Misregulation of the TDP-43 RNA target, Sortilin, in neurodegeneration

TDP-43 RNA 靶点分拣蛋白在神经退行性疾病中的错误调节

• 批准号: 8319320
• 负责人: LEONARD PETRUCELLI
• 金额: $ 33.91万
• 依托单位: MAYO CLINIC JACKSONVILLE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-15 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8319320
• 关键词: 3' Splice Site; Amyotrophic Lateral Sclerosis; Attenuated; Base Pairing; Binding; Brain; Cell Culture System; Cells; Cultured Cells; Data; Development; Disease; Endocytosis; Exons; Frontotemporal Lobar Degenerations; Functional disorder; Genetic Transcription; Goals; Human; Immunoprecipitation; In Vitro; Introns; Link; Maintenance; Maps; Mediating; Messenger RNA; Morphology; Mus; Mutation; Nerve Degeneration; Neurites; Neuroblastoma; Neurons; Nonsense Codon; Pathogenesis; Pathology; Pathway interactions; Phenotype; Phosphotransferases; Progranulin; Protein Isoforms; Proteins; RNA; RNA Binding; RNA Processing; RNA Splicing; Reading Frames; Recombinants; Regulation; Signal Transduction; Suggestion; Toxic effect; Transcript; Transmembrane Domain; Variant; extracellular; in vivo; in vivo Model; insight; knock-down; mRNA Expression; mouse model; mutation carrier; neuroblastoma cell; neurotoxic; novel; overexpression; prevent; protein TDP-43; protein expression; receptor; sortilin; therapeutic target; uptake

DESCRIPTION (provided by applicant): TDP-43 is the principal component of neuronal inclusions in frontotemporal lobar degeneration with TDP-43-positive inclusions (FTLD-TDP) and amyotrophic lateral sclerosis (ALS). Since the major actions of TDP-43 derive from its RNA binding activity, determining if TDP-43 RNA targets are altered in disease will provide insight on the mechanisms of TDP-43 toxicity. RNA-immunoprecipitation studies in human HEK293T cells identified sortilin (SORT1), a neuronal progranulin (PGRN) receptor, as a TDP-43 target. Mutations in GRN are a major cause of FTLD-TDP. That TDP-43 regulates SORT1 RNA now provides a putative link between TDP-43 dysfunction and altered PGRN signaling in sporadic FTLD-TDP and ALS. Preliminary data shows that Tdp-43 regulates Sort1 mRNA splicing in murine primary neurons and mouse brain. Tdp-43 knockdown leads to the retention of 99 base pairs within intron 17 of Sort1, referred to as "exon 17b". Exon 17b is highly conserved between mouse and human, so it is very probable that human SORT1 is regulated by TDP-43. Indeed, an alternatively spliced SORT1 variant lacking exon 18 (SORT1 Ex18) was identified as a TDP-43-bound transcript in human neuroblastoma cells. It encodes a SORT1 (SORT1 Ex18) isoform with a truncated C-terminus. Given that the C-terminus is required for PGRN endocytosis, expression of SORT1 Ex18 is likely to have detrimental consequences on PGRN signaling. Likewise, Sort1+Ex17b expression may alter Pgrn function. We have shown that neurite outgrowth and branching are decreased in Pgrn-/- cortical neurons, a phenotype rescued by recombinant human PGRN. Overall, we presume there exists a bidirectional relationship between TDP-43 and SORT1/PGRN, such that abnormalities in TDP-43 promote SORT1/PGRN dysregulation and, conversely, SORT1/PGRN dysregulation causes TDP-43 pathology, thus perpetuating a neurotoxic cycle. The latter is supported by the finding that TDP-43 aggregation is attenuated in cultured cells when SORT1 is overexpressed, suggestion that loss of SORT1 function may indeed contribute to TDP-43 pathology. Our objective is thus to investigate this relationship in more detail. To this end, we will use a combination of in vitro and in vivo models to investigate: 1) the functional mechanisms underlying TDP-43-mediated SORT1 regulation; 2) whether SORT1 RNA expression and/or splicing, as well as SORT1 protein expression, are altered in FTLD-TDP; 3) whether various SORT1 isoforms differentially influence PGRN signaling; and 4) the influence of SORT1 isoforms on TDP-43 aggregation and toxicity.

描述（由申请人提供）：TDP-43是额颞叶变性（FTLD-TDP）和肌萎缩侧索硬化症（ALS）中神经元包涵体的主要成分。由于 TDP-43 的主要作用源自其 RNA 结合活性，因此确定 TDP-43 RNA 靶标在疾病中是否发生改变将为了解 TDP-43 毒性机制提供见解。人 HEK293T 细胞中的 RNA 免疫沉淀研究确定分拣蛋白 (SORT1)（一种神经元颗粒体蛋白前体 (PGRN) 受体）作为 TDP-43 靶标。 GRN 突变是 FTLD-TDP 的主要原因。 TDP-43 调节 SORT1 RNA 现在提供了 TDP-43 功能障碍与散发性 FTLD-TDP 和 ALS 中 PGRN 信号改变之间的假定联系。初步数据显示，Tdp-43 调节小鼠原代神经元和小鼠大脑中的 Sort1 mRNA 剪接。 Tdp-43 敲低导致 Sort1 的内含子 17（称为“外显子 17b”）内保留 99 个碱基对。外显子 17b 在小鼠和人类之间高度保守，因此人类 SORT1 很可能受 TDP-43 调节。事实上，缺乏外显子 18 的选择性剪接 SORT1 变体 (SORT1 Ex18) 被鉴定为人神经母细胞瘤细胞中 TDP-43 结合的转录物。它编码具有截短 C 末端的 SORT1 (SORT1 Ex18) 同工型。鉴于 PGRN 内吞作用需要 C 末端，SORT1 Ex18 的表达可能会对 PGRN 信号传导产生不利影响。同样，Sort1+Ex17b 表达式可能会改变 Pgrn 函数。我们已经证明 Pgrn-/- 皮质神经元的神经突生长和分支减少，这是重组人 PGRN 挽救的一种表型。总体而言，我们推测 TDP-43 和 SORT1/PGRN 之间存在双向关系，因此 TDP-43 的异常会促进 SORT1/PGRN 失调，反之，SORT1/PGRN 失调会导致 TDP-43 病理，从而使神经毒性循环永久存在。后者得到了以下发现的支持：当 SORT1 过表达时，培养细胞中 TDP-43 聚集减弱，这表明 SORT1 功能的丧失确实可能导致 TDP-43 病理学。因此，我们的目标是更详细地研究这种关系。为此，我们将结合体外和体内模型来研究：1）TDP-43介导的SORT1调节的功能机制； 2) FTLD-TDP 中 SORT1 RNA 表达和/或剪接以及 SORT1 蛋白表达是否发生改变； 3) 不同的SORT1亚型是否对PGRN信号传导有不同的影响； 4) SORT1亚型对TDP-43聚集和毒性的影响。