Unbiased proteomic analyses of plasma and brain tissue from TBI and AD mouse models II

The relationship between repetitive mild traumatic brain injury (r-mTBI) and Alzheimer’s disease (AD) is well-recognized. However, the precise nature of how r-mTBI leads to or precipitates AD pathogenesis is currently not understood. Part A: Plasma biomarkers potentially provide non-invasive tools for detecting neurological changes in the brain, and can reveal overlaps between long-term consequences of r-mTBI and AD. In this study we address this by generating time-dependent molecular profiles of response to r-mTBI and AD pathogenesis in mouse models using unbiased proteomic analyses. To model AD, we used the well-validated hTau and PSAPP(APP/PS1) mouse models that develop age-related tau and amyloid pathological features respectively, and our well-established model of r-mTBI in C57BL/6 mice. Plasma were collected at different ages (3, 9, and 15 months-old for hTau and PSAPP mice), encompassing pre-, peri- and post-“onset” of the cognitive and neuropathological phenotypes, or at different timepoints after r-mTBI (24hrs, 3, 6, 9 and 12 months post-injury). Liquid chromatography/mass spectrometry (LC-MS) approaches coupled with Tandem Mass Tag labeling technology were applied to develop molecular profiles of protein species that were significantly differentially expressed as a consequence of mTBI or AD. Mixed model ANOVA after Benjamini-Hochberg correction, and a stringent cut-off identified 31 proteins significantly changing in r-mTBI groups over time and, when compared with changes over time in sham mice, 13 of these were unique to the injured mice. The canonical pathways predicted to be modulated by these changes were LXR/RXR activation, production of nitric oxide and reactive oxygen species and complement systems. We identified 18 proteins significantly changing in PSAPP mice and 19 proteins in hTau mice compared to their wildtype littermates with ageing. Six proteins were found to be significantly regulated in all three models i.e. r-mTBI, hTau and PSAPP mice compared to their controls. The top canonical pathways coincidently changing in all three models were LXR/RXR activation, and production of nitric oxide and reactive oxygen species. This work suggests potential biomarkers for TBI and AD pathogenesis and for the overlap between these two, and warrant targeted investigation in human populations. Part B: In this part of the study we address the above problem by utilizing our unbiased proteomic approach to generate detailed time-dependent brain molecular profiles of response to repetitive mTBI and AD pathogenesis in established mouse models. The same animal models described above were used herein. A LC/MS approach coupled with TMT labeling was also employed. Results: Mixed model ANOVA after Benjamin Hochberg correction identified 30 and 47 proteins that were specifically unique and changing in the hippocampus and cortex, respectively, within the r-mTBI group alone when compared with changes overtime in sham mice. PI3K/AKT signaling, Protein Kinase A signaling and PPAR/RXR activation in the hippocampus, and Protein Kinase A signaling, GNRH signaling and B cell receptor signaling in the cortex were the top canonical systems significantly altered in injury groups compared to sham mice. Mixed model AONVA identified 19 proteins significantly changing in the cortex of PSAPP mice and 7 proteins in hTau mice compared to their relative wildtype littermates respectively. In addition to the heterogeneous changes observed in the TBI and AD mouse models, there was a notable convergence and coincidental change in 6 unique proteins identified in the repetitive mTBI model and the hTau and PSAPP model. These proteins ostensibly indicate significant common pathobiological responses involving alterations in mitochondrial bioenergetics and energy metabolism, aberrant cytoskeletal reorganization and alterations in intracellular signaling transduction cascades. Conclusion: We believe that this work could help identify the common molecular substrates responsible for the precipitation of AD pathogenesis following repetitive mTBI, and also help to identify novel biological targets for therapeutic modulation in mTBI and AD.

重复性轻度创伤性脑损伤（repetitive mild traumatic brain injury, r-mTBI）与阿尔茨海默病（Alzheimer’s disease, AD）之间的关联已得到广泛认可，但目前学界尚未明确r-mTBI诱发或促进AD发病的确切机制。第一部分：血浆生物标志物（plasma biomarkers）可作为无创工具检测脑部神经系统变化，同时能够揭示r-mTBI长期后遗症与AD之间的重叠病理机制。本研究通过无偏倚蛋白质组学分析，构建小鼠模型中r-mTBI应答与AD发病的时间依赖性分子谱，以此解决上述科学问题。为构建AD模型，本研究采用已得到充分验证的hTau与PSAPP(APP/PS1)小鼠模型，二者可分别模拟年龄相关性tau蛋白与淀粉样蛋白病理特征；同时采用我们已建立的C57BL/6小鼠r-mTBI模型。分别在不同年龄（hTau与PSAPP小鼠为3、9、15月龄）采集血浆，覆盖认知与神经病理表型的“发病前”“发病期”及“发病后”阶段；或在r-mTBI造模后不同时间点（伤后24小时、3、6、9及12个月）采集血浆。本研究采用液相色谱/质谱（liquid chromatography/mass spectrometry, LC-MS）结合串联质量标签（Tandem Mass Tag, TMT）标记技术，对因mTBI或AD出现显著差异表达的蛋白质分子进行谱图构建。经Benjamini-Hochberg校正的混合模型方差分析（mixed model ANOVA）及严格阈值筛选后，r-mTBI组中共鉴定出31个随时间变化的差异表达蛋白；与假手术小鼠的时间依赖性变化相比，其中13个蛋白仅在损伤小鼠中出现特异性改变。上述蛋白调控的经典通路包括LXR/RXR激活、一氧化氮与活性氧生成及补体系统。与同龄野生型同窝仔鼠相比，PSAPP小鼠中鉴定出18个差异表达蛋白，hTau小鼠中则鉴定出19个差异表达蛋白。在r-mTBI、hTau及PSAPP三种模型中，均存在6个显著调控的共有蛋白。三种模型共同富集的经典通路为LXR/RXR激活、一氧化氮与活性氧生成。本研究鉴定出可用于TBI与AD发病机制研究的潜在生物标志物，以及二者重叠病理的相关靶点，为后续人群队列的靶向研究提供了依据。第二部分：本部分研究采用上述已建立的小鼠模型，通过无偏倚蛋白质组学方法构建重复性mTBI应答与AD发病的时间依赖性脑部分子谱，以此解决前述科学问题。实验同样采用LC/MS结合TMT标记技术。结果：经Benjamini-Hochberg校正的混合模型方差分析显示，与假手术小鼠的时间依赖性变化相比，仅r-mTBI组的海马体（hippocampus）与大脑皮层（cortex）中分别鉴定出30个与47个特异性差异表达蛋白。与假手术组相比，损伤组显著改变的顶部经典通路包括：海马体中的PI3K/AKT信号通路、蛋白激酶A信号通路及PPAR/RXR激活；大脑皮层中的蛋白激酶A信号通路、GNRH信号通路及B细胞受体信号通路。混合模型方差分析显示，与对应野生型同窝仔鼠相比，PSAPP小鼠大脑皮层中鉴定出19个差异表达蛋白，hTau小鼠中则鉴定出7个差异表达蛋白。除TBI与AD小鼠模型中观察到的异质性变化外，重复性mTBI模型与hTau、PSAPP模型中共存在6个特异性共有差异蛋白。上述蛋白提示二者存在共同的病理生物学应答，涉及线粒体生物能学与能量代谢改变、异常细胞骨架重构及细胞内信号转导级联变化。结论：本研究有望明确重复性mTBI后AD发病的共同分子基础，同时为TBI与AD的治疗调控提供新型生物学靶点。