Handara Gerry, Hetsch Florian J A, Jüttner René, Schick Anna, Haupt Corinna, Rathjen Fritz G, Kröger Stephan
Department of Physiological Genomics, Biomedical Center, Ludwig-Maximilians-University, Großhaderner Str. 9, D-82152 Planegg-Martinsried, Germany; Institute for Stem Cell Research, German Research Center for Environmental Health, Helmholtz Centre Munich, Ingolstädter Landstraße 1, D-85764 Neuherberg, Germany.
Max-Delbrück-Center for Molecular Medicine (MDC), Robert-Rössle-Str. 10, D-13092 Berlin, Germany.
Dev Biol. 2019 Jan 1;445(1):54-67. doi: 10.1016/j.ydbio.2018.10.017. Epub 2018 Oct 29.
The role of agrin, Lrp4 and MuSK, key organizers of neuromuscular synaptogenesis, in the developing CNS is only poorly understood. We investigated the role of these proteins in cultured mouse embryonic cortical neurons from wildtype and from Lrp4- and MuSK-deficient mice. Neurons from Lrp4-deficient mice had fewer but longer primary dendrites and a decreased density of puncta containing excitatory and inhibitory synapse-associated proteins. Neurons from MuSK-deficient mice had an altered dendritic branching pattern but no change in the density of puncta stained by antibodies against synapse-associated proteins. Transfection of TM-agrin compensated the dendritic branching deficits in Lrp4-deficient but not in MuSK-deficient neurons. TM-agrin transfection increased the density of excitatory synaptic puncta in MuSK-deficient but not in Lrp4-deficient mice and reduced the number of inhibitory synaptic puncta irrespective of MuSK and Lrp4 expression. Addition of purified soluble agrin to microisland cultures of cortical neurons revealed an Lrp4-dependent increase in the size and density of glutamatergic synaptic puncta and in mEPSC but not in mIPSC frequency and amplitude. Thus, agrin induced an Lrp4-independent increase in dendritic branch complexity, an Lrp4-dependent increase of excitatory synaptic puncta and an Lrp4- and MuSK-independent decrease in the density of puncta containing inhibitory synapse-associated proteins. These results establish selective roles for agrin, Lrp4 and MuSK during dendritogenesis and synaptogenesis in cultured CNS neurons.
聚集蛋白、低密度脂蛋白受体相关蛋白4（Lrp4）和肌肉特异性激酶（MuSK）作为神经肌肉突触形成的关键组织者，它们在中枢神经系统发育中的作用目前仍知之甚少。我们研究了这些蛋白在来自野生型、Lrp4基因缺陷型和MuSK基因缺陷型小鼠的培养胚胎皮质神经元中的作用。Lrp4基因缺陷型小鼠的神经元初级树突数量减少但长度增加，且含有兴奋性和抑制性突触相关蛋白的突触小体密度降低。MuSK基因缺陷型小鼠的神经元树突分支模式改变，但针对突触相关蛋白的抗体染色的突触小体密度没有变化。转染截短型聚集蛋白（TM - agrin）可补偿Lrp4基因缺陷型神经元的树突分支缺陷，但不能补偿MuSK基因缺陷型神经元的缺陷。TM - agrin转染增加了MuSK基因缺陷型小鼠而非Lrp4基因缺陷型小鼠中兴奋性突触小体的密度，并且无论MuSK和Lrp4的表达情况如何，都减少了抑制性突触小体的数量。向皮质神经元微岛培养物中添加纯化的可溶性聚集蛋白，结果显示谷氨酸能突触小体的大小和密度以及微小兴奋性突触后电流（mEPSC）频率呈Lrp4依赖性增加，但微小抑制性突触后电流（mIPSC）的频率和幅度未增加。因此，聚集蛋白可诱导树突分支复杂性的增加，且这种增加不依赖于Lrp4；可诱导兴奋性突触小体数量的增加，且这种增加依赖于Lrp4；还可诱导含有抑制性突触相关蛋白的突触小体密度的降低，且这种降低不依赖于Lrp4和MuSK。这些结果确定了聚集蛋白、Lrp4和MuSK在培养的中枢神经系统神经元树突形成和突触形成过程中的选择性作用。
