TNF deficiency causes changes in the spatial organization of neurogenic zones and the number of microglia and neurons in the cerebral cortex

Background: Although TNF inhibitors are used to treat chronic inflammatory diseases, there is little information about how long-term inhibition of TNF affects the homeostatic functions that TNF maintains in the intact CNS. TNF is known to modulate neurogenesis by decreasing cell proliferation, increasing apoptosis of precursor cells, and impairing neuronal differentiation. TNF can also influence the formation of the hippocampus, with long-lasting effects on cognition. Materials and methods: To clarify whether developmental TNF deficiency causes alterations in the naïve CNS, we estimated the number of proliferating cells, microglia, and neurons in the brains of E13.5, P7, and adult TNF +/+ and TNF-/- mice and measured changes in gene and protein expression and monoamine levels in adult TNF+/+ and TNF-/- mice. To evaluate long-term effects of TNF inhibitors, we treated healthy adult C57BL/6 mice with either saline, selective soluble TNF inhibitor XPro1595, or nonselective TNF inhibitor etanercept. We estimated changes in cell number and protein expression after two months of treatment. We assessed the effects of TNF deficiency on cognition by testing adult TNF+/+ and TNF-/- mice and anti-TNF treated mice with behavioral tasks. Results: TNF deficiency initially decreased the number of proliferating cells and microglia, but subsequently increased the number of neurons. At the same time, TNF deficiency decreased the expression of WNT signaling-related proteins, specifically Collagen Triple Helix Repeat Containing 1 (CTHRC1) and Frizzled receptor 6 (FZD6). In contrast to XPro1595, longterm inhibition of TNF with etanercept in adult C57BL/6 mice decreased the number of proliferating cells in the granule cell layer of the dentate gyrus. Etanercept, but not XPro1595, also impaired spatial learning and memory in the Barnes maze memory test. Conclusion: TNF deficiency impacts the organization of neurogenic zones and alters the cell composition in brain. Long-term inhibition of TNF with the nonselective TNF inhibitor etanercept, but not the soluble TNF inhibitor XPro1595, decreases neurogenesis in the adult mouse hippocampus and impairs learning and memory after two months of treatment. We first characterized the possible effects of developmental TNF deficiency and long-term inhibition of TNF in adulthood on gene and protein expression, monoamine levels, neurogenesis, and cell composition in the brain, and on the function and permeability of the blood-brain barrier (BBB). We then investigated the effect of TNF deficiency on cognitive functions. Adult male TNF+/+ and TNF-/- brain lysates were used for RNA isolation. Isolated RNA was quality assessed using the Agilent RNA 6000 Nano Kit (Agilent Technologies, Santa Clara, CA, USA) with an Agilent 2100 bioanalyzer (Agilent Technologies). Isolated RNA was hybridized to the Affymetrix GeneChip Mouse Gene 1.0 ST Array according to the manufactorer protocol (Thermo Fisher Scientific, Waltham, MA, USA). We used the Significance Analysis of Microarray (SAM) method to identify probe sets with target in transcripts that were significantly differentially expressed between TNF+/+ and TNF-/- mice (Tusher et al., 2001). SAM analysis was performed using MultiExperimentViewer Software v. 4.5.1. (Dana- Farber Cancer Institute, Boston, MA, USA), 70 unique permutations and an s0 value selected by Tusher’s method (Saeed et al., 2006; Saeed et al., 2003; Tusher et al., 2001). A false discovery rate (FDR) < 5% was considered evidence of statistical significance.

背景：尽管肿瘤坏死因子（TNF）抑制剂已被用于治疗慢性炎症性疾病，但关于长期抑制TNF如何维持完整中枢神经系统（CNS）内稳态功能的相关信息仍十分匮乏。已知TNF可通过降低细胞增殖、增加前体细胞凋亡以及损害神经元分化来调控神经发生。此外，TNF还可影响海马体形成，并对认知功能产生长期影响。
材料与方法：为明确发育阶段TNF缺陷是否会导致未致敏中枢神经系统出现异常，我们对E13.5胎龄、P7龄以及成年TNF+/+和TNF-/-小鼠的脑组织中增殖细胞、小胶质细胞与神经元数量进行了定量，并检测了成年TNF+/+与TNF-/-小鼠的基因与蛋白质表达水平以及单胺类物质含量。为评估TNF抑制剂的长期作用效果，我们将健康成年C57BL/6小鼠分为三组，分别给予生理盐水、选择性可溶性TNF抑制剂XPro1595或非选择性TNF抑制剂依那西普（etanercept）处理。持续给药两个月后，我们对细胞数量与蛋白质表达的变化进行了评估。通过行为学实验检测成年TNF+/+、TNF-/-小鼠以及抗TNF处理小鼠的认知功能，以此分析TNF缺陷对认知的影响。
结果：TNF缺陷最初会降低增殖细胞与小胶质细胞的数量，但后续会增加神经元的数量。与此同时，TNF缺陷会降低WNT信号通路相关蛋白的表达，具体为胶原蛋白三螺旋重复结构域包含蛋白1（CTHRC1, Collagen Triple Helix Repeat Containing 1）以及卷曲蛋白受体6（FZD6, Frizzled receptor 6）。与XPro1595不同，采用非选择性TNF抑制剂依那西普对成年C57BL/6小鼠进行长期TNF抑制处理后，其海马齿状回颗粒细胞层的增殖细胞数量出现下降。仅依那西普（而非XPro1595）会在巴恩斯迷宫记忆实验中损害小鼠的空间学习与记忆能力。
结论：TNF缺陷会影响神经发生区域的组织结构，并改变脑组织内的细胞组成。采用非选择性TNF抑制剂依那西普（而非可溶性TNF抑制剂XPro1595）进行长期TNF抑制，在持续给药两个月后，会降低成年小鼠海马体的神经发生水平并损害其学习记忆能力。本研究首次对发育阶段TNF缺陷以及成年阶段长期TNF抑制对脑组织内基因与蛋白质表达、单胺类物质含量、神经发生、细胞组成，以及血脑屏障（BBB）的功能与通透性的潜在影响进行了系统表征。随后我们探究了TNF缺陷对认知功能的影响。我们采用成年雄性TNF+/+与TNF-/-小鼠的脑组织裂解物进行RNA提取。提取的RNA质量通过安捷伦RNA 6000 Nano试剂盒（Agilent Technologies，美国加利福尼亚州圣克拉拉市安捷伦科技公司）以及安捷伦2100生物分析仪（Agilent Technologies）进行评估。按照制造商方案，将提取的RNA与Affymetrix GeneChip Mouse Gene 1.0 ST芯片进行杂交（赛默飞世尔科技，美国马萨诸塞州沃尔瑟姆市）。我们采用微阵列显著性分析（SAM, Significance Analysis of Microarray）方法，筛选出TNF+/+与TNF-/-小鼠之间转录本表达存在显著差异的探针集（Tusher等，2001）。SAM分析采用MultiExperiment Viewer软件v.4.5.1版本（丹娜-法伯癌症研究所，美国马萨诸塞州波士顿市）进行，包含70次独立置换，并通过Tusher方法选取s0值（Saeed等，2006；Saeed等，2003；Tusher等，2001）。错误发现率（FDR, false discovery rate）<5%被视为具有统计学显著性的依据。