In-vivo biotinylation to capture neuron-specific proteomic changes in mouse models of Alzheimer's disease pathology

项目摘要 阿尔茨海默病（AD）是一种毁灭性的和日益增长的流行病，没有明确的诊断手段， 预防或改善疾病的治疗。AD的特征是一个长达数十年的前驱期，其中淀粉样蛋白-β （Aβ）蓄积先于神经变性和认知障碍。这段时间代表了一个关键的 在不可逆的神经变性之前阻断治疗干预的窗口。突触丧失是 最强的病理学与认知能力下降相关，尽管神经元特异性和小胶质细胞依赖性 导致突触丧失的机制尚不清楚。为此，我们的理解存在着关键的差距， （1）由Aβ病理驱动的突触中发生的早期分子变化，（2）年龄对Aβ- 诱发突触的分子变化，以及（3）小胶质细胞在这些早期突触变化中的作用。的 本提案的总体目标是确定小鼠中Aβ沉积的年龄依赖性和突触特异性效应 AD模型，并确定小胶质细胞在Aβ驱动的突触蛋白质组学变化中的作用。我的中枢 一种假说认为，小胶质细胞是早期突触蛋白质组变化的关键介质， 进行性Aβ病理学。我将通过两个具体目标严格检验我的假设。第一（目标1），我会 表征Aβ病理学对神经元突触中蛋白水平变化的早期和年龄依赖性影响。我 将研究Aβ病理学小鼠模型（5xFAD）中神经元和突触特异性变化， 表达混杂的生物素连接酶（TurboID）以特异性标记神经元中表达的蛋白质，然后 突触分离这将使我们能够从突触分离的制备物中纯化神经元蛋白质 而不需要细胞类型分离。通过对这些样品进行质谱分析，我将定义蛋白质 作为Aβ病理学和衰老（1.5、3和6个月）的结果，使用 差异表达和网络方法的组合（目标1）。这些研究将揭示蛋白质 在突触丧失和认知损害之前出现的淀粉样蛋白病理学特异性改变的特征。 接下来（目标2），我将从机制上确定小胶质细胞如何促进早期Aβ驱动的病理变化 在神经元中使用体内小胶质细胞耗竭策略（CSF 1 R抑制）。神经元标记的5xFAD和WT小鼠 将经历小胶质细胞耗竭，我们将应用MS来识别早期神经元蛋白质的变化， 小胶质细胞依赖性和小胶质细胞非依赖性。通过整合来自两个目标的小鼠蛋白质组数据， 通过加速药物伙伴关系（AMP-AD）产生的现有人脑蛋白质组，我将 能够识别AD进展早期症状前阶段神经元中发生的分子变化。 这些目标的成功完成将解决神经蛋白质组学和AD领域的关键空白， 特别是需要解决由于老化而在神经元和突触中发生的早期分子变化， Aβ病理学和免疫机制。

PROJECT ABSTRACT Alzheimer’s disease (AD) is a devastating and growing epidemic without means of definitive diagnosis, prevention, or disease-modifying treatment. AD features a decades-long prodromal period in which amyloid-β (Aβ) accumulation precedes neurodegeneration and cognitive impairment. This time period represents a critical window of therapeutic intervention to intercept before irreversible neurodegeneration. Synaptic loss is the strongest pathological correlate to cognitive decline, although neuron specific and microglial dependent mechanisms contributing to synaptic loss are unknown. To this end, there are critical gaps in our understanding of (1) early molecular changes occurring in synapses driven by Aβ pathology, (2) the impact of age on Aβ- induced molecular changes in the synapse, and (3) the role of microglia in these early synaptic changes. The overall goal of this proposal is to identify age-dependent and synapse-specific effects of Aβ deposition in mouse models of AD, and to determine the role microglia play in Aβ-driven synaptic proteomic changes. My central hypothesis is that microglia are key mediators of early synaptic proteomic changes that occur as a result of progressive Aβ pathology. I will rigorously test my hypothesis through two specific aims. First (Aim 1), I will characterize early and age-dependent effects of Aβ pathology on protein-level changes in neuronal synapses. I will study neuronal as well as synapse-specific changes in a mouse model of Aβ pathology (5xFAD) by expressing a promiscuous biotin ligase (TurboID) to specifically label proteins expressed in neurons followed by synaptic fractionation. This will allow us to purify neuronal proteins from synapse-fractionated preparations without requiring cell type isolation. By applying mass spectrometry (MS) to these samples, I will define protein changes occurring specifically in neurons as a result of Aβ pathology and aging (1.5, 3 and 6 months) using a combination of differential expression and network approaches (Aim 1). These studies will reveal protein signatures altered specifically by amyloid pathology which emerge before synaptic loss and cognitive impairment. Next (Aim 2), I will mechanistically determine how microglia contribute to early Aβ-driven pathological changes in neurons using an in-vivo microglia depletion strategy (CSF1R inhibition). Neuron-labeled 5xFAD and WT mice will undergo microglia depletion and we will apply MS to identify early neuronal protein changes that are microglia-dependent and microglia-independent. By integrating mouse proteomic data from both aims with existing human brain proteomic generated through the accelerating medicines partnership (AMP-AD), I will be able to identify molecular changes occurring in neurons in early pre-symptomatic stages of AD progression. Successful completion of these aims will address critical gaps in the fields of neuroproteomics and AD, particularly the need to resolve early molecular changes occurring in neurons and synapses as a result of aging, Aβ pathology and immune mechanisms.