Busceti Carla Letizia, Di Menna Luisa, Bianchi Franca, Mastroiacovo Federica, Di Pietro Paola, Traficante Anna, Bozza Giovanna, Niehrs Christof, Battaglia Giuseppe, Bruno Valeria, Fornai Francesco, Volpe Massimo, Rubattu Speranza, Nicoletti Ferdinando
IRCCS Neuromed, Pozzilli, Italy.
Division of Molecular Embryology, DKFZ-ZMBH Allianz, German Cancer Research Center, Heidelberg, Germany.
Front Cell Neurosci. 2018 Sep 11;12:292. doi: 10.3389/fncel.2018.00292. eCollection 2018.
Dickkopf-3 (Dkk3) is an atypical member of the Dkk family of Wnt inhibitors, which has been implicated in the pathophysiology of neurodegenerative disorders. However, the role of Dkk3 in mechanisms of cell degeneration and protection is unknown. We used Dkk3 knockout mice to examine how endogenous Dkk3 influences ischemic brain damage. In addition, we used primary cultures of astrocytes or mixed cultures of astrocytes and neurons to investigate the action of Dkk3 on cell damage and dissect the underlying molecular mechanisms. In a model of focal brain ischemia induced by permanent middle cerebral artery (MCA) occlusion (MCAO) Dkk3 mice showed a significantly greater infarct size with respect to their wild-type counterparts at all time points investigated (1, 3 and 7 days after MCAO). Immunohistochemical analysis showed that Dkk3 expression was enhanced at the borders of the ischemic focus, and was predominantly detected in astrocytes. This raised the possibility that Dkk3 produced by astrocytes acted as a protective molecule. We tested this hypothesis using either primary cultures of cortical astrocytes or mixed cortical cultures containing both neurons and astrocytes. Genetic deletion of Dkk3 was permissive to astrocyte damage induced by either oxidative stress or glucose deprivation. In addition, application of human recombinant Dkk3 (hrDkk3) was highly protective against oxidative stress in cultured astrocytes. We tested the hypothesis that the protective activity of Dkk3 was mediated byvascular endothelial growth factor (VEGF). Interestingly, glucose deprivation up-regulated both Dkk3 and VEGF in cultured astrocytes prepared from wild-type mice. VEGF induction was not observed in astrocytes lacking Dkk3 (i.e., in cultures prepared from Dkk3 mice). In mixed cultures of cortical cells, excitotoxic neuronal death induced by a brief pulse with -methyl-D-aspartate (NMDA) was significantly enhanced when Dkk3 was lacking in astrocytes, whereas post-NMDA addition of hrDkk3 was neuroprotective. Neuroprotection by hrDkk3 was significantly reduced by pharmacological blockade of type-2 VEGF receptors and was mimicked by hrVEGF. These data offer the first evidence that Dkk3 protects both neurons and astrocytes against a variety of toxic insults, and at least in culture, protection involves VEGF induction.
Dickkopf-3（Dkk3）是Wnt抑制剂Dkk家族的一个非典型成员，它与神经退行性疾病的病理生理学有关。然而，Dkk3在细胞变性和保护机制中的作用尚不清楚。我们使用Dkk3基因敲除小鼠来研究内源性Dkk3如何影响缺血性脑损伤。此外，我们使用星形胶质细胞原代培养物或星形胶质细胞与神经元的混合培养物来研究Dkk3对细胞损伤的作用，并剖析其潜在的分子机制。在永久性大脑中动脉（MCA）闭塞（MCAO）诱导的局灶性脑缺血模型中，在所有研究时间点（MCAO后1、3和7天），Dkk3基因敲除小鼠相对于野生型对照显示出明显更大的梗死面积。免疫组织化学分析表明，Dkk3表达在缺血灶边缘增强，且主要在星形胶质细胞中检测到。这增加了星形胶质细胞产生的Dkk3作为一种保护分子的可能性。我们使用皮质星形胶质细胞原代培养物或包含神经元和星形胶质细胞的皮质混合培养物来验证这一假设。Dkk3的基因缺失会导致氧化应激或葡萄糖剥夺诱导的星形胶质细胞损伤。此外，应用人重组Dkk3（hrDkk3）对培养的星形胶质细胞中的氧化应激具有高度保护作用。我们验证了Dkk3的保护活性是由血管内皮生长因子（VEGF）介导的这一假设。有趣的是，葡萄糖剥夺上调了野生型小鼠制备的培养星形胶质细胞中的Dkk3和VEGF。在缺乏Dkk3的星形胶质细胞（即从Dkk3基因敲除小鼠制备的培养物）中未观察到VEGF的诱导。在皮质细胞混合培养物中，当星形胶质细胞中缺乏Dkk3时，由短暂脉冲给予N-甲基-D-天冬氨酸（NMDA）诱导的兴奋性毒性神经元死亡显著增强，而在NMDA给药后添加hrDkk3具有神经保护作用。hrDkk3的神经保护作用通过2型VEGF受体的药理学阻断显著降低，并被hrVEGF模拟。这些数据首次证明Dkk3保护神经元和星形胶质细胞免受多种毒性损伤，并且至少在培养中，保护作用涉及VEGF的诱导。
