23. In a longitudinal study, we noninvasively demonstrated the reinforced formation of microvascular pathology during passive amyloid ß immunotherapy of APP23 mice. Histology confirmed that foci of signal attenuation reflected an increase in CAA-related lesions. Our data demonstrate that MRI has the sensitivity to noninvasively monitor the development of vascular pathology and its possible enhancement by amyloid ß immunotherapy in transgenic mice modeling AD.
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Enfermedad de Alzheimer/patología , Precursor de Proteína beta-Amiloide/genética , Angiopatía Amiloide Cerebral/patología , Corteza Cerebral/patología , Nanopartículas de Magnetita , Enfermedad de Alzheimer/genética , Análisis de Varianza , Animales , Angiopatía Amiloide Cerebral/genética , Modelos Animales de Enfermedad , Femenino , Inmunización Pasiva , Imagen por Resonancia Magnética , Masculino , Ratones , Ratones Transgénicos , Estadísticas no ParamétricasRESUMEN
Huntington's disease is caused by a gain-of-function neurotoxic mutation in normally neuroprotective huntingtin. Sensitive assays are required to discriminate mutant huntingtin from wild-type huntingtin. We have developed a normalized 384-plate assay for determination of mutant and wild-type huntingtin. Based on a single pipetting step, the sensitive assay uses two antibody pairs for simultaneous mutant and wild-type huntingtin time-resolved fluorescence resonance energy transfer detection combined with PicoGreen quantification of double-stranded DNA. The assay can be used for discovery of drugs reducing mutant huntingtin over wild-type huntingtin and for assessing the value of huntingtin as a disease progression marker, and it is adaptable to other proteins of interest.
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Anticuerpos/análisis , Transferencia Resonante de Energía de Fluorescencia/métodos , Proteínas Mutantes/análisis , Proteínas Mutantes/química , Proteínas del Tejido Nervioso/análisis , Proteínas del Tejido Nervioso/química , Proteínas Nucleares/análisis , Proteínas Nucleares/química , Recuento de Células , Línea Celular , ADN , Fibroblastos/citología , Humanos , Proteína Huntingtina , Enfermedad de Huntington/genética , Compuestos OrgánicosRESUMEN
Uveal melanoma is a rare and aggressive cancer that originates in the eye. Currently, there are no approved targeted therapies and very few effective treatments for this cancer. Although activating mutations in the G protein alpha subunits, GNAQ and GNA11, are key genetic drivers of the disease, few additional drug targets have been identified. Recently, studies have identified context-specific roles for the mammalian SWI/SNF chromatin remodeling complexes (also known as BAF/PBAF) in various cancer lineages. Here, we find evidence that the SWI/SNF complex is essential through analysis of functional genomics screens and further validation in a panel of uveal melanoma cell lines using both genetic tools and small-molecule inhibitors of SWI/SNF. In addition, we describe a functional relationship between the SWI/SNF complex and the melanocyte lineage-specific transcription factor Microphthalmia-associated Transcription Factor, suggesting that these two factors cooperate to drive a transcriptional program essential for uveal melanoma cell survival. These studies highlight a critical role for SWI/SNF in uveal melanoma, and demonstrate a novel path toward the treatment of this cancer.
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Cromatina/metabolismo , Melanoma/genética , Neoplasias de la Úvea/genética , Animales , Línea Celular Tumoral , Proteínas Cromosómicas no Histona , Humanos , Ratones , Factores de TranscripciónRESUMEN
The genetic mutation causing Huntington's disease is a polyglutamine expansion in the huntingtin protein where more than 37 glutamines cause disease by formation of toxic intracellular fragments, aggregates, and cell death. Despite a clear pathogenic role for mutant huntingtin, understanding huntingtin expression during the presymptomatic phase of the disease or during disease progression has remained obscure. Central to clarifying the role in the pathomechanism of disease is the ability to easily and accurately measure mutant huntingtin in accessible human tissue samples as well as cell and animal models. Here we describe a highly sensitive time-resolved Förster resonance energy transfer (FRET) assay for quantification of soluble mutant huntingtin in brain, plasma, and cerebrospinal fluid. Surprisingly, in mice, soluble huntingtin levels decrease during disease progression, inversely correlating with brain aggregate load. Mutant huntingtin is easily detected in human brain and blood-derived fractions, providing a utility to assess mutant huntingtin expression during disease course as well as a pharmacodynamic marker for disease-modifying therapeutics targeting expression, cleavage, or degradation of mutant huntingtin. The design of the homogeneous one-step method for huntingtin detection is such that it can be easily applied to measure other proteins of interest.
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Transferencia Resonante de Energía de Fluorescencia/métodos , Enfermedad de Huntington/diagnóstico , Proteínas Mutantes/aislamiento & purificación , Proteínas del Tejido Nervioso/aislamiento & purificación , Proteínas Nucleares/aislamiento & purificación , Adulto , Análisis de Varianza , Animales , Encéfalo/metabolismo , Línea Celular , Progresión de la Enfermedad , Células Madre Embrionarias/metabolismo , Exones , Femenino , Expresión Génica , Humanos , Proteína Huntingtina , Enfermedad de Huntington/sangre , Enfermedad de Huntington/líquido cefalorraquídeo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Persona de Mediana Edad , Músculo Esquelético/metabolismo , Proteínas Mutantes/metabolismo , Neocórtex/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Recombinantes de Fusión , Sensibilidad y Especificidad , Factores de Tiempo , Adulto JovenRESUMEN
Human beta-amyloid precursor protein (APP) transgenic mice are commonly used to test potential therapeutics for Alzheimer's disease. We have characterized the dynamics of beta-amyloid (Abeta) generation and deposition following gamma-secretase inhibition with compound LY-411575 [N(2)-[(2S)-2-(3,5-difluorophenyl)-2-hydroxyethanoyl]-N(1)-[(7S)-5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl]-L-alaninamide]. Kinetic studies in preplaque mice distinguished a detergent-soluble Abeta pool in brain with rapid turnover (half-lives for Abeta40 and Abeta42 were 0.7 and 1.7 h) and a much more stable, less soluble pool. Abeta in cerebrospinal fluid (CSF) reflected the changes in the soluble brain Abeta pool, whereas plasma Abeta turned over more rapidly. In brain, APP C-terminal fragments (CTF) accumulated differentially. The half-lives for gamma-secretase degradation were estimated as 0.4 and 0.1 h for C99 and C83, respectively. Three different APP transgenic lines responded very similarly to gamma-secretase inhibition regardless of the familial Alzheimer's disease mutations in APP. Amyloid deposition started with Abeta42, whereas Abeta38 and Abeta40 continued to turn over. Chronic gamma-secretase inhibition lowered amyloid plaque formation to a different degree in different brain regions of the same mice. The extent was inversely related to the initial amyloid load in the region analyzed. No evidence for plaque removal below baseline was obtained. gamma-Secretase inhibition led to a redistribution of intracellular Abeta and an elevation of CTFs in neuronal fibers. In CSF, Abeta showed a similar turnover as in preplaque animals demonstrating its suitability as marker of newly generated, soluble Abeta in plaque-bearing brain. This study supports the use of APP transgenic mice as translational models to characterize Abeta-lowering therapeutics.
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Alanina/análogos & derivados , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Azepinas/farmacología , Encéfalo/metabolismo , Inhibidores Enzimáticos/farmacología , Alanina/farmacología , Precursor de Proteína beta-Amiloide/análisis , Precursor de Proteína beta-Amiloide/genética , Animales , Semivida , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
Amyloid precursor protein (APP) processing and the generation of beta-amyloid peptide (Abeta) are important in the pathogenesis of Alzheimer's disease. Although this has been studied extensively at the molecular and cellular levels, much less is known about the mechanisms of amyloid accumulation in vivo. We transplanted transgenic APP23 and wild-type B6 embryonic neural cells into the neocortex and hippocampus of both B6 and APP23 mice. APP23 grafts into wild-type hosts did not develop amyloid deposits up to 20 months after grafting. In contrast, both transgenic and wild-type grafts into young transgenic hosts developed amyloid plaques as early as 3 months after grafting. Although largely diffuse in nature, some of the amyloid deposits in wild-type grafts were congophilic and were surrounded by neuritic changes and gliosis, similar to the amyloid-associated pathology previously described in APP23 mice. Our results indicate that diffusion of soluble Abeta in the extracellular space is involved in the spread of Abeta pathology, and that extracellular amyloid formation can lead to neurodegeneration.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/biosíntesis , Precursor de Proteína beta-Amiloide/metabolismo , Espacio Extracelular/metabolismo , Hipocampo/metabolismo , Neuronas/metabolismo , Placa Amiloide/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/fisiopatología , Péptidos beta-Amiloides/genética , Precursor de Proteína beta-Amiloide/genética , Animales , Trasplante de Tejido Encefálico , Difusión , Modelos Animales de Enfermedad , Espacio Extracelular/genética , Gliosis/genética , Gliosis/metabolismo , Gliosis/patología , Supervivencia de Injerto/genética , Hipocampo/fisiopatología , Hipocampo/trasplante , Ratones , Ratones Transgénicos , Neuronas/patología , Placa Amiloide/genética , Placa Amiloide/patología , Transporte de Proteínas/genética , Tiempo de Reacción/genética , Solubilidad , Regulación hacia Arriba/genéticaRESUMEN
The E693Q mutation in the amyloid beta precursor protein (APP) leads to cerebral amyloid angiopathy (CAA), with recurrent cerebral hemorrhagic strokes and dementia. In contrast to Alzheimer disease (AD), the brains of those affected by hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D) show few parenchymal amyloid plaques. We found that neuronal overexpression of human E693Q APP in mice (APPDutch mice) caused extensive CAA, smooth muscle cell degeneration, hemorrhages and neuroinflammation. In contrast, overexpression of human wild-type APP (APPwt mice) resulted in predominantly parenchymal amyloidosis, similar to that seen in AD. In APPDutch mice and HCHWA-D human brain, the ratio of the amyloid-beta40 peptide (Abeta40) to Abeta42 was significantly higher than that seen in APPwt mice or AD human brain. Genetically shifting the ratio of AbetaDutch40/AbetaDutch42 toward AbetaDutch42 by crossing APPDutch mice with transgenic mice producing mutated presenilin-1 redistributed the amyloid pathology from the vasculature to the parenchyma. The understanding that different Abeta species can drive amyloid pathology in different cerebral compartments has implications for current anti-amyloid therapeutic strategies. This HCHWA-D mouse model is the first to develop robust CAA in the absence of parenchymal amyloid, highlighting the key role of neuronally produced Abeta to vascular amyloid pathology and emphasizing the differing roles of Abeta40 and Abeta42 in vascular and parenchymal amyloid pathology.
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Péptidos beta-Amiloides/metabolismo , Amiloidosis/metabolismo , Vasos Sanguíneos/metabolismo , Hemorragia Cerebral/metabolismo , Modelos Animales de Enfermedad , Factores de Edad , Anciano , Anciano de 80 o más Años , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Amiloidosis/complicaciones , Animales , Vasos Sanguíneos/patología , Vasos Sanguíneos/ultraestructura , Western Blotting/métodos , Encéfalo/metabolismo , Encéfalo/patología , Hemorragia Cerebral/complicaciones , Circulación Cerebrovascular , Encefalitis/etiología , Encefalitis/metabolismo , Encefalitis/patología , Ensayo de Inmunoadsorción Enzimática/métodos , Ácido Glutámico/genética , Glutamina/genética , Humanos , Inmunohistoquímica/métodos , Hibridación in Situ , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Electrónica/métodos , Persona de Mediana Edad , Mutación/genética , Fragmentos de Péptidos/metabolismo , Piamadre/metabolismo , Cambios Post Mortem , Antígenos Thy-1/genéticaRESUMEN
The amyloid beta-protein (Abeta) is the main component of Alzheimer's disease-related senile plaques. Although Abeta is associated with the development of Alzheimer's disease, it has not been shown which forms of Abeta induce neurodegeneration in vivo and which types of neurons are vulnerable. To address these questions, we implanted DiI crystals into the left frontocentral cortex of APP23 transgenic mice overexpressing mutant human APP (amyloid precursor protein gene) and of littermate controls. Traced commissural neurons in layer III of the right frontocentral cortex were quantified in 3-, 5-, 11- and 15-month-old mice. Three different types of commissural neurons were traced. At 3 months of age no differences in the number of labelled commissural neurons were seen in APP23 mice compared with wild-type mice. A selective reduction of the heavily ramified type of neurons was observed in APP23 mice compared with wild-type animals at 5, 11 and 15 months of age, starting when the first Abeta-deposits occurred in the frontocentral cortex at 5 months. The other two types of commissural neurons did not show alterations at 5 and 11 months. At 15 months, the number of traced sparsely ramified pyramidal neurons was reduced in addition to that of the heavily ramified neurons in APP23 mice compared with wild-type mice. At this time Abeta-deposits were seen in the neo- and allocortex as well as in the basal ganglia and the thalamus. In summary, our results show that Abeta induces progressive degeneration of distinct types of commissural neurons. Degeneration of the most vulnerable neurons starts in parallel with the occurrence of the first fibrillar Abeta-deposits in the neocortex, that is, with the detection of aggregated Abeta. The involvement of additional neuronal subpopulations is associated with the expansion of Abeta-deposition into further brain regions. The vulnerability of different types of neurons to Abeta, thereby, is presumably related to the complexity of their dendritic morphology.
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Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/fisiología , Células Dendríticas/patología , Neuronas/patología , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Animales , Axones/patología , Western Blotting , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Ratones , Ratones Transgénicos , Neocórtex/patología , Placa Amiloide/patología , Prosencéfalo/metabolismoRESUMEN
UNLABELLED: CRISPR/Cas9 has emerged as a powerful new tool to systematically probe gene function. We compared the performance of CRISPR to RNAi-based loss-of-function screens for the identification of cancer dependencies across multiple cancer cell lines. CRISPR dropout screens consistently identified more lethal genes than RNAi, implying that the identification of many cellular dependencies may require full gene inactivation. However, in two aneuploid cancer models, we found that all genes within highly amplified regions, including nonexpressed genes, scored as lethal by CRISPR, revealing an unanticipated class of false-positive hits. In addition, using a CRISPR tiling screen, we found that sgRNAs targeting essential domains generate the strongest lethality phenotypes and thus provide a strategy to rapidly define the protein domains required for cancer dependence. Collectively, these findings not only demonstrate the utility of CRISPR screens in the identification of cancer-essential genes, but also reveal the need to carefully control for false-positive results in chromosomally unstable cancer lines. SIGNIFICANCE: We show in this study that CRISPR-based screens have a significantly lower false-negative rate compared with RNAi-based screens, but have specific liabilities particularly in the interrogation of regions of genome amplification. Therefore, this study provides critical insights for applying CRISPR-based screens toward the systematic identification of new cancer targets. Cancer Discov; 6(8); 900-13. ©2016 AACR.See related commentary by Sheel and Xue, p. 824See related article by Aguirre et al., p. 914This article is highlighted in the In This Issue feature, p. 803.
Asunto(s)
Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Amplificación de Genes , Genoma Humano , Genómica , Neoplasias/genética , Línea Celular Tumoral , Estudios de Asociación Genética , Genómica/métodos , Genómica/normas , Ensayos Analíticos de Alto Rendimiento , Humanos , Fenotipo , Polimorfismo de Nucleótido Simple , Sitios de Carácter Cuantitativo , ARN Guía de Kinetoplastida/genética , ARN Interferente Pequeño/genética , Reproducibilidad de los ResultadosRESUMEN
Brain-derived neurotrophic factor (BDNF) is a versatile neurotrophic factor that has been implicated in cell survival, cell differentiation, axonal growth, and activity-dependent synaptic plasticity. Changes in BDNF expression have also been reported during the course of several neurological disorders, including Alzheimer's disease (AD). The role of BDNF in AD, however, has remained elusive. To learn more about this neurotrophic factor, we investigated BDNF expression in brain of amyloid precursor protein overexpressing mice (APP23 transgenic mice). In situ hybridization revealed BDNF mRNA signals associated with amyloid plaques. Laser microdissection in combination with quantitative RT-PCR demonstrated a sixfold increase of BDNF mRNA in the immediate plaque vicinity, a threefold increase in a tissue ring surrounding the plaque, and control levels in interplaque areas comparable with those measured in age-matched nontransgenic mice. Double immunofluorescence localized BDNF to microglial cells and astrocytes surrounding the plaque. Cortical BDNF protein levels were quantified by ELISA demonstrating a >10-fold increase compared with age-matched controls. This upregulation of BDNF protein significantly correlated with the beta-amyloid load in the transgenic animals. Taken together, our data demonstrate a plaque-associated upregulation of BDNF in APP23 transgenic mice and implicate this neurotrophin in the regulation of inflammatory and axonal growth processes in the plaque vicinity.
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Enfermedad de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/genética , Factor Neurotrófico Derivado del Encéfalo/biosíntesis , Neuroglía/metabolismo , Placa Amiloide/metabolismo , Factores de Edad , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Factor Neurotrófico Derivado del Encéfalo/genética , Modelos Animales de Enfermedad , Lóbulo Frontal/metabolismo , Lóbulo Frontal/patología , Hibridación in Situ , Ratones , Ratones Transgénicos , Neuroglía/patología , Fragmentos de Péptidos/metabolismo , Placa Amiloide/patología , Terminales Presinápticos/metabolismo , Terminales Presinápticos/patología , ARN Mensajero/metabolismoRESUMEN
Alzheimer's Disease (AD) is a neurodegenerative disorder that is characterized by extracellular deposits of amyloid-beta peptide (Abeta) and a severe depletion of the cholinergic system, although the relationship between these two events is poorly understood. In the neocortex, there is a loss of cholinergic fibers and receptors and a decrease of both choline acetyltransferase (ChAT) and acetylcholinesterase enzyme activities. The nucleus basalis of Meynert (NBM), which provides the major cholinergic input to the neocortex, undergoes profound neuron loss in AD. In the present study, we have examined the cholinergic alterations in amyloid precursor protein transgenic mice (APP23), a mouse model of cerebral beta-amyloidosis. In aged APP23 mice, our results reveal modest decreases in cortical cholinergic enzyme activity compared with age-matched wild-type mice. Total cholinergic fiber length was more severely affected, with 29 and 35% decreases in the neocortex of aged APP23 mice compared with age-matched wild-type mice and young transgenic mice, respectively. However, there was no loss of cholinergic basal forebrain neurons in these aged APP23 mice, suggesting that the cortical cholinergic deficit in APP23 mice is locally induced by the deposition of amyloid and is not caused by a loss of cholinergic basal forebrain neurons. To study the impact of cholinergic basal forebrain degeneration on cortical amyloid deposition, we performed unilateral NBM lesions in adult APP23 mice. Three to 8 months after lesioning, a 38% reduction in ChAT activity and significant cholinergic fiber loss were observed in the ipsilateral frontal cortex. There was a 19% decrease in Abeta levels of the ipsilateral compared with contralateral frontal cortex with no change in the ratio of Abeta40 to Abeta42. We conclude that the severe cholinergic deficit in AD is caused by both the loss of cholinergic basal forebrain neurons and locally by cerebral amyloidosis in the neocortex. Moreover, our results suggest that disruption of the basal cholinergic forebrain system does not promote cerebral amyloidosis in APP23 transgenic mice.
Asunto(s)
Precursor de Proteína beta-Amiloide/metabolismo , Amiloidosis/patología , Fibras Colinérgicas/patología , Acetilcolinesterasa/metabolismo , Envejecimiento/metabolismo , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/fisiopatología , Amiloide/análisis , Precursor de Proteína beta-Amiloide/genética , Amiloidosis/fisiopatología , Animales , Núcleo Basal de Meynert/patología , Recuento de Células , Tamaño de la Célula , Colina O-Acetiltransferasa/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Lóbulo Frontal/enzimología , Lóbulo Frontal/patología , Inmunohistoquímica , Masculino , Ratones , Ratones Transgénicos , Neocórtex/química , Neocórtex/patología , Neuronas/enzimología , Neuronas/patología , Prosencéfalo/enzimología , Prosencéfalo/patologíaRESUMEN
RATIONALE: APP23 mice are a promising model of Alzheimer's disease, expressing several histopathological, cognitive and behavioural hallmarks of the human condition. A valid animal model should respond to therapeutic interventions in an equivalent manner as human patients. OBJECTIVES: To further validate the APP23 model, we examined whether cognitive deficits could be antagonised by donepezil, rivastigmine, galantamine or memantine, which are approved drugs for symptomatic treatment of dementia. METHODS: Animals were tested at an age at which untreated APP23 mice display severe deficits in visual-spatial learning. Four-month-old APP23 mice and control littermates were administered donepezil (0.3 or 0.6 mg kg(-1)), rivastigmine (0.5 or 1.0 mg kg(-1)), galantamine (1.25 or 2.5 mg kg(-1)), memantine (2 or 10 mg kg(-1)) or saline through daily i.p. injections. After 1 week of treatment, acquisition phase commenced, with daily treatment continuing during cognitive testing. RESULTS: All cholinesterase inhibitors reduced cognitive deficits with the following optimal daily doses: galantamine 1.25 mg kg(-1), rivastigmine 0.5 mg kg(-1) and donepezil 0.3 mg kg(-1). Higher dosages often did not exert beneficial effects in accordance with inverted U-shaped dose-response curves described for cholinomimetics. Symptomatic efficacy of memantine on cognition was mild, with significant amelioration manifesting during probe trial. CONCLUSIONS: This is the first study to simultaneously evaluate the efficacy of therapeutically relevant doses of these four compounds in one particular learning and memory paradigm, being the Morris water maze. The fact that symptomatic intervention was able to diminish cognitive impairment, substantially adds to the validity of the APP23 model as a valuable tool to evaluate future therapeutic approaches.
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Enfermedad de Alzheimer/tratamiento farmacológico , Inhibidores de la Colinesterasa/uso terapéutico , Cognición/efectos de los fármacos , Precursor de Proteína beta-Amiloide/genética , Animales , Modelos Animales de Enfermedad , Donepezilo , Relación Dosis-Respuesta a Droga , Galantamina/uso terapéutico , Humanos , Indanos/uso terapéutico , Masculino , Memantina/uso terapéutico , Ratones , Ratones Mutantes , Fenilcarbamatos/uso terapéutico , Piperidinas/uso terapéutico , Rivastigmina , Sensibilidad y EspecificidadRESUMEN
Animal models are considered essential in research ensuing elucidation of human disease processes and subsequently, testing of potential therapeutic strategies. This is especially true for neurodegenerative disorders, in which the first steps in pathogenesis are often not accessible in human patients. Alzheimer's disease is vastly becoming a major medical and socioeconomic problem in our aging society. Valid animal models for this uniquely human condition should exhibit histopathological, biochemical, cognitive, and behavioral alterations observed in Alzheimer's disease patients. Major progress has been made since the understanding of the genetic basis of Alzheimer's disease and the development and improvement of transgenic mouse models. All present Alzheimer's disease models developed are partial but nevertheless essential in further unraveling the nature and spatial and temporal development of the complex molecular pathology underlying this condition. One of the more recent transgenic attempts to model Alzheimer's disease is the APP23 transgenic mouse. This article describes the development and assessment of this human amyloid precursor protein overexpression model. We summarize histopathological and biochemical, cognitive and behavioral observations made in heterozygous APP23 mice, thereby emphasizing the model's contribution to clarification of neurodegenerative disease mechanisms. In addition, the first therapeutic interventions in the APP23 model are included.
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Enfermedad de Alzheimer/genética , Modelos Animales de Enfermedad , Ratones Transgénicos/genética , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animales , Cruzamientos Genéticos , Hipocampo/patología , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Neocórtex/patología , Nexinas de Proteasas , Receptores de Superficie Celular/genéticaRESUMEN
INTRODUCTION: The deposition of the amyloid ß protein (Aß) in the brain is a hallmark of Alzheimer's disease (AD). Removal of Aß by Aß-antibody treatment has been developed as a potential treatment strategy against AD. First clinical trials showed neither a stop nor a reduction of disease progression. Recently, we have shown that the formation of soluble and insoluble Aß aggregates in the human brain follows a hierarchical sequence of three biochemical maturation stages (B-Aß stages). To test the impact of the B-Aß stage on Aß immunotherapy, we treated transgenic mice expressing human amyloid precursor protein (APP) carrying the Swedish mutation (KM670/671NL; APP23) with the Aß-antibody ß1 or phosphate-buffered saline (PBS) beginning 1) at 3 months, before the onset of dendrite degeneration and plaque deposition, and 2) at 7 months, after the start of Aß plaque deposition and dendrite degeneration. RESULTS: At 5 months of age, first Aß aggregates in APP23 brain consisted of non-modified Aß (representing B-Aß stage 1) whereas mature Aß-aggregates containing N-terminal truncated, pyroglutamate-modified AßN3pE and phosphorylated Aß (representing B-Aß stage 3) were found at 11 months of age in both ß1- and PBS-treated animals. Protective effects on commissural neurons with highly ramified dendritic trees were observed only in 3-month-old ß1-treated animals sacrificed at 5 months. When treatment started at 7 months of age, no differences in the numbers of healthy commissural neurons were observed between ß1- and PBS-treated APP23 mice sacrificed with 11 months. CONCLUSIONS: Aß antibody treatment was capable of protecting neurons from dendritic degeneration as long as Aß aggregation was absent or represented B-Aß stage 1 but had no protective or curative effect in later stages with mature Aß aggregates (B-Aß stage 3). These data indicate that the maturation stage of Aß aggregates has impact on potential treatment effects in APP23 mice.
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Enfermedad de Alzheimer/terapia , Péptidos beta-Amiloides/inmunología , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Anticuerpos/uso terapéutico , Encéfalo/metabolismo , Factores de Edad , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/sangre , Análisis de Varianza , Animales , Encéfalo/patología , Encéfalo/ultraestructura , Humanos , Inmunoprecipitación , Inmunoterapia/métodos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Electrónica , Vacunación/métodosRESUMEN
Early diagnosis of Alzheimer`s disease (AD) is currently difficult and involves a complex approach including clinical assessment, neuroimaging, and measurement of amyloid-ß (Aß) and tau levels in cerebrospinal fluid (CSF). A better mechanistic understanding is needed to develop more accurate and even presymptomatic diagnostic tools. It has been shown that Aß derived from amyloid-containing brain tissue has prion-like properties: it induces misfolding and aggregation of Aß when injected into human amyloid precursor protein (APP) transgenic mice. In contrast, Aß in the CSF has been less studied, and it is not clear whether it also exhibits prion-like characteristics, which might provide a sensitive diagnostic tool. Therefore, we collected CSF from APP transgenic mice carrying the Swedish mutation (APP23 mice), and injected it intracerebrally into young mice from the same transgenic line. We found that CSF derived Aß did not induce increased ß-amyloidosis, even after long incubation periods and additional concentration. This suggests that Aß present in the CSF does not have the same prion-like properties as the Aß species in the brain.
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Enfermedad de Alzheimer/líquido cefalorraquídeo , Péptidos beta-Amiloides/administración & dosificación , Péptidos beta-Amiloides/líquido cefalorraquídeo , Hipocampo/metabolismo , Priones/metabolismo , Enfermedad de Alzheimer/patología , Amiloidosis/metabolismo , Animales , Hipocampo/patología , Humanos , Inmunohistoquímica , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
Huntington's disease (HD) is an autosomal dominant, progressive and fatal neurological disorder caused by an expansion of CAG repeats in exon-1 of the huntingtin gene. The encoded poly-glutamine stretch renders mutant huntingtin prone to aggregation. HdhQ150 mice genocopy a pathogenic repeat (â¼150 CAGs) in the endogenous mouse huntingtin gene and model predominantly pre-manifest HD. Treating early is likely important to prevent or delay HD, and HdhQ150 mice may be useful to assess therapeutic strategies targeting pre-manifest HD. This requires appropriate markers and here we demonstrate, that pre-symptomatic HdhQ150 mice show several dramatic mutant huntingtin gene-dose dependent pathological changes including: (i) an increase of neuronal intra-nuclear inclusions (NIIs) in brain, (ii) an increase of extra-nuclear aggregates in dentate gyrus, (iii) a decrease of DARPP32 protein and (iv) an increase in glial markers of neuroinflammation, which curiously did not correlate with local neuronal mutant huntingtin inclusion-burden. HdhQ150 mice developed NIIs also in all retinal neuron cell-types, demonstrating that retinal NIIs are not specific to human exon-1 R6 HD mouse models. Taken together, the striking and robust mutant huntingtin gene-dose related changes in aggregate-load, DARPP32 levels and glial activation markers should greatly facilitate future testing of therapeutic strategies in the HdhQ150 HD mouse model.
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Modelos Animales de Enfermedad , Fosfoproteína 32 Regulada por Dopamina y AMPc/metabolismo , Dosificación de Gen/genética , Regulación de la Expresión Génica/genética , Enfermedad de Huntington/genética , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/genética , Animales , Western Blotting , Técnica del Anticuerpo Fluorescente , Genotipo , Proteína Huntingtina , Enfermedad de Huntington/patología , Inmunohistoquímica , Cuerpos de Inclusión Intranucleares/patología , Ratones , Ratones Mutantes , Oligonucleótidos/genética , Retina/patología , Estadísticas no ParamétricasRESUMEN
BACKGROUND: The deposition of the amyloid ß-peptide (Aß) in the brain is one of the hallmarks of Alzheimer's disease (AD). It is not yet clear whether Aß always leads to similar changes or whether it induces different features of neurodegeneration in relation to its intra- and/or extracellular localization or to its intracellular trafficking routes. To address this question, we have analyzed two transgenic mouse models: APP48 and APP23 mice. The APP48 mouse expresses Aß1-42 with a signal sequence in neurons. These animals produce intracellular Aß independent of amyloid precursor protein (APP) but do not develop extracellular Aß plaques. The APP23 mouse overexpresses human APP with the Swedish mutation (KM670/671NL) in neurons and produces APP-derived extracellular Aß plaques and intracellular Aß aggregates. RESULTS: Tracing of commissural neurons in layer III of the frontocentral cortex with the DiI tracer revealed no morphological signs of dendritic degeneration in APP48 mice compared to littermate controls. In contrast, the dendritic tree of highly ramified commissural frontocentral neurons was altered in 15-month-old APP23 mice. The density of asymmetric synapses in the frontocentral cortex was reduced in 3- and 15-month-old APP23 but not in 3- and 18-month-old APP48 mice. Frontocentral neurons of 18-month-old APP48 mice showed an increased proportion of altered mitochondria in the soma compared to wild type and APP23 mice. Aß was often seen in the membrane of neuronal mitochondria in APP48 mice at the ultrastructural level. CONCLUSIONS: These results indicate that APP-independent intracellular Aß accumulation in APP48 mice is not associated with dendritic and neuritic degeneration but with mitochondrial alterations whereas APP-derived extra- and intracellular Aß pathology in APP23 mice is linked to dendrite degeneration and synapse loss independent of obvious mitochondrial alterations. Thus, Aß aggregates in APP23 and APP48 mice induce neurodegeneration presumably by different mechanisms and APP-related production of Aß may, thereby, play a role for the degeneration of neurites and synapses.