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

不可溶和错误折叠的蛋白质的积累通常与神经元的退化有关 肌萎缩侧索硬化症(ALS)和额颞部痴呆(FTD)患者。热休克蛋白(HSPs) 通过促进有效的折叠、运输和调节，在调节蛋白质的动态平衡中发挥核心作用 新生多肽和老化多肽的降解。虽然越来越明显的是， 在蛋白平衡网络在ALS/FTD中发挥重要作用方面，人们对其研究的重视程度有限 热休克蛋白功能与疾病发病机制之间的直接因果关系。杂合子，丢失- 编码Hsp40蛋白DNAJC7的DNAJC7基因的功能外突变最近被认为是 被确认为罕见形式的肌萎缩侧索硬化症的原因。DNAJC7蛋白作为HSP70伴侣的辅助伴侣， 从而促进HSP70-多肽的相互作用和适当的多肽折叠。然而，人们对此知之甚少 关于DNAJC7在中枢神经系统和运动神经元中的特定功能，特别是细胞类型 这在ALS患者中主要是退化的。我们的主要假设是DNAJC7单倍体不足导致 对错误折叠的HSP70客户蛋白的积累导致生物过程的中断至关重要 对脆弱的MNS的功能和生存的影响。在这里，我们将使用突变的DNAJC7细胞模型，患者诱导 多能干细胞(IPSC)来源的皮质和脊髓运动神经元与CRISPR/Cas9基因编辑 结合基于质谱仪(MS)的定量蛋白质组学和RNA测序来阐明 DNAJC7的ALS/FTD相关突变导致神经元功能障碍和变性。在目标1中 我们将在突变和同基因对照的人神经元中确定DNAJC7的内源性相互作用组，以 更好地了解它在有丝分裂后细胞中所起的功能作用。在目标2中，我们将系统地描述 用两个独立的基于MS的蛋白质组学研究致病DNAJC7引起的蛋白质折叠中断 接近了。这些实验将提供对基本方面的无偏见的蛋白质组范围的询问 ALS-DNAJC7突变型和同基因对照神经元的蛋白稳定性。在目标3中，这是基于我们的初步 发现DNAJC7与ALS临时RNA代谢蛋白FUS和MATR3相互作用，我们将 研究DNAJC7突变如何影响患者神经元中的RNA处理。总而言之，我们的建议 AIMS将阐明不同的DNAJC7单倍体不足所损害的细胞机制 人类大脑皮层和脊髓神经元。我们的发现将对该领域产生影响，有助于理解 人类神经元中热休克蛋白依赖的蛋白稳定机制以及罕见的ALS基因突变 导致神经元功能障碍和丧失。鉴定人类易错折叠的新蛋白质 神经元可能突出对ALS/FTD病理生理学至关重要的细胞通路。

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.