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Lobar变性中神经元内包含物的主要成分，具有TDP-43阳性夹杂物（FTLD-TDP）和肌萎缩性侧面硬化症（ALS）。由于TDP-43的主要作用来自其RNA结合活性，因此确定TDP-43 RNA靶标在疾病中是否改变了TDP-43毒性机制。人HEK293T细胞中的RNA免疫沉淀研究鉴定出神经元前素（PGRN）受体作为TDP-43靶标的Torsilin（Sort1）。 GRN中的突变是FTLD-TDP的主要原因。 TDP-43调节Sort1 RNA现在提供了TDP-43功能障碍与零星FTLD-TDP和ALS中PGRN信号改变之间的推定联系。初步数据表明，TDP-43调节鼠原发性神经元和小鼠脑中的Sort1 mRNA剪接。 TDP-43敲低导致在Sort1的内含子17中保留99个基对，称为“外显子17b”。外显子17B在小鼠和人之间高度保守，因此人类sort1很可能受TDP-43调节。实际上，在人神经母细胞瘤细胞中，缺乏外显子18（sort1 ex18）的剪接的sort1变体被确定为TDP-43结合的转录本。它用截断的C端编码sort1（stort1 ex18）同工型。鉴于C端是PGRN内吞作用所必需的，因此Sort1 Ex18的表达可能对PGRN信号产生不利的后果。同样，Sort1+Ex17b表达可能会改变PGRN函数。我们已经表明，PGRN - / - 皮质神经元的神经突生长和分支降低，这是由重组人PGRN挽救的表型。总体而言，我们认为TDP-43和Sort1/PGRN之间存在双向关系，因此TDP-43中的异常促进了Sort1/PGRN失调，相反，Sort1/PGRN失调会导致TDP-43病理学，从而使神经毒性循环永久化。后者得到了以下发现，即当Sort1过表达时TDP-43聚集在培养细胞中会减弱，这表明Sort1功能的丧失确实可能有助于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，我们将使用体外和体内模型的组合来研究：1）TDP-43介导的Sort1调节的功能机制； 2）在FTLD-TDP中，Sort1 RNA表达和/或剪接以及sort1蛋白表达是否会改变； 3）各种排序1同工型是否差异影响PGRN信号传导； 4）Sort1同工型对TDP-43聚集和毒性的影响。