RESUMEN
Huntington disease (HD) is an autosomal dominant neurodegenerative disorder caused by expanded CAG repeats in the huntingtin gene (HTT). Although mutant HTT is expressed during embryonic development and throughout life, clinical HD usually manifests later in adulthood. A number of studies document neurodevelopmental changes associated with mutant HTT, but whether these are reversible under therapy remains unclear. Here, we identify very early behavioral, molecular, and cellular changes in preweaning transgenic HD rats and mice. Reduced ultrasonic vocalization, loss of prepulse inhibition, and increased risk taking are accompanied by disturbances of dopaminergic regulation in vivo, reduced neuronal differentiation capacity in subventricular zone stem/progenitor cells, and impaired neuronal and oligodendrocyte differentiation of mouse embryo-derived neural stem cells in vitro. Interventional treatment of this early phenotype with the histone deacetylase inhibitor (HDACi) LBH589 led to significant improvement in behavioral changes and markers of dopaminergic neurotransmission and complete reversal of aberrant neuronal differentiation in vitro and in vivo. Our data support the notion that neurodevelopmental changes contribute to the prodromal phase of HD and that early, presymptomatic intervention using HDACi may represent a promising novel treatment approach for HD.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Enfermedad de Huntington/fisiopatología , Ácidos Hidroxámicos/farmacología , Indoles/farmacología , Neuronas/efectos de los fármacos , Animales , Animales Modificados Genéticamente , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Modelos Animales de Enfermedad , Femenino , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Proteína Huntingtina/genética , Enfermedad de Huntington/genética , Ventrículos Laterales/patología , Masculino , Ratones Transgénicos , Mutación , Neuronas/metabolismo , Neuronas/fisiología , Panobinostat , RatasRESUMEN
Glucose hypometabolism is the earliest symptom observed in the brains of Alzheimer disease (AD) patients. In a former study, we analyzed the cortical proteome of the APP23 mouse model of AD at presymptomatic age (1 month) using a 2-D electrophoresis-based approach. Interestingly, long before amyloidosis can be observed in APP23 mice, proteins associated with energy metabolism were predominantly altered in transgenic as compared to wild-type mice indicating presymptomatic changes in energy metabolism. In the study presented here, we analyzed whether the observed changes were associated with oxidative stress and confirmed our previous findings in primary cortical neurons, which exhibited altered ADP/ATP levels if transgenic APP was expressed. Reactive oxygen species produced during energy metabolism have important roles in cell signaling and homeostasis as they modify proteins. We observed an overall up-regulation of protein oxidation status as shown by increased protein carbonylation in the cortex of presymptomatic APP23 mice. Interestingly, many carbonylated proteins, such as Vilip1 and Syntaxin were associated to synaptic plasticity. This demonstrates an important link between energy metabolism and synaptic function, which is altered in AD. In summary, we demonstrate that changes in cortical energy metabolism and increased protein oxidation precede the amyloidogenic phenotype in a mouse model for AD. These changes might contribute to synaptic failure observed in later disease stages, as synaptic transmission is particularly dependent on energy metabolism.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/genética , Metabolismo Energético , Estrés Oxidativo , Animales , Enfermedades Asintomáticas , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación , Carbonilación Proteica , Proteoma/metabolismo , Sinapsis/fisiologíaRESUMEN
Kainate, a glutamate analogue, activates kainate and AMPA receptors inducing strong synaptic activation. Systemic kainate application to rodents results in seizures, neurodegeneration, and neuronal remodeling in the brain. It is therefore used to investigate molecular mechanisms responsible for these conditions. We analyzed proteome alterations in murine primary cortical neurons after 24 h of kainate treatment. Our 2-D gel based proteomics approach revealed 91 protein alterations, some already associated with kainate-induced pathology. In addition, we found a large number of proteins which have not previously been reported to be associated with kainate-induced pathology. Functional classification of altered proteins revealed that they predominantly participate in mRNA splicing and cytoskeleton remodeling.
Asunto(s)
Ácido Kaínico/farmacología , Neuronas/fisiología , Empalme del ARN/efectos de los fármacos , Animales , Células Cultivadas , Electroforesis en Gel Bidimensional , Ratones , Ratones Endogámicos BALB C , Neuronas/química , Neuronas/citología , Proteoma/análisis , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Espectrometría de Masas en Tándem/métodosRESUMEN
Parkinson's disease (PD) is histologically well defined by its characteristic degeneration of dopaminergic neurons in the substantia nigra pars compacta. Remarkably, divergent PD-related mutations can generate comparable brain region specific pathologies. This indicates that some intrinsic region-specificity respecting differential neuron vulnerability exists, which codetermines the disease progression. To gain insight into the pathomechanism of PD, we investigated protein expression and protein oxidation patterns of three different brain regions in a PD mouse model, the PINK1 knockout mice (PINK1-KO), in comparison to wild type control mice. The dysfunction of PINK1 presumably affects mitochondrial turnover by disturbing mitochondrial autophagic pathways. The three brain regions investigated are the midbrain, which is the location of substantia nigra; striatum, the major efferent region of substantia nigra; and cerebral cortex, which is more distal to PD pathology. In all three regions, mitochondrial proteins responsible for energy metabolism and membrane potential were significantly altered in the PINK1-KO mice, but with very different region specific accents in terms of up/down-regulations. This suggests that disturbed mitophagy presumably induced by PINK1 knockout has heterogeneous impacts on different brain regions. Specifically, the midbrain tissue seems to be most severely hit by defective mitochondrial turnover, whereas cortex and striatum could compensate for mitophagy nonfunction by feedback stimulation of other catabolic programs. In addition, cerebral cortex tissues showed the mildest level of protein oxidation in both PINK1-KO and wild type mice, indicating either a better oxidative protection or less reactive oxygen species (ROS) pressure in this brain region. Ultra-structural histological examination in normal mouse brain revealed higher incidences of mitophagy vacuoles in cerebral cortex than in striatum and substantia nigra. Taken together, the delicate balance between oxidative protection and mitophagy capacity in different brain regions could contribute to brain region-specific pathological patterns in PD.
RESUMEN
Huntington disease (HD) is fatal in humans within 15-20 years of symptomatic disease. Although late stage HD has been studied extensively, protein expression changes that occur at the early stages of disease and during disease progression have not been reported. In this study, we used a large two-dimensional gel/mass spectrometry-based proteomics approach to investigate HD-induced protein expression alterations and their kinetics at very early stages and during the course of disease. The murine HD model R6/2 was investigated at 2, 4, 6, 8, and 12 weeks of age, corresponding to absence of disease and early, intermediate, and late stage HD. Unexpectedly the most HD stage-specific protein changes (71-100%) as well as a drastic alteration (almost 6% of the proteome) in protein expression occurred already as early as 2 weeks of age. Early changes included mainly the up-regulation of proteins involved in glycolysis/gluconeogenesis and the down-regulation of the actin cytoskeleton. This suggests a period of highly variable protein expression that precedes the onset of HD phenotypes. Although an up-regulation of glycolysis/gluconeogenesis-related protein alterations remained dominant during HD progression, late stage alterations at 12 weeks showed an up-regulation of proteins involved in proteasomal function. The early changes in HD coincide with a peak in protein alteration during normal mouse development at 2 weeks of age that may be responsible for these massive changes. Protein and mRNA data sets showed a large overlap on the level of affected pathways but not single proteins/mRNAs. Our observations suggest that HD is characterized by a highly dynamic disease pathology not represented by linear protein concentration alterations over the course of disease.
Asunto(s)
Enfermedad de Huntington/metabolismo , Enfermedad de Huntington/patología , Proteínas/metabolismo , Animales , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Desarrollo Embrionario , Metabolismo Energético , Femenino , Regulación de la Expresión Génica , Enfermedad de Huntington/genética , Cinética , Masculino , Redes y Vías Metabólicas , Ratones , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Tiempo , Extractos de TejidosRESUMEN
Biological aging is often described by its phenotypic effect on individuals. Still, its causes are more likely found on the molecular level. Biological organisms can be considered as reliability-engineered, robust systems and applying reliability theory to their basic nonaging components, proteins, could provide insight into the aging mechanism. Reliability theory suggests that aging is an obligatory trade-off in a fault-tolerant system such as the cell which is constructed based on redundancy design. Aging is the inevitable redundancy loss of functional system components, that is proteins, over time. In our study, we investigated mouse brain development, adulthood, and aging from embryonic day 10 to 100 weeks. We determined redundancy loss of different protein categories with age using reliability theory. We observed a near-linear decrease of protein redundancy during aging. Aging may therefore be a phenotypic manifestation of redundancy loss caused by nonfunctional protein accumulation. This is supported by a loss of proteasome system components faster than dictated by reliability theory. This loss is highly detrimental to biological self-renewal and seems to be a key contributor to aging and therefore could represent a major target for therapies for aging and age-related diseases.
Asunto(s)
Envejecimiento/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteoma/metabolismo , Proteómica/métodos , Análisis de Varianza , Animales , Electroforesis en Gel Bidimensional/métodos , Ratones , Ratones Endogámicos C57BL , Proteínas/química , Análisis de Regresión , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodosRESUMEN
Neurotransmitter signaling is essential for physiologic brain development. Sedative and anticonvulsant agents that reduce neuronal excitability via antagonism at N-methyl-D-aspartate receptors (NMDARs) and/or agonism at gamma-aminobutyric acid subtype A receptors (GABA(A)Rs) are applied frequently in obstetric and pediatric medicine. We demonstrated that a 1-day treatment of infant mice at postnatal day 6 (P6) with the NMDAR antagonist dizocilpine or the GABA(A)R agonist phenobarbital not only has acute but also long term effects on the cerebral cortex. Changes of the cerebral cortex proteome 1 day (P7), 1 week (P14), and 4 weeks (P35) following treatment at P6 suggest that a suppression of synaptic neurotransmission during brain development dysregulates proteins associated with apoptosis, oxidative stress, inflammation, cell proliferation, and neuronal circuit formation. These effects appear to be age-dependent as most protein changes did not occur in mice subjected to such pharmacological treatment in adulthood. Previously performed histological evaluations of the brains revealed widespread apoptosis and decreased cell proliferation following such a drug treatment in infancy and are thus consistent with brain protein changes reported in this study. Our results point toward several pathways modulated by a reduction of neuronal excitability that might interfere with critical developmental events and thus affirm concerns about the impact of NMDAR- and/or GABA(A)R-modulating drugs on human brain development.
Asunto(s)
Corteza Cerebral/crecimiento & desarrollo , Corteza Cerebral/metabolismo , Receptores de GABA-A/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Transmisión Sináptica/fisiología , Animales , Animales Recién Nacidos , Movimiento Celular/efectos de los fármacos , Corteza Cerebral/efectos de los fármacos , Maleato de Dizocilpina/farmacología , Electroforesis en Gel Bidimensional , Agonistas de Receptores de GABA-A , Masculino , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Neuronas/citología , Neuronas/efectos de los fármacos , Fenobarbital/farmacología , Proteoma/química , Proteoma/metabolismo , Ratas , Ratas Wistar , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Transmisión Sináptica/efectos de los fármacos , Factores de TiempoRESUMEN
In recent years proteomics became increasingly important to functional genomics. Although a large amount of data is generated by high throughput large-scale techniques, a connection of these mostly heterogeneous data from different analytical platforms and of different experiments is limited. Data mining procedures and algorithms are often insufficient to extract meaningful results from large datasets and therefore limit the exploitation of the generated biological information. In our proteomic core facility, which almost exclusively focuses on 2-DE/MS-based proteomics, we developed a proteomic database custom tailored to our needs aiming at connecting MS protein identification information to 2-DE derived protein expression profiles. The tools developed should not only enable an automatic evaluation of single experiments, but also link multiple 2-DE experiments with MS-data on different levels and thereby helping to create a comprehensive network of our proteomics data. Therefore the key feature of our "PROTEOMER" database is its high cross-referencing capacity, enabling integration of a wide range of experimental data. To illustrate the workflow and utility of the system, two practical examples are provided to demonstrate that proper data cross-referencing can transform information into biological knowledge.
Asunto(s)
Sistemas de Administración de Bases de Datos , Bases de Datos de Proteínas , Electroforesis en Gel Bidimensional , Perfilación de la Expresión Génica , Espectrometría de Masas , Animales , Diseño de Equipo , Humanos , Ratones , Enfermedades Neurodegenerativas/genética , Polimorfismo Genético/fisiología , Programas Informáticos , Interfaz Usuario-ComputadorRESUMEN
Our protein extraction protocol for two-dimensional gel electrophoresis (2DE) was updated to meet current needs in the field of proteomics. This protocol summarizes our experience using this method since its introduction over 30 years ago. We provide a total as well as fractionated extraction protocol. The former is easy and fast to use, suitable for most standard 2DE applications, whereas the latter is used for special applications such as the extraction of membrane or nuclear proteins.Both extraction protocols stress the need that protease inhibitors are added early to still deep frozen tissue to preclude an activation of proteases which destroy proteins and make them inaccessible to analysis. We also emphasize that, to remain soluble, proteins need to stay in an environment resembling a living cell as closely as possible. Sample dilution is therefore kept to a minimum and the pH of the extract is close to in vivo conditions at pH 7.1. In addition there are no precipitation/resolubilization steps which could irreversibly remove proteins from the extract. Furthermore, the total extraction does not even require centrifugation. Our extraction protocol is compatible with recent advances in 2DE-staining techniques such as differential in gel electrophoresis and fluorescence staining as well as mass spectrometry.
Asunto(s)
Electroforesis en Gel Bidimensional/métodos , Proteínas/aislamiento & purificación , Animales , Química Encefálica , Humanos , Hígado/química , Ratones , Miocardio/química , Proteínas/análisis , Proteómica/métodosRESUMEN
Our two-dimensional gel electrophoresis (2DE) protocol has been continuously improved in our laboratory since its inception 30 years ago. An updated version is presented here. This protocol is a result of our experience in proteome analysis of tissue extracts, cultured cells (mammalian, yeast, and bacteria), cellular organelles, and subcellular fractions. Many modifications and suggestions emerging in our lab as well as in the literature were tested and integrated into our improved protocol if helpful. Importantly we use (a) large (46 x 30 cm) gels to achieve a high resolution and (b) ready-made gel solutions produced in large batches and stored frozen, a prerequisite, among others, for our very high reproducibility. Employing the 2DE method described here we demonstrated that protein patterns separating more than 10,000 protein spots can be obtained from mouse tissue. This is the highest resolution reported in the literature for 2DE of complex protein mixtures so far. Our 2DE patterns are of high quality with regard to spot shape and intensity as well as background. The reproducibility of the protein patterns is shown to be extremely satisfactory. New staining methods such as differential in gel electrophoresis (DIGE) and the latest 2DE gel evaluation software are compatible to our 2DE protocol. Using suitable staining protocols proteins can easily be identified by mass spectrometry.
Asunto(s)
Electroforesis en Gel Bidimensional/métodos , Proteínas/análisis , Animales , Tampones (Química) , Electroforesis en Gel Bidimensional/instrumentación , Procesamiento de Imagen Asistido por Computador , Focalización Isoeléctrica/instrumentación , Focalización Isoeléctrica/métodos , Ratones , Proteoma/análisis , Reproducibilidad de los Resultados , Programas Informáticos , Coloración y Etiquetado/métodosRESUMEN
Treatment of mice by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridene hydrochloride (MPTP) is a well established animal model for Parkinson's disease (PD), while overexpression of L1 cell adhesion molecule (L1cam) has been proposed to attenuate the degeneration of dopaminergic neurons induced by MPTP. To gain insight into the role of L1cam in the pathomechanism of PD, we investigated protein expression patterns after MPTP-treatment in both C57BL/6 (wild-type) and transgenic mice overexpressing L1cam in astrocytes. Our results showed that during the acute phase, proteins in functional complexes responsible for mitochondrial, glycolysis, and cytoskeletal function were down-regulated in MPTP-treated wild-type mice. After a recovery phase, proteins that were down-regulated in the acute phase reverted to normal levels. In L1cam transgenic mice, a much higher number of proteins was altered during the acute phase and this number even increased after the recovery phase. Many proteins involved in oxidative phosphorylation were still down-regulated and glycolysis related protein were still up-regulated. This pattern indicates a lasting severely impaired energy production in L1cam mice after MPTP treatment.
Asunto(s)
Intoxicación por MPTP/metabolismo , Mesencéfalo/metabolismo , Molécula L1 de Adhesión de Célula Nerviosa/metabolismo , Proteoma/análisis , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/administración & dosificación , Animales , Northern Blotting , Citoesqueleto/metabolismo , Electroforesis en Gel Bidimensional , Glucólisis , Intoxicación por MPTP/genética , Intoxicación por MPTP/patología , Mesencéfalo/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mitocondrias/metabolismo , Molécula L1 de Adhesión de Célula Nerviosa/genética , Fosforilación , Reacción en Cadena de la Polimerasa , Ubiquitina/metabolismoRESUMEN
Mouse embryonic brain development involves sequential differentiation of multipotent progenitors into neurons and glia cells. Using microarrays and large 2-DE, we investigated the mouse brain transcriptome and proteome of embryonic days 9.5, 11.5, and 13.5. During this developmental period, neural progenitor cells shift from proliferation to neuronal differentiation. As expected, we detected numerous expression changes between all time points investigated, but interestingly, the rate of alteration remained in a similar range within 2 days of development. Furthermore, up- and down-regulation of gene products was balanced at each time point which was also seen at embryonic days 16-18. We hypothesize that during embryonic development, the rate of gene expression alteration is rather constant due to limited cellular resources such as energy, space, and free water. A similar complexity in terms of expressed genes and proteins suggests that changes in relative concentrations rather than an increase in the number of gene products dominate cellular differentiation. In general, expression of metabolism and cell cycle related gene products was down-regulated when precursor cells switched from proliferation to neuronal differentiation (days 9.5-11.5), whereas neuron specific gene products were up-regulated. A detailed functional analysis revealed their implication in differentiation related processes such as rearrangement of the actin cytoskeleton as well as Notch- and Wnt-signaling pathways.
Asunto(s)
Encéfalo/metabolismo , Desarrollo Embrionario/genética , Perfilación de la Expresión Génica/métodos , Proteoma/análisis , Animales , Encéfalo/citología , Encéfalo/embriología , Electroforesis en Gel Bidimensional , Regulación del Desarrollo de la Expresión Génica , Ratones , Proteoma/genética , Factores de TiempoRESUMEN
In recent years, a large number of proteomics studies for various diseases were conducted, such as for cancer, cardiovascular and neurodegenerative disorders (NDs). The availability of huge data sets with a large number of differentially expressed proteins showed for the first time that not all protein changes between a diseased and a control state were specific. This review focuses on this protein expression overlap, specifically between NDs, and tries to investigate the possible reasons for this overlap by investigating 14 ND proteomics studies of Alzheimer's (six studies), Parkinson's (four studies) and Huntington's disease (three studies), as well as amyotrophic lateral sclerosis (one study). Studies were selected according to the availability of quantitative changes, number of (biological) repeats and numbers of proteins changed. The studies include investigations of human tissue and mouse, as well as cell culture, models. A change in metabolism-related proteins was found to be common among all disorders. These changes can be explained by alterations in key regulatory proteins, such as those involved in transcription. Since most NDs affect, at least initially, very specific areas of the brain, the location of the changes may be more important than the kind of protein alterations that occur, since they are very similar among NDs.
Asunto(s)
Expresión Génica , Enfermedades Neurodegenerativas/metabolismo , Proteínas/análisis , Proteómica/métodos , Animales , Susceptibilidad a Enfermedades , Humanos , Metabolismo , Proteínas/genéticaRESUMEN
The accumulation of mitochondria containing mutated genomes was proposed to be an important factor involved in aging. Although the level of mutated mtDNA has shown to increase over time, it is currently not possible to directly measure the mtDNA mutation rate within living cells. The combination of mathematical modeling and controlled experiments is an alternative approach to obtain an estimate for the mutation rate in a well-defined system. In order to judge the relevance of mitochondrial mutations for the aging process, we used a mouse model to study age-related alterations of the mitochondrial proteins. Based on these experimental data we constructed a mathematical model of the mitochondrial population dynamics to estimate mtDNA mutation rates. Mitochondria were isolated from mouse brain and liver at six different ages (newborn to 24-months). A large-gel 2D-electrophoresis-based proteomics approach was used to analyze the mitochondrial proteins. The expression of two respiratory chain complex I subunits and one complex IV subunit decreased significantly with age. One subunit of complex III and one subunit of complex V increased in expression during aging. Together, these data indicate that complex I and IV deficiency in aged tissues might be accompanied by feedback regulation of other protein complexes in the respiratory chain. When we fitted our experimental data to the mathematical model, mtDNA mutation rate was estimated to be 2.7x10(-8) per mtDNA per day for brain and 3.2x10(-9) per mtDNA per day for liver. According to our model and in agreement with the mitochondrial theory of aging, mtDNA mutations could cause the detrimental changes seen in mitochondrial populations during the normal lifespan of mice, while at the same time ensure that the mitochondrial population remains functional during the developmental and reproductive period of mice.
Asunto(s)
Envejecimiento/genética , ADN Mitocondrial/genética , Proteínas Mitocondriales/metabolismo , Modelos Genéticos , Mutación , Animales , Encéfalo/metabolismo , Transporte de Electrón/fisiología , Electroforesis en Gel Bidimensional/métodos , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Mitocondrias Hepáticas/metabolismo , Mitocondrias Hepáticas/ultraestructura , Proteínas Mitocondriales/genética , Proteoma , Regulación hacia ArribaRESUMEN
Neurodegenerative disorders (ND) belong to the most devastating diseases in the industrialized western world. Alzheimer disease (AD) is the most prevalent among these disorders followed by Parkinson disease (PD). Huntington disease (HD) is an autosomal dominantly inherited condition with a single mutation that causes disease in almost 100% of all cases. In this review we used previously published proteomics studies on AD, PD and HD to find cellular pathways changed similarly in ND and aging. All studies employed large gel two dimensional gel electrophoresis for protein separation and mass spectrometry for protein identification. Altered proteins were subjected to a KEGG pathway analysis and altered pathways determined for each disorder and aging. We found that besides the mitochondrial oxidative phosphorylation, the proteasome system are altered in aging and ND. The proteasome facilitates protein degradation which is commonly perturbed in ND which may link neurodegeneration to its largest risk factor-aging.
Asunto(s)
Envejecimiento/patología , Enfermedades Neurodegenerativas/patología , Fosforilación Oxidativa , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteómica/métodos , Envejecimiento/metabolismo , Animales , Enfermedades Neurodegenerativas/clasificación , Enfermedades Neurodegenerativas/metabolismo , Factores de RiesgoRESUMEN
Mutations in the human gene MCPH1 cause primary microcephaly associated with a unique cellular phenotype with premature chromosome condensation (PCC) in early G2 phase and delayed decondensation post-mitosis (PCC syndrome). The gene encodes the BRCT-domain containing protein microcephalin/BRIT1. Apart from its role in the regulation of chromosome condensation, the protein is involved in the cellular response to DNA damage. We report here on the first mouse model of impaired Mcph1-function. The model was established based on an embryonic stem cell line from BayGenomics (RR0608) containing a gene trap in intron 12 of the Mcph1 gene deleting the C-terminal BRCT-domain of the protein. Although residual wild type allele can be detected by quantitative real-time PCR cell cultures generated from mouse tissues bearing the homozygous gene trap mutation display the cellular phenotype of misregulated chromosome condensation that is characteristic for the human disorder, confirming defective Mcph1 function due to the gene trap mutation. While surprisingly the DNA damage response (formation of repair foci, chromosomal breakage, and G2/M checkpoint function after irradiation) appears to be largely normal in cell cultures derived from Mcph1(gt/gt) mice, the overall survival rates of the Mcph1(gt/gt) animals are significantly reduced compared to wild type and heterozygous mice. However, we could not detect clear signs of premature malignant disease development due to the perturbed Mcph1 function. Moreover, the animals show no obvious physical phenotype and no reduced fertility. Body and brain size are within the range of wild type controls. Gene expression on RNA and protein level did not reveal any specific pattern of differentially regulated genes. To the best of our knowledge this represents the first mammalian transgenic model displaying a defect in mitotic chromosome condensation and is also the first mouse model for impaired Mcph1-function.
Asunto(s)
Proteínas Cromosómicas no Histona/fisiología , Rotura Cromosómica , Cromosomas de los Mamíferos/genética , Daño del ADN , Animales , Encéfalo/metabolismo , Encéfalo/patología , Proteínas de Ciclo Celular , Proliferación Celular , Células Cultivadas , Proteínas Cromosómicas no Histona/deficiencia , Proteínas Cromosómicas no Histona/genética , Proteínas del Citoesqueleto , Electroforesis en Gel Bidimensional , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Humanos , Imagen por Resonancia Magnética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Ratones Noqueados , Modelos Animales , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteómica , Análisis de SupervivenciaRESUMEN
Gel electrophoresis is known for its often unsatisfactory precision. Percental relative standard deviations (RSD%) in a range of 15-70% have been reported. Therefore, an improvement of precision in quantitative 2-DE is necessary. In the present, study we have analyzed the work flow of 2-DE in detail to assess the main error sources. Potential major sources of variability for this technique include the transfer between first and second dimension, the analyst's expertise, and the staining or rather detection of separated proteins. The remarkable and completely irregular changes of the background signal from gel to gel were identified as one of the governing error sources. These background changes can be strongly reduced by the direct detection of the separated proteins using native fluorescence. More than a 3-fold better signal-to-noise ratio was found compared to Ruthenium-(II)-tris-(bathophenanthroline disulfonat) (RuBPS) and Coomassie staining, although the sample was used in an 800-fold lower concentration. This improvement together with well-defined peaks resulted in a better quantitative spot reproducibility of approximately 12-16% RSD%. Possibly, the variabilities due to detection and evaluation were already reduced to minor error components. However, according to the law of error propagation, the major error sources dominate the total error. To really prove the good detection and evaluation, these other sources of variability such as sample preparation, strip rehydration, protein loading, transfer between dimensions, interactions between gel and proteins, gel scanning, and spot integration have to be reduced next.
Asunto(s)
Electroforesis en Gel de Poliacrilamida/normas , Focalización Isoeléctrica , Reproducibilidad de los ResultadosRESUMEN
Cell volume alteration represents an important factor contributing to the pathology of late-onset diseases. Previously, it was reported that protein biosynthesis and degradation are inversely (trans) regulated during cell volume regulation. Upon cell shrinkage, protein biosynthesis was up-regulated and protein degradation down-regulated. Cell swelling showed opposite regulation. Recent evidence suggests a decrease of protein biodegradation activity in many neurodegenerative diseases and even during aging; both also show prominent cell shrinkage. To clarify the effect of cell volume regulation on the overall protein turnover dynamics, we investigated mouse embryonic stem cells under hyper- and hypotonic osmotic conditions using a 2-D gel based proteomics approach. These conditions cause cell swelling and shrinkage, respectively. Our results demonstrate that the adaption to altered osmotic conditions and therefore cell volume alterations affects a broad spectrum of cellular pathways, including stress response, cytoskeleton remodeling and importantly, cellular metabolism and protein degradation. Interestingly, protein synthesis and degradation appears to be cis-regulated (same direction) on a global level. Our findings also support the hypothesis that protein alterations due to osmotic stress contribute to the pathology of neurodegenerative diseases due to a 60% expression overlap with proteins found altered in Alzheimer's, Huntington's, or Parkinson's disease. Eighteen percent of the proteins altered are even shared with all three disorders.
Asunto(s)
Células Madre Embrionarias/metabolismo , Ósmosis , Animales , División Celular , Línea Celular , Cromatografía Liquida , Electroforesis en Gel Bidimensional , Células Madre Embrionarias/citología , Ratones , Estrés Oxidativo , Proteínas/metabolismo , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en TándemRESUMEN
The amyloid precursor protein (APP) was assumed to be an important neuron-morphoregulatory protein and plays a central role in Alzheimer's disease (AD) pathology. In the study presented here, we analyzed the APP-transgenic mouse model APP23 using 2-dimensional gel electrophoresis technology in combination with DIGE and mass spectrometry. We investigated cortex and hippocampus of transgenic and wildtype mice at 1, 2, 7 and 15 months of age. Furthermore, cortices of 16 days old embryos were analyzed. When comparing the protein patterns of APP23 with wildtype mice, we detected a relatively large number of altered protein spots at all age stages and brain regions examined which largely preceded the occurrence of amyloid plaques. Interestingly, in hippocampus of adolescent, two-month old mice, a considerable peak in the number of protein changes was observed. Moreover, when protein patterns were compared longitudinally between age stages, we found that a large number of proteins were altered in wildtype mice. Those alterations were largely absent in hippocampus of APP23 mice at two months of age although not in other stages compared. Apparently, the large difference in the hippocampal protein patterns between two-month old APP23 and wildtype mice was caused by the absence of distinct developmental changes in the hippocampal proteome of APP23 mice. In summary, the absence of developmental proteome alterations as well as a down-regulation of proteins related to plasticity suggest the disturption of a normally occurring peak of hippocampal plasticity during adolescence in APP23 mice. Our findings are in line with the observation that AD is preceded by a clinically silent period of several years to decades. We also demonstrate that it is of utmost importance to analyze different brain regions and different age stages to obtain information about disease-causing mechanisms.