Generation of dystrophin short product-specific tag-insertion mouse: distinct Dp71 glycoprotein complexes at inhibitory postsynapse and glia limitans
Generation of dystrophin short product-specific tag-insertion mouse: distinct Dp71 glycoprotein complexes at inhibitory postsynapse and glia limitans
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DOI：
10.1007/s00018-022-04151-2
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发表时间：
2022-02-01
影响因子：
8
通讯作者：
Itoh, Kyoko
中科院分区：
文献类型：
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作者：
Fujimoto, Takahiro;Yaoi, Takeshi;Itoh, Kyoko
Duchenne肌营养不良症是一种以进行性肌肉变性和不同程度的智能障碍为特征的致死性X连锁隐性神经肌肉疾病，是最严重的肌营养不良症。由于多个dystrophin产物的存在，负责基因dystrophin在大脑中的生理和病理作用仍然难以捉摸，主要是全长dystrophin Dp427和短产物Dp71。在这项研究中，我们产生了Dp71特异的血凝素(HA)肽标签插入小鼠，以使抗HA标签抗体能够特异性地检测Dp71的内在表达。免疫组织化学检测表明，Dp71不仅在血脑屏障(星形细胞终足环绕微血管的地方)表达，而且在海马齿状颗粒神经元的突触后抑制部位也有表达。有趣的是，虽然抗dystrophin抗体检测到的Dp427明显存在于抑制性突触后，但海马角氨酸区(CA)1锥体神经元的Dp71呈阴性，这表明Dstrophin的表达与细胞类型有关。使用原代海马区培养的精确检查证实了Dp71在神经元中仅定位于抑制性突触后隔室，而不是兴奋性突触。我们进一步进行相互作用组分析，发现Dp71形成了不同的分子复合体，即突触相关的Dp71与Dstroglan(DG)和dystrobrevin beta(Dtnb)相互作用，而胶质细胞相关的Dp71与DG和Dystrobrevin Alpha(DTNA)相互作用。因此，我们的数据表明Dp71及其结合伙伴与海马颗粒神经元的突触后抑制功能有关，新型的Dp71转基因小鼠为了解Dp71及其相互作用蛋白在体内和体外的精确生理表达和功能提供了有价值的工具。
Duchenne muscular dystrophy (DMD), the most severe form of dystrophinopathies, is a fatal X-linked recessive neuromuscular disorder characterized by progressive muscle degeneration and various extents of intellectual disabilities. Physiological and pathological roles of the responsible gene, dystrophin, in the brain remain elusive due to the presence of multiple dystrophin products, mainly full-length dystrophin, Dp427, and the short product, Dp71. In this study, we generated a Dp71-specific hemagglutinin (HA) peptide tag-insertion mice to enable specific detection of intrinsic Dp71 expression by anti-HA-tag antibodies. Immunohistochemical detections in the transgenic mice demonstrated Dp71 expression not only at the blood-brain barrier, where astrocytic endfeet surround the microvessels, but also at the inhibitory postsynapse of hippocampal dentate granule neurons. Interestingly, hippocampal cornu ammonis (CA)1 pyramidal neurons were negative for Dp71, although Dp427 detected by anti-dystrophin antibody was clearly present at the inhibitory postsynapse, suggesting cell-type dependent dystrophin expressions. Precise examination using the primary hippocampal culture validated exclusive localization of Dp71 at the inhibitory postsynaptic compartment but not at the excitatory synapse in neurons. We further performed interactome analysis and found that Dp71 formed distinct molecular complexes, i.e. synapse-associated Dp71 interacted with dystroglycan (Dg) and dystrobrevin beta (Dtnb), whereas glia-associated Dp71 did with Dg and dystrobrevin alpha (Dtna). Thus, our data indicate that Dp71 and its binding partners are relevant to the inhibitory postsynaptic function of hippocampal granule neurons and the novel Dp71-transgenic mouse provides a valuable tool to understand precise physiological expressions and functions of Dp71 and its interaction proteins in vivo and in vitro.