Proteomic studies using postmortem human brain tissue samples have yielded robust assessments of the aging and neurodegenerative disease(s) proteomes. While these analyses provide lists of molecular alterations in human conditions, like Alzheimer's disease (AD), identifying individual proteins that affect biological processes remains a challenge. To complicate matters, protein targets may be highly understudied and have limited information on their function. To address these hurdles, we sought to establish a blueprint to aid selection and functional validation of targets from proteomic datasets. A cross-platform pipeline was engineered to focus on synaptic processes in the entorhinal cortex (EC) of human patients, including controls, preclinical AD, and AD cases. Label-free quantification mass spectrometry (MS) data (n = 2260 proteins) was generated on synaptosome fractionated tissue from Brodmann area 28 (BA28; n = 58 samples). In parallel, dendritic spine density and morphology was measured in the same individuals. Weighted gene co-expression network analysis was used to construct a network of protein co-expression modules that were correlated with dendritic spine metrics. Module-trait correlations were used to guide unbiased selection of Twinfilin-2 (TWF2), which was the top hub protein of a module that positively correlated with thin spine length. Using CRISPR-dCas9 activation strategies, we demonstrated that boosting endogenous TWF2 protein levels in primary hippocampal neurons increased thin spine length, thus providing experimental validation for the human network analysis. Collectively, this study describes alterations in dendritic spine density and morphology as well as synaptic proteins and phosphorylated tau from the entorhinal cortex of preclinical and advanced stage AD patients.

SIGNIFICANCE STATEMENT Proteomic studies can yield vast lists of molecules that are altered under various experimental or disease conditions. Here, we provide a blueprint to facilitate mechanistic validation of protein targets from human brain proteomic datasets. We conducted a proteomic analysis of human entorhinal cortex (EC) samples spanning cognitively normal and Alzheimer's disease (AD) cases with a comparison of dendritic spine morphology in the same samples. Network integration of proteomics with dendritic spine measurements allowed for unbiased discovery of Twinfilin-2 (TWF2) as a regulator of dendritic spine length. A proof-of-concept experiment in cultured neurons demonstrated that altering Twinfilin-2 protein level induced corresponding changes in dendritic spine length, thus providing experimental validation for the computational framework.

使用死后人类脑组织样本进行的蛋白质组学研究对衰老和神经退行性疾病蛋白质组进行了可靠的评估。虽然这些分析提供了人类疾病（如阿尔茨海默氏病（AD））的分子变化列表，但识别影响生物过程的单个蛋白质仍然是一个挑战。更复杂的是，蛋白质靶点的研究可能还很不够，并且关于其功能的信息也很有限。为了解决这些障碍，我们试图建立一个蓝图来帮助从蛋白质组数据集中选择和功能验证目标。跨平台管道的设计重点关注人类患者内嗅皮层 (EC) 的突触过程，包括对照、临床前 AD 和 AD 病例。无标记定量质谱 (MS) 数据（n = 2260 个蛋白质）是在来自 Brodmann 区域 28（BA28； n = 58 个样本）的突触体分级组织上生成的。同时，测量同一个体的树突棘密度和形态。使用加权基因共表达网络分析来构建与树突棘指标相关的蛋白质共表达模块网络。模块-性状相关性用于指导 Twinfilin-2 (TWF2) 的无偏选择，TWF2 是模块的顶部中心蛋白，与细脊柱长度呈正相关。使用 CRISPR-dCas9 激活策略，我们证明提高初级海马神经元内源性 TWF2 蛋白水平会增加细脊柱长度，从而为人类网络分析提供实验验证。总的来说，这项研究描述了临床前和晚期 AD 患者的内嗅皮层树突棘密度和形态以及突触蛋白和磷酸化 tau 蛋白的变化。

意义声明蛋白质组学研究可以产生大量在各种实验或疾病条件下发生改变的分子。在这里，我们提供了一个蓝图，以促进人脑蛋白质组数据集中蛋白质靶标的机械验证。我们对认知正常和阿尔茨海默病 (AD) 病例的人类内嗅皮层 (EC) 样本进行了蛋白质组学分析，并比较了同一样本中的树突棘形态。蛋白质组学与树突棘测量的网络集成允许公正地发现 Twinfilin-2 (TWF2) 作为树突棘长度的调节剂。在培养神经元中进行的概念验证实验表明，改变 Twinfilin-2 蛋白水平会引起树突棘长度的相应变化，从而为计算框架提供了实验验证。