Mutação eqlb causa alteração na expressão de genes do controle do ciclo celular de precursores de neurônios cerebelares
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2011-04-27
Autores
Ariza, Carolina Batista [UNIFESP]
Orientadores
Porcionatto, Marimélia Aparecida [UNIFESP]
Tipo
Tese de doutorado
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Várias doenças humanas atualmente conhecidas têm causas genéticas. Uma maneira de se entender as alterações moleculares que levam a essas doenças é através da análise da disfunção de um gene, que pode ser feito usando modelos animais que recapitulem tais patologias. O camundongo desempenha muito bem essa função, devido à alta porcentagem de homologia entre os genomas murino e humano. Mutações espontâneas ou quimicamente induzidas são ferramentas úteis para elucidar como um gene mutado dá origem ao fenótipo da doença. Apesar do atual banco de dados de camundongos mutantes, que é composto na sua maioria por animais nocautes, ser uma fonte valiosa de modelos animais, apenas uma pequena proporção do total de genes mamíferos é representada. Esse vazio está sendo, em parte, completado pelo estabelecimento de novos programas de mutagênese utilizando-se potentes agentes mutagênicos como N-ethyl-N-nitrosourea (ENU). Através de um projeto de mutagênese por ENU, foram isolados mutantes recessivos apresentando vários fenótipos, sendo um deles um mutante apresentando problemas de equilíbrio com características de falta de coordenação motora, denominado equilibrio (eqlb). O fenótipo apresentado por esse mutante nos levou à hipótese de que a mutação gerada poderia estar afetando o desenvolvimento do cerebelo, região do sistema nervoso que controla a execução dos movimentos e o equilíbrio. A avaliação do comportamento de camundongos adultos selvagem e mutantes em teste de desempenho em barra rotatória mostrou que o tempo de permanência dos mutantes eqlb estava significantemente diminuído em relação aos camundongos controle BALB/c. Análises histológicas de cerebelo de camundongos mutantes revelaram formação anormal das camadas celulares, com desorganização da camada de células de Purkinje, o principal tipo de neurônio do cerebelo. As análises histológicas também revelaram uma camada granular externa (CGE) mais espessa nos camundongos mutantes, quando comparados com cerebelos de camundongos selvagem de mesma idade. Ensaio de incorporação de [3H]-timidina mostrou que o espessamento dessa camada pode ser atribuído ao alto índice de proliferação observado nos cerebelos dos camundongos mutantes. Esse aumento no índice de proliferação de precursores de neurônios granulares não foi observado em outros órgãos e outras regiões do SNC, bem com também não foi observado células tronco neurais e mesenquimais derivadas da medula óssea. O mapeamento genético da mutação, usando-se marcadores microssatélites polimórficos, localizou a mutação no cromossomo 17, em uma sub-região delimitada pelo marcador D17Mit267 (3,31Mb) e o limite centromérico. Essa região possui sintenia conservada com a região telomérica do cromossomo 6 humano, a qual apresenta loci relacionados ao aparecimento de ataxias recessivas. Dois genes candidatos foram sequenciados: ENSMUSG00000044697 foi sequenciado completamente e a mutação não foi encontrada, e ENSMUSG00000046201 (Rbm16) e o sequenciamento não foi completo. Análise de expressão gênica diferencial revelou alteração na expressão de genes relacionados a doenças neurológicas e crescimento celular e proliferação nos camundongos mutantes de 6 dias pós natal, e alterações na expressão de genes relacionadas a tumor cerebral e meduloblastoma em camundongos mutantes de 15 dias de desenvolvimento pós natal.
Several human diseases currently known are of genetic cause. One way to understand the molecular alterations that lead to a given disease is through the analysis of gene dysfunction, which can be done using animal models that recapitulate such pathologies. The mouse performs very well this function, because there is a high percentage of homology between its genome and that of man. Spontaneous or chemically induced mutations are useful to elucidate how a mutated gene gives rise to a given illness phenotype. Although the current mouse mutant database, which is composed largely of knockout animals, has been a valuable source of models, only a small proportion of the likely total number of mammalian genes is represented. This gap is being partially filled by the establishment of new mutagenesis programs using potent mammalian mutagens like N-ethyl-N-nitrosourea (ENU). Through an ENU mutagenesis project, recessive mutant mice bearing varied phenotypes were isolated, one being a mutant mouse presenting balance problems, with characteristics of lack of motor coordination, named equilibrio (eqlb). The phenotype led us to hypothesize that the generated mutation could be affecting the development of the cerebellum, region of the central nervous system that controls the execution of the movements and balance. The evaluation of mutant and wild-type adults behavior in the test of permanence on a rotating bar showed that the time of permanence of eqlb was significantly diminished in relation the wild-type. Histological analysis of eqlb mice cerebella shows abnormal formation of the cellular layers, with disorganization of the Purkinje cell layer, the main type of neuron in the cerebellum. Histological analysis also revealed that the external granular cell layer is thicker in eqlb mutant mice, when compared to same age wild-type mice cerebellum. [3H]-thymidine incorporation showed that the thickness of this layer can be attributed to the high proliferation rate observed in the cerebellum of mutant mice. This increased rate of proliferation of precursors of granular neurons was not observed in other organs and other regions of the CNS and was not observed with neural stem cells and adult stem cells derived from bone marrow. Genetic mapping of the mutation, using polymorphic microsatellite markers, located at chromosome 17 in a subregion bounded by the marker D17Mit267 (3.31 Mb) and the centromeric boundary. This region has conserved synteny with the telomeric region of human chromosome 6, which presents loci related to the appearance of recessive ataxias. Two candidate genes were sequenced: ENSMUSG00000044697 was sequenced completely and the mutation was not found in that gene, and ENSMUSG00000046201 (Rbm16), but the sequencing of this gene was not complete. Differential gene expression analysis revealed abnormal expression of genes related to neurological diseases and cell growth and proliferation in the mutant mice 6 days after birth, and changes in gene expression related to brain tumor and medulloblastoma in mice mutant for 15 days of postnatal development.
Several human diseases currently known are of genetic cause. One way to understand the molecular alterations that lead to a given disease is through the analysis of gene dysfunction, which can be done using animal models that recapitulate such pathologies. The mouse performs very well this function, because there is a high percentage of homology between its genome and that of man. Spontaneous or chemically induced mutations are useful to elucidate how a mutated gene gives rise to a given illness phenotype. Although the current mouse mutant database, which is composed largely of knockout animals, has been a valuable source of models, only a small proportion of the likely total number of mammalian genes is represented. This gap is being partially filled by the establishment of new mutagenesis programs using potent mammalian mutagens like N-ethyl-N-nitrosourea (ENU). Through an ENU mutagenesis project, recessive mutant mice bearing varied phenotypes were isolated, one being a mutant mouse presenting balance problems, with characteristics of lack of motor coordination, named equilibrio (eqlb). The phenotype led us to hypothesize that the generated mutation could be affecting the development of the cerebellum, region of the central nervous system that controls the execution of the movements and balance. The evaluation of mutant and wild-type adults behavior in the test of permanence on a rotating bar showed that the time of permanence of eqlb was significantly diminished in relation the wild-type. Histological analysis of eqlb mice cerebella shows abnormal formation of the cellular layers, with disorganization of the Purkinje cell layer, the main type of neuron in the cerebellum. Histological analysis also revealed that the external granular cell layer is thicker in eqlb mutant mice, when compared to same age wild-type mice cerebellum. [3H]-thymidine incorporation showed that the thickness of this layer can be attributed to the high proliferation rate observed in the cerebellum of mutant mice. This increased rate of proliferation of precursors of granular neurons was not observed in other organs and other regions of the CNS and was not observed with neural stem cells and adult stem cells derived from bone marrow. Genetic mapping of the mutation, using polymorphic microsatellite markers, located at chromosome 17 in a subregion bounded by the marker D17Mit267 (3.31 Mb) and the centromeric boundary. This region has conserved synteny with the telomeric region of human chromosome 6, which presents loci related to the appearance of recessive ataxias. Two candidate genes were sequenced: ENSMUSG00000044697 was sequenced completely and the mutation was not found in that gene, and ENSMUSG00000046201 (Rbm16), but the sequencing of this gene was not complete. Differential gene expression analysis revealed abnormal expression of genes related to neurological diseases and cell growth and proliferation in the mutant mice 6 days after birth, and changes in gene expression related to brain tumor and medulloblastoma in mice mutant for 15 days of postnatal development.
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Citação
ARIZA, Carolina Batista. Mutação eqlb causa alteração na expressão de genes do controle do ciclo celular de precursores de neurônios cerebelares. 2011. Tese (Doutorado) - Universidade Federal de São Paulo (UNIFESP), São Paulo, 2011.