Targeting RNA polymerase-1 (POL1) machinery is a new strategy for suppression of multiple sclerosis (MS) relapse activity. Oral administration of POL1 inhibitor RAM-589.555, which is characterized by high permeability and bioavailability in naïve mice, ameliorates proteolipid protein (PLP)-induced experimental autoimmune encephalomyelitis (EAE) by suppressing activated autoreactive lymphocytes. We assessed the accessibility of RAM-589.555 to the central nervous system (CNS) of EAE-mice and further investigated its immunomodulatory effects on CNS-resident astro- and micro-glial cells in-vitro and in-vivo. Effects of RAM-589.555 on activated microglia and astrocyte viability, proliferation, and secretion of neurotrophic factors were assessed in-vitro. The pharmacokinetic of RAM-589.555 was evaluated in the blood and central nervous system (CNS) of EAE-affected mice. High-dimensional single-cell mass cytometry was applied to characterize the effect of RAM-589.555 on EAE-affected mice’s CNS-resident micro- and astroglial cells and CNS-infiltrating immune cells, which were obtained seven days after RAM-589.555 administration at EAE onset. Simultaneously, the expression level of pre-rRNA, the POL1 end product, was assessed in blood cells, microglia, and astrocytes to monitor RAM-589.555 effects. RAM-589.555 demonstrated blood and CNS permeability in EAE mice. In-vitro, incubation with 400 nM of RAM-589.555 significantly reduced viability and proliferation of lipopolysaccharide (LPS)-activated microglia by 70% and 45% (p < 0.05), respectively, while tumor necrosis factor α (TNFα)-activated astrocytes were not affected. The secretion of neurotrophic factors was preserved. Furthermore, 7 days after administration of RAM-589.555 at EAE onset, the level of pre-rRNA transcript in peripheral blood mononuclear cells (PBMC) was decreased by 38.6% (p = 0.02), while levels of pre-rRNA transcript in microglia and astrocytes remained unchanged. The high-dimensional single-cell mass cytometry analysis showed decreased percentages of CNS-resident microglia and astrocytes, diminished pro-inflammatory cytokines (IL-1β, IL-6, IL-12, IL-17, TNFα, and IFNγ), and an increase of their anti-inflammatory cytokines (IL-4, IL-10, and TGFβ) in RAM-589.555-treated compared to vehicle-treated mice (p < 0.05). These data correlate RAM-589.555-induced clinical amelioration and its CNS-permeability to decreased CNS-inflammation, and decreased micro- and astrogliosis, while restoring micro- and astroglial anti-inflammatory and neuroprotective capacity.

中文翻译：

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RAM-589.555 有利于急性复发性 EAE 小鼠中 CNS 驻留神经胶质细胞的神经保护和抗炎特性

靶向 RNA 聚合酶 1 (POL1) 机制是抑制多发性硬化 (MS) 复发活动的新策略。POL1 抑制剂 RAM-589.555 的口服给药具有高渗透性和生物利用度，可通过抑制活化的自身反应性淋巴细胞来改善蛋白脂蛋白 (PLP) 诱导的实验性自身免疫性脑脊髓炎 (EAE)。我们评估了 RAM-589.555 对 EAE 小鼠中枢神经系统 (CNS) 的可及性，并进一步研究了其在体外和体内对 CNS 驻留星形和小胶质细胞的免疫调节作用。体外评估 RAM-589.555 对活化小胶质细胞和星形胶质细胞活力、增殖和神经营养因子分泌的影响。RAM-589 的药代动力学。在受 EAE 影响的小鼠的血液和中枢神经系统 (CNS) 中评估了 555。应用高维单细胞质量流式细胞术来表征 RAM-589.555 对 EAE 影响小鼠的中枢神经系统驻留微和星形胶质细胞以及中枢神经系统浸润免疫细胞的影响，这些细胞是在 EAE 施用 RAM-589.555 后 7 天获得的发作。同时，在血细胞、小胶质细胞和星形胶质细胞中评估了 POL1 终产物 pre-rRNA 的表达水平，以监测 RAM-589.555 的影响。RAM-589.555 证明了 EAE 小鼠的血液和 CNS 通透性。在体外，与 400 nM RAM-589.555 孵育显着降低了脂多糖 (LPS) 激活的小胶质细胞的活力和增殖 70% 和 45% (p < 0.05)，而肿瘤坏死因子 α (TNFα) 激活的星形胶质细胞没有受到影响。神经营养因子的分泌得以保留。此外，在 EAE 发病时施用 RAM-589.555 7 天后，外周血单核细胞 (PBMC) 中的 pre-rRNA 转录水平降低了 38.6% (p = 0.02)，而小胶质细胞和星形胶质细胞保持不变。高维单细胞质量流式细胞术分析显示中枢神经系统驻留小胶质细胞和星形胶质细胞的百分比降低，促炎细胞因子（IL-1β、IL-6、IL-12、IL-17、TNFα 和 IFNγ）减少，并且与载体治疗的小鼠相比，RAM-589.555 治疗的小鼠的抗炎细胞因子（IL-4、IL-10 和 TGFβ）增加（p < 0.05）。这些数据将 RAM-589.555 诱导的临床改善及其 CNS 通透性与减少 CNS 炎症以及减少微量和星形胶质细胞增生相关联，