Characterizing TDP-43 isoforms in neurodegenerative disease

描述（由申请人提供）：神经退行性疾病中主要聚集蛋白的发现首次揭示了疾病的发病机制。最近，在额颞叶变性（FTLD-U）和散发性肌萎缩侧索硬化（ALS）的泛素阳性包涵体内的主要蛋白组分被发现是TAR DNA结合蛋白43（TDP-43）。在这些疾病中观察到的共同病理学提示基于对TDP-43聚集事件的理解的平行治疗方法。在正常细胞中，TDP-43是参与转录调节的核RNA结合蛋白。然而，据报道，病理性TDP-43从细胞核重新分布到细胞质，在细胞质中其被聚集、磷酸化、泛素化和/或切割。我们的初步结果表明，过度表达的人TDP-43和选择性剪接异构体（TDP-S6）在哺乳动物细胞培养中被磷酸化，泛素化和切割。表达的TDP-43弥漫性定位，几乎只在细胞核中。相反，TDP-S6亚型主要聚集在细胞质中，重现了疾病特异性病理学标志。因此，我们的假设是TDP-43聚集和毒性取决于剪接和/或后修饰的TDP-43同种型（即切割、磷酸化和泛素化）的特异性产生。为了验证这一假设，我们将使用RT-PCR和定量质谱（MS）方法研究剪接的TDP-43亚型在正常脑和FTLD-U组织中的表达。我们还将使用MS来表征磷酸化和切割的TDP-43亚型。最后，我们将通过在细胞系和原代神经元培养物中表达全长TDP-43和S6亚型来评估TDP-43介导的聚集和神经毒性。本研究将首次提供从FTLD-U脑组织中分离或在培养物中表达的TDP-43亚型的详细表征。额颞叶痴呆（FTD）和肌萎缩侧索硬化症（ALS）是严重的神经退行性疾病，在美国共影响约30，000人。TDP-43的研究将为这些疾病的发病机制提供更深入的了解，并可能导致新的治疗策略。

DESCRIPTION (provided by applicant): The discovery of major aggregated proteins in neurodegenerative diseases provides the first insight into disease pathogenesis. Recently, the dominant protein component within ubiquitin positive inclusions of frontotemporal lobar degeneration (FTLD-U) and sporadic amyotrophic lateral sclerosis (ALS) was found to be TAR DNA-binding protein 43 (TDP-43). The common pathology observed in these diseases suggests parallel approaches to treatment based on the understanding of TDP-43 aggregation events. In normal cells TDP-43 is a nuclear RNA binding protein involved transcriptional regulation. However, pathologic TDP-43 has been reported to redistribute from the nucleus to the cytoplasm where it is aggregated, phosphorylated, ubiquitinated and/or cleaved. Our preliminary results show that over-expressed human TDP-43 and the alternative splicing isoform (TDP-S6) are phosphorylated, ubiquitinated and cleaved in mammalian cell cultures. The expressed TDP-43 is localized diffusely and almost exclusively in the nucleus. In contrast, the TDP-S6 isoform is predominantly aggregated in the cytoplasm, recapitulating the disease-specific pathological hallmark. Therefore, our hypothesis is that TDP-43 aggregation and toxicity depends on the specific production of spliced and/or posttranslationally modified TDP-43 isoforms (i.e. cleavage, phosphorylation and ubiquitination). To test this hypothesis,we will investigate the expression of spliced TDP-43 isoforms in normal brain and FTLD-U tissue using RT-PCR and quantitative mass spectrometry (MS) approaches. We will also use MS to characterize phosphorylated and cleaved TDP-43 isoforms. Finally, we will assess TDP-43 mediated aggregation and neurotoxicity by expressing full-length TDP-43 and the S6 isoform in cell lines and primary neuronal cultures. This study will provide the first detailed characterization of TDP-43 isoforms either isolated from FTLD-U brain tissue or expressed in culture. Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are severe neurodegenerative disorders that together affect approximately 30,000 people in the United States. The study of TDP-43 will provide deeper understanding into the pathogenesis of these diseases and potentially lead to novel therapeutic strategies.