Defining the mechanisms by which mutations in DNAJC7 increase susceptibility to ALS/FTD

确定 DNAJC7 突变增加 ALS/FTD 易感性的机制

基本信息

批准号：
10679697
负责人：
Andrew Fleming
金额：
$ 4.77万
依托单位：
NORTHWESTERN UNIVERSITY AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-09-01 至 2025-02-28
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10679697
关键词：
ALS patients Affect Amyotrophic Lateral Sclerosis Appearance Binding Biochemical Biological Process CRISPR/Cas technology Case Study Cell model Cell physiology Cells Cellular Stress Central Nervous System Client Clinical Complex Coupled Critical Pathways DNA Sequence Alteration Data Disease Dissociation Exhibits Fractionation Frontotemporal Dementia Functional disorder Genes Genetic Genetic Transcription HSF1 Heat shock proteins Heat-Shock Proteins 70 Heat-Shock Response Heterozygote Human Induced pluripotent stem cell derived neurons Lead Light Mass Spectrum Analysis Measures Mediating Messenger RNA Molecular Chaperones Molecular Weight Motor Neurons Mutation Nerve Degeneration Neurodegenerative Disorders Neuronal Dysfunction Neurons Pathogenesis Pathogenicity Patients Play Predisposition Process Protein Truncation Proteins Proteome Proteomics RNA RNA Processing RNA Splicing RNA metabolism RNA-Binding Proteins Rare Diseases Regulation Role Solubility Spinal Stress Testing Transcript Up-Regulation Vertebral column Work aged biological adaptation to stress cell type exome sequencing experimental study frontotemporal lobar dementia amyotrophic lateral sclerosis functional loss genetic variant heat-shock proteins 40 induced pluripotent stem cell loss of function loss of function mutation mRNA Stability misfolded protein motor control mutant neuron loss novel polypeptide protein metabolism proteostasis response sporadic amyotrophic lateral sclerosis trafficking transcriptome sequencing

项目摘要

ABSTRACT 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 spinal motor neurons (MNS) 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, which is based on our preliminary finding that DNAJC7 interacts with the ALS-causal RNA metabolism protein MATR3, we will determine if pathogenic mutations in DNAJC7 impede MATR3 folding. Next, we will investigate the functional consequences of disruptions in DNAJC7-substrate interface by measuring how DNAJC7 mutations affect MATR3-associated RNA processing in patient MNs. In Aim 2, which is based on our preliminary finding that DNAJC7 impedes the HSF1 response, we will define the mechanistic role DNAJC7 plays in the HSF1- mediated stress response in patient derived mutant and isogenic control MNs. We will directly measure DNAJC7- dependent loss of HSF1 transcriptional activity upon stress, determine if DNAJC7 is involved in the dissociation of the inhibitory HSP70-HSF1 complex, and methodically characterize disruptions in protein solubility caused by pathogenic DNAJC7 using MS-based proteomics approaches. Taken together, our proposed aims will shed light upon the cellular mechanisms that are compromised by DNAJC7 haploinsufficiency. 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.

抽象的不溶性和错误折叠蛋白的积累通常与神经元的变性有关肌萎缩性外侧硬化症（ALS）和额颞痴呆（FTD）患者。热休克蛋白（HSP）通过促进有效的折叠，贩运和新生和老化多肽的降解。虽然越来越清楚地扰动在Proteostasis网络中在ALS/FTD中起着重要作用，对研究有限的重点 HSP的功能与疾病发病机理之间的直接因果关系。杂合，损失 - DNAJC7基因中的功能突变，该基因编码Hsp40蛋白DNAJC7已最近已有被确定为稀有形式的ALS的原因。 DNAJC7蛋白充当HSP70伴侣的副酮，从而促进HSP70-多肽相互作用和适当的多肽折叠。但是，鲜为人知关于中枢神经系统和运动神经元中DNAJC7的特定功能，特别是细胞类型这主要在ALS患者中退化。我们的主要假设是DNAJC7单倍性引导为了积累错误折叠的HSP70客户蛋白，导致生物过程中断至关重要易受伤害的MN的功能和生存。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）衍生的脊柱运动神经元（MNS）和CRISPR/CAS9基因编辑组合具有质谱（MS）的定量蛋白质组学和RNA顺序，以阐明ALS/FTD-如何 DNAJC7中的相关突变有助于神经元功能障碍和变性。在AIM 1中，是基于我们的初步发现，DNAJC7与Als-Causal RNA代谢蛋白Matr3相互作用，我们将确定DNAJC7中的致病突变是否阻碍MATR3折叠。接下来，我们将调查通过测量DNAJC7突变的方式影响患者MNS中与MATR3相关的RNA处理。在AIM 2中，这是基于我们的初步发现 DNAJC7阻碍了HSF1响应，我们将定义机械作用DNAJC7在HSF1-中的作用患者衍生的突变体和同基因对照MN中介导的应激反应。我们将直接测量DNAJC7- 应力时HSF1转录活性的依赖损失，确定DNAJC7是否参与解离抑制性HSP70-HSF1复合物的抑制作用，并有条不紊地表征了由使用基于MS的蛋白质组学方法的致病DNAJC7。综上所述，我们提出的目标将熄灭在被DNAJC7单倍弥补损害的细胞机制上。我们的发现将影响通过对人神经元中HSP依赖性蛋白抑制机制的理解有助于领域以及罕见的ALS基因突变导致神经元功能障碍和丧失。识别新型蛋白质容易在人神经元中脱折叠的折叠可能突出显示对ALS/FTD至关重要的细胞途径病理生理学。