RESUMO
In humans, mutations in the fused in sarcoma (FUS) gene have been identified in sporadic and familial forms of amyotrophic lateral sclerosis (ALS). Cabeza (Caz) is the Drosophila ortholog of human FUS. Previously, we established Drosophila models of ALS harboring Caz-knockdown. These flies develop locomotive deficits and anatomical defects in motoneurons (MNs) at neuromuscular junctions; these phenotypes indicate that loss of physiological FUS functions in the nucleus can cause MN degeneration similar to that seen in FUS-related ALS. Here, we aimed to explore molecules that affect these ALS-like phenotypes of our Drosophila models with eye-specific and neuron-specific Caz-knockdown. We examined several previously reported ALS-related genes and found genetic links between Caz and ter94, the Drosophila ortholog of human Valosin-containing protein (VCP). Genetic crossing the strongest loss-of-function allele of ter94 with Caz-knockdown strongly enhanced the rough-eye phenotype and the MN-degeneration phenotype caused by Caz-knockdown. Conversely, the overexpression of wild-type ter94 in the background of Caz-knockdown remarkably suppressed those phenotypes. Our data demonstrated that expression levels of Drosophila VCP ortholog dramatically modified the phenotypes caused by Caz-knockdown in either direction, exacerbation or remission. Our results indicate that therapeutic agents that up-regulate the function of human VCP could modify the pathogenic processes that lead to the degeneration of MNs in ALS.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Drosophila/metabolismo , Neurônios Motores/metabolismo , Proteína FUS de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fator de Transcrição TFIID/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas de Ciclo Celular/genética , Sistema Nervoso Central/citologia , Sistema Nervoso Central/metabolismo , Olho Composto de Artrópodes/metabolismo , Olho Composto de Artrópodes/patologia , Drosophila , Proteínas de Drosophila/genética , Mutação , Proteína FUS de Ligação a RNA/genética , Proteínas de Ligação a RNA/genética , Fator de Transcrição TFIID/genética , Proteína com ValosinaRESUMO
Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease that causes progressive muscular weakness. Fused in Sarcoma (FUS) that has been identified in familial ALS is an RNA binding protein that is normally localized in the nucleus. However, its function in vivo is not fully understood. Drosophila has Cabeza (Caz) as a FUS homologue and specific knockdown of Caz in the eye imaginal disc and pupal retina using a GMR-GAL4 driver was here found to induce an abnormal morphology of the adult compound eyes, a rough eye phenotype. This was partially suppressed by expression of the apoptosis inhibitor P35. Knockdown of Caz exerted no apparent effect on differentiation of photoreceptor cells. However, immunostaining with an antibody to Cut that marks cone cells revealed fusion of these and ommatidia of pupal retinae. These results indicate that Caz knockdown induces apoptosis and also inhibits differentiation of cone cells, resulting in abnormal eye morphology in adults. Mutation in EGFR pathway-related genes, such as rhomboid-1, rhomboid-3 and mirror suppressed the rough eye phenotype induced by Caz knockdown. Moreover, the rhomboid-1 mutation rescued the fusion of cone cells and ommatidia observed in Caz knockdown flies. The results suggest that Caz negatively regulates the EGFR signaling pathway required for determination of cone cell fate in Drosophila.
Assuntos
Animais Geneticamente Modificados/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Receptores ErbB/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteína FUS de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Receptores de Peptídeos de Invertebrados/metabolismo , Retina/metabolismo , Fator de Transcrição TFIID/metabolismo , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/crescimento & desenvolvimento , Apoptose , Western Blotting , Diferenciação Celular , Drosophila/genética , Drosophila/crescimento & desenvolvimento , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/genética , Receptores ErbB/genética , Feminino , Técnicas Imunoenzimáticas , Masculino , Microscopia Eletrônica de Varredura , RNA Interferente Pequeno/genética , Proteína FUS de Ligação a RNA/genética , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Receptores de Peptídeos de Invertebrados/genética , Retina/citologia , Transdução de Sinais , Fator de Transcrição TFIID/antagonistas & inibidores , Fator de Transcrição TFIID/genéticaRESUMO
Charcot-Marie-Tooth disease (CMT) is the most common hereditary neuropathy, and more than 80 CMT-causing genes have been identified to date. CMT4J is caused by a loss-of-function mutation in the Factor-Induced-Gene 4 (FIG4) gene, the product of which plays important roles in endosome-lysosome homeostasis. We hypothesized that Mammalian sterile 20-like kinase (MST) 1 and 2, tumor-suppressor genes, are candidate modifiers of CMT4J. We therefore examined the interaction between dFIG4 and Hippo (hpo), Drosophila counterparts of FIG4 and MSTs, respectively, using the Drosophila CMT4J model with the knockdown of dFIG4. The loss-of-function allele of hpo improved the rough eye morphology, locomotive dysfunction accompanied by structural defects in the presynaptic terminals of motoneurons, and the enlargement of lysosomes caused by the knockdown of dFIG4. Therefore, we identified hpo as a modifier of phenotypes induced by the knockdown of dFIG4. These results in Drosophila may provide an insight into the pathogenesis of CMT4J and contribute toward the development of disease-modifying therapy for CMT. We also identified the regulation of endosome-lysosome homeostasis as a novel probable function of Hippo/MST.
Assuntos
Doença de Charcot-Marie-Tooth/genética , Proteínas de Drosophila/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lisossomos/genética , Mutação/genética , Proteínas Serina-Treonina Quinases/genética , Alelos , Animais , Drosophila melanogaster , Técnicas de Silenciamento de Genes/métodos , Neurônios Motores/metabolismo , FenótipoRESUMO
Mutations in Factor-Induced-Gene 4 (FIG4) gene have been identified in Charcot-Marie-Tooth disease type 4J (CMT4J), Yunis-Varon syndrome and epilepsy with polymicrogyria. FIG4 protein regulates a cellular abundance of phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2), a signaling lipid on the cytosolic surface of membranes of the late endosomal compartment. PI(3,5)P2 is required for retrograde membrane trafficking from lysosomal and late endosomal compartments to the Golgi. However, it is still unknown how the neurodegeneration that occurs in these diseases is related to the loss of FIG4 function. Drosophila has CG17840 (dFIG4) as a human FIG4 homolog. Here we specifically knocked down dFIG4 in various tissues, and investigated their phenotypes. Neuron-specific knockdown of dFIG4 resulted in axonal targeting aberrations of photoreceptor neurons, shortened presynaptic terminals of motor neurons in 3rd instar larvae and reduced climbing ability in adulthood and life span. Fat body-specific knockdown of dFIG4 resulted in enlarged lysosomes in cells that were detected by staining with LysoTracker. In addition, eye imaginal disk-specific knockdown of dFIG4 disrupted differentiation of pupal ommatidial cell types, such as cone cells and pigment cells, suggesting an additional role of dFIG4 during eye development.