Sharma Kamal, Choi Se-Young, Zhang Yong, Nieland Thomas J F, Long Shunyou, Li Min, Huganir Richard L
Department of Neuroscience, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA.
Department of Physiology, Seoul National University School of Dentistry, Seoul 110-749, South Korea.
Cell Rep. 2013 Dec 12;5(5):1330-41. doi: 10.1016/j.celrep.2013.11.008. Epub 2013 Dec 5.
Genetic screens in invertebrates have discovered many synaptogenic genes and pathways. However, similar genetic studies have not been possible in mammals. We have optimized an automated high-throughput platform that employs automated liquid handling and imaging of primary mammalian neurons. Using this platform, we have screened 3,200 shRNAs targeting 800 proteins. One of the hits identified was LRP6, a coreceptor for canonical Wnt ligands. LRP6 regulates excitatory synaptogenesis and is selectively localized to excitatory synapses. In vivo knockdown of LRP6 leads to a reduction in the number of functional synapses. Moreover, we show that the canonical Wnt ligand, Wnt8A, promotes synaptogenesis via LRP6. These results provide a proof of principle for using a high-content approach to screen for synaptogenic factors in the mammalian nervous system and identify and characterize a Wnt ligand receptor complex that is critical for the development of functional synapses in vivo.
在无脊椎动物中进行的遗传筛选发现了许多突触生成基因和信号通路。然而，在哺乳动物中进行类似的遗传研究是不可能的。我们优化了一个自动化高通量平台，该平台采用了自动化液体处理和对原代哺乳动物神经元进行成像的技术。利用这个平台，我们筛选了针对800种蛋白质的3200个短发夹RNA（shRNA）。其中一个被鉴定出的命中靶点是低密度脂蛋白受体相关蛋白6（LRP6），它是经典Wnt配体的共受体。LRP6调节兴奋性突触的形成，并选择性地定位于兴奋性突触。在体内敲低LRP6会导致功能性突触数量减少。此外，我们表明经典Wnt配体Wnt8A通过LRP6促进突触形成。这些结果为使用高内涵方法筛选哺乳动物神经系统中的突触生成因子提供了原理证明，并鉴定和表征了一种对体内功能性突触发育至关重要的Wnt配体受体复合物。
