Understanding Roles for Protein Homeostasis Machinery in Aging Brain Vasculature

项目摘要 衰老是大多数神经退行性疾病的最大风险因素，包括阿尔茨海默病（AD） 以及相关的痴呆症尽管这类疾病的发病率不断增加，但没有任何治疗方法能够逆转 在疾病和健康老龄化中观察到的与衰老相关的认知衰退的进展。通过使用 然而，异时共生，或者年轻小鼠和老年小鼠之间的循环系统的手术连接， 我们的研究小组证明，来自年轻动物的血液传播因子能够逆转许多有害的 在老年大脑中看到的表型。了解认知改善的分子机制 后联体可能揭示衰老相关神经变性的新的治疗途径。 与AD发病相关的衰老的一个标志是蛋白质稳态（proteostasis）的崩溃 通过分子伴侣调节新合成蛋白质折叠的网络， 病理性错误折叠蛋白质的降解。在衰老过程中，细胞的蛋白质稳定能力下降，导致 蛋白质聚集体增加，具有由应激反应激活驱动的生理后果 下游细胞类型的通路。最近，通过分析衰老小鼠中发生的转录变化， 在大脑中，我们的研究小组确定了随着年龄增长脑内皮细胞（BEC）减少的伴侣蛋白， 同时，在相同的细胞中，应激诱导基因的水平已知在感知错误折叠时增加。 蛋白质（例如Hspa 1a、Hsp 90 aa 1）升高-这是逆转联体后的特征。虽然BEC 是AD中最脆弱的细胞，许多患者表现出改变的血脑屏障（BBB） 完整性，这些关键屏障细胞的蛋白质稳定机制尚未研究。 总之，在老化的BEC中，（1）分子伴侣减少，而（2）应激诱导的热休克 热休克蛋白（HSP）增加，可能反映了整个老年大脑中错误折叠蛋白的存在。到 为此，我们首先试图确定在衰老过程中和在通过质量共生时在大脑中聚集的蛋白质。 光谱法在此过程中，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，但 可逆的方式。然后将评估鉴定出的易聚集蛋白激活应激的能力 BECs的反应途径。而我们的第一个目标是问，什么是负责激活应激诱导HSP 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Hspa 1a（衰老中最差异表达的HSP）水平来评估，使用主要的 鼠和人BEC以及含有AD突变的人诱导多能干细胞（iPSC）衍生的 通过慢病毒构建体的BEC。与此同时，我们将在体内特异性地调节Hspa 1a在BEC中的水平， 小鼠通过腺相关病毒载体。然后测量BEC标志物的表达和功能。 本文概述的实验研究蛋白质稳态和相关应激反应机制的作用 在老化的大脑中，可能确定BEC/BBB靶向AD和痴呆治疗的新途径。

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.