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、不断增长的流行病，没有明确的诊断手段， 预防，或疾病修正治疗。AD具有长达数十年的前驱期，在此期间淀粉样蛋白-β (aβ)积聚先于神经变性和认知障碍。这段时间代表着一个关键的 不可逆性神经变性前阻断的治疗干预窗口。突触丢失是 尽管神经元特异性和小胶质细胞依赖，但最强的病理与认知能力下降有关 导致突触丢失的机制尚不清楚。为此，我们在认识上存在严重差距。 (1)由β病理驱动的突触中发生的早期分子变化，(2)年龄对β的影响- 诱导突触的分子变化；(3)小胶质细胞在这些早期突触变化中的作用。这个 这项建议的总体目标是确定β沉积对小鼠的年龄依赖性和突触特异性的影响 AD模型，并确定小胶质细胞在β驱动的突触蛋白质组变化中所起的作用。我的中央 假设小胶质细胞是早期突触蛋白质组变化的关键媒介，这种变化是由于 进行性Aβ病理。我将通过两个具体目标严格检验我的假设。首先(目标1)，我会 Aβ病理对神经元突触蛋白质水平变化的早期和年龄依赖性影响的特征。我 将通过以下方法研究β病理(5xFAD)小鼠模型中神经元和突触的特异性变化 表达混杂生物素连接酶(TurboID)以特异性标记神经元中表达的蛋白质 突触分裂。这将使我们能够从突触分离的制剂中提纯神经元蛋白质 而不需要细胞类型隔离。通过将质谱学(MS)应用于这些样本，我将定义蛋白质 β病理和老化(1.5个月、3个月和6个月)导致的神经元变化 差异表达和网络方法的结合(目标1)。这些研究将揭示蛋白质 在突触丧失和认知障碍之前出现的由淀粉样蛋白病理改变的特征。 下一步(目标2)，我将机械地确定小胶质细胞如何在早期β驱动的病理改变中起作用 使用体内小胶质细胞耗竭策略(CSF1R抑制)的神经元。神经元标记的5xFAD和WT小鼠 将经历小胶质细胞的枯竭，我们将应用MS来识别早期神经元蛋白的变化 小胶质细胞依赖和非小胶质细胞依赖。通过将来自两个AIMS的小鼠蛋白质组数据与 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进展的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， 尤其是需要解决因衰老而在神经元和突触中发生的早期分子变化， Aβ的病理和免疫机制。

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.