Ferreira A, Niclas J, Vale R D, Banker G, Kosik K S
Department of Neuroscience, University of Virginia School of Medicine, Charlottesville 22908.
J Cell Biol. 1992 May;117(3):595-606. doi: 10.1083/jcb.117.3.595.
Kinesin, a microtubule-based force-generating molecule, is thought to translocate organelles along microtubules. To examine the function of kinesin in neurons, we sought to suppress kinesin heavy chain (KHC) expression in cultured hippocampal neurons using antisense oligonucleotides and study the phenotype of these KHC "null" cells. Two different antisense oligonucleotides complementary to the KHC sequence reduced the protein levels of the heavy chain by greater than 95% within 24 h after application and produced identical phenotypes. After inhibition of KHC expression for 24 or 48 h, neurons extended an array of neurites often with one neurite longer than the others; however, the length of all these neurites was significantly reduced. Inhibition of KHC expression also altered the distribution of GAP-43 and synapsin I, two proteins thought to be transported in association with membranous organelles. These proteins, which are normally localized at the tips of growing neurites, were confined to the cell body in antisense-treated cells. Treatment of the cells with the corresponding sense oligonucleotides affected neither the distribution of GAP-43 and synapsin I, nor the length of neurites. A full recovery of neurite length occurred after removal of the antisense oligonucleotides from the medium. These data indicate that KHC plays a role in the anterograde translocation of vesicles containing GAP-43 and synapsin I. A deficiency in vesicle delivery may also explain the inhibition of neurite outgrowth. Despite the inhibition of KHC and the failure of GAP-43 and synapsin I to move out of the cell body, hippocampal neurons can extend processes and acquire as asymmetric morphology.
驱动蛋白是一种基于微管产生力的分子，被认为可沿微管转运细胞器。为了研究驱动蛋白在神经元中的功能，我们试图使用反义寡核苷酸抑制培养的海马神经元中驱动蛋白重链（KHC）的表达，并研究这些KHC“缺失”细胞的表型。两种与KHC序列互补的不同反义寡核苷酸在应用后24小时内可将重链蛋白水平降低95%以上，并产生相同的表型。在抑制KHC表达24或48小时后，神经元长出一系列神经突，其中一条神经突通常比其他神经突长；然而，所有这些神经突的长度都显著缩短。抑制KHC表达还改变了GAP-43和突触素I的分布，这两种蛋白被认为与膜性细胞器相关运输。这些蛋白通常位于生长中的神经突尖端，在反义处理的细胞中则局限于细胞体。用相应的正义寡核苷酸处理细胞既不影响GAP-43和突触素I的分布，也不影响神经突的长度。从培养基中去除反义寡核苷酸后，神经突长度完全恢复。这些数据表明，KHC在含有GAP-43和突触素I的囊泡的顺行转运中起作用。囊泡运输缺陷也可能解释神经突生长的抑制。尽管KHC受到抑制，GAP-43和突触素I无法移出细胞体，但海马神经元仍可延伸突起并获得不对称形态。
