RESUMO
Dp71 is the major product of the Duchenne muscular dystrophy gene in the brain. In order to study the function of Dp71 in the nervous system we examined the expression of Dp71 isoforms in PC12 rat pheochromocytoma cell line, a well-established system to study neuronal differentiation. We show by reverse transcriptase-polymerase chain reaction and Western blot assays that PC12 cells express two Dp71 isoforms. One isoform lacks exon 71 and the other isoform lacks exons 71 and 78 (Dp71d and Dp71f isoforms respectively). Nerve growth factor-induced neuronal differentiation of PC12 cells results in differential regulation of the expression and subcellular localization of Dp71 isoforms: a) the amount of Dp71f protein increases nine-fold in total extracts while Dp71d increases up to seven-fold in nuclear extracts; b) Dp71f relocates from the cytoplasm to neuritic processes, being prominent at varicosities and the growth cone; c) Dp71d relocates almost entirely to the nucleus and is detected to a lower extent in the cytoplasm and neuritic processes. Dp71f co-localizes with beta-dystroglycan and synaptophysin while Dp71d co-localizes with beta-dystroglycan in the nucleus. Dp71d accumulates at cell-cell contacts where Dp71f is absent. These results suggest that Dp71d and Dp71f associate with different subcellular complexes and therefore may have distinct functions in PC12 cells.
Assuntos
Diferenciação Celular/fisiologia , Distrofina/análogos & derivados , Distrofina/metabolismo , Células PC12/metabolismo , Isoformas de Proteínas/metabolismo , Animais , Western Blotting/métodos , Diferenciação Celular/genética , Proteínas do Citoesqueleto/metabolismo , Distroglicanas , Distrofina/genética , Imunofluorescência/métodos , Expressão Gênica , Glicoproteínas de Membrana/metabolismo , Fator de Crescimento Neural/fisiologia , Isoformas de Proteínas/genética , RNA Mensageiro/biossíntese , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Frações Subcelulares/metabolismo , Sinaptofisina/metabolismo , Fatores de TempoRESUMO
BACKGROUND: Myotonic dystrophy (DM) is an autosomal dominant neuromuscular disorder with defects in many tissues, including skeletal muscle myotonia, progressive myopathy, and abnormalities in heart, brain, and endocrine systems. It is associated with a trinucleotide repeat occurring in the 3' (UTR) untranslated region of the myotonic dystrophy protein kinase (DMPK) gene. Several studies have been carried out to determine DMPK gene expression in muscle and non-muscle tissues. METHODS: DMPK gene expression was determined in lymphocytes of adult-onset patients with DM and normal controls. To quantitate total locus expression as well as allele-specific mRNA levels, semiquantitative RT-PCR assay was used. Allele-specific expression was analyzed using a Bpm1 polymorphism located at exon 10 of the DMPK gene. RESULTS: In heterozygous patients with DM, we observed a fourfold difference between mRNA levels produced by the Bpm1-undigested allele (187 bp) compared to the Bpm1-digested allele (136 bp). By using (CTG) trinucleotide (with cytosine, thymine, and guanine) expansion polymorphism, it was shown that the down-regulated allele corresponds to the mutant allele. Interestingly, the reduction in the mutant allele-transcript levels is compensated by an increase of the wild-type allele, yielding no significant differences in total locus mRNA amount between patients and normal individuals. CONCLUSIONS: These results suggest that the expression of the two alleles at the DMPK locus in lymphocytes is coordinated. The reduction in mutant-allele transcript levels is compensated by an increase in wild-type allele mRNA levels.