RESUMO
Spinal muscular atrophy (SMA) is a progressive neurodegenerative disease affecting lower motor neurons. SMA is caused by mutations in the Survival Motor Neuron 1 (SMN1) gene, which result in reduced levels of functional SMN protein. Biochemical studies have linked the ubiquitously expressed SMN protein to the assembly of pre-mRNA processing U snRNPs, raising the possibility that aberrant splicing is a major defect in SMA. Accordingly, several transcripts affected upon SMN deficiency have been reported. A second function for SMN in axonal mRNA transport has also been proposed that may likewise contribute to the SMA phenotype. The underlying etiology of SMA, however, is still not fully understood. Here, we have used a combination of genomics and live Ca(2+) imaging to investigate the consequences of SMN deficiency in a zebrafish model of SMA. In a transcriptome analyses of SMN-deficient zebrafish, we identified neurexin2a (nrxn2a) as strongly down-regulated and displaying changes in alternative splicing patterns. Importantly, the knock-down of two distinct nrxn2a isoforms phenocopies SMN-deficient fish and results in a significant reduction of motor axon excitability. Interestingly, we observed altered expression and splicing of Nrxn2 also in motor neurons from the Smn(-/-);SMN2(+/+) mouse model of SMA, suggesting conservation of nrxn2 regulation by SMN in mammals. We propose that SMN deficiency affects splicing and abundance of nrxn2a. This may explain the pre-synaptic defects at neuromuscular endplates in SMA pathophysiology.
Assuntos
Atrofia Muscular Espinal/genética , Proteínas do Tecido Nervoso/genética , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Processamento Alternativo/genética , Animais , Cálcio/metabolismo , Sinalização do Cálcio , Células Cultivadas , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Hibridização In Situ , Microdissecção e Captura a Laser , Camundongos , Camundongos Transgênicos , Morfolinos/genética , Neurônios Motores/metabolismo , Proteínas do Tecido Nervoso/biossíntese , Isoformas de Proteínas/genética , RNA Mensageiro/genética , Medula Espinal/metabolismo , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Peixe-ZebraRESUMO
The lamin B receptor (LBR) is an integral membrane protein of the inner nuclear membrane that is interacting with B-type lamins, chromatin and DNA. The complete loss of the protein in mouse mutants causes a reduced viability of embryos, and viable animals develop abnormalities of the skeleton. Here, we present the molecular characterization of the zebrafish LBR (zLBR) gene and the functional analysis of LBR during zebrafish embryogenesis. We found that the coding region of the LBR mRNA of zebrafish as well as of mammals is contained in 13 exons. At the protein level, human and zebrafish LBR exhibit a high sequence identity (57% and higher) in 8 of the 13 exons. Knockdown of zLBR by microinjection of 0.5-1.0 mM morpholino antisense oligonucleotides (MO) into 1- to 2-cell stage embryos reduced the amount of endogenous zLBR protein to approximately 10-20%. The viability of MO-injected embryos within 24 h was reduced to 70-77%. Surviving 1-day-old embryos exhibited morphological alterations including reduced growth of head structures, retardation of tail growth and a bent backbone and tail. Expression analysis of the transcription factors no tail (ntl) and goosecoid (gsc) by in situ hybridization suggests that these malformations are caused by altered cell migration during gastrulation. Our data indicate that the LBR of zebrafish and mammals are both required for correct development.
Assuntos
Receptores Citoplasmáticos e Nucleares/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Complementar/química , DNA Complementar/genética , Eletroforese em Gel de Poliacrilamida , Embrião não Mamífero/citologia , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Éxons/genética , Proteínas Fetais , Regulação da Expressão Gênica no Desenvolvimento , Inativação Gênica , Proteína Goosecoid/genética , Proteína Goosecoid/metabolismo , Humanos , Immunoblotting , Íntrons/genética , Dados de Sequência Molecular , Mutação , Receptores Citoplasmáticos e Nucleares/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Receptor de Lamina BRESUMO
Spinal muscular atrophy (SMA) is a motoneuron disease caused by reduced levels of survival motoneuron (SMN) protein. Previous studies have assigned SMN to uridine-rich small nuclear ribonucleoprotein particle (U snRNP) assembly, splicing, transcription, and RNA localization. Here, we have used gene silencing to assess the effect of SMN protein deficiency on U snRNP metabolism in living cells and organisms. In HeLa cells, we show that reduction of SMN to levels found in SMA patients impairs U snRNP assembly. In line with this, induced silencing of SMN expression in Xenopus laevis or zebrafish arrested embryonic development. Under less severe knock-down conditions, zebrafish embryos proceeded through development yet exhibited dramatic SMA-like motor axon degeneration. The same was observed after silencing two other essential factors in the U snRNP assembly pathway, Gemin2 and pICln. Importantly, the injection of purified U snRNPs into either SMN- or Gemin2-deficient embryos of Xenopus and zebrafish prevented developmental arrest and motoneuron degeneration, respectively. These findings suggest that motoneuron degeneration in SMA patients is a direct consequence of impaired production of U snRNPs.