Center for Neuroscience and Cell Biology, University of Coimbra, Largo Marquês de Pombal, Coimbra, Portugal.
Stem Cells Dev. 2013 Jun 1;22(11):1693-708. doi: 10.1089/scd.2012.0161. Epub 2013 Mar 1.
Neural stem cells of the subventricular zone (SVZ) represent a potentially important source of surrogate cells for the treatment of brain damage. Proper use of these cells for neuronal replacement depends on the ability to drive neuronal differentiation. Several neuromodulators stimulate neurogenesis. Here we examined the effects of the neuropeptide galanin, on neuronal differentiation in murine SVZ cultures. SVZ neurospheres obtained from early postnatal mice were treated with 10 nM to 2 μM galanin. Galanin promoted neuronal differentiation, increasing numbers of NeuN-, vesicular GABA transporter- and tyrosine hydroxylase-expressing neurons. In contrast, galanin neither affected cell proliferation assessed by BrdU incorporation nor cell death evaluated by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). Neuronal differentiation was further confirmed at the functional level by measuring [Ca(2+)]i variations in single SVZ cells after KCl and histamine stimulations to distinguish neurons from immature cells. Galanin treatment increased the numbers of neuronal-like responding cells compared to immature cells. Using selective agonists (M617, AR-M1896) and antagonists (galantide, M871) for galanin receptors 1 and 2, we showed that both galanin receptors mediated neuronal differentiation. Early proneuronal effects of galanin included positive regulation of the transcription factor neurogenin-1 (Ngn1). In addition, galanin promoted axonogenesis and dendritogenesis, increasing both the length of phosphorylated stress-activated protein kinase- and Tau-positive axons and the numbers of microtubule associated protein-2 (MAP-2)-labelled dendrites. Moreover, galanin inhibited SVZ cell migration in the transwell assay. Our results show a proneurogenic effect of galanin and open new perspectives for future applications in stem cell-based therapies for neuronal replacement.
室管膜下区（SVZ）的神经干细胞代表了用于治疗脑损伤的替代细胞的潜在重要来源。这些细胞用于神经元替代的适当用途取决于驱动神经元分化的能力。几种神经调节剂刺激神经发生。在这里，我们研究了神经肽甘丙肽对鼠 SVZ 培养物中神经元分化的影响。从小鼠出生后早期获得的 SVZ 神经球用 10 nM 至 2 μM 甘丙肽处理。甘丙肽促进神经元分化，增加 NeuN、囊泡 GABA 转运体和酪氨酸羟化酶表达神经元的数量。相比之下，甘丙肽既不影响通过 BrdU 掺入评估的细胞增殖，也不影响通过末端脱氧核苷酸转移酶 dUTP 缺口末端标记法（TUNEL）评估的细胞死亡。通过测量 KCl 和组氨酸刺激后单个 SVZ 细胞中的 [Ca(2+)]i 变化来区分神经元和未成熟细胞，在功能水平上进一步证实了神经元分化。与未成熟细胞相比，甘丙肽处理增加了神经元样反应细胞的数量。使用甘丙肽受体 1 和 2 的选择性激动剂（M617、AR-M1896）和拮抗剂（甘丙肽、M871），我们表明两种甘丙肽受体都介导了神经元分化。甘丙肽的早期神经前效应包括转录因子神经生成素-1（Ngn1）的正调节。此外，甘丙肽促进轴突发生和树突发生，增加磷酸化应激激活蛋白激酶和 Tau 阳性轴突的长度以及微管相关蛋白-2（MAP-2）标记的树突的数量。此外，甘丙肽在 Transwell 测定中抑制 SVZ 细胞迁移。我们的结果显示甘丙肽具有神经前体效应，并为基于干细胞的神经元替代治疗的未来应用开辟了新的前景。
