RESUMO
Spinal muscular atrophy (SMA) is an autosomal recessive neurodegenerative disease. Loss of the survival motor neuron (SMN1) gene, in the presence of the SMN2 gene causes SMA. SMN functions in snRNP assembly in all cell types, however, it is unclear how this function results in specifically motor neuron cell death. Lack of endogenous mouse SMN (Smn) in mice results in embryonic lethality. Introduction of two copies of human SMN2 results in a mouse with severe SMA, while one copy of SMN2 is insufficient to overcome embryonic lethality. We show that SMN(A111G), an allele capable of snRNP assembly, can rescue mice that lack Smn and contain either one or two copies of SMN2 (SMA mice). The correction of SMA in these animals was directly correlated with snRNP assembly activity in spinal cord, as was correction of snRNA levels. These data support snRNP assembly as being the critical function affected in SMA and suggests that the levels of snRNPs are critical to motor neurons. Furthermore, SMN(A111G) cannot rescue Smn-/- mice without SMN2 suggesting that both SMN(A111G) and SMN from SMN2 undergo intragenic complementation in vivo to function in heteromeric complexes that have greater function than either allele alone. The oligomer composed of limiting full-length SMN and SMN(A111G) has substantial snRNP assembly activity. Also, the SMN(A2G) and SMN(A111G) alleles in vivo did not complement each other leading to the possibility that these mutations could affect the same function.
Assuntos
Atrofia Muscular Espinal/fisiopatologia , Mutação de Sentido Incorreto , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Animais , Células Cultivadas , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Neurônios Motores/metabolismo , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/metabolismo , Atrofia Muscular Espinal/mortalidade , Ribonucleoproteínas Nucleares Pequenas/genética , Medula Espinal/metabolismo , Medula Espinal/fisiopatologia , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Proteína 2 de Sobrevivência do Neurônio Motor/metabolismoRESUMO
Spinal muscular atrophy (SMA) is a motor neuron degenerative disease caused by low levels of the survival motor neuron (SMN) protein and is linked to mutations or loss of SMN1 and retention of SMN2. How low levels of SMN cause SMA is unclear. SMN functions in small nuclear ribonucleoprotein (snRNP) biogenesis, but recent studies indicate that SMN may also function in axons. We showed previously that decreasing Smn levels in zebrafish using morpholinos (MO) results in motor axon defects. To determine how Smn functions in motor axon outgrowth, we coinjected smn MO with various human SMN RNAs and assayed the effect on motor axons. Wild-type SMN rescues motor axon defects caused by Smn reduction in zebrafish. Consistent with these defects playing a role in SMA, SMN lacking exon 7, the predominant form from the SMN2 gene, and human SMA mutations do not rescue defective motor axons. Moreover, the severity of the motor axon defects correlates with decreased longevity. We also show that a conserved region in SMN exon 7, QNQKE, is critical for motor axon outgrowth. To address the function of SMN important for motor axon outgrowth, we determined the ability of different SMN forms to oligomerization and bind Sm protein, functions required for snRNP biogenesis. We identified mutations that failed to rescue motor axon defects but retained snRNP function. Thus, we have dissociated the snRNP function of SMN from its function in motor axons. These data indicate that SMN has a novel function in motor axons that is relevant to SMA and is independent of snRNP biosynthesis.
Assuntos
Axônios/fisiologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Neurônios Motores/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Proteínas de Ligação a RNA/fisiologia , Ribonucleoproteínas Nucleares Pequenas/biossíntese , Sequência de Aminoácidos , Animais , Células Cultivadas , Embrião de Galinha , Humanos , Dados de Sequência Molecular , Mutação , Fenótipo , Ribonucleoproteínas Nucleares Pequenas/genética , Proteínas do Complexo SMN , Proteína 1 de Sobrevivência do Neurônio Motor , Proteína 2 de Sobrevivência do Neurônio Motor , Peixe-ZebraRESUMO
Motoneuron diseases cause paralysis and death due to loss of motoneurons that innervate skeletal muscle. Spinal muscular atrophy is a human motoneuron disease that is genetically linked to the survival motor neuron gene (SMN). Although SMN was identified more than a decade ago, it remains unclear how decreased levels of the SMN protein cause spinal muscular atrophy. The use of animal models, however, offers a crucial tool in determining the function of SMN in this disease. In this review, we discuss our efforts to develop a zebrafish model of spinal muscular atrophy.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Predisposição Genética para Doença/genética , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas de Ligação a RNA/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Animais , Diferenciação Celular/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Modelos Animais de Doenças , Humanos , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Músculo Esquelético/embriologia , Músculo Esquelético/inervação , Músculo Esquelético/fisiopatologia , Atrofia Muscular Espinal/fisiopatologia , Mutação/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas do Complexo SMN , Proteína 1 de Sobrevivência do Neurônio Motor , Peixe-Zebra/metabolismoRESUMO
Xenopus Zic3 (zinc finger in the cerebellum-3) is expressed in the dorsal neural tube of tailbud embryos and tadpoles. We have isolated a 3.1kb DNA fragment from the Xenopus laevis Zic3 locus that drives proper expression of a GFP reporter in transgenic frog tailbud embryos and tadpoles. This fragment contains regions that are highly similar among frogs, mice, and humans. One extremely conserved region contains a predicted Pbx binding site. We found that the transcription factors Pbx1b and Meis1 can bind this site and synergistically transactivate expression of a reporter containing the conserved region. Finally, we found that an intact Pbx site is essential for normal Zic3 promoter activity in transgenic frog embryos. Our data strongly suggest that a highly conserved region of the Zic3 promoter functions by direct interaction with Pbx1b and Meis1.