Wnt signaling plays key roles in cellular and physiological processes, including cell proliferation, differentiation and migration during development and tissue homeostasis in adults. This pathway can be defined as Wnt/β-catenin-dependent or β-catenin-independent or “non-canonical”, both signaling are involved in neurite and synapse development/maintenance. Porcupine (PORCN), an acylase that o-acylates Wnt ligands, a major modification in secretion and interaction with its receptors. We use Wnt-C59, a specific PORCN inhibitor, to block the secretion of endogenous Wnts in embryonic hippocampal neurons (DIV 4). Under these conditions, the activity of exogenous Wnt ligands on the complexity of the dendritic tree and axonal polarity were evaluated Cultured primary embryonic hippocampal neurons obtained from Sprague–Dawley rat fetuses (E18), were cultured until day in vitro (DIV) 4 (according to Banker´s protocol) and treated with Wnt-C59 for 24 h, Wnt ligands were added to the cultures on DIV 3 for 24 h. Dendritic arbors and neurites were analysis by fluorescence microscopy. Transfection with Lipofectamine 2000 on DIV 2 of plasmid expressing eGFP and KIF5-Cherry was carried out to evaluate neuronal polarity. Immunostaining was performed with MAP1B and Tau protein. Immunoblot analysis was carried out with Wnt3a, β-catenin and GSK-3β (p-Ser9). Quantitative analysis of dendrite morphology was carried out with ImageJ (NIH) software with Neuron J Plugin. We report, here, that Wnt-C59 treatment changed the morphology of the dendritic arbors and neurites of embryonic hippocampal neurons, with decreases β-catenin and Wnt3a and an apparent increase in GSK-3β (p-Ser9) levels. No effect was observed on axonal polarity. In sister cultures, addition of exogenous Wnt3a, 5a and 7a ligands rescued the changes in neuronal morphology. Wnt3a restored the length of neurites to near that of the control, but Wnt7a increased the neurite length beyond that of the control. Wnt5a also restored the length of neurites relative to Wnt concentrations. Results indicated that Wnt ligands, added exogenously, restored dendritic arbor complexity in embryonic hippocampal neurons, previously treated with a high affinity specific Porcupine inhibitor. We proposed that PORCN is an emerging molecular target of interest in the search for preclinical options to study and treat Wnt-related diseases.

中文翻译：

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豪猪抑制诱导的形态神经突变化被 Wnt 配体拯救

Wnt 信号传导在细胞和生理过程中发挥关键作用，包括成人发育过程中的细胞增殖、分化和迁移以及组织稳态。该途径可以定义为 Wnt/β-连环蛋白依赖性或 β-连环蛋白非依赖性或“非典型”，这两种信号传导都参与神经突和突触的发育/维持。豪猪 (PORCN)，一种酰化 Wnt 配体的酰化酶，是分泌和与其受体相互作用的主要修饰。我们使用 Wnt-C59（一种特定的 PORCN 抑制剂）来阻断胚胎海马神经元 (DIV 4) 中内源性 Wnts 的分泌。在这些条件下，评估了外源 Wnt 配体对树突树复杂性和轴突极性的活性。培养至体外 (DIV) 4 天（根据 Banker 的方案）并用 Wnt-C59 处理 24 小时，将 Wnt 配体添加到 DIV 3 的培养物中 24 小时。通过荧光显微镜分析树突乔木和神经突。在表达 eGFP 和 KIF5-Cherry 的质粒的 DIV 2 上用 Lipofectamine 2000 转染以评估神经元极性。用 MAP1B 和 Tau 蛋白进行免疫染色。用 Wnt3a、β-连环蛋白和 GSK-3β (p-Ser9) 进行免疫印迹分析。使用带有 Neuron J 插件的 ImageJ (NIH) 软件对枝晶形态进行定量分析。我们在此报告，Wnt-C59 治疗改变了胚胎海马神经元的树突乔木和神经突的形态，β-catenin 和 Wnt3a 减少，GSK-3β (p-Ser9) 水平明显增加。没有观察到对轴突极性的影响。在姐妹文化中，添加外源 Wnt3a、5a 和 7a 配体挽救了神经元形态的变化。Wnt3a 将轴突的长度恢复到接近对照的长度，但 Wnt7a 增加的轴突长度超过了对照。Wnt5a 还恢复了相对于 Wnt 浓度的神经突长度。结果表明，外源添加的 Wnt 配体恢复了先前用高亲和力特异性豪猪抑制剂处理的胚胎海马神经元中的树突乔木复杂性。我们提出 PORCN 是一个新兴的分子靶标，用于寻找研究和治疗 Wnt 相关疾病的临床前选择。Wnt3a 将轴突的长度恢复到接近对照的长度，但 Wnt7a 增加的轴突长度超过了对照。Wnt5a 还恢复了相对于 Wnt 浓度的神经突长度。结果表明，外源添加的 Wnt 配体恢复了先前用高亲和力特异性豪猪抑制剂处理的胚胎海马神经元中的树突乔木复杂性。我们提出 PORCN 是一个新兴的分子靶标，用于寻找研究和治疗 Wnt 相关疾病的临床前选择。Wnt3a 将轴突的长度恢复到接近对照的长度，但 Wnt7a 增加的轴突长度超过了对照。Wnt5a 还恢复了相对于 Wnt 浓度的神经突长度。结果表明，外源添加的 Wnt 配体恢复了先前用高亲和力特异性豪猪抑制剂处理的胚胎海马神经元中的树突乔木复杂性。我们提出 PORCN 是一个新兴的分子靶标，用于寻找研究和治疗 Wnt 相关疾病的临床前选择。恢复了胚胎海马神经元的树突乔木复杂性，之前用高亲和力特异性豪猪抑制剂治疗。我们提出 PORCN 是一个新兴的分子靶标，用于寻找研究和治疗 Wnt 相关疾病的临床前选择。恢复了胚胎海马神经元的树突乔木复杂性，之前用高亲和力特异性豪猪抑制剂治疗。我们提出 PORCN 是一个新兴的分子靶标，用于寻找研究和治疗 Wnt 相关疾病的临床前选择。