Departments of Neurobiology and Pathology, University of Pittsburgh, PA, USA.
Acta Neuropathol. 2012 Nov;124(5):717-32. doi: 10.1007/s00401-012-1045-x. Epub 2012 Sep 21.
RNA-binding protein pathology now represents one of the best characterized pathologic features of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration patients with TDP-43 or FUS pathology (FTLD-TDP and FTLD-FUS). Using liquid chromatography tandem mass spectrometry, we identified altered levels of the RNA-binding motif 45 (RBM45) protein in the cerebrospinal fluid (CSF) of ALS patients. This protein contains sequence similarities to TAR DNA-binding protein 43 (TDP-43) and fused-in-sarcoma (FUS) that are contained in cytoplasmic inclusions of ALS and FTLD-TDP or FTLD-FUS patients. To further characterize RBM45, we first verified the presence of RBM45 in CSF and spinal cord tissue extracts of ALS patients by immunoblot. We next used immunohistochemistry to examine the subcellular distribution of RBM45 and observed in a punctate staining pattern within nuclei of neurons and glia in the brain and spinal cord. We also detected RBM45 cytoplasmic inclusions in 91 % of ALS, 100 % of FTLD-TDP and 75 % of Alzheimer's disease (AD) cases. The most extensive RBM45 pathology was observed in patients that harbor the C9ORF72 hexanucleotide repeat expansion. These RBM45 inclusions were observed in spinal cord motor neurons, glia and neurons of the dentate gyrus. By confocal microscopy, RBM45 co-localizes with ubiquitin and TDP-43 in inclusion bodies. In neurons containing RBM45 cytoplasmic inclusions we often detected the protein in a punctate pattern within the nucleus that lacked either TDP-43 or ubiquitin. We identified RBM45 using a proteomic screen of CSF from ALS and control subjects for candidate biomarkers, and link this RNA-binding protein to inclusion pathology in ALS, FTLD-TDP and AD.
RNA 结合蛋白病理学现在是肌萎缩侧索硬化症 (ALS) 和具有 TDP-43 或 FUS 病理学 (FTLD-TDP 和 FTLD-FUS) 的额颞叶变性患者的最佳特征病理特征之一。使用液相色谱串联质谱法，我们在 ALS 患者的脑脊液 (CSF) 中鉴定出 RNA 结合基序 45 (RBM45) 蛋白水平改变。该蛋白与 TAR DNA 结合蛋白 43 (TDP-43) 和肉瘤融合 (FUS) 具有序列相似性，这些蛋白存在于 ALS 和 FTLD-TDP 或 FTLD-FUS 患者的细胞质包涵体中。为了进一步表征 RBM45，我们首先通过免疫印迹法验证了 CSF 和 ALS 患者脊髓组织提取物中 RBM45 的存在。接下来，我们使用免疫组织化学技术检查了 RBM45 的亚细胞分布，并在大脑和脊髓神经元和神经胶质细胞核内观察到点状染色模式。我们还在 91%的 ALS 病例、100%的 FTLD-TDP 病例和 75%的阿尔茨海默病 (AD) 病例中检测到 RBM45 细胞质包涵体。在携带 C9ORF72 六核苷酸重复扩展的患者中观察到最广泛的 RBM45 病理学。这些 RBM45 包涵体存在于脊髓运动神经元、神经胶质细胞和齿状回神经元中。通过共聚焦显微镜，RBM45 与包涵体中的泛素和 TDP-43 共定位。在含有 RBM45 细胞质包涵体的神经元中，我们经常在缺乏 TDP-43 或泛素的核内观察到点状蛋白模式。我们使用 ALS 和对照 CSF 的蛋白质组学筛选来鉴定 RBM45，以确定候选生物标志物，并将这种 RNA 结合蛋白与 ALS、FTLD-TDP 和 AD 的包涵体病理学联系起来。
