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1.
Dis Model Mech ; 3(11-12): 743-51, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20829563

RESUMO

Mutations in the SPAST (SPG4) gene, which encodes the microtubule-severing protein spastin, are the most common cause of autosomal dominant hereditary spastic paraplegia (HSP). Following on from previous work in our laboratory showing that spastin is required for axon outgrowth, we report here that the related microtubule-severing protein katanin is also required for axon outgrowth in vivo. Using confocal time-lapse imaging, we have identified requirements for spastin and katanin in maintaining normal axonal microtubule dynamics and growth cone motility in vivo, supporting a model in which microtubule severing is required for concerted growth of neuronal microtubules. Simultaneous knockdown of spastin and katanin caused a more severe phenotype than did individual knockdown of either gene, suggesting that they have different but related functions in supporting axon outgrowth. In addition, the microtubule-destabilising drug nocodazole abolished microtubule dynamics and growth cone motility, and enhanced phenotypic severity in spast-knockdown zebrafish embryos. Thus, disruption of microtubule dynamics might underlie neuronal dysfunction in this model, and this system could be used to identify compounds that modulate microtubule dynamics, some of which might have therapeutic potential in HSP.


Assuntos
Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Axônios/metabolismo , Embrião não Mamífero/enzimologia , Microtúbulos/patologia , Paraplegia Espástica Hereditária/enzimologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Axônios/efeitos dos fármacos , Embrião não Mamífero/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Proteínas de Fluorescência Verde/metabolismo , Cones de Crescimento/efeitos dos fármacos , Cones de Crescimento/patologia , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/efeitos dos fármacos , Modelos Biológicos , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/enzimologia , Neurônios Motores/patologia , Nocodazol/farmacologia , Oligonucleotídeos Antissenso/farmacologia , Fenótipo , Splicing de RNA/efeitos dos fármacos , Splicing de RNA/genética , Paraplegia Espástica Hereditária/patologia , Espastina
2.
Hum Mol Genet ; 15(18): 2763-71, 2006 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-16893913

RESUMO

Hereditary spastic paraplegia (HSP) is a collection of neurological disorders characterized by developmental failure or degeneration of motor axons in the corticospinal tract and progressive lower limb spasticity. SPG4 mutations are the most common cause of autosomal dominant HSP and Spastin (the SPG4 gene product) is a microtubule severing protein that shares homology with katanin, the microtubule severing activity of which promotes axon growth in cultured neurons. Given the sequence and functional similarity between spastin and katanin, we hypothesized that spastin promotes the dynamic disassembly and remodelling of microtubules required for robust, properly directed motor axon outgrowth. To investigate this hypothesis, we cloned the zebrafish spg4 orthologue and used morpholino antisense oligonucleotides directed against the translation start site and the intron 7-8 splice donor site to knock down spastin function in the developing zebrafish embryo. Reduced spg4 function caused dramatic defects in motor axon outgrowth without affecting the events driving the initial specification of motor neurones. Other neuronal subtypes also exhibited a requirement for spg4 function, since spg4 knock down caused both widespread defects in neuronal connectivity and extensive CNS-specific apoptosis. Our results reveal a critical requirement for spastin to promote axonal outgrowth during embryonic development, and they validate the zebrafish embryo as a novel model system to dissect the pathogenetic mechanisms underlying HSP. Taken together with other recent studies, our findings suggest that axon outgrowth defects may be a common feature of childhood SPG3A and SPG4 cases.


Assuntos
Adenosina Trifosfatases/metabolismo , Axônios/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Adenosina Trifosfatases/deficiência , Adenosina Trifosfatases/genética , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , Humanos , Microtúbulos/metabolismo , Dados de Sequência Molecular , Neurônios Motores/metabolismo , Oligodesoxirribonucleotídeos Antissenso/genética , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Paraplegia Espástica Hereditária/genética , Paraplegia Espástica Hereditária/metabolismo , Sinapses/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genética
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