TDP-43 Proteinopathy in ALS-FTD: Mechanism, Target Validation and Biomarker

 描述（由申请人提供）：肌萎缩性侧索硬化症（ALS）是一种致死性成人发病运动神经元疾病，其特征为上下运动神经元的选择性丧失，额颞叶痴呆（FTD）是一种常见的痴呆形式，其特征为行为、个性和/或语言的进行性恶化，具有共同的疾病谱。涉及反式激活反应元件DNA结合蛋白43（TDP-43）的神经病理学发生在几乎所有ALS病例和大部分FTD病例中，这些神经退行性疾病目前尚无有效治疗。该提案的总体目标是阐明疾病机制，验证新的治疗策略，并开发ALS-FTD的生物标志物。我们最近的发现确定了TDP-43（一种被认为是ALS-FTD发病机制中的核心蛋白）是非保守隐蔽外显子的剪接抑制因子，这种功能的丧失通过无义介导的衰变导致一组对细胞功能至关重要的mRNA下调。支持TDP-43蛋白质病反映TDP-43功能丧失的假设，我们发现在显示TDP-43蛋白质病的ALS-FTD脑中，隐蔽外显子的抑制受损。我们假设存在神经元特异性TDP-43依赖性隐蔽外显子，其与阐明疾病机制相关，以解释ALS- FTD中神经元的选择性脆弱性。我们将通过鉴定缺乏TDP-43的人类神经元中的神经元特异性隐藏外显子来解决这个关键问题。然后，我们将确认ALS-FTD病例的脑中是否也会损害这种隐蔽外显子的抑制。我们进一步假设，一组特定的隐藏外显子易患ALS或FTD病例的运动或额叶皮质。为了测试这种可能性，我们将确定一组独特的隐藏外显子是否与ALS或FTD病例相关。重要的是，我们在缺乏TDP-43的细胞模型中证明，这些隐藏的外显子可以被抑制，并且通过强制表达由TDP-43的N-末端结构域融合到充分表征的抑制剂的剪接阻遏物结构域组成的杂合蛋白来防止细胞死亡。我们的研究结果提供了一种新的治疗策略，使用这种杂合蛋白抑制隐藏外显子的剪接，以改善ALS-FTD的神经变性。我们建议进行一系列临床前主要研究，以验证这种方法的有效性，这些信息将是至关重要的，或将这种有前途的治疗策略转化为临床。用于患者选择和临床试验监测的生物标志物，特别是症状前的生物标志物，仍然是一个关键的未满足的需求。我们假设，针对表达的隐蔽外显子的新抗原代表ALS和FTD的新生物标志物。我们将产生单克隆抗血清的新表位对应的几个隐藏的外显子，并评估其潜在的症状前的生物标志物。总之，我们拟定研究的结果将对理解疾病机制、验证治疗策略和开发ALS-FTD的功能性生物标志物具有重要意义。

 DESCRIPTION (provided by applicant): Amyotrophic Lateral Sclerosis (ALS), a fatal adult onset motor neuron disease characterized by selective loss of upper and lower motor neurons, and Fronto-Temporal Dementia (FTD), a common form of dementia characterized by a progressive deterioration in behavior, personality and/or language, share a common disease spectrum. The neuropathology involving Transactivation response element DNA-binding protein 43 (TDP-43) occurs in nearly all cases of ALS and large proportion of FTD, neurodegenerative diseases currently without effective therapy. The overall goals of this proposal are to clarify disease mechanism, validate a novel therapeutic strategy, and develop biomarkers for ALS-FTD. Our recent discovery established that TDP-43, a protein thought to be central in the pathogenesis of ALS-FTD, is a splicing suppressor of non-conserved cryptic exons and that loss of such function leads to down-regulation of a set of mRNA critical for cellular function via nonsense-mediated decay. Supporting the hypothesis that TDP-43 proteinopathy reflects a loss of TDP-43 function, we showed that in brains of ALS-FTD exhibiting TDP-43 proteinopathy, suppression of cryptic exon is impaired. We hypothesize that there exists neuron-specific TDP-43 dependent cryptic exons that would be relevant towards clarifying disease mechanisms to account for the selective vulnerability of neurons in ALS- FTD. We will address this critical question by identifying neuron specific cryptic exons in human neurons lacking TDP-43. We will then confirm whether suppression of such cryptic exons is also compromised in brains of cases of ALS-FTD. We further hypothesize that a specific set of cryptic exons predispose motor or frontal cortex in ALS or FTD cases. To test this possibility, we will determine whether a unique set of cryptic exons is linked either to cases of ALS or FTD. Importantly, we demonstrated in a cell model lacking Tdp-43 that these cryptic exons can be suppressed and cell death prevented by forced expression of a hybrid protein comprised of the N-terminal domain of TDP-43 fused to the splicing repressor domain of a well-characterized suppressor. Our findings offer a novel therapeutic strategy to suppress splicing of cryptic exons using this hybrid protein in an effort t ameliorate neurodegeneration in ALS-FTD. We propose to perform a series of preclinical proof-of- principal studies to validate the efficacy of this approach, information that will be critical or translating such a promising therapeutic strategy to the clinic. Biomarkers, particularly pre-symptomatic ones, for patient selection and monitoring of clinical trials remain a critical unmet need. We hypothesize that neoantigens against expressed cryptic exons represent novel biomarkers for ALS and FTD. We will generate monoclonal antisera to novel epitopes corresponding to several cryptic exons and evaluate their potential as pre-symptomatic biomarkers. Together, results from our proposed studies will have important implications for understanding disease mechanism, validating therapeutic strategy and developing functional biomarkers for ALS-FTD.