Probing extracellular Sonic hedgehog in neurons.
Probing extracellular Sonic hedgehog in neurons.
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DOI：
10.1242/bio.019422
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发表时间：
2016-08-15
期刊：
影响因子：
2.4
通讯作者：
Yao PJ
中科院分区：
文献类型：
--
作者：
Eitan E;Petralia RS;Wang YX;Indig FE;Mattson MP;Yao PJ
Sonic hedgehog（Shh）的生物活性取决于特定的脂质修饰;在其N-末端的棕榈酸酯和在其C-末端的胆固醇。这种双脂质修饰使Shh分子具有亲脂性，从而阻止它们在细胞外空间中自由扩散。多种证据表明Shh蛋白由各种形式的细胞外囊泡（EV）携带。例如，在某些组织中，Shh蛋白通过丝状伪足直接转运到邻近细胞。我们以前报道过Shh蛋白在海马神经元中表达。在这项研究中，我们发现，在海马和小脑的出生后（P）2天的大鼠，Shh主要是发现附近或膜表面的小神经突或丝状伪足。我们还检查了培养的海马神经元，我们观察到明显的和广泛的Shh-免疫标记的囊泡位于神经元外。通过免疫电镜和生化分析，我们发现含有Shh的EV具有广泛的大小范围。与从过表达N-末端Shh片段构建体的细胞中分离的EV中的稳健Shh活性不同，我们在从培养的海马神经元的培养基中纯化的EV中没有检测到可测量的Shh活性。这些结果表明神经元中跨细胞Shh信号传导机制的复杂性。在发育中的大脑中，Sonic hedgehog分子定位于神经元丝状伪足中，并优先定位于细胞与细胞的接触。在培养的神经元中，Sonic hedgehog也存在于细胞外囊泡中。
The bioactivity of Sonic hedgehog (Shh) depends on specific lipid modifications; a palmitate at its N-terminus and a cholesterol at its C-terminus. This dual-lipid modification makes Shh molecules lipophilic, which prevents them from diffusing freely in extracellular space. Multiple lines of evidence indicate that Shh proteins are carried by various forms of extracellular vesicles (EVs). It also has been shown, for instance, that in some tissues Shh proteins are transported to neighboring cells directly via filopodia. We have previously reported that Shh proteins are expressed in hippocampal neurons. In this study we show that, in the hippocampus and cerebellum of postnatal day (P)2 rats, Shh is mostly found near or on the membrane surface of small neurites or filopodia. We also examined cultured hippocampal neurons where we observed noticeable and widespread Shh-immunolabeled vesicles located outside neurons. Through immunoelectron microscopy and biochemical analysis, we find Shh-containing EVs with a wide range of sizes. Unlike robust Shh activity in EVs isolated from cells overexpressing an N-terminal Shh fragment construct, we did not detect measurable Shh activity in EVs purified from the medium of cultured hippocampal neurons. These results suggest the complexity of the transcellular Shh signaling mechanisms in neurons. Summary: In the developing brain, Sonic hedgehog molecules localize in neuronal filopodia and preferentially position at cell-to-cell contacts. In cultured neurons, Sonic hedgehog is also found in extracellular vesicles.
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