Understanding Roles for Protein Homeostasis Machinery in Aging Brain Vasculature

了解蛋白质稳态机制在衰老脑血管中的作用

• 批准号: 10537760
• 负责人: Richard Giadone
• 金额: $ 6.76万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10537760
• 关键词: Age; Aging; Alzheimer' s Disease; Alzheimer' s disease related dementia; Amyloid beta-Protein; Animals; Biochemical; Biological Assay; Blood; Blood - brain barrier anatomy; Brain; Cardiovascular system; Cell Culture Techniques; Cell physiology; Cells; Cerebrovascular system; Cognitive; Complement; Disease; Electrical Resistance; Endothelium; Exhibits; Exposure to; Functional disorder; Gene Expression Profiling; Genes; Heat shock proteins; Heat-Shock Response; Hippocampus (Brain); Homeostasis; Human; Impaired cognition; Impairment; In Vitro; Incidence; Link; Mass Spectrum Analysis; Measures; Modeling; Molecular; Molecular Chaperones; Mus; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Operative Surgical Procedures; Outcome; Parabiosis; Pathogenesis; Pathologic; Pathway interactions; Patients; Permeability; Phenotype; Physiological; Proteins; Proteomics; Risk Factors; Role; Sampling; Serotyping; Stress; System; Techniques; Therapeutic; Viral; Work; adeno-associated viral vector; aged; aging brain; base; biological adaptation to stress; brain cell; brain endothelial cell; cell type; cytokine; differential expression; experimental study; functional decline; functional improvement; functional restoration; healthy aging; in vitro Model; in vivo; induced pluripotent stem cell; juvenile animal; knock-down; misfolded protein; normal aging; novel; novel therapeutics; overexpression; protein aggregation; protein expression; proteostasis; restoration

PROJECT SUMMARY Aging is the largest risk factor for a majority of neurodegenerative disorders, including Alzheimer’s disease (AD) and related dementias. Despite growing incidence for such disorders, zero therapeutics are capable of reversing progression of aging-related cognitive decline seen in disease and healthy aging. Through the use of heterochronic parabiosis however, or the surgical joining of circulatory systems between young and old mice, our group demonstrated that blood-borne factors from young animals are capable of reversing many deleterious phenotypes seen in the aged brain. Understanding the molecular mechanisms underlying cognitive improvement post-parabiosis may uncover novel therapeutic avenues for aging-related neurodegeneration. One hallmark of aging linked to the onset of AD is the collapse of protein homeostasis (proteostasis) networks that regulate the folding of newly synthesized proteins via molecular chaperones as well as the degradation of pathologic misfolded proteins. During aging, cellular proteostatic capacity declines, leading to an increase in protein aggregates with physiological consequences driven by activation of stress response pathways in downstream cell types. Recently, by profiling transcriptional changes occurring in the aging mouse brain, our group identified chaperones that decrease in brain endothelial cells (BECs) with age, while simultaneously, in the same cells, levels of stress-inducible genes known to increase upon sensing misfolded proteins (e.g. Hspa1a, Hsp90aa1) were elevated – signatures which reversed post-parabiosis. Although BECs are some of the most vulnerable cells in AD, with many patients exhibiting altered blood-brain barrier (BBB) integrity, proteostasis machinery of these critical barrier cells has not been investigated. Taken together, in aging BECs, (1) molecular chaperones decrease, while (2) stress-inducible heat shock proteins (HSPs) increase, potentially reflecting the presence of misfolded proteins throughout the aged brain. To this end, we first seek to identify proteins that aggregate in the brain during aging and upon parabiosis via mass spectrometry. In doing so, we will profile aging-associated proteins which aggregate in a deleterious, yet reversible manner. Will then assess the ability of identified aggregation-prone proteins to activate stress response pathways in BECs. While our first aim asks, what is responsible for activation of stress-inducible HSP expression in aged BECs, our second asks what impact does increased HSP expression have on BEC function? This will be assessed by modulating Hspa1a (the most differentially expressed HSP in aging) levels using primary murine and human BECs alongside AD mutation-containing human induced pluripotent stem cell (iPSC)-derived BECs via lentiviral constructs. In parallel, we will modulate levels of Hspa1a in vivo specifically within BECs of mice via adeno-associated viral vectors. Expression of BEC markers and function will then be measured. The experiments outlined herein investigate roles for proteostasis and related stress response machinery in the aging brain, potentially identifying novel avenues for BEC/BBB-targeting AD and dementia therapeutics.

项目总结 衰老是包括阿尔茨海默病(AD)在内的大多数神经退行性疾病的最大风险因素 以及相关的痴呆症。尽管此类疾病的发病率不断上升，但零疗法能够逆转 疾病和健康衰老中与衰老相关的认知衰退的进展。通过使用 然而，异慢性异种共生，即幼鼠和老年鼠之间循环系统的外科连接， 我们的小组证明了幼年动物的血液传播因子能够逆转许多有害的 在衰老的大脑中可见的表型。理解认知改善的分子机制 异种复苏后可能为衰老相关的神经退行性变找到新的治疗途径。 与阿尔茨海默病发病有关的衰老的一个标志是蛋白质稳态的崩溃(蛋白质稳态) 通过分子伴侣调节新合成蛋白质折叠的网络以及 病理性错误折叠蛋白的降解。在衰老过程中，细胞的蛋白抑制能力下降，导致 由应激反应的激活引起的具有生理后果的蛋白质聚集体的增加 下游细胞类型中的通路。最近，通过描绘衰老小鼠中发生的转录变化 大脑，我们的团队发现随着年龄的增长，脑内皮细胞(BECs)中的伴侣蛋白会减少，而 同时，在同一细胞中，已知的在感觉到错误折叠时压力诱导基因的水平会增加 蛋白质(如Hspa1a、Hsp90aa1)升高--这是逆转后异种共生的信号。虽然BEC 是阿尔茨海默病中最脆弱的细胞，许多患者表现出血脑屏障(BBB)的改变 这些关键屏障细胞的完整性、蛋白平衡机制尚未得到研究。 综上所述，在老化的BEC中，(1)分子伴侣减少，而(2)应激诱导的热休克 蛋白质(热休克蛋白)增加，潜在地反映了整个衰老大脑中存在错误折叠的蛋白质。至 为此，我们首先寻求确定在衰老过程中和在异种共生时通过质量聚集在大脑中的蛋白质。 光谱分析。在这样做的过程中，我们将分析与衰老相关的蛋白质，这些蛋白质聚集在一个有害的、尚未 可逆方式。然后将评估已识别的易于聚集的蛋白质激活压力的能力 BEC中的反应通路。虽然我们的第一个目标是问，是什么导致了应激诱导的热休克蛋白的激活 在老年BEC中的表达，我们的第二个问题是HSP表达增加对BEC功能有什么影响？ 这将通过调节Hspa1a(衰老过程中表达差异最大的HSP)水平来进行评估 含AD突变的人诱导多能干细胞(IPSC)来源的人和鼠BECs 通过慢病毒构建的BEC。同时，我们将在体内调节Hspa1a的水平，特别是在BEC中 小鼠通过腺相关病毒载体。然后将测量BEC标志物的表达和功能。 本文概述的实验研究了蛋白平衡和相关的应激反应机制的作用。 在老化的大脑中，潜在地确定了BEC/BBB-靶向AD和痴呆治疗的新途径。