RESUMO
At least eight dominant human neurodegenerative diseases are due to the expansion of a polyglutamine within the disease proteins. This confers toxicity on the proteins and is associated with nuclear inclusion formation. Recent findings indicate that molecular chaperones can modulate polyglutamine pathogenesis, but the basis of polyglutamine toxicity and the mechanism by which chaperones suppress neurodegeneration remains unknown. In a Drosophila: disease model, we demonstrate that chaperones show substrate specificity for polyglutamine protein, as well as synergy in suppression of neurotoxicity. Our analysis also reveals that chaperones alter the solubility properties of the protein, indicating that chaperone modulation of neurodegeneration in vivo is associated with altered biochemical properties of the mutant polyglutamine protein. These findings have implications for these and other human neurodegenerative diseases associated with abnormal protein aggregation.
Assuntos
Drosophila melanogaster , Proteínas de Choque Térmico/metabolismo , Transtornos Heredodegenerativos do Sistema Nervoso/metabolismo , Chaperonas Moleculares/metabolismo , Peptídeos/metabolismo , Expansão das Repetições de Trinucleotídeos/genética , Animais , Modelos Animais de Doenças , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Genótipo , Proteínas de Choque Térmico HSP40 , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Transtornos Heredodegenerativos do Sistema Nervoso/genética , Histocitoquímica , Humanos , Proteína Huntingtina , Proteínas de Insetos/química , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Mutação , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Peptídeos/química , Peptídeos/genética , Fenótipo , Retina/metabolismo , Retina/patologia , Solubilidade , Especificidade por SubstratoRESUMO
Genes involved in eye development are highly conserved between vertebrates and Drosophila. Given the complex genetic network controlling early eye development, identification of regulatory sequences controlling gene expression will provide valuable insights toward understanding central events of early eye specification. We have focused on defining regulatory elements critical for Drosophila eyes absent (eya) expression. Although eya has a complex expression pattern during development, analysis of eye-specific mutations in the gene revealed a region selectively deleted in the eye-specific alleles. Here we have performed detailed analysis of the region deleted in the eye-specific eya(2) allele. This analysis shows that this region can direct early eya gene expression in a pattern consistent with that of normal eya in eye progenitor cells. Functional studies indicate that this element will restore appropriate eya transcript expression to rescue the eye-specific allele. We have examined regulation of this element during eye specification, both in normal eye development and in ectopic eye formation. These studies demonstrate that the element was activated upon ectopic expression of the eye specification genes eyeless and dachshund, but does not respond to ectopic expression of eya or sine oculis. The differential regulation of this element by genes involved during early retinal formation reveals new aspects of the genetic hierarchy of eye development.
Assuntos
Proteínas de Drosophila , Drosophila/embriologia , Drosophila/genética , Elementos Facilitadores Genéticos , Proteínas do Olho/genética , Olho/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Animais , Animais Geneticamente Modificados , Cruzamentos Genéticos , Proteínas de Ligação a DNA/genética , Olho/transplante , Genes de Insetos , Mutagênese , Proteínas Nucleares/genética , Sequências Reguladoras de Ácido Nucleico , Deleção de SequênciaRESUMO
At least eight inherited human neurodegenerative diseases are caused by expansion of a polyglutamine domain within the respective proteins. This confers dominant toxicity on the proteins, leading to dysfunction and loss of neurons. Expanded polyglutamine proteins form aggregates, including nuclear inclusions (NI), within neurons, possibly due to misfolding of the proteins. NI are ubiquitinated and sequester molecular chaperone proteins and proteasome components, suggesting that disease pathogenesis includes activation of cellular stress pathways to help refold, disaggregate or degrade the mutant disease proteins. Overexpression of specific chaperone proteins reduces polyglutamine aggregation in transfected cells, but whether this alters toxicity is unknown. Using a Drosophila melanogaster model of polyglutamine disease, we show that directed expression of the molecular chaperone HSP70 suppresses polyglutamine-induced neurodegeneration in vivo. Suppression by HSP70 occurred without a visible effect on NI formation, indicating that polyglutamine toxicity can be dissociated from formation of large aggregates. Our studies indicate that HSP70 or related molecular chaperones may provide a means of treating these and other neurodegenerative diseases associated with abnormal protein conformation and toxicity.
Assuntos
Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/fisiologia , Degeneração Neural/genética , Degeneração Neural/prevenção & controle , Peptídeos/genética , Peptídeos/fisiologia , Animais , Ataxina-3 , Modelos Animais de Doenças , Drosophila melanogaster/genética , Drosophila melanogaster/fisiologia , Olho/patologia , Feminino , Expressão Gênica , Humanos , Doença de Machado-Joseph/genética , Doença de Machado-Joseph/terapia , Masculino , Degeneração Neural/etiologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Doenças Neurodegenerativas/terapia , Proteínas Nucleares , Estrutura Terciária de Proteína/genética , Proteínas Repressoras , TransfecçãoRESUMO
Spinocerebellar ataxia type 3 (SCA3/MJD) is one of at least eight human neurodegenerative diseases caused by glutamine-repeat expansion. We have recreated glutamine-repeat disease in Drosophila using a segment of the SCA3/MJD protein. Targeted expression of the protein with an expanded polyglutamine repeat led to nuclear inclusion (NI) formation and late-onset cell degeneration. Differential sensitivity to the mutant transgene was observed among different cell types, with neurons being particularly susceptible; NI formation alone was not sufficient for degeneration. The viral antiapoptotic gene P35 mitigated polyglutamine-induced degeneration in vivo. Our results demonstrate that cellular mechanisms of human glutamine-repeat disease are conserved in invertebrates. This fly model will aid in identifying additional factors that modulate neurodegeneration.
Assuntos
Corpos de Inclusão/genética , Doença de Machado-Joseph/patologia , Degeneração Neural/genética , Proteínas do Tecido Nervoso/genética , Peptídeos , Animais , Animais Geneticamente Modificados , Apoptose , Ataxina-3 , Baculoviridae , Núcleo Celular/patologia , Drosophila , Olho/patologia , Marcação de Genes , Humanos , Proteínas Inibidoras de Apoptose , Larva , Doença de Machado-Joseph/genética , Proteínas Nucleares , Especificidade de Órgãos , Peptídeos/genética , Proteínas Recombinantes de Fusão , Proteínas Repressoras , Repetições de Trinucleotídeos , Proteínas Virais/genética , Proteínas Virais/fisiologiaRESUMO
The fly eyes absent (eya) gene which is essential for compound eye development in Drosophila, was shown to be functionally replaceable in eye development by a vertebrate Eya homolog. The relationship between eya and that of the eyeless gene, a Pax-6 homolog, critical for eye formation in both flies and man, was defined: eya was found to be essential for eye formation by eyeless. Moreover, eya could itself direct ectopic eye formation, indicating that eya has the capacity to function as a master control gene for eye formation. Finally, we show that eya and eyeless together were more effective in eye formation than either gene alone. These data indicate conservation of the pathway of eya function between flies and vertebrates; they suggest a model whereby eya/Eya gene function is essential for eye formation by eyeless/Pax-6, and that eya/Eya can in turn mediate, via a regulatory loop, the activity of eyeless/Pax-6 in eye formation.
