Department of Experimental and Clinical Biomedical Sciences "Mario Serio", University of Florence, Florence, Italy.
Institute of Applied Physics "Nello Carrara", National Research Council, Sesto Fiorentino, Italy.
Ann Med. 2023 Dec;55(1):72-88. doi: 10.1080/07853890.2022.2148734.
Several neurodegenerative conditions are associated with a common histopathology within neurons of the central nervous system, consisting of the deposition of cytoplasmic inclusions of TAR DNA-binding protein 43 (TDP-43). Such inclusions have variably been described as morphologically and molecularly ordered aggregates having amyloid properties, as filaments without the cross-β-structure and dye binding specific for amyloid, or as amorphous aggregates with no defined structure and fibrillar morphology. Here we have expressed human full-length TDP-43 in neuroblastoma x spinal cord 34 (NSC-34) cells to investigate the morphological, structural, and tinctorial properties of TDP-43 inclusions . We have used last-generation amyloid diagnostic probes able to cross the cell membrane and detect amyloid in the cytoplasm and have adopted Raman and Fourier transform infrared microspectroscopies to study the secondary structure of the TDP-43 protein in the inclusions. We have then used transmission electron microscopy to study the morphology of the TDP-43 inclusions. The results show the absence of amyloid dye binding, the lack of an enrichment of cross-β structure in the inclusions, and of a fibrillar texture in the round inclusions. The aggregates formed from the purified protein under conditions in which it is initially native also lack all these characteristics, ruling out a clear amyloid-like signature. These findings indicate a low propensity of TDP-43 to form amyloid fibrils and even non-amyloid filaments, under conditions in which the protein is initially native and undergoes its typical nucleus-to-cell mislocalization. It cannot be excluded that filaments emerge on the long time scale from such inclusions, but the high propensity of the protein to form initially other types of inclusions appear to be an essential characteristic of TDP-43 proteinopathies.KEY MESSAGESCytoplasmic inclusions of TDP-43 formed in NSC-34 cells do not stain with amyloid-diagnostic dyes, are not enriched with cross-β structure, and do not show a fibrillar morphology.TDP-43 assemblies formed from pure TDP-43 do not have any hallmarks of amyloid.
几种神经退行性疾病与中枢神经系统神经元中的细胞质包含物有关，这些包含物由 TAR DNA 结合蛋白 43（TDP-43）的细胞质包含物组成。这些包含物在形态和分子上被描述为具有淀粉样特性的有序聚集物，具有无交叉-β 结构的纤维和对淀粉样蛋白具有特异性的染料结合，或者是无定义结构和纤维状形态的无定形聚集物。在这里，我们在神经母细胞瘤 x 脊髓 34（NSC-34）细胞中表达了全长人类 TDP-43，以研究 TDP-43 包含物的形态、结构和染色特性。我们使用了能够穿过细胞膜并在细胞质中检测淀粉样蛋白的最新一代淀粉样蛋白诊断探针，并采用拉曼和傅里叶变换红外显微镜研究了包含物中 TDP-43 蛋白的二级结构。然后，我们使用透射电子显微镜研究了 TDP-43 包含物的形态。结果表明，不存在淀粉样蛋白染料结合，包含物中不存在交叉-β 结构的富集，也不存在圆形包含物中的纤维纹理。在最初为天然状态的蛋白质的条件下，从纯化的蛋白质中形成的聚集体也缺乏所有这些特征，排除了明确的淀粉样特征。这些发现表明，在蛋白质最初为天然状态并经历其典型的核到细胞质定位错误的情况下，TDP-43 形成淀粉样纤维甚至非淀粉样纤维的倾向较低。不能排除这些包含物在长时间尺度上会出现纤维，但该蛋白质最初形成其他类型包含物的高倾向似乎是 TDP-43 蛋白病的一个重要特征。
关键信息
在 NSC-34 细胞中形成的 TDP-43 细胞质包含物用淀粉样蛋白诊断染料染色，不富含交叉-β 结构，也没有纤维状形态。
从纯 TDP-43 形成的 TDP-43 组装物没有任何淀粉样特征。
