Translation dysregulation in neurodegeneration

肌萎缩侧索硬化症(ALS)和额颞叶痴呆(FTD)构成谱系障碍 以进行性神经元退化为特征的。ALS/FTD的一个病理特征是存在 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)。至 阐明ALS/FTD的分子基础，我们建立了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。 这些模型与人类疾病有显著的相似之处，包括细胞质聚集体。 (ALS/FTD)，神经肌肉缺陷伴随运动功能障碍和寿命缩短(ALS) 作为记忆和睡眠缺陷(FTD)。使用ALS苍蝇模型，我们确定了随后发生的 在患者来源的IPSC运动神经元(IPSC MN)或脊髓中得到验证，包括在 微管相关蛋白Futsch/MAP1B和突触小泡伴侣Hsc70-4/HSPA8。这些 研究结果支持核素平衡假说，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。要进一步探讨这一点 假设我们使用了RNA免疫沉淀(RIP)和标记核糖体亲和力的组合 TDP-43蛋白病果蝇模型的纯化(TRAP)。功能注释工具突出显示多个目标 以及TDP-43蛋白病中核转录抑制改变的途径，包括Dally-like Protein(DLP/GPC6)，一个调节因子 神经肌肉接头(NMJ)跨突触信号和可塑性与阿尔茨海默病的危险因素 疾病。我们发现DLP/GPC6在果蝇的NMJ处减少，但在果蝇的腹神经索中积聚， ALS患者的IPSC、MNS和脊髓。有趣的是，DLP在果蝇的MBS中显著减少 年龄依赖的方式。综上所述，我们发表的工作和这些发现使我们假设TDP- 神经元的毒性部分是由于特定的信使核糖核酸靶标的翻译失调引起的，包括 DLP/GPC6。为了验证这一假设，我们将在果蝇中使用遗传学和分子方法来阐明 DLP/GPC6在ALS患者TDP-43依赖、神经元和突触功能障碍中的作用机制。 这些发现将在患者来源的IPSC MNS、NMJ和组织中得到验证(目标1)。我们还将建立 利用果蝇和患者来源的皮质神经元、器官和组织研究DLP/GPC6在FTD中的作用 (目标2)。最后，我们将在体内使用非典型氨基酸标记(NCAT)，在果蝇中识别特定的TDP- 43新蛋白质合成的依赖变化将在IPSC、NMJ和有机类化合物中得到验证。这些研究 在ALS和FTD的相关模型有望填补一个关键的知识空白，关于TDP-43的S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，以提供 探索神经元特异性脆弱性的机会，并可能发现新的分子靶点和 ALS/FTD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.