Translation dysregulation in neurodegeneration

肌萎缩侧索硬化症（ALS）和额颞叶痴呆（FTD）包括一种谱系障碍， 以进行性神经元变性为特征。ALS/FTD的病理学标志是存在 含有RNA结合蛋白TDP-43的细胞质聚集体（97%的ALS和45%的FTD病例）。到 为了阐明ALS/FTD的分子基础，我们开发了TDP-43蛋白病的果蝇模型 基于人TDP-43在运动神经元（ALS）或蘑菇体（MB）神经元（FTD）中的过表达。 这些模型表现出与人类疾病的显著相似性，包括细胞质聚集 (ALS/FTD）、神经肌肉缺陷伴运动功能障碍和寿命缩短（ALS） 记忆和睡眠缺陷（FTD）。使用ALS苍蝇模型，我们确定了随后 在患者来源的iPSC运动神经元（iPSC MN）或脊髓中验证，包括在神经元中的改变。 微管相关蛋白Futsch/MAP 1B和突触囊泡伴侣蛋白Hsc 70 -4/HSPA 8。这些 研究结果支持核糖体抑制假说，该假说认为在疾病中，TDP-43错误定位于细胞质 在那里它隔离RNA，使它们不能被核糖体翻译。为了进一步调查 假设我们使用了RNA免疫层析（RIP）和标记核糖体亲和力的组合 TDP-43蛋白质病的苍蝇模型中的TRAP纯化。功能注释工具突出多个目标 TDP-43蛋白质病中核糖体平衡改变的途径，包括dally样蛋白（Dlp/GPC 6），一种调节剂， 神经肌肉接头（NMJ）的跨突触信号传导和可塑性以及阿尔茨海默氏症的危险因素 疾病我们发现Dlp/GPC 6在果蝇的NMJ处减少，但在果蝇的腹神经索中积累， 以及ALS患者iPSC MN和脊髓。有趣的是，Dlp在果蝇MBs中显著降低， 年龄依赖的方式。总之，我们发表的工作和这些发现使我们假设TDP- 神经元中的毒性部分是由特定mRNA靶的翻译失调引起的， Dlp/GPC 6.为了验证这一假设，我们将使用遗传和分子方法在苍蝇，以阐明 Dlp/GPC 6导致ALS中TDP-43依赖性神经元和突触功能障碍的机制。 这些发现将在患者来源的iPSC MN、NMJ和组织中得到验证（目的1）。我们还将建立 使用果蝇和患者源性皮质神经元（CN）、类器官和组织研究Dlp/GPC 6对FTD的贡献 (Aim 2）。最后，我们将使用非规范氨基酸标记（NCAT）在体内，在苍蝇，以确定具体的，TDP- 新蛋白质合成的43种依赖性变化将在iPSC NMJ和类器官中得到验证。这些研究 在ALS和FTD中，相关模型有望填补知识的关键空白，关于TDP-43在翻译中的作用， 从机制上确定TDP-43蛋白病中轴突和突触功能障碍的基础， 一个探测神经元特异性脆弱性的机会，并可能发现新的分子靶点， 对ALS/FTD具有治疗潜力的途径。

Amyotrophic lateral sclerosis (ALS) and fronto-temporal dementia (FTD) comprise a spectrum disorder characterized by progressive neuronal degeneration. A pathological hallmark of ALS/FTD is the presence of cytoplasmic aggregates containing the RNA binding protein TDP-43 (97% of ALS and 45% of FTD cases). To elucidate the molecular underpinnings of ALS/FTD, we developed Drosophila models of TDP-43 proteinopathy based on overexpression of human TDP-43 in motor neurons (ALS) or mushroom body (MB) neurons (FTD). These models exhibit remarkable similarities to the human disease, including cytoplasmic aggregates (ALS/FTD), neuromuscular deficits accompanied by locomotor dysfunction and reduced lifespan (ALS) as well as memory and sleep deficits (FTD). Using ALS fly models we identified phenotypes that were subsequently validated in patient derived iPSC motor neurons (iPSC MNs) or spinal cords, including alterations in the microtubule associated protein Futsch/MAP1B and the synaptic vesicle chaperone Hsc70-4/HSPA8. These findings support the ribostasis hypothesis, which posits that in disease, TDP-43 mis-localizes to the cytoplasm where it sequesters RNAs rendering them unavailable to ribosomes for translation. To further probe this hypothesis we used a combination of RNA ImmunoPrecipitations (RIP) and Tagged Ribosome Affinity Purifications (TRAP) in fly models of TDP-43 proteinopathy. Functional annotation tools highlight multiple targets and pathways with altered ribostasis in TDP-43 proteinopathy, including dally-like protein (Dlp/GPC6), a regulator of trans-synaptic signaling and plasticity at the neuromuscular junction (NMJ) and a risk factor for Alzheimer’s Disease. We found that Dlp/GPC6 is reduced at the fly NMJ but accumulates in the ventral nerve cord in flies, and ALS patient iPSC MNs and spinal cords. Interestingly, Dlp is significantly reduced in Drosophila MBs in an age dependent manner. Taken together, our published work and these findings led us to hypothesize that TDP- 43 toxicity in neurons is caused in part by translation dysregulation of specific mRNA targets including Dlp/GPC6. To test this hypothesis we will use genetic and molecular approaches in flies to elucidate the mechanism by which Dlp/GPC6 contributes to TDP-43 dependent, neuronal and synaptic dysfunction in ALS. These findings will be validated in patient derived iPSC MNs, NMJs and tissues (Aim 1). We will also establish the contribution of Dlp/GPC6 to FTD using flies and patient derived cortical neurons (CNs), organoids and tissues (Aim 2). Finally, we will use Non-Canonical Aminoacid Tagging (NCAT) in vivo, in flies to identify specific, TDP- 43 dependent changes in new protein synthesis that will be validated in iPSC NMJs and organoids. These studies in ALS and FTD relevant models are expected to fill a critical gap of knowledge, on TDP-43’s role in translation, to mechanistically determine the basis of axonal and synaptic dysfunction in TDP-43 proteinopathy, to provide an opportunity for probing neuronal specific vulnerabilities and may uncover novel molecular targets and pathways with therapeutic potential for ALS/FTD.