Brancia Carla, Noli Barbara, Boido Marina, Boi Andrea, Puddu Roberta, Borghero Giuseppe, Marrosu Francesco, Bongioanni Paolo, Orrù Sandro, Manconi Barbara, D'Amato Filomena, Messana Irene, Vincenzoni Federica, Vercelli Alessandro, Ferri Gian-Luca, Cocco Cristina
NEF-Laboratory, Department of Biomedical Sciences, University of Cagliari, Cagliari, Italy.
Neuroscience Institute Cavalieri Ottolenghi, Department of Neuroscience, University of Turin, Turin, Italy.
PLoS One. 2016 Oct 13;11(10):e0164689. doi: 10.1371/journal.pone.0164689. eCollection 2016.
VGF mRNA is widely expressed in areas of the nervous system known to degenerate in Amyotrophic Lateral Sclerosis (ALS), including cerebral cortex, brainstem and spinal cord. Despite certain VGF alterations are reported in animal models, little information is available with respect to the ALS patients. We addressed VGF peptide changes in fibroblast cell cultures and in plasma obtained from ALS patients, in parallel with spinal cord and plasma samples from the G93A-SOD1 mouse model. Antisera specific for the C-terminal end of the human and mouse VGF proteins, respectively, were used in immunohistochemistry and enzyme-linked immunosorbent assay (ELISA), while gel chromatography and HPLC/ESI-MS/MS were used to identify the VGF peptides present. Immunoreactive VGF C-terminus peptides were reduced in both fibroblast and plasma samples from ALS patients in an advanced stage of the disease. In the G93A-SOD1 mice, the same VGF peptides were also decreased in plasma in the late-symptomatic stage, while showing an earlier down-regulation in the spinal cord. In immunohistochemistry, a large number of gray matter structures were VGF C-terminus immunoreactive in control mice (including nerve terminals, axons and a few perikarya identified as motoneurons), with a striking reduction already in the pre-symptomatic stage. Through gel chromatography and spectrometry analysis, we identified one form likely to be the VGF precursor as well as peptides containing the NAPP- sequence in all tissues studied, while in the mice and fibroblasts, we revealed also AQEE- and TLQP- peptides. Taken together, selective VGF fragment depletion may participate in disease onset and/or progression of ALS.
VGF mRNA在已知会在肌萎缩侧索硬化症（ALS）中退化的神经系统区域广泛表达，包括大脑皮层、脑干和脊髓。尽管在动物模型中报道了某些VGF的改变，但关于ALS患者的信息却很少。我们研究了ALS患者成纤维细胞培养物和血浆中VGF肽的变化，并与G93A-SOD1小鼠模型的脊髓和血浆样本进行了对比。分别针对人和小鼠VGF蛋白C末端的抗血清用于免疫组织化学和酶联免疫吸附测定（ELISA），同时使用凝胶色谱法和HPLC/ESI-MS/MS来鉴定存在的VGF肽。在疾病晚期的ALS患者的成纤维细胞和血浆样本中，免疫反应性VGF C末端肽减少。在G93A-SOD1小鼠中，相同的VGF肽在症状后期的血浆中也减少，而在脊髓中则显示出更早的下调。在免疫组织化学中，对照小鼠的大量灰质结构对VGF C末端具有免疫反应性（包括神经末梢、轴突和一些被鉴定为运动神经元的核周体），在症状前期就已经显著减少。通过凝胶色谱法和光谱分析，我们在所有研究的组织中鉴定出一种可能是VGF前体的形式以及含有NAPP-序列的肽，而在小鼠和成纤维细胞中，我们还发现了AQEE-和TLQP-肽。综上所述，选择性VGF片段的消耗可能参与了ALS的疾病发生和/或进展。
