TDP-43 is an aggregation-prone protein which accumulates in the hallmark pathological inclusions of amyotrophic lateral sclerosis (ALS). However, the analysis of deeply phenotyped human post-mortem samples has shown that TDP-43 aggregation, revealed by standard antibody methods, correlates poorly with symptom manifestation. Recent identification of cryptic-splicing events, such as the detection of Stathmin-2 (STMN-2) cryptic exons, are providing evidence implicating TDP-43 loss-of-function as a potential driving pathomechanism but the temporal nature of TDP-43 loss and its relation to the disease process and clinical phenotype is not known. To address these outstanding questions, we used a novel RNA aptamer, TDP-43^APT, to detect TDP-43 pathology and used single molecule in situ hybridization to sensitively reveal TDP-43 loss-of-function and applied these in a deeply phenotyped human post-mortem tissue cohort. We demonstrate that TDP-43^APT identifies pathological TDP-43, detecting aggregation events that cannot be detected by classical antibody stains. We show that nuclear TDP-43 pathology is an early event, occurring prior to cytoplasmic accumulation and is associated with loss-of-function measured by coincident STMN-2 cryptic splicing pathology. Crucially, we show that these pathological features of TDP-43 loss-of-function precede the clinical inflection point and are not required for region specific clinical manifestation. Furthermore, we demonstrate that gain-of-function in the form of extensive cytoplasmic accumulation, but not loss-of-function, is the primary molecular correlate of clinical manifestation. Taken together, our findings demonstrate implications for early diagnostics as the presence of STMN-2 cryptic exons and early TDP-43 aggregation events could be detected prior to symptom onset, holding promise for early intervention in ALS.

TDP-43 是一种易于聚集的蛋白质，可在肌萎缩侧索硬化症 (ALS) 的标志性病理包涵体中积累。然而，对深度表型人类死后样本的分析表明，标准抗体方法揭示的 TDP-43 聚集与症状表现的相关性较差。最近对隐秘剪接事件的识别，例如Stathmin-2 ( STMN-2 ) 隐秘外显子的检测，提供了证据表明 TDP-43 功能丧失是一种潜在的驱动病理机制，但 TDP-43 丧失的时间性质其与疾病过程和临床表型的关系尚不清楚。为了解决这些悬而未决的问题，我们使用了一种新型 RNA 适体 TDP-43 ^APT来检测 TDP-43 病理学，并使用单分子原位杂交灵敏地揭示 TDP-43 功能丧失，并将其应用于深度表型人类死后组织队列。我们证明 TDP-43 ^APT可以识别病理性 TDP-43，检测经典抗体染色无法检测到的聚集事件。我们表明，核 TDP-43 病理学是一种早期事件，发生在细胞质积累之前，并且与通过一致的STMN-2隐性剪接病理学测量的功能丧失相关。至关重要的是，我们表明 TDP-43 功能丧失的这些病理特征早于临床拐点，并且对于区域特异性临床表现来说并不是必需的。此外，我们证明，以广泛的细胞质积累形式出现的功能获得，而不是功能丧失，是临床表现的主要分子相关性。总而言之，我们的研究结果证明了对早期诊断的影响，因为可以在症状出现之前检测到STMN-2隐性外显子和早期 TDP-43 聚集事件的存在，这为 ALS 的早期干预带来了希望。