Defining the Mechanisms by Which Mutations in DNAJC7 Increase Susceptibility to ALS/FTD

不溶性和错误折叠的蛋白质的积累通常与神经元的变性有关， 肌萎缩侧索硬化症（ALS）和额颞叶痴呆（FTD）患者。热休克蛋白 通过促进有效的折叠、运输和运输，在调节蛋白质稳态中发挥核心作用。 新生和老化多肽的降解。虽然越来越清楚的是， 在ALS/FTD中发挥重要作用，但研究重点有限 热休克蛋白的功能与疾病发病机制之间的直接因果关系。杂合子，缺失- 编码HSP 40蛋白DNAJC 7的DNAJC 7基因中的功能缺失突变最近已被发现。 被认为是导致罕见ALS的原因。DNAJC 7蛋白充当HSP 70伴侣的共伴侣， 从而促进HSP 70-多肽相互作用和适当的多肽折叠。然而， 关于DNAJC 7在中枢神经系统和运动神经元中的特殊功能， 主要在ALS患者中退化。我们的主要假设是DNAJC 7单倍不足导致 错误折叠的HSP 70客户蛋白的积累导致生物过程的破坏， 脆弱的MN的功能和生存。在这里，我们将使用患者诱导的突变DNAJC 7细胞模型， 多能干细胞（iPSC）衍生的皮质和脊髓运动神经元和CRISPR/Cas9基因编辑， 结合基于质谱（MS）的定量蛋白质组学和RNA测序，以阐明 DNAJC 7中的ALS/FTD相关突变导致神经元功能障碍和变性。目标1 我们将确定突变体和同基因对照人类神经元中DNAJC 7的内源性相互作用组， 更好地了解它在有丝分裂后细胞中的功能作用。在目标2中，我们将系统地描述 使用两个独立的基于MS的蛋白质组学研究致病性DNAJC 7引起的蛋白质折叠破坏 接近。这些实验将提供一个公正的蛋白质组范围内的基本方面的审讯 ALS-DNAJC 7突变体和同基因对照神经元中的蛋白质稳态。在目标3中，这是基于我们的初步 发现DNAJC 7与ALS-因果RNA代谢蛋白FUS和MATR 3相互作用，我们将 研究DNAJC 7突变如何影响患者神经元中的RNA加工。综上所述，我们的建议 目的将揭示细胞机制，是由DNAJC 7单倍不足，在不同的损害。 人类皮层和脊髓神经元。我们的研究结果将通过促进理解而影响该领域 人类神经元中HSP依赖的蛋白质稳定机制以及ALS基因突变的罕见性 导致神经元功能障碍和损失。鉴定在人类中易于错误折叠的新蛋白质 神经元可能突出ALS/FTD病理生理学的关键细胞通路。

The accumulation of insoluble and misfolded proteins is commonly associated with degeneration of neurons in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) patients. Heat shock proteins (HSPs) play a central role in the regulation of protein homeostasis by facilitating effective folding, trafficking, and degradation of both nascent and aged polypeptides. While it has become increasingly clear that perturbations in in the proteostasis network play a significant role in ALS/FTD, limited emphasis has been placed on investigating the direct causal relationship between the functionality of HSPs and disease pathogenesis. Heterozygous, loss- of-function mutations in the DNAJC7 gene, which encodes for the HSP40 protein DNAJC7 have recently been identified as a cause for rare forms of ALS. The DNAJC7 protein acts as a co-chaperone for HSP70 chaperones, thereby facilitating HSP70-polypeptide interactions and appropriate polypeptide folding. However, little is known about the specific function of DNAJC7 in the central nervous system and motor neurons specifically, the cell type that predominantly degenerates in ALS patients. Our primary hypothesis is that DNAJC7 haploinsufficiency leads to the accumulation of misfolded HSP70 client proteins resulting in the disruption of biological processes critical to the function and survival of vulnerable MNs. Here, we will use mutant DNAJC7 cellular models, patient induced pluripotent stem cell (iPSC)-derived cortical and spinal motor neurons and CRISPR/Cas9 gene-editing, in combination with mass spectrometry (MS)-based quantitative proteomics and RNA-Sequencing to elucidate how ALS/FTD-associated mutations in DNAJC7 contribute towards neuronal dysfunction and degeneration. In Aim 1 we will determine the endogenous interactome of DNAJC7 in mutant and isogenic control human neurons to better understand the functional role that it plays in postmitotic cells. In Aim 2 we will methodically characterize disruptions in protein folding caused by pathogenic DNAJC7 using two independent MS-based proteomics approaches. These experiments will provide an unbiased proteome-wide interrogation of fundamental aspects of proteostasis in ALS-DNAJC7 mutant and isogenic control neurons. In Aim 3, which is based on our preliminary finding that DNAJC7 interacts with the ALS-casual RNA-metabolism proteins FUS and MATR3, we will investigate how DNAJC7 mutations affect RNA processing in patient neurons. Taken together, our proposed aims will shed light upon the cellular mechanisms that are compromised by DNAJC7 haploinsufficiency in distinct human cortical and spinal neurons. Our findings will impact the field by contributing towards the understanding of HSP-dependent proteostasis mechanisms in human neurons as well as to how rare ALS genetic mutations lead to neuron dysfunction and loss. Identifying novel proteins that are susceptible to misfolding in human neurons might highlight cellular pathways critical for ALS/FTD pathophysiology.