Andersson A M, Olsen M, Zhernosekov D, Gaardsvoll H, Krog L, Linnemann D, Bock E
Research Centre for Medical Biotechnology, University of Copenhagen, Panum Institute, Denmark.
Biochem J. 1993 Mar 15;290 ( Pt 3)(Pt 3):641-8. doi: 10.1042/bj2900641.
Neural cell adhesion molecule (NCAM) is expressed by muscle and involved in muscle-neuron and muscle-muscle cell interactions. The expression in muscle is regulated during myogenesis and by the state of innervation. In aged muscle, both neurogenic and myogenic degenerative processes occur. We here report quantitative and qualitative changes in NCAM protein and mRNA forms during aging in normal rat skeletal muscle. Determination of the amount of NCAM by e.l.i.s.a. showed that the level decreased from perinatal to adult age, followed by a considerable increase in 24-month-old rat muscle. Thus NCAM concentration in aged muscle was sixfold higher than in young adult muscle. In contrast with previous reports, NCAM polypeptides of 200, 145, 125 and 120 kDa were observed by immunoblotting throughout postnatal development and aging, the relative proportions of the individual NCAM polypeptides remaining virtually unchanged at all ages examined. However, changes in the extent of sialylation of NCAM were demonstrated. Even though the relative amounts of the various NCAM polypeptides were unchanged during aging, distinct changes in NCAM mRNA classes were observed. Three NCAM mRNA classes of 6.7, 5.2 and 2.9 kb were present in perinatal and young adult skeletal muscle, whereas only the 5.2 and 2.9 kb mRNA classes could be demonstrated in aged muscle. This indicates that metabolism of the various NCAM polypeptides is individually regulated during aging. Alternative splicing of NCAM mRNA in skeletal muscle was studied by Northern blotting using DNA oligonucleotide probes specifically hybridizing to selected exons or exon combinations. Exon VASE, which has previously been shown to be present in both brain and heart NCAM mRNA, was virtually absent from skeletal muscle at all ages studied. In contrast, the majority of NCAM mRNA in postnatal skeletal muscle was shown to contain extra exons inserted between exons 12 and 13. Of the various possible exon combinations at this splice site, the combinations 12-a-AAG-13 and 12-a-b seemed to be prevalent in postnatal skeletal muscle. No significant change in the relative proportion of these two exon combinations occurred during aging. The observed upregulation of NCAM protein in aged muscle supports the assumption that an increasing proportion of muscle fibres are denervated in aged muscle. Selective upregulation of the 5.2 and 2.9 kb mRNA forms have previously been demonstrated in muscle cell lines and in primary cultures of muscle cells during formation of myotubes in vitro, and this switch in NCAM mRNA classes has been suggested to correlate with myogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)
神经细胞黏附分子（NCAM）由肌肉表达，并参与肌肉与神经元以及肌肉与肌肉细胞之间的相互作用。其在肌肉中的表达在肌生成过程以及受神经支配状态下受到调控。在衰老肌肉中，神经源性和肌源性退化过程都会发生。我们在此报告正常大鼠骨骼肌衰老过程中NCAM蛋白和mRNA形式的定量及定性变化。通过酶联免疫吸附测定法（ELISA）测定NCAM的量，结果显示其水平从围产期到成年期下降，随后在24月龄大鼠肌肉中显著增加。因此，衰老肌肉中NCAM的浓度比年轻成年肌肉高六倍。与先前报道不同，通过免疫印迹法在整个出生后发育和衰老过程中均观察到200、145、125和120 kDa的NCAM多肽，在所检测的所有年龄段，各个NCAM多肽的相对比例几乎保持不变。然而，已证实NCAM的唾液酸化程度发生了变化。尽管在衰老过程中各种NCAM多肽的相对量未变，但观察到NCAM mRNA类别有明显变化。围产期和年轻成年骨骼肌中存在6.7、5.2和2.9 kb的三种NCAM mRNA类别，而在衰老肌肉中仅能检测到5.2和2.9 kb的mRNA类别。这表明在衰老过程中各种NCAM多肽的代谢是分别受到调控的。利用与选定外显子或外显子组合特异性杂交的DNA寡核苷酸探针，通过Northern印迹法研究了骨骼肌中NCAM mRNA的可变剪接。先前已证明存在于脑和心脏NCAM mRNA中的外显子VASE，在所研究的所有年龄段的骨骼肌中几乎都不存在。相反，出生后骨骼肌中的大多数NCAM mRNA显示在第12和13外显子之间插入了额外的外显子。在这个剪接位点的各种可能的外显子组合中，组合12 - a - AAG - 13和12 - a - b在出生后骨骼肌中似乎占主导地位。在衰老过程中，这两种外显子组合的相对比例没有显著变化。在衰老肌肉中观察到的NCAM蛋白上调支持了这样一种假设，即衰老肌肉中去神经支配的肌纤维比例增加。先前已证明在体外肌管形成过程中，肌肉细胞系和原代培养的肌肉细胞中5.2和2.9 kb mRNA形式有选择性上调，并且有人提出这种NCAM mRNA类别的转变与肌生成相关。（摘要截短至250字）
