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1.
Brain ; 142(8): 2380-2401, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31237944

RESUMO

α-Synuclein misfolding and aggregation plays a major role in the pathogenesis of Parkinson's disease. Although loss of function mutations in the ubiquitin ligase, parkin, cause autosomal recessive Parkinson's disease, there is evidence that parkin is inactivated in sporadic Parkinson's disease. Whether parkin inactivation is a driver of neurodegeneration in sporadic Parkinson's disease or a mere spectator is unknown. Here we show that parkin in inactivated through c-Abelson kinase phosphorylation of parkin in three α-synuclein-induced models of neurodegeneration. This results in the accumulation of parkin interacting substrate protein (zinc finger protein 746) and aminoacyl tRNA synthetase complex interacting multifunctional protein 2 with increased parkin interacting substrate protein levels playing a critical role in α-synuclein-induced neurodegeneration, since knockout of parkin interacting substrate protein attenuates the degenerative process. Thus, accumulation of parkin interacting substrate protein links parkin inactivation and α-synuclein in a common pathogenic neurodegenerative pathway relevant to both sporadic and familial forms Parkinson's disease. Thus, suppression of parkin interacting substrate protein could be a potential therapeutic strategy to halt the progression of Parkinson's disease and related α-synucleinopathies.

2.
J Clin Invest ; 128(7): 2927-2943, 2018 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-29863500

RESUMO

Although nonmalignant stromal cells facilitate tumor growth and can occupy up to 90% of a solid tumor mass, better strategies to exploit these cells for improved cancer therapy are needed. Here, we describe a potent MMAE-linked antibody-drug conjugate (ADC) targeting tumor endothelial marker 8 (TEM8, also known as ANTXR1), a highly conserved transmembrane receptor broadly overexpressed on cancer-associated fibroblasts, endothelium, and pericytes. Anti-TEM8 ADC elicited potent anticancer activity through an unexpected killing mechanism we term DAaRTS (drug activation and release through stroma), whereby the tumor microenvironment localizes active drug at the tumor site. Following capture of ADC prodrug from the circulation, tumor-associated stromal cells release active MMAE free drug, killing nearby proliferating tumor cells in a target-independent manner. In preclinical studies, ADC treatment was well tolerated and induced regression and often eradication of multiple solid tumor types, blocked metastatic growth, and prolonged overall survival. By exploiting TEM8+ tumor stroma for targeted drug activation, these studies reveal a drug delivery strategy with potential to augment therapies against multiple cancer types.


Assuntos
Imunoconjugados/farmacologia , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Receptores de Superfície Celular/antagonistas & inibidores , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/deficiência , Biomarcadores Tumorais/genética , Brentuximab Vedotin , Linhagem Celular Tumoral , Feminino , Humanos , Imunoconjugados/farmacocinética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Nus , Camundongos SCID , Proteínas dos Microfilamentos , Neoplasias/metabolismo , Receptores de Peptídeos/antagonistas & inibidores , Receptores de Peptídeos/deficiência , Receptores de Peptídeos/genética , Células Estromais/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Proc Natl Acad Sci U S A ; 115(7): 1635-1640, 2018 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-29386392

RESUMO

Mutations in LRRK2 are known to be the most common genetic cause of sporadic and familial Parkinson's disease (PD). Multiple lines of LRRK2 transgenic or knockin mice have been developed, yet none exhibit substantial dopamine (DA)-neuron degeneration. Here we develop human tyrosine hydroxylase (TH) promoter-controlled tetracycline-sensitive LRRK2 G2019S (GS) and LRRK2 G2019S kinase-dead (GS/DA) transgenic mice and show that LRRK2 GS expression leads to an age- and kinase-dependent cell-autonomous neurodegeneration of DA and norepinephrine (NE) neurons. Accompanying the loss of DA neurons are DA-dependent behavioral deficits and α-synuclein pathology that are also LRRK2 GS kinase-dependent. Transmission EM reveals that that there is an LRRK2 GS kinase-dependent significant reduction in synaptic vesicle number and a greater abundance of clathrin-coated vesicles in DA neurons. These transgenic mice indicate that LRRK2-induced DA and NE neurodegeneration is kinase-dependent and can occur in a cell-autonomous manner. Moreover, these mice provide a substantial advance in animal model development for LRRK2-associated PD and an important platform to investigate molecular mechanisms for how DA neurons degenerate as a result of expression of mutant LRRK2.


Assuntos
Modelos Animais de Doenças , Dopamina/metabolismo , Neurônios Dopaminérgicos/patologia , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/fisiologia , Doenças Neurodegenerativas/patologia , Norepinefrina/metabolismo , Fatores Etários , Animais , Comportamento Animal , Neurônios Dopaminérgicos/metabolismo , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Atividade Motora , Mutação , Doenças Neurodegenerativas/metabolismo , alfa-Sinucleína/metabolismo
4.
Genetics ; 207(4): 1335-1345, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-29021281

RESUMO

BRCA2 loss-of-heterozygosity (LOH) is frequently observed in BRCA2-mutated tumors, but its biallelic loss causes embryonic lethality in mice and inhibits proliferation of normal somatic cells. Therefore, it remains unclear how loss of BRCA2 contributes to tumorigenesis. One possibility is that mutation in potential genetic interactors of BRCA2, such as TRP53, is required for cell survival/proliferation in the absence of BRCA2. In this study, using an insertional mutagenesis screen in mouse embryonic stem cells (mESC), we have identified GIPC3 (GAIP-interacting protein C-terminus 3) as a BRCA2 genetic interactor that contributes to survival of Brca2-null mESC. GIPC3 does not compensate for BRCA2 loss in the repair of double-strand breaks. Mass-spectrometric analysis resulted in the identification of G-protein signaling transducers, APPL1 and APPL2, as potential GIPC3-binding proteins. A mutant GIPC3 (His155Ala) that does not bind to APPL1/2 failed to rescue the lethality of Brca2-null mESC, suggesting that the cell viability by GIPC3 is mediated via APPL1/2. Finally, the physiological significance of GIPC3 as a genetic interactor of BRCA2 is supported by the observation that Brca2-null embryos with Gipc3 overexpression are developmentally more advanced than their control littermates. Taken together, we have uncovered a novel role for GIPC3 as a BRCA2 genetic interactor.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteína BRCA2/genética , Neoplasias da Mama/genética , Carcinogênese/genética , Animais , Proteína BRCA2/deficiência , Neoplasias da Mama/patologia , Proteínas de Transporte/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Perda de Heterozigosidade/genética , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Mutagênese Insercional , Mutação
5.
Cancer Cell ; 31(4): 501-515.e8, 2017 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-28399408

RESUMO

Targeting the tumor vasculature with antibody-drug conjugates (ADCs) is a promising anti-cancer strategy that in order to be realized must overcome several obstacles, including identification of suitable targets and optimal warheads. Here, we demonstrate that the cell-surface protein CD276/B7-H3 is broadly overexpressed by multiple tumor types on both cancer cells and tumor-infiltrating blood vessels, making it a potentially ideal dual-compartment therapeutic target. In preclinical studies CD276 ADCs armed with a conventional MMAE warhead destroyed CD276-positive cancer cells, but were ineffective against tumor vasculature. In contrast, pyrrolobenzodiazepine-conjugated CD276 ADCs killed both cancer cells and tumor vasculature, eradicating large established tumors and metastases, and improving long-term overall survival. CD276-targeted dual-compartment ablation could aid in the development of highly selective broad-acting anti-cancer therapies.


Assuntos
Antígenos B7/genética , Antígenos B7/metabolismo , Imunoconjugados/farmacologia , Neoplasias/irrigação sanguínea , Animais , Antineoplásicos/imunologia , Antineoplásicos/farmacologia , Antígenos B7/imunologia , Benzodiazepinas/farmacologia , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/patologia , Linhagem Celular Tumoral , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/patologia , Feminino , Humanos , Imunoconjugados/imunologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Terapia de Alvo Molecular/métodos , Neoplasias/patologia , Neoplasias/terapia , Oligopeptídeos/farmacologia , Pirróis/farmacologia , Coelhos
6.
Cell Rep ; 16(3): 793-804, 2016 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-27373150

RESUMO

The neural network of the temporal lobe is thought to provide a cognitive map of our surroundings. Functional analysis of this network has been hampered by coarse tools that often result in collateral damage to other circuits. We developed a chemogenetic system to temporally control electrical input into the hippocampus. When entorhinal input to the perforant path was acutely silenced, hippocampal firing patterns became destabilized and underwent extensive remapping. We also found that spatial memory acquired prior to neural silencing was impaired by loss of input through the perforant path. Together, our experiments show that manipulation of entorhinal activity destabilizes spatial coding and disrupts spatial memory. Moreover, we introduce a chemogenetic model for non-invasive neuronal silencing that offers multiple advantages over existing strategies in this setting.


Assuntos
Hipocampo/fisiologia , Rede Nervosa/fisiologia , Memória Espacial/fisiologia , Lobo Temporal/fisiologia , Animais , Córtex Entorrinal/fisiologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Modelos Neurológicos , Via Perfurante/fisiologia
7.
Neuron ; 90(3): 535-50, 2016 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-27112497

RESUMO

Hexanucleotide expansions in C9ORF72 are the most frequent genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Disease mechanisms were evaluated in mice expressing C9ORF72 RNAs with up to 450 GGGGCC repeats or with one or both C9orf72 alleles inactivated. Chronic 50% reduction of C9ORF72 did not provoke disease, while its absence produced splenomegaly, enlarged lymph nodes, and mild social interaction deficits, but not motor dysfunction. Hexanucleotide expansions caused age-, repeat-length-, and expression-level-dependent accumulation of RNA foci and dipeptide-repeat proteins synthesized by AUG-independent translation, accompanied by loss of hippocampal neurons, increased anxiety, and impaired cognitive function. Single-dose injection of antisense oligonucleotides (ASOs) that target repeat-containing RNAs but preserve levels of mRNAs encoding C9ORF72 produced sustained reductions in RNA foci and dipeptide-repeat proteins, and ameliorated behavioral deficits. These efforts identify gain of toxicity as a central disease mechanism caused by repeat-expanded C9ORF72 and establish the feasibility of ASO-mediated therapy.


Assuntos
Esclerose Amiotrófica Lateral/tratamento farmacológico , Demência Frontotemporal/tratamento farmacológico , Fatores de Troca do Nucleotídeo Guanina/genética , Oligonucleotídeos Antissenso/farmacologia , RNA/metabolismo , Esclerose Amiotrófica Lateral/genética , Animais , Proteína C9orf72 , Expansão das Repetições de DNA/genética , Demência Frontotemporal/genética , Camundongos Transgênicos , Neurônios/metabolismo , Oligonucleotídeos Antissenso/efeitos adversos , Oligonucleotídeos Antissenso/genética
8.
Proc Natl Acad Sci U S A ; 112(46): E6321-30, 2015 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-26578792

RESUMO

Centrosomes are microtubule-organizing centers that facilitate bipolar mitotic spindle assembly and chromosome segregation. Recognizing that centrosome amplification is a common feature of aneuploid cancer cells, we tested whether supernumerary centrosomes are sufficient to drive tumor development. To do this, we constructed and analyzed mice in which centrosome amplification can be induced by a Cre-recombinase-mediated increase in expression of Polo-like kinase 4 (Plk4). Elevated Plk4 in mouse fibroblasts produced supernumerary centrosomes and enhanced the expected mitotic errors, but proliferation continued only after inactivation of the p53 tumor suppressor. Increasing Plk4 levels in mice with functional p53 produced centrosome amplification in liver and skin, but this did not promote spontaneous tumor development in these tissues or enhance the growth of chemically induced skin tumors. In the absence of p53, Plk4 overexpression generated widespread centrosome amplification, but did not drive additional tumors or affect development of the fatal thymic lymphomas that arise in animals lacking p53. We conclude that, independent of p53 status, supernumerary centrosomes are not sufficient to drive tumor formation.


Assuntos
Divisão Celular Assimétrica/fisiologia , Centrossomo/metabolismo , Animais , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Regulação da Expressão Gênica , Linfoma/genética , Linfoma/metabolismo , Linfoma/patologia , Camundongos , Camundongos Knockout , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Neoplasias do Timo/genética , Neoplasias do Timo/metabolismo , Neoplasias do Timo/patologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
9.
Dev Cell ; 35(3): 322-32, 2015 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-26555052

RESUMO

The mammalian lung forms its elaborate tree-like structure following a largely stereotypical branching sequence. While a number of genes have been identified to play essential roles in lung branching, what coordinates the choice between branch growth and new branch formation has not been elucidated. Here we show that loss of FGF-activated transcription factor genes, Etv4 and Etv5 (collectively Etv), led to prolonged branch tip growth and delayed new branch formation. Unexpectedly, this phenotype is more similar to mutants with increased rather than decreased FGF activity. Indeed, an increased Fgf10 expression is observed, and reducing Fgf10 dosage can attenuate the Etv mutant phenotype. Further evidence indicates that ETV inhibits Fgf10 via directly promoting Shh expression. SHH in turn inhibits local Fgf10 expression and redirects growth, thereby initiating new branches. Together, our findings establish ETV as a key node in the FGF-ETV-SHH inhibitory feedback loop that dictates branching periodicity.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento/genética , Pulmão/metabolismo , Proteínas Proto-Oncogênicas c-ets/metabolismo , Fatores de Transcrição/metabolismo , Animais , Padronização Corporal/genética , Fator 10 de Crescimento de Fibroblastos/metabolismo , Proteínas Hedgehog/metabolismo , Camundongos , Camundongos Transgênicos , Morfogênese/genética , Transdução de Sinais/genética
10.
J Mol Cell Cardiol ; 88: 1-13, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26386426

RESUMO

Gap junctions (GJ) are intercellular channels composed of connexin subunits that play a critical role in a diverse number of cellular processes in all tissue types. In the heart, GJs mediate electrical coupling between cardiomyocytes and display mislocalization and/or downregulation in cardiac disease (a process known as GJ remodeling), producing an arrhythmogenic substrate. The main constituent of GJs in the ventricular myocardium is Connexin 43 (Cx43), an integral membrane protein that is rapidly turned over and shows decreased expression or function with age. We hypothesized that Wwp1, an ubiquitin ligase whose expression in known to increase in aging-related pathologies, may regulate Cx43 in vivo by targeting it for ubiquitylation and degradation and yield tissue-specific Cx43 loss of function phenotypes. When Wwp1 was globally overexpressed in mice under the control of a ß-actin promoter, the highest induction of Wwp1 expression was observed in the heart which was associated with a 90% reduction in cardiac Cx43 protein levels, left ventricular hypertrophy (LVH), and the development of lethal ventricular arrhythmias around 8weeks of age. This phenotype was completely penetrant in two independent founder lines. Cardiomyocyte-specific overexpression of Wwp1 confirmed that this phenotype was cell autonomous and delineated Cx43-dependent and -independent roles for Wwp1 in arrhythmogenesis and LVH, respectively. Using a cell-based system, it was determined that Wwp1 co-immunoprecipitates with and ubiquitylates Cx43, causing a decrease in the steady state levels of Cx43 protein. These findings offer new mechanistic insights into the regulation of Cx43 which may be exploitable in various gap junctionopathies.


Assuntos
Arritmias Cardíacas/genética , Conexina 43/genética , Hipertrofia Ventricular Esquerda/genética , Miócitos Cardíacos/metabolismo , Ubiquitina-Proteína Ligases/genética , Actinas/genética , Actinas/metabolismo , Animais , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/patologia , Conexina 43/metabolismo , Modelos Animais de Doenças , Feminino , Junções Comunicantes/metabolismo , Junções Comunicantes/patologia , Regulação da Expressão Gênica , Ventrículos do Coração/metabolismo , Ventrículos do Coração/patologia , Hipertrofia Ventricular Esquerda/metabolismo , Hipertrofia Ventricular Esquerda/patologia , Masculino , Camundongos , Camundongos Transgênicos , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/patologia , Fenótipo , Regiões Promotoras Genéticas , Complexo de Endopeptidases do Proteassoma/metabolismo , Estabilidade Proteica , Proteólise , Transdução de Sinais , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
11.
Cell Rep ; 10(2): 123-30, 2015 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-25558062

RESUMO

G protein-coupled receptor 124 (GPR124) is an orphan receptor in the adhesion family of GPCRs, and previous global or endothelial-specific disruption of Gpr124 in mice led to defective CNS angiogenesis and blood-brain barriergenesis. Similar developmental defects were observed following dual deletion of Wnt7a/Wnt7b or deletion of ß-catenin in endothelial cells, suggesting a possible relationship between GPR124 and canonical WNT signaling. Here, we show using in vitro reporter assays, mutation analysis, and genetic interaction studies in vivo that GPR124 functions as a WNT7A/WNT7B-specific costimulator of ß-catenin signaling in brain endothelium. WNT7-stimulated ß-catenin signaling was dependent upon GPR124's intracellular PDZ binding motif and a set of leucine-rich repeats in its extracellular domain. This study reveals a vital role for GPR124 in potentiation of WNT7-induced canonical ß-catenin signaling with important implications for understanding and manipulating CNS-specific angiogenesis and blood-brain barrier-genesis.


Assuntos
Receptores Acoplados a Proteínas-G/metabolismo , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , beta Catenina/metabolismo , Motivos de Aminoácidos , Animais , Barreira Hematoencefálica/metabolismo , Encéfalo/citologia , Encéfalo/metabolismo , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Domínios PDZ , Proteínas Proto-Oncogênicas/metabolismo , Receptores Acoplados a Proteínas-G/química , Receptores Acoplados a Proteínas-G/deficiência
12.
Sci Transl Med ; 6(242): 242ra84, 2014 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-24964992

RESUMO

Antiangiogenic agents that block vascular endothelial growth factor (VEGF) signaling are important components of current cancer treatment modalities but are limited by alternative ill-defined angiogenesis mechanisms that allow persistent tumor vascularization in the face of continued VEGF pathway blockade. We identified prostaglandin E2 (PGE2) as a soluble tumor-derived angiogenic factor associated with VEGF-independent angiogenesis. PGE2 production in preclinical breast and colon cancer models was tightly controlled by cyclooxygenase-2 (COX-2) expression, and COX-2 inhibition augmented VEGF pathway blockade to suppress angiogenesis and tumor growth, prevent metastasis, and increase overall survival. These results demonstrate the importance of the COX-2/PGE2 pathway in mediating resistance to VEGF pathway blockade and could aid in the rapid development of more efficacious anticancer therapies.


Assuntos
Inibidores da Angiogênese/uso terapêutico , Inibidores de Ciclo-Oxigenase 2/uso terapêutico , Neoplasias Hepáticas/prevenção & controle , Neoplasias Hepáticas/secundário , Neoplasias Mamárias Experimentais/prevenção & controle , Neoplasias Mamárias Experimentais/secundário , Ensaios Antitumorais Modelo de Xenoenxerto , Inibidores da Angiogênese/farmacologia , Animais , Axitinibe , Carcinogênese/patologia , Linhagem Celular Tumoral , Células Clonais , Ciclo-Oxigenase 2/metabolismo , Inibidores de Ciclo-Oxigenase 2/farmacologia , Dinoprostona/metabolismo , Feminino , Humanos , Imidazóis/farmacologia , Indazóis/farmacologia , Neoplasias Hepáticas/irrigação sanguínea , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Mamárias Experimentais/irrigação sanguínea , Neoplasias Mamárias Experimentais/tratamento farmacológico , Camundongos , Terapia Neoadjuvante , Transdução de Sinais/efeitos dos fármacos , Análise de Sobrevida , Fator A de Crescimento do Endotélio Vascular/metabolismo
13.
PLoS One ; 9(1): e85883, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24465765

RESUMO

The LMO2 oncogene is deregulated in the majority of human T-cell leukemia cases and in most gene therapy-induced T-cell leukemias. We made transgenic mice with enforced expression of Lmo2 in T-cells by the CD2 promoter/enhancer. These transgenic mice developed highly penetrant T-ALL by two distinct patterns of gene expression: one in which there was concordant activation of Lyl1, Hhex, and Mycn or alternatively, with Notch1 target gene activation. Most strikingly, this gene expression clustering was conserved in human Early T-cell Precursor ALL (ETP-ALL), where LMO2, HHEX, LYL1, and MYCN were most highly expressed. We discovered that HHEX is a direct transcriptional target of LMO2 consistent with its concordant gene expression. Furthermore, conditional inactivation of Hhex in CD2-Lmo2 transgenic mice markedly attenuated T-ALL development, demonstrating that Hhex is a crucial mediator of Lmo2's oncogenic function. The CD2-Lmo2 transgenic mice offer mechanistic insight into concordant oncogene expression and provide a model for the highly treatment-resistant ETP-ALL subtype.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Carcinogênese/metabolismo , Proteínas com Domínio LIM/metabolismo , Leucemia de Células T/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Antígenos CD2/metabolismo , Carcinogênese/patologia , Linhagem Celular Tumoral , Elementos E-Box/genética , Regulação Leucêmica da Expressão Gênica , Proteínas de Homeodomínio/genética , Humanos , Leucemia de Células T/genética , Leucemia de Células T/patologia , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Proteínas de Neoplasias/metabolismo , Oncogenes , Penetrância , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Regiões Promotoras Genéticas/genética , Ligação Proteica , Fatores de Transcrição/genética , Transcrição Genética , Regulação para Cima/genética
14.
PLoS One ; 8(4): e62479, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23638095

RESUMO

The study of axis extension and somitogenesis has been greatly advanced through the use of genetic tools such as the TCre mouse line. In this line, Cre is controlled by a fragment of the T (Brachyury) promoter that is active in progenitor cells that reside within the primitive streak and tail bud and which give rise to lineages emerging from these tissues as the embryonic axis extends. However, because TCre-mediated recombination occurs early in development, gene inactivation can result in an axis truncation that precludes the study of gene function in later or more posterior tissues. To address this limitation, we have generated an inducible TCre transgenic mouse line, called TCreERT2, that provides temporal control, through tamoxifen administration, in all cells emerging from the primitive streak or tail bud throughout development. TCreERT2 activity is mostly silent in the absence of tamoxifen and, in its presence, results in near complete recombination of emerging mesoderm from E7.5 through E13.5. We demonstrate the utility of the TCreERT2 line for determining rate of posterior axis extension and somite formation, thus providing the first in vivo tool for such measurements. To test the usefulness of TCreERT2 for genetic manipulation, we demonstrate that an early deletion of ß-Catenin via TCreERT2 induction phenocopies the TCre-mediated deletion of ß-Catenin defect, whereas a later induction bypasses this early phenotype and produces a similar defect in more caudal tissues. TCreERT2 provides a useful and novel tool for the control of gene expression of emerging embryonic lineages throughout development.


Assuntos
Camundongos Transgênicos/genética , Linha Primitiva/embriologia , Recombinação Genética , Animais , Antagonistas de Estrogênios/administração & dosagem , Feminino , Proteínas Fetais/genética , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Linha Primitiva/citologia , Linha Primitiva/metabolismo , Proteínas com Domínio T/genética , Tamoxifeno/administração & dosagem
15.
Genome Res ; 22(5): 870-84, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22367191

RESUMO

Endogenous retrotransposons have caused extensive genomic variation within mammalian species, but the functional implications of such mobilization are mostly unknown. We mapped thousands of endogenous retrovirus (ERV) germline integrants in highly divergent, previously unsequenced mouse lineages, facilitating a comparison of gene expression in the presence or absence of local insertions. Polymorphic ERVs occur relatively infrequently in gene introns and are particularly depleted from genes involved in embryogenesis or that are highly expressed in embryonic stem cells. Their genomic distribution implies ongoing negative selection due to deleterious effects on gene expression and function. A polymorphic, intronic ERV at Slc15a2 triggers up to 49-fold increases in premature transcriptional termination and up to 39-fold reductions in full-length transcripts in adult mouse tissues, thereby disrupting protein expression and functional activity. Prematurely truncated transcripts also occur at Polr1a, Spon1, and up to ∼5% of other genes when intronic ERV polymorphisms are present. Analysis of expression quantitative trait loci (eQTLs) in recombinant BxD mouse strains demonstrated very strong genetic associations between the polymorphic ERV in cis and disrupted transcript levels. Premature polyadenylation is triggered at genomic distances up to >12.5 kb upstream of the ERV, both in cis and between alleles. The parent of origin of the ERV is associated with variable expression of nonterminated transcripts and differential DNA methylation at its 5'-long terminal repeat. This study defines an unexpectedly strong functional impact of ERVs in disrupting gene transcription at a distance and demonstrates that ongoing retrotransposition can contribute significantly to natural phenotypic diversity.


Assuntos
Retrovirus Endógenos/genética , Regulação da Expressão Gênica , Transcrição Genética , Animais , Sequência de Bases , Mapeamento Cromossômico , Metilação de DNA , Feminino , Variação Genética , Heterozigoto , Íntrons , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Polimorfismo Genético , Biossíntese de Proteínas/genética , Locos de Características Quantitativas , Análise de Sequência de DNA , Simportadores/genética , Simportadores/metabolismo , Sequências Repetidas Terminais
16.
Genesis ; 50(2): 112-8, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21898766

RESUMO

The Notch1 receptor plays a critical role in cell fate decisions during development. Activation of Notch signaling has been implicated in several types of cancer, particularly T-cell acute lymphoblastic leukemia (T-ALL). Consequently, several transgenic mouse strains have been made to study the role of Notch1 in T-ALL. However, the existing Notch1 transgenic lines mimic a translocation event found in only ∼1% of T-ALL cases. Here we describe three novel NOTCH1 transgenic mouse strains that have Cre-inducible expression of the entire human NOTCH1 locus, each possessing a common mutation found in T-ALL. Unlike existing Notch1 transgenic strains, these NOTCH1 transgenic strains express full-length receptors from an endogenous human promoter that should be susceptible to a number of Notch antagonists that have recently been developed. These strains will allow researchers to modulate Notch signaling to study both normal development and cancer biology.


Assuntos
Regulação Neoplásica da Expressão Gênica , Camundongos Transgênicos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Receptor Notch1/genética , Animais , Western Blotting , Diferenciação Celular , Imunofluorescência , Humanos , Camundongos , Mutação , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Receptor Notch1/metabolismo , Transdução de Sinais
17.
PLoS One ; 6(4): e18568, 2011 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-21494637

RESUMO

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause late-onset, autosomal dominant familial Parkinson's disease (PD) and also contribute to idiopathic PD. LRRK2 mutations represent the most common cause of PD with clinical and neurochemical features that are largely indistinguishable from idiopathic disease. Currently, transgenic mice expressing wild-type or disease-causing mutants of LRRK2 have failed to produce overt neurodegeneration, although abnormalities in nigrostriatal dopaminergic neurotransmission have been observed. Here, we describe the development and characterization of transgenic mice expressing human LRRK2 bearing the familial PD mutations, R1441C and G2019S. Our study demonstrates that expression of G2019S mutant LRRK2 induces the degeneration of nigrostriatal pathway dopaminergic neurons in an age-dependent manner. In addition, we observe autophagic and mitochondrial abnormalities in the brains of aged G2019S LRRK2 mice and markedly reduced neurite complexity of cultured dopaminergic neurons. These new LRRK2 transgenic mice will provide important tools for understanding the mechanism(s) through which familial mutations precipitate neuronal degeneration and PD.


Assuntos
Substituição de Aminoácidos/genética , Autofagia , Dopamina/metabolismo , Proteínas Mutantes/metabolismo , Neuritos/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Comportamento Animal , Cromatografia Líquida de Alta Pressão , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Mesencéfalo/metabolismo , Mesencéfalo/patologia , Mesencéfalo/ultraestrutura , Camundongos , Camundongos Transgênicos , Atividade Motora , Neuritos/ultraestrutura , Técnicas de Cultura de Órgãos , Transporte Proteico
18.
Genetics ; 183(2): 581-94, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19635938

RESUMO

The microphthalmia-associated transcription factor (Mitf) has emerged as an important model for gene regulation in eukaryotic organisms. In vertebrates, it regulates the development of several cell types including melanocytes and has also been shown to play an important role in melanoma. In vitro, the activity of MITF is regulated by multiple signaling pathways, including the KITL/KIT/B-Raf pathway, which results in phosphorylation of MITF on serine residues 73 and 409. However, the precise role of signaling to MITF in vivo remains largely unknown. Here, we use a BAC transgene rescue approach to introduce specific mutations in MITF to study the importance of specific phospho-acceptor sites and protein domains. We show that mice that carry a BAC transgene where single-amino-acid substitutions have been made in the Mitf gene rescue the phenotype of the loss-of-function mutations in Mitf. This may indicate that signaling from KIT to MITF affects other phospho-acceptor sites in MITF or that alternative sites can be phosphorylated when Ser73 and Ser409 have been mutated. Our results have implications for understanding signaling to transcription factors. Furthermore, as MITF and signaling mechanisms have been shown to play an important role in melanomas, our findings may lead to novel insights into this resilient disease.


Assuntos
Cromossomos Artificiais Bacterianos/genética , Olho/metabolismo , Cor de Cabelo/genética , Fator de Transcrição Associado à Microftalmia/genética , Transgenes/genética , Processamento Alternativo , Animais , Sítios de Ligação/genética , Éxons/genética , Olho/crescimento & desenvolvimento , Feminino , Deleção de Genes , Masculino , Melanócitos/metabolismo , Camundongos , Camundongos Transgênicos , Fator de Transcrição Associado à Microftalmia/metabolismo , Mutação , Miocárdio/metabolismo , Fenótipo , Fosforilação , Serina/genética , Serina/metabolismo , Pele/crescimento & desenvolvimento , Pele/metabolismo
19.
Dev Biol ; 325(1): 33-42, 2009 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-18926813

RESUMO

Homozygous ataxia (ax(J)) mice have reduced expression of ubiquitin-specific protease 14 (Usp14), resulting in severe neuromuscular defects and death by 2 months of age. Transgenic expression of Usp14 exclusively in the nervous system of ax(J) mice (ax(J)-Tg) prevents early lethality and restores motor system function to the ax(J) mice, enabling an analysis of the reproductive capabilities of Usp14-deficient mice. Although female ax(J)-Tg mice had a 75% reduction of Usp14 in the ovaries, they were able to produce normal litters. Ovary transfer experiments also demonstrated that the ovaries of ax(J) mice were capable of producing viable pups. In contrast, male ax(J) and ax(J)-Tg mice displayed a 50% reduction in testicular Usp14 levels and were infertile, indicating that Usp14 is required for development and function of the male reproductive system. Immunohistochemistry experiments showed that Usp14 is found in the redundant nuclear envelope and cytoplasmic droplet of epididymal spermatozoa. Analysis of ax(J) testes demonstrated a 50% reduction in testis weight, a 100-fold reduction in sperm number and the presence of abnormal spermatozoa in the epididymis. Histological examination of the Usp14-deficient testes revealed abnormal spermatogenesis and the presence of degenerating germ cells, indicating that Usp14 and the ubiquitin proteasome system are required for spermatid differentiation during spermiogenesis.


Assuntos
Ataxia/complicações , Ataxia/patologia , Infertilidade Masculina/complicações , Infertilidade Masculina/patologia , Animais , Compensação de Dosagem (Genética) , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/patologia , Endopeptidases/metabolismo , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Dosagem de Genes , Perfilação da Expressão Gênica , Heterozigoto , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Tamanho do Órgão , Transporte Proteico , Espermatozoides/metabolismo , Testículo/embriologia , Testículo/enzimologia , Testículo/metabolismo , Testículo/patologia , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo
20.
Proc Natl Acad Sci U S A ; 101(48): 16831-6, 2004 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-15550542

RESUMO

MYO5A is a major actin-based vesicle transport motor that binds to one of its cargos, the melanosome, by means of a RAB27A/MLPH receptor. When one of the members of this receptor-motor complex is mutated, the melanosomes clump in the perinuclear region of the melanocyte and are transferred unevenly to the developing hair, leading to a dilution of coat color. Mutation of a fourth gene, dilute suppressor (dsu), suppresses this coat color dilution. MYO5A is required for the peripheral accumulation of melanosomes in melanocytes, but its role in melanosome transfer to neighboring keratinocytes and the hair is unknown. Here, we show that MYO5A is nonessential for melanosome transfer, although pigment incorporation into the hair in MYO5A-deficient mice is uneven, probably due to the clumping of melanosomes that occurs in the perinuclear region of mutant melanocytes. We also show that dsu is caused by a loss-of-function mutation in a unique vertebrate-specific protein that appears to function in an MYO5A-independent pathway to alter pigment incorporation into the hair. Therefore, dsu identifies a unique protein involved in pigmentation of the mammalian hair.


Assuntos
Cor de Cabelo/genética , Cadeias Pesadas de Miosina/fisiologia , Miosina Tipo V/fisiologia , Animais , Western Blotting , Cromossomos Bacterianos , Teste de Complementação Genética , Camundongos , Dados de Sequência Molecular , Cadeias Pesadas de Miosina/genética , Miosina Tipo V/genética
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