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Up-regulated spinal microRNAs induce aggregation of superoxide dismutase 1 protein in canine degenerative myelopathy
Research in Veterinary Science ( IF 2.2 ) Pub Date : 2020-11-25 , DOI: 10.1016/j.rvsc.2020.11.018
Kohei Nakata , Mariko Namiki , Yui Kobatake , Hidetaka Nishida , Hiroki Sakai , Osamu Yamato , Makoto Urushitani , Sadatoshi Maeda , Hiroaki Kamishina

Canine degenerative myelopathy (DM) is a fatal progressive neurodegenerative disease. Mutations in the superoxide dismutase 1 (SOD1) gene have been shown to be the major risk factor for DM, and it is hypothesized that neural degeneration is caused by a “gain of toxic function” of mutant SOD1. In this study, the spinal cord microRNA (miRNA) profiles of DM-affected dogs were investigated to elucidate the pathomechanisms of DM. Quantification of 277 miRNAs identified three up-regulated miRNAs and 18 down-regulated miRNAs in the spinal cords of DM-affected dogs. Based on gene ontology analysis, the target cluster of up-regulated miRNAs was associated with protein expression or modification and cellular response, and that of down-regulated miRNAs was associated with tissue development. In these clusters, we focused on the mechanism of protein ubiquitination. Polyubiquitination assay demonstrated that canine SOD1 proteins were polyubiquitinated and degraded by proteasomes. Immunohistochemistry of the spinal cords of DM-affected dogs showed that mutant SOD1 aggregations were not ubiquitin immunopositive. Using cultured cells, co-transfection of canine SOD1 and up-regulated miRNA in DM-affected dogs demonstrated that miR-23a, miR-142 and miR-221 significantly increased the proportion of cells with mutant SOD1 aggregation. These results suggested that up-regulated miRNAs in the spinal cords of DM-affected dogs may inhibit ubiquitination of misfolded SOD1 protein and induce mutant SOD1 aggregations, leading to further progression of degenerative processes in the DM pathology.



中文翻译:

脊髓microRNA上调诱导犬变性脊髓病中超氧化物歧化酶1蛋白的聚集。

犬变性脊髓病(DM)是一种致命的进行性神经退行性疾病。超氧化物歧化酶1(SOD1)基因中的突变已被证明是DM的主要危险因素,据推测神经变性是由突变体SOD1的“毒性功能获得”引起的。在这项研究中,研究了受DM影响的狗的脊髓microRNA(miRNA)图谱,以阐明DM的发病机制。对277个miRNA的定量分析发现,在受DM影响的狗的脊髓中,有3个上调的miRNA和18个下调的miRNA。基于基因本体分析,上调的miRNA的目标簇与蛋白质表达或修饰和细胞反应相关,下调的miRNA的目标簇与组织发育相关。在这些集群中 我们专注于蛋白质泛素化的机制。多聚泛素化试验表明,犬SOD1蛋白被多聚泛素化并被蛋白酶体降解。患有DM的狗的脊髓的免疫组织化学表明,突变的SOD1聚集体不是泛素免疫阳性的。使用培养的细胞,在受DM感染的犬中共转染犬SOD1和上调的miRNA,证明miR-23a,miR-142和miR-221显着增加了具有突变SOD1聚集的细胞比例。这些结果表明,受DM影响的狗的脊髓中的miRNA上调可能抑制错误折叠的SOD1蛋白的泛素化并诱导突变的SOD1聚集,从而导致DM病理过程中变性过程的进一步发展。多聚泛素化试验表明,犬SOD1蛋白被多聚泛素化并被蛋白酶体降解。患有DM的狗的脊髓的免疫组织化学表明,突变的SOD1聚集体不是泛素免疫阳性的。使用培养的细胞,在受DM感染的犬中共转染犬SOD1和上调的miRNA,证明miR-23a,miR-142和miR-221显着增加了具有突变SOD1聚集的细胞比例。这些结果表明,受DM影响的狗的脊髓中的miRNA上调可能抑制错误折叠的SOD1蛋白的泛素化并诱导突变的SOD1聚集,从而导致DM病理过程中变性过程的进一步发展。多聚泛素化试验表明,犬SOD1蛋白被多聚泛素化并被蛋白酶体降解。患有DM的狗的脊髓的免疫组织化学表明,突变的SOD1聚集体不是泛素免疫阳性的。使用培养的细胞,在受DM感染的犬中共转染犬SOD1和上调的miRNA,证明miR-23a,miR-142和miR-221显着增加了具有突变SOD1聚集的细胞比例。这些结果表明,受DM影响的狗的脊髓中的miRNA上调可能抑制错误折叠的SOD1蛋白的泛素化并诱导突变的SOD1聚集,从而导致DM病理过程中变性过程的进一步发展。患有DM的狗的脊髓的免疫组织化学表明,突变的SOD1聚集体不是泛素免疫阳性的。使用培养的细胞,在受DM感染的犬中共转染犬SOD1和上调的miRNA,证明miR-23a,miR-142和miR-221显着增加了具有突变SOD1聚集的细胞比例。这些结果表明,受DM影响的狗的脊髓中的miRNA上调可能抑制错误折叠的SOD1蛋白的泛素化并诱导突变的SOD1聚集,从而导致DM病理过程中变性过程的进一步发展。患有DM的狗的脊髓的免疫组织化学表明,突变的SOD1聚集体不是泛素免疫阳性的。使用培养的细胞,在受DM感染的犬中共转染犬SOD1和上调的miRNA,证明miR-23a,miR-142和miR-221显着增加了具有突变SOD1聚集的细胞比例。这些结果表明,受DM影响的狗的脊髓中的miRNA上调可能抑制错误折叠的SOD1蛋白的泛素化并诱导突变的SOD1聚集,从而导致DM病理过程中变性过程的进一步发展。miR-142和miR-221显着增加了具有突变SOD1聚集的细胞比例。这些结果表明,受DM影响的狗的脊髓中的miRNA上调可能抑制错误折叠的SOD1蛋白的泛素化并诱导突变的SOD1聚集,从而导致DM病理过程中变性过程的进一步发展。miR-142和miR-221显着增加了具有突变SOD1聚集的细胞比例。这些结果表明,受DM影响的狗的脊髓中的miRNA上调可能抑制错误折叠的SOD1蛋白的泛素化并诱导突变的SOD1聚集,从而导致DM病理过程中变性过程的进一步发展。

更新日期:2020-11-25
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