RESUMO
Huntington's disease (HD) is an autosomal-dominant neurodegenerative disorder caused by a CAG repeat expansion that lengthens a glutamine segment in the novel huntingtin protein. To elucidate the molecular basis of HD, we extended the polyglutamine tract of the mouse homologue, Hdh, by targetted introduction of an expanded human HD CAG repeat, creating mutant HdhneoQ50 and HdhQ50 alleles that express reduced and wild-type levels of altered huntingtin, respectively. Mice homozygous for reduced levels displayed characteristic aberrant brain development and perinatal lethality, indicating a critical function for Hdh in neurogenesis. However, mice with normal levels of mutant huntingtin did not display these abnormalities, indicating that the expanded CAG repeat does not eliminate or detectably impair huntingtin's neurogenic function. Thus, the HD defect in man does not mimic complete or partial Hdh inactivation and appears to cause neurodegenerative disease by a gain-of-function mechanism.
Assuntos
Doença de Huntington/genética , Doença de Huntington/patologia , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Sequências Repetitivas de Ácido Nucleico/genética , Alelos , Animais , Diferenciação Celular/genética , Desenvolvimento Embrionário e Fetal/genética , Deleção de Genes , Heterozigoto , Homozigoto , Humanos , Proteína Huntingtina , Masculino , Camundongos , Camundongos Mutantes , Camundongos Transgênicos , Mutagênese Insercional , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , FenótipoRESUMO
Mice that have been made deficient for the cystic fibrosis transmembrane conductance regulator (Cftr) usually die of intestinal obstruction. We have created Cftr-deficient mice and demonstrate prolonged survival among backcross and intercross progeny with different inbred strains, suggesting that modulation of disease severity is genetically determined. A genome scan showed that the major modifier locus maps near the centromere of mouse chromosome 7. Electrophysiological studies on mice with prolonged survival show that the partial rectification of Cl- and Na+ ion transport abnormalities can be explained in part by up-regulation of a calcium-activated Cl- conductance. Identification of modifier genes in our Cftr(m1HSC)/Cftr(m1HSC) mice should provide important insight into the heterogeneous disease presentation observed among CF patients.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/deficiência , Fibrose Cística/genética , Animais , Sequência de Bases , Linhagem Celular , Cloretos/metabolismo , Mapeamento Cromossômico , Colo/patologia , Fibrose Cística/patologia , Fibrose Cística/fisiopatologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Primers do DNA , Modelos Animais de Doenças , Feminino , Íleo/patologia , Masculino , Potenciais da Membrana , Camundongos , Camundongos Endogâmicos , Camundongos Mutantes , Dados de Sequência Molecular , Mutagênese , Técnicas de Patch-Clamp , Sobreviventes , Aumento de PesoRESUMO
Fanconi anaemia (FA) is an autosomal recessive disease characterized by bone marrow failure, variable congenital malformations and predisposition to malignancies. Cells derived from FA patients show elevated levels of chromosomal breakage and an increased sensitivity to bifunctional alkylating agents such as mitomycin C (MMC) and diepoxybutane (DEB). Five complementation groups have been identified by somatic cell methods, and we have cloned the gene defective in group C (FAC)(7). To understand the in vivo role of this gene, we have disrupted murine Fac and generated mice homozygous for the targeted allele. The -/- mice did not exhibit developmental abnormalities nor haematologic defects up to 9 months of age. However, their spleen cells had dramatically increased numbers of chromosomal aberrations in response to MMC and DEB. Homozygous male and female mice also had compromised gametogenesis, leading to markedly impaired fertility, a characteristic of FA patients. Thus, inactivation of Fac replicates some of the features of the human disease.
Assuntos
Anemia de Fanconi/genética , Infertilidade/genética , Mutação , Animais , Clonagem Molecular , Feminino , Marcação de Genes , Genes Recessivos , Vetores Genéticos , Homozigoto , Infertilidade/patologia , Masculino , Camundongos , Ovário/patologia , Testículo/patologiaRESUMO
This article provides a review of the literature on anteroposterior differences in the growth and functional expression of normal and neoplastic cells. Diverse studies in immunology, wound healing, carcinogenesis, transplantation biology, and developmental biology are brought together to emphasize the practical implications of regional differences for research protocols in all these areas of research. The more fundamental question of mechanisms is approached by consideration of variations in the vascular supply, pattern of nervous system development, expression of temperature differentials, and the establishment of metabolic gradients during development.
Assuntos
Neoplasias Experimentais/patologia , Animais , Temperatura Corporal , Carcinógenos , Feminino , Neoplasias Mamárias Experimentais/patologia , Camundongos , Transplante de Neoplasias , Neoplasias Experimentais/etiologia , Fenômenos Fisiológicos do Sistema Nervoso , Fluxo Sanguíneo Regional , CicatrizaçãoRESUMO
In this review we discuss the concept of anti-angiogenesis, which is the inhibition of neovascularization. Anti-angiogenic agents are viewed from the standpoint of their effect on various elements of the angiogenic process, including induction of vascular discontinuity, endothelial cell movement, endothelial cell proliferation, and three-dimensional restructuring of patent vessels. An effort is made to place the many different approaches to anti-angiogenesis research into a comprehensible structure, in order to identify problems of evaluation and interpretation, thereby providing a clearer basis for determining promising and needed directions for further investigation.
Assuntos
Neovascularização Patológica/prevenção & controle , Animais , Membrana Basal/metabolismo , Comunicação Celular , Divisão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , HumanosRESUMO
Accurate, reliable quantitation of the neovascular (angiogenic) response, both in vitro and in vivo, is an essential requirement for the study of new blood vessel growth. Over many years, ingenious ways have been developed for measuring this process, and they have contributed much to our present understanding of the vasculogenesis and angiogenesis that accompany normal embryonic development, lactation and wound healing, as well as tumor growth and a variety of other disease states ranging from diabetic retinopathy to autoimmune vasculitis. In this review we describe and evaluate the methodology and specific features of some of the most frequently used of these assays.
Assuntos
Neovascularização Patológica , Animais , MétodosRESUMO
The Sonic hedgehog (Shh)-Gli signaling pathway regulates development of many organs, including teeth. We cloned a novel gene encoding a transcription factor that contains a zinc finger domain with highest homology to the Gli family of proteins (61-64% amino acid sequence identity) from incisor pulp. Consistent with this sequence conservation, gel mobility shift assays demonstrated that this new Gli homologous protein, GliH1, could bind previously characterized Gli DNA binding sites. Furthermore, transfection assays in dental pulp cells showed that whereas Gli1 induces a nearly 50-fold increase in activity of a luciferase reporter containing Gli DNA binding sites, coexpression of Gli1 with Gli3 and/or GliH1 results in inhibition of the Gli1-stimulated luciferase activity. In situ hybridization analysis of mouse embryos demonstrated that GliH1 expression is initiated later than the three Gli genes and has a more restricted expression pattern. GliH1 is first detected diffusely in the limb buds at 10.0 days post coitus and later is expressed in the branchial arches, craniofacial interface, ventral part of the tail, whisker follicles and hair, intervertebral discs, teeth, eyes and kidney. LacZ was inserted into the GliH1 allele in embryonic stem cells to produce mice lacking GliH1 function. While this produced indicator mice for GliH1-expression, analysis of mutant mice revealed no discernible phenotype or required function for GliH1. A search of the Celera Genomics and associated databases identified possible gene sequences encoding a zinc finger domain with approximately 90% homology to that of GliH1, indicating there is a family of GliH genes and raising the possibility of overlapping functions during development.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/fisiologia , Proteínas Oncogênicas/química , Proteínas Oncogênicas/fisiologia , Fatores de Transcrição/química , Fatores de Transcrição/fisiologia , Alelos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Northern Blotting , Clonagem Molecular , DNA/metabolismo , DNA Complementar/metabolismo , Proteínas de Ligação a DNA/biossíntese , Bases de Dados como Assunto , Polpa Dentária/metabolismo , Marcação de Genes , Glutationa Transferase/metabolismo , Proteínas Hedgehog , Hibridização In Situ , Luciferases/metabolismo , Camundongos , Modelos Genéticos , Dados de Sequência Molecular , Filogenia , Regiões Promotoras Genéticas , Ligação Proteica , Estrutura Terciária de Proteína , RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Fatores de Tempo , Transativadores/metabolismo , Transfecção , Proteína GLI1 em Dedos de Zinco , Dedos de ZincoRESUMO
Hundreds of new mutant mouse lines are being produced annually using gene targeting and gene trap approaches in embryonic stem (ES) cells, and the number is expected to continue to grow as the human and mouse genome projects progress. The availability of robust ES cell lines and a simple technology for making chimeras is more attractive now than ever before. We established several new ES cell lines from 129/SvEv and C57BL/6 mice and tested their ability to contribute to the germline following blastocyst injections and/or the less expensive and easier method of morula-ES cell aggregation. Using morula aggregation to produce chimeras, five newly derived 129/SvEv and two C57BL/6 ES cell lines tested at early passages were found to contribute extensively to chimeras and produce germline-transmitting male chimeras. Furthermore, the two 129S/vEv ES cell lines that were tested and one of the C57BL/6 ES cell lines were able to maintain these characteristics after many passages in vitro. Our results indicate that the ability of ES cells to contribute strongly to chimeras following aggregation with outbred embryos is a general property of early passage ES cells and can be maintained for many passages. C56BL/6-derived ES cell lines, however, have a greater tendency than 129-derived ES cell lines to lose their ability to colonize the germline.
Assuntos
Quimera/embriologia , Quimera/genética , Células-Tronco/citologia , Células-Tronco/metabolismo , Animais , Blastocisto/citologia , Blastocisto/metabolismo , Agregação Celular , Técnicas de Cultura de Células/métodos , Células Cultivadas , Células Clonais/citologia , Células Clonais/metabolismo , Feminino , Mutação em Linhagem Germinativa , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Transgênicos , Microinjeções , Mórula/citologia , Mórula/metabolismoRESUMO
One hundred and thirteen patients with non-seminoma testis and without clinical evidence of distant metastasis (category M0) have been analyzed with regard to the risk of subsequent metastasis after subdiaphragmatic irradiation. Important prognostic factors were: histology (malignant teratoma intermediate (MTI), malignant teratoma undifferentiated (MTU), T-category (T4, T less than 4), clinical regional and juxta-regional subdiaphragmatic lymph node involvement (N0, N1,2, N3,4) and vascular invasion in the orchidectomy specimen (V-, V+). If vascular invasion was considered, the histological type MTI or MTU lost its prognostic impact. Categories T4 and N greater than or equal to 1 worsen prognosis and if vascular invasion could be observed metastasis-risk would be additionally increased. Watch-and-wait policy is probably most justified in categories T less than 4, N0, V-. Elective chemotherapy might be most justified in categories T4N0V+ and T less than 4N greater than or equal to 1V+.
Assuntos
Neoplasias Testiculares/radioterapia , Humanos , Linfonodos/efeitos da radiação , Masculino , Invasividade Neoplásica , Metástase Neoplásica , Risco , Teratoma/radioterapia , Doenças Vasculares/patologiaRESUMO
As a first step in our efforts to delineate the role of CFTR in cellular phenotypes we have studied its expression in cultured human intestinal epithelial cells. In particular we have examined the effect of cellular differentiation on CFTR gene expression. CFTR mRNA was measured by quantitative densitometry of Northern blots and normalized to the amounts of pyruvate dehydrogenase message. We have found that in T84 cells the levels of CFTR mRNA do not change as the cells grow to confluence. In contrast, levels of CFTR mRNA increase by a factor of 10-20 as Caco2 cells grow after subculture. This change in the levels of CFTR mRNA is correlated with the morphological differentiation that occurs in Caco2 cells during culture. The potential significance of this observation is discussed.
Assuntos
Fibrose Cística/metabolismo , Mucosa Intestinal/metabolismo , Proteínas de Membrana/metabolismo , Diferenciação Celular , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística , Células Epiteliais , Epitélio/metabolismo , Regulação da Expressão Gênica , Humanos , Intestinos/citologia , Proteínas de Membrana/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células Tumorais Cultivadas/metabolismo , Células Tumorais Cultivadas/patologiaRESUMO
The mechanism by which alcohol increases plasma total high density lipoproteins (HDLs) and HDL-cholesterol is unknown, but it may involve modulation of the lipolytic enzymes, hepatic triglyceride lipase (HTGL) and/or lipoprotein lipase (LPL) in hepatic and extrahepatic tissues. The modulation of HDL metabolism by alcohol may also be related to its potential to induce mixed function oxidases in liver microsomes. These possibilities were examined by a pair-feeding protocol in which rats were fed diets with 35% of the caloric content as ethanol; control groups received a diet with an isocaloric amount of sucrose or were fed chow ad libitum. Alcohol caused a significant decrease in HTGL activity of liver microsomes, but there was no significant effect of alcohol upon the activities of LPL in adipose tissue and heart muscle. The relative rates of mixed function oxidases, assayed in control liver microsomes using ethoxy-,pentoxy- and benzyloxy-resorufin as substrates, were benzyloxy greater than ethoxy greater than pentoxy. This order was not affected by alcohol, but the oxidation of ethoxy- and pentoxyresorufin was reduced in liver microsomes from the ethanol-fed group. HTGL synthesis and secretion were also measured using primary rat hepatocyte cultures isolated from animals on the above dietary regimes and maintained for up to 3 days in basal medium alone or supplemented with 10 mmol/l ethanol. In basal media the order of activity of extracellular HTGL, released by the addition of heparin, was sucrose-fed greater than chow-fed greater than ethanol-fed. The rate of HTGL secretion from hepatocytes was stimulated in ethanol-containing medium, and was greater in hepatocytes from the sucrose-fed controls.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Alcoolismo/metabolismo , Etanol/farmacologia , Lipase/metabolismo , Lipólise/efeitos dos fármacos , Animais , Células Cultivadas , Cinética , Fígado/efeitos dos fármacos , Fígado/enzimologia , Fígado/metabolismo , Masculino , Oxigenases de Função Mista/metabolismo , Ratos , Ratos Endogâmicos , Valores de ReferênciaRESUMO
A case of a large, completely excised left-sided primary ovarian chondrosarcoma occurring in a 61-year-old woman is described. There was no evidence of metastases. Histologically the tumour was a pure chondrosarcoma, which was mainly well differentiated. The patient is well and disease free at 4 years. Review of the literature revealed no other reported cases of pure chondrosarcoma of the ovary. The possible histogenetic origin of this neoplasm is discussed.
Assuntos
Condrossarcoma/patologia , Neoplasias Ovarianas/patologia , Feminino , Humanos , Pessoa de Meia-IdadeRESUMO
Caco-2 human colonic carcinoma cells were transfected with an expression vector encoding a mutant form of RI (regulatory subunit of the type 1 cAMP-dependent protein kinase), driven by the metallothionein 1 promoter. A stable transformant was isolated that expressed the mutant RI gene in a Zn(2+)-inducible manner. The consequences of the RI mutation on cAMP-dependent protein kinase activity, cell division, and regulation of chloride efflux were examined. When grown in the absence of ZnSO4, protein kinase activity in the transformant was stimulated 2.5-fold by cAMP and approached the levels of cAMP-dependent protein kinase activity seen in parental Caco-2 cells; when treated with ZnSO4, cAMP-dependent protein kinase activity in the transformant was inhibited by 60%. In the absence of ZnSO4 the transformant grew with the same doubling time and to the same saturation density as the untransformed parent. In the presence of ZnSO4 the transformant exhibited a cAMP-reversible inhibition of cell division, indicating that a functional cAMP-dependent protein kinase was required for the growth of these cells in culture. Induction of the mutant RI gene also abolished forskolin-stimulated chloride efflux from these cells, suggesting obligatory roles for cAMP and cAMP-dependent protein kinase in forskolin's actions on chloride channel activity. We anticipate that this transformant will be useful for further studies on the roles of cAMP and cAMP-dependent protein kinase in the regulation of intestinal epithelial cells, including regulation of cell proliferation and differentiation, and regulation of chloride channel activity by neurohormones and neurotransmitters.
Assuntos
Proteínas de Transporte/fisiologia , AMP Cíclico/fisiologia , Mucosa Intestinal/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana/fisiologia , Proteínas Quinases/fisiologia , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Divisão Celular/fisiologia , Canais de Cloreto , Cloretos/metabolismo , Neoplasias do Colo , Humanos , Intestinos/citologia , Ativação do Canal Iônico/fisiologia , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Sistemas do Segundo Mensageiro/fisiologia , Células Tumorais CultivadasRESUMO
The Pax6 gene has a central role in development of the eye. We show, through targeted deletion in the mouse, that an ectoderm enhancer in the Pax6 gene is required for normal lens formation. Ectoderm enhancer-deficient embryos exhibit distinctive defects at every stage of lens development. These include a thinner lens placode, reduced placodal cell proliferation, and a small lens pit and lens vesicle. In addition, the lens vesicle fails to separate from the surface ectoderm and the maturing lens is smaller and shows a delay in fiber cell differentiation. Interestingly, deletion of the ectoderm enhancer does not eliminate Pax6 production in the lens placode but results in a diminished level that, in central sections, is apparent primarily on the nasal side. This argues that Pax6 expression in the lens placode is controlled by the ectoderm enhancer and at least one other transcriptional control element. It also suggests that Pax6 enhancers active in the lens placode drive expression in distinct subdomains, an assertion that is supported by the expression pattern of a lacZ reporter transgene driven by the ectoderm enhancer. Interestingly, deletion of the ectoderm enhancer causes loss of expression of Foxe3, a transcription factor gene mutated in the dysgenetic lens mouse. When combined, these data and previously published work allow us to assemble a more complete genetic pathway describing lens induction. This pathway features (1) a pre-placodal phase of Pax6 expression that is required for the activity of multiple, downstream Pax6 enhancers; (2) a later, placodal phase of Pax6 expression regulated by multiple enhancers; and (3) the Foxe3 gene in a downstream position. This pathway forms a basis for future analysis of lens induction mechanism.
Assuntos
Ectoderma/citologia , Indução Embrionária , Elementos Facilitadores Genéticos , Proteínas de Homeodomínio/genética , Cristalino/embriologia , Animais , Diferenciação Celular , Proteínas do Olho , Fatores de Transcrição Forkhead , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Cristalino/anormalidades , Cristalino/citologia , Camundongos , Camundongos Mutantes , Modelos Biológicos , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados , Proteínas Repressoras , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
CFTR, the protein defective in cystic fibrosis is regulated during differentiation of intestinal epithelial cells. The undifferentiated cells (Caco-2 and HT-29) show a lower level of CFTR mRNA, while a 10-fold increase is seen in differentiated cells. These differences correlate well with those of other intestinal-specific genes, including sucrase-isomaltase, villin and alpha 1-antitrypsin, indicating that the regulation is cell specific. In Caco-2 cells the increase in CFTR mRNA cannot be accounted for by increased transcription of the gene. These data indicate that CFTR mRNA stabilizing factor(s) might be present in differentiated cells. The higher levels of CFTR mRNA in differentiated cells are accompanied by decreased protein levels, indicating, as well, involvement of translational control in the regulation of CFTR in these cells. Finally, fully differentiated cells show lowered levels of cyclic AMP-activated C1- transport, the characteristic function of CFTR. Thus, CFTR function in differentiated cells is modulated by a complex interaction of regulatory elements. Caco-2 and HT-29 cells provide a suitable in vitro system in which to study the mechanism of regulation of CFTR.
Assuntos
Colo/metabolismo , Fibrose Cística/metabolismo , Regulação da Expressão Gênica , Proteínas de Membrana/genética , Western Blotting , Proteínas de Transporte/genética , Diferenciação Celular , Colo/citologia , Regulador de Condutância Transmembrana em Fibrose Cística , Células Epiteliais , Epitélio/metabolismo , Radioisótopos do Iodo/metabolismo , Proteínas dos Microfilamentos/genética , RNA Mensageiro/metabolismo , Transcrição Gênica , Células Tumorais CultivadasRESUMO
We undertook to extend the in vitro lifespan of epithelial cell cultures useful for the study of the cellular defect underlying cystic fibrosis (CF). Primary cultures from sweat glands of four CF and four non-CF and from nasal polyps of one non-CF and two CF individuals were transformed using a chimaeric virus, Ad5/SV40 1613 ori-. The extended lifespans ranged from 20 to more than 250 population doublings beyond that of the primary cultures. Despite some degree of aneuploidy (as assayed by total cellular DNA content) all samples tested retained at least one copy of the region of chromosome 7 containing the CF gene (as assayed by probing with flanking DNA markers). Epithelial characteristics, including an epithelioid morphology, tight junctions and desmosomes, apical microvilli, keratin networks, and dome formation were positive in the majority of cells examined, although variably expressed. All cells tested demonstrated outwardly rectifying chloride channels by patch clamp, with some from non-CF cells responsive to the catalytic subunit of cyclic AMP-dependent protein kinase. The cells were used for DNA transfection assays with selectable marker genes in appropriate vectors, in order to develop methodology for assaying the function of the CF gene product and the effects of mutations.
Assuntos
Transformação Celular Viral/fisiologia , Fibrose Cística/patologia , Pólipos Nasais/patologia , Glândulas Sudoríparas/citologia , Divisão Celular/fisiologia , Linhagem Celular Transformada , Sobrevivência Celular , DNA/análise , Células Epiteliais , Humanos , Queratinas/análise , Microscopia Eletrônica , Pólipos Nasais/ultraestrutura , Glândulas Sudoríparas/ultraestruturaRESUMO
Huntingtin is an essential protein that with mutant polyglutamine tracts initiates dominant striatal neurodegeneration in Huntington's disease (HD). To assess the consequences of mutant protein when huntingtin is limiting, we have studied three lines of compound heterozygous mice in which both copies of the HD gene homolog (Hdh) were altered, resulting in greatly reduced levels of huntingtin with a normal human polyglutamine length (Q20) and/or an expanded disease-associated segment (Q111): Hdh(neoQ20)/Hdh(neoQ20), Hdh(neoQ20)/Hdh(null) and Hdh(neoQ20)/Hdh(neoQ111). All surviving mice in each of the three lines were small from birth, and had variable movement abnormalities. Magnetic resonance micro-imaging and histological evaluation showed enlarged ventricles in approximately 50% of the Hdh(neoQ20)/Hdh(neoQ111) and Hdh(neoQ20)/Hdh(null) mice, revealing a developmental defect that does not worsen with age. Only Hdh(neoQ20)/Hdh(neoQ111) mice exhibited a rapidly progressive movement disorder that, in the absence of striatal pathology, begins with hind-limb clasping during tail suspension and tail stiffness during walking by 3-4 months of age, and then progresses to paralysis of the limbs and tail, hypokinesis and premature death, usually by 12 months of age. Thus, dramatically reduced huntingtin levels fail to support normal development in mice, resulting in reduced body size, movement abnormalities and a variable increase in ventricle volume. On this sensitized background, mutant huntingtin causes a rapid neurological disease, distinct from the HD-pathogenic process. These results raise the possibility that therapeutic elimination of huntingtin in HD patients could lead to unintended neurological, as well as developmental side-effects.
Assuntos
Proteínas do Tecido Nervoso/metabolismo , Doenças do Sistema Nervoso/genética , Proteínas Nucleares/metabolismo , Animais , Comportamento Animal/fisiologia , Encéfalo/metabolismo , Encéfalo/patologia , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Progressão da Doença , Feminino , Proteína Huntingtina , Masculino , Camundongos , Camundongos Knockout , Transtornos dos Movimentos/genética , Transtornos dos Movimentos/mortalidade , Transtornos dos Movimentos/fisiopatologia , Mutação , Proteínas do Tecido Nervoso/genética , Doenças do Sistema Nervoso/mortalidade , Doenças do Sistema Nervoso/fisiopatologia , Proteínas Nucleares/genética , Taxa de Sobrevida , Fatores de TempoRESUMO
The CAG repeats in the human Huntington's disease (HD) gene exhibit striking length-dependent intergenerational instability, typically small size increases or decreases of one to a few CAGs, but little variation in somatic tissues. In a subset of male transmissions, larger size increases occur to produce extreme HD alleles that display somatic instability and cause juvenile onset of the disorder. Initial efforts to reproduce these features in a mouse model transgenic for HD exon 1 with 48 CAG repeats revealed only mild intergenerational instability ( approximately 2% of meioses). A similar pattern was obtained when this repeat was inserted into exon 1 of the mouse Hdh gene. However, lengthening the repeats in Hdh to 90 and 109 units produced a graded increase in the mutation frequency to >70%, with instability being more evident in female transmissions. No large jumps in CAG length were detected in either male or female transmissions. Instead, size changes were modest increases and decreases, with expansions typically emanating from males and contractions from females. Limited CAG variation in the somatic tissues gave way to marked mosaicism in liver and striatum for the longest repeats in older mice. These results indicate that gametogenesis is the primary source of inherited instability in the Hdh knock-in mouse, as it is in man, but that the underlying repeat length-dependent mechanism, which may or may not be related in the two species, operates at higher CAG numbers. Moreover, the large CAG repeat increases seen in a subset of male HD transmissions are not reproduced in the mouse, suggesting that these arise by a different fundamental mechanism than the small size fluctuations that are frequent during gametogenesis in both species.
Assuntos
Doença de Huntington/genética , Expansão das Repetições de Trinucleotídeos , Fatores Etários , Animais , Modelos Animais de Doenças , Éxons , Feminino , Humanos , Proteína Huntingtina , Masculino , Meiose/genética , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Caracteres SexuaisRESUMO
Huntington's disease (HD) is caused by an expanded N-terminal glutamine tract that endows huntingtin with a striatal-selective structural property ultimately toxic to medium spiny neurons. In precise genetic models of juvenile HD, HdhQ92 and HdhQ111 knock-in mice, long polyglutamine segments change huntingtin's physical properties, producing HD-like in vivo correlates in the striatum, including nuclear localization of a version of the full-length protein predominant in medium spiny neurons, and subsequent formation of N-terminal inclusions and insoluble aggregate. These changes show glutamine length dependence and dominant inheritance with recruitment of wild-type protein, critical features of the altered HD property that strongly implicate them in the HD disease process and that suggest alternative pathogenic scenarios: the effect of the glutamine tract may act by altering interaction with a critical cellular constituent or by depleting a form of huntingtin essential to medium spiny striatal neurons.