DataSheet5_Prenatal methadone exposure selectively alters protein expression in primary motor cortex: Implications for synaptic function.xlsx

As problematic opioid use has reached epidemic levels over the past 2 decades, the annual prevalence of opioid use disorder (OUD) in pregnant women has also increased 333%. Yet, how opioids affect the developing brain of offspring from mothers experiencing OUD remains understudied and not fully understood. Animal models of prenatal opioid exposure have discovered many deficits in the offspring of prenatal opioid exposed mothers, such as delays in the development of sensorimotor function and long-term locomotive hyperactivity. In attempt to further understand these deficits and link them with protein changes driven by prenatal opioid exposure, we used a mouse model of prenatal methadone exposure (PME) and preformed an unbiased multi-omic analysis across many sensoriomotor brain regions known to interact with opioid exposure. The effects of PME exposure on the primary motor cortex (M1), primary somatosensory cortex (S1), the dorsomedial striatum (DMS), and dorsolateral striatum (DLS) were assessed using quantitative proteomics and phosphoproteomics. PME drove many changes in protein and phosphopeptide abundance across all brain regions sampled. Gene and gene ontology enrichments were used to assess how protein and phosphopeptide changes in each brain region were altered. Our findings showed that M1 was uniquely affected by PME in comparison to other brain regions. PME uniquely drove changes in M1 glutamatergic synapses and synaptic function. Immunohistochemical analysis also identified anatomical differences in M1 for upregulating the density of glutamatergic and downregulating the density of GABAergic synapses due to PME. Lastly, comparisons between M1 and non-M1 multi-omics revealed conserved brain wide changes in phosphopeptides associated with synaptic activity and assembly, but only specific protein changes in synapse activity and assembly were represented in M1. Together, our studies show that lasting changes in synaptic function driven by PME are largely represented by protein and anatomical changes in M1, which may serve as a starting point for future experimental and translational interventions that aim to reverse the adverse effects of PME on offspring.

过去二十年间，阿片类药物问题性使用已攀升至流行病级别的严峻态势，孕妇群体中阿片类药物使用障碍（Opioid Use Disorder, OUD）的年患病率亦增长了333%。然而，针对阿片类药物如何对罹患OUD的母亲所孕育后代的发育中大脑产生影响这一问题，现有研究仍较为匮乏，且尚未得到充分阐明。产前阿片类药物暴露的动物模型研究已发现，产前暴露于阿片类药物的母鼠后代存在多方面发育缺陷，例如感觉运动功能发育延迟以及长期运动过度活跃。为进一步明晰此类缺陷，并将其与产前阿片类药物暴露所驱动的蛋白质变化建立关联，我们采用了产前美沙酮暴露（Prenatal Methadone Exposure, PME）小鼠模型，对已知与阿片类药物暴露存在相互作用的多个感觉运动脑区开展了无偏多组学分析。我们借助定量蛋白质组学与磷酸化蛋白质组学技术，评估了PME暴露对初级运动皮层（Primary Motor Cortex, M1）、初级躯体感觉皮层（Primary Somatosensory Cortex, S1）、背内侧纹状体（Dorsomedial Striatum, DMS）以及背外侧纹状体（Dorsolateral Striatum, DLS）的影响。研究发现，PME在所检测的全部脑区中均引发了大量蛋白质与磷酸肽丰度的改变。我们通过基因及基因本体（Gene Ontology, GO）富集分析，评估了各脑区内蛋白质与磷酸肽变化的调控模式。结果显示，相较于其他脑区，初级运动皮层（M1）受到PME的影响具有独特性：PME特异性地驱动了M1内谷氨酸能突触及突触功能的相关变化。免疫组织化学分析进一步证实，PME暴露会使M1内出现解剖学层面的差异——谷氨酸能突触密度上调，而γ-氨基丁酸能（GABAergic）突触密度下调。此外，对M1与非M1多组学数据的对比分析显示，与突触活动及组装相关的磷酸肽在全脑范围内存在保守性变化，但仅在M1中观测到突触活动与组装相关的特异性蛋白质改变。综合而言，本研究表明，PME所驱动的突触功能持久性改变，主要体现为M1内的蛋白质与解剖学层面的变化，这可为未来旨在逆转PME对后代产生的不良影响的实验性及转化性干预研究提供关键起点。