RESUMO
Posttranslational modifications of tubulin are emerging regulators of microtubule functions. We have shown earlier that upregulated polyglutamylation is linked to rapid degeneration of Purkinje cells in mice with a mutation in the deglutamylating enzyme CCP1. How polyglutamylation leads to degeneration, whether it affects multiple neuron types, or which physiological processes it regulates in healthy neurons has remained unknown. Here, we demonstrate that excessive polyglutamylation induces neurodegeneration in a cell-autonomous manner and can occur in many parts of the central nervous system. Degeneration of selected neurons in CCP1-deficient mice can be fully rescued by simultaneous knockout of the counteracting polyglutamylase TTLL1. Excessive polyglutamylation reduces the efficiency of neuronal transport in cultured hippocampal neurons, suggesting that impaired cargo transport plays an important role in the observed degenerative phenotypes. We thus establish polyglutamylation as a cell-autonomous mechanism for neurodegeneration that might be therapeutically accessible through manipulation of the enzymes that control this posttranslational modification.
Assuntos
Doenças Neurodegenerativas/metabolismo , Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Células de Purkinje/metabolismo , Tubulina (Proteína)/metabolismo , Animais , Transporte Biológico Ativo/genética , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismo , Peptídeos/genética , Células de Purkinje/patologia , Tubulina (Proteína)/genéticaRESUMO
Tubulin is subject to a wide variety of posttranslational modifications, which, as part of the tubulin code, are involved in the regulation of microtubule functions. Glycylation has so far predominantly been found in motile cilia and flagella, and absence of this modification leads to ciliary disassembly. Here, we demonstrate that the correct functioning of connecting cilia of photoreceptors, which are non-motile sensory cilia, is also dependent on glycylation. In contrast to many other tissues, only one glycylase, TTLL3, is expressed in retina. Ttll3-/- mice lack glycylation in photoreceptors, which results in shortening of connecting cilia and slow retinal degeneration. Moreover, absence of glycylation results in increased levels of tubulin glutamylation in photoreceptors, and inversely, the hyperglutamylation observed in the Purkinje cell degeneration (pcd) mouse abolishes glycylation. This suggests that both posttranslational modifications compete for modification sites, and that unbalancing the glutamylation-glycylation equilibrium on axonemes of connecting cilia, regardless of the enzymatic mechanism, invariably leads to retinal degeneration.
Assuntos
Ácido Glutâmico/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/patologia , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Tubulina (Proteína)/metabolismo , Animais , Apoptose , Cílios/metabolismo , Glicosilação , Camundongos Endogâmicos C57BL , Neuroglia/metabolismo , Neuroglia/patologia , Peptídeo Sintases/metabolismo , Fenótipo , Células de Purkinje/metabolismo , Células de Purkinje/patologia , Retina/metabolismo , Retina/patologia , Rodopsina/metabolismo , Fatores de TempoRESUMO
TTLL3 and TTLL8 are tubulin glycine ligases catalyzing posttranslational glycylation of microtubules. We show here for the first time that these enzymes are required for robust formation of primary cilia. We further discover the existence of primary cilia in colon and demonstrate that TTLL3 is the only glycylase in this organ. As a consequence, colon epithelium shows a reduced number of primary cilia accompanied by an increased rate of cell division in TTLL3-knockout mice. Strikingly, higher proliferation is compensated by faster tissue turnover in normal colon. In a mouse model for tumorigenesis, lack of TTLL3 strongly promotes tumor development. We further demonstrate that decreased levels of TTLL3 expression are linked to the development of human colorectal carcinomas. Thus, we have uncovered a novel role for tubulin glycylation in primary cilia maintenance, which controls cell proliferation of colon epithelial cells and plays an essential role in colon cancer development.
Assuntos
Proliferação de Células , Cílios/metabolismo , Colo/metabolismo , Neoplasias do Colo/metabolismo , Glicina/metabolismo , Peptídeo Sintases/fisiologia , Tubulina (Proteína)/fisiologia , Animais , Western Blotting , Carcinógenos/toxicidade , Células Cultivadas , Colo/patologia , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/patologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Modelos Animais de Doenças , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Técnicas Imunoenzimáticas , Camundongos , Camundongos Knockout , Microtúbulos/metabolismo , Processamento de Proteína Pós-Traducional , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Microtubules are subject to a variety of posttranslational modifications that potentially regulate cytoskeletal functions. Two modifications, glutamylation and glycylation, are highly enriched in the axonemes of most eukaryotes, and might therefore play particularly important roles in cilia and flagella. Here we systematically analyze the dynamics of glutamylation and glycylation in developing mouse ependymal cilia and the expression of the corresponding enzymes in the brain. By systematically screening enzymes of the TTLL family for specific functions in ependymal cilia, we demonstrate that the glycylating enzymes TTLL3 and TTLL8 were required for stability and maintenance of ependymal cilia, whereas the polyglutamylase TTLL6 was necessary for coordinated beating behavior. Our work provides evidence for a functional separation of glutamylating and glycylating enzymes in mammalian ependymal cilia. It further advances the elucidation of the functions of tubulin posttranslational modifications in motile cilia of the mammalian brain and their potential importance in brain development and disease.