Effect of fluoxetine treatment on translational profiles of S100a10 cortical pyramidal cells in p11 KOs

Molecular phenotyping of cell types and neural circuits underlying pathological neuropsychiatric conditions and their responses to therapy provides one avenue for the development of more specific and effective treatments. In this study, we identify a cell population in the cerebral cortex that shows robust and specific molecular adaptations following long-term SSRI treatment. We employed the bacTRAP strategy, which uses BAC transgenic mice expressing EGFP-tagged ribosomal protein L10a in specific cell populations, to affinity purify polysome-bound mRNAs from S100a10-expressing corticostriatal projection neurons. We show that the S100a10 cells are a unique population of cortical cells that are strongly and specifically responsive to chronic SSRI administration and that this response requires p11 (the protein product of S100a10). Our data demonstrate that the beneficial actions of antidepressant therapy can be mediated by a single cell type that is positioned to normalize activity between cortical and subcortical sites, and suggest that development of drugs that specifically target the activity of these cells may result in improved therapies to treat depression. S100a10 bacTRAP mice crossed to constitutive p11 KO mice (S100a10 bacTRAP/p11 KO) were administered either fluoxetine (FLX) or vehicle (VEH) in their drinking water for 15-18 days. Three independent TRAP replicates from the cortex of each group were then collected. Total RNA from the immunoprecipitates was amplified and hybridized. Data were normalized with the GCRMA algorithm and replicates were averaged across conditions. We recommend filtering data to remove probe sets with normalized expression values less than 50 in at least one condition. Because the S100a10 BAC labels some non-neuronal cells, we recommend only probe sets listed in Table S1 of the accompanying paper be included in the analysis.

针对病理性神经精神疾病及其治疗响应的细胞类型与神经环路开展分子表型分析，是开发更具特异性与有效性治疗手段的重要途径之一。本研究中，我们在大脑皮层中鉴定出一类细胞群，其在长期选择性5-羟色胺再摄取抑制剂（Selective Serotonin Reuptake Inhibitor, SSRI）给药后会出现显著且特异性的分子适应性改变。我们采用了细菌人工染色体介导的核糖体亲和纯化（bacterial artificial chromosome-based translating ribosome affinity purification, bacTRAP）技术：该技术利用在特定细胞群中表达增强绿色荧光蛋白（Enhanced Green Fluorescent Protein, EGFP）标记的核糖体蛋白L10a的细菌人工染色体（Bacterial Artificial Chromosome, BAC）转基因小鼠，从表达S100钙结合蛋白A10（S100a10）的皮质纹状体投射神经元中亲和纯化结合于多聚核糖体的信使RNA（mRNA）。我们证实，S100a10阳性细胞是一类独特的皮层细胞群，对慢性SSRI给药呈现强烈且特异性的响应，且该响应依赖于p11（即S100a10编码的蛋白产物）。本研究数据表明，抗抑郁治疗的获益效应可由一类特定细胞类型介导：该细胞群能够调节皮层与皮层下脑区之间的活动稳态，同时提示，特异性靶向这类细胞活性的药物开发或可获得更优的抑郁症治疗方案。将S100a10 bacTRAP小鼠与组成型p11基因敲除（knockout, KO）小鼠杂交得到S100a10 bacTRAP/p11 KO小鼠，向其饮用水中添加氟西汀（Fluoxetine, FLX）或赋形剂（Vehicle, VEH），给药时长为15至18天。随后从每组小鼠的皮层中采集3个独立的TRAP实验重复样本。对免疫沉淀物中的总RNA进行扩增并用于杂交实验。使用基因芯片稳健多阵列分析（GeneChip Robust Multiarray Analysis, GCRMA）算法对数据进行标准化处理，并对各实验条件下的重复样本数据取平均值。我们建议对数据进行过滤，移除至少在一个实验条件下标准化表达值低于50的探针集。由于S100a10 BAC会标记部分非神经元细胞，因此建议仅将伴随论文表S1中列出的探针集纳入分析。