Neurocellular Endoplasmic Reticulum Stress Response in Alzheimer's Disease and Related Dementias (ADRD) Risk

Currently, more than 55 million people around the world suffer from dementia, and Alzheimer's disease-related dementia (ADRD) accounts for nearly 60-70% of all those cases. The spread of AD pathology and progressive neurodegeneration in the hippocampus and cerebral cortex is strongly correlated with cognitive decline in AD patients; however, the molecular underpinning of the ADRD causality is still unclear. Studies of postmortem AD brains and animal models of AD suggest that elevated endoplasmic reticulum (ER) stress may have a role in ADRD pathology through altered neurocellular homeostasis in brain regions associated with learning and memory. To study the ER stress-associated neurocellular response and its effects on neurocellular homeostasis and neurogenesis, we modeled an ER stress challenge using Thapsigargin (TG), a specific inhibitor of sarco/endoplasmic reticulum Ca2+ ATPase (SERCA), in induced pluripotent stem cell (iPSC)-derived neural stem cells (NSCs) of two individuals from our Mexican American Family Study (MAFS). High-content screening and transcriptomic analysis of the control and ER stress-challenged NSCs showed that NSC’s ER stress response resulted in a significant decline in NSC self-renewal and an increase in apoptosis and cellular oxidative stress. A total of 2300 genes were significantly (moderated t statistics FDR corrected p-value ≤ 0.05 and Fold Change absolute ≥ 2.0) differentially expressed (DE). Pathway enrichment and gene network analysis of DE genes suggests that all three unfolded protein response (UPR) pathways (PERK, IRE1, and ATF6) were significantly activated and cooperatively regulated the NSC’s transcriptional response to ER stress. Our results show that IRE1/XBP1 mediated transcriptional regulation of E2F1 and its downstream targets have a dominant role in inducing G1/S-phase cell cycle arrest in the ER stress-challenged NSCs. The ER stress-challenged NSCs also showed activation of CHOP-mediated apoptosis and dysregulation of synaptic plasticity and neurotransmitter homeostasis-associated genes. Overall, our results suggest that ER stress-associated attenuation of NSC self-renewal, increased apoptosis, and dysregulated synaptic plasticity and neurotransmitter homeostasis plausibly play a role in the causation of ADRD. Well-characterized NSC lines generated from validated iPSCs of 2 different individuals were ER stress-challenged using Thapsigargin (TG) for 24 hours. TG is a specific inhibitor of sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) and is a classical tool commonly used to study ER stress and UPR biology in mammalian cells. Both, vehicle-treated controls and ER stress-challenged NSCs were then quantitatively measured for cell proliferation, apoptotic cell death, protein aggregation, oxidative stress, mitochondrial function cellular phenotypes by high content screening (HCS) analysis and for genome-wide gene expression using deep mRNA sequencing.

目前，全球已有超过5500万痴呆症患者，其中阿尔茨海默病相关性痴呆（Alzheimer's Disease-Related Dementia, ADRD）占总病例数的近60%~70%。AD病理的扩散以及海马体与大脑皮层的进行性神经退行性变，与AD患者的认知衰退密切相关，但ADRD致病的分子机制仍未明确。研究人员对AD死后脑组织及AD动物模型的研究表明，内质网（Endoplasmic Reticulum, ER）应激水平升高，可能通过改变学习记忆相关脑区的神经细胞稳态，参与ADRD的病理进程。为研究ER应激相关的神经细胞应答及其对神经细胞稳态与神经发生的影响，本研究利用毒胡萝卜素（Thapsigargin, TG）——一种肌质网/内质网钙离子ATP酶（sarco/endoplasmic reticulum Ca2+ ATPase, SERCA）的特异性抑制剂——对来自墨西哥裔美国人家庭研究（Mexican American Family Study, MAFS）的2名受试者的诱导多能干细胞（induced pluripotent stem cell, iPSC）分化而来的神经干细胞（neural stem cells, NSCs）构建ER应激模型。对对照组与ER应激处理组NSCs的高内涵筛选（high content screening, HCS）及转录组分析显示，NSCs的ER应激应答会显著降低其自我更新能力，并增加细胞凋亡与氧化应激水平。共计2300个基因呈现显著差异表达（DE），筛选标准为：调节t检验的错误发现率（FDR）校正P值≤0.05，且折叠变化绝对值≥2.0。对差异表达基因的通路富集与基因网络分析显示，三条未折叠蛋白反应（unfolded protein response, UPR）通路（PERK、IRE1与ATF6）均被显著激活，并协同调控NSCs对ER应激的转录应答。本研究结果显示，IRE1/XBP1介导的E2F1及其下游靶基因的转录调控，在ER应激处理的NSCs中诱导G1/S期细胞周期阻滞的过程中发挥主导作用。ER应激处理的NSCs还表现出CHOP介导的细胞凋亡激活，以及突触可塑性与神经递质稳态相关基因的表达失调。综上，本研究结果表明，ER应激相关的NSCs自我更新能力减弱、细胞凋亡增加、突触可塑性与神经递质稳态失调，可能在ADRD的致病过程中发挥作用。本研究对来自2名不同受试者的经验证的iPSCs分化得到的特性明确的NSCs系，使用毒胡萝卜素（TG）进行24小时的ER应激处理。TG是SERCA的特异性抑制剂，也是研究哺乳动物细胞中ER应激与UPR生物学功能的经典工具。随后，本研究通过高内涵筛选（HCS）分析，对溶剂处理对照组与ER应激处理组NSCs的细胞增殖、细胞凋亡、蛋白质聚集、氧化应激、线粒体功能等细胞表型进行定量检测，并通过深度mRNA测序开展全基因组基因表达分析。