Metachromatic leukodystrophy (MLD) is a severe demyelinating, autosomal recessive genetic leukodystrophy, with no curative treatment. The disease is underpinned by mutations in the arylsulfatase A gene (ARSA), resulting in deficient activity of this lysosomal enzyme, and consequential accumulation of galactosylceramide-3-O-sulfate (sulfatide) in the brain. Most of the effects in the brain have been attributed to the accumulation of sulfatides in oligodendrocytes and their cell damage. In contrast, less is known regarding sulfatide toxicity in astrocytes. Poly (ADP-ribose) polymerase (PARP) inhibitors are anti-cancer therapeutics that have proven efficacy in preclinical models of many neurodegenerative and inflammatory diseases, but have never been tested for MLD. Here, we examined the toxic effect of sulfatides on human astrocytes and restoration of this cell damage by the marketed PARP-1 inhibitor, Olaparib. Cultured human astrocytes were treated with increasing concentrations of sulfatides (5–100 μM) with or without Olaparib (100 nM). Cell viability assays were used to ascertain whether sulfatide-induced toxicity was rescued by Olaparib. Immunofluorescence, calcium (Ca²⁺) imaging, ROS, and mitochondrial damage assays were also used to explore the effects of sulfatides and Olaparib. ELISAs were performed and chemotaxis of peripheral blood immune cells was measured to examine the effects of Olaparib on sulfatide-induced inflammation in human astrocytes. Here, we established a concentration-dependent (EC₅₀∼20 μM at 24 h) model of sulfatide-induced astrocyte toxicity. Our data demonstrate that sulfatide-induced astrocyte toxicity involves (i) PARP-1 activation, (ii) pro-inflammatory cytokine release, and (iii) enhanced chemoattraction of peripheral blood immune cells. Moreover, these sulfatide-induced effects were attenuated by Olaparib (IC₅₀∼100 nM). In addition, sulfatide caused impairments of ROS production, mitochondrial stress, and Ca²⁺ signaling in human astrocytes, that were indicative of metabolic alterations and that were also alleviated by Olaparib (100 nM) treatment. Our data support the hypothesis that sulfatides can drive astrocyte cell death and demonstrate that Olaparib can dampen many facets of sulfatide-induced toxicity, including, mitochondrial stress, inflammatory responses, and communication between human astrocytes and peripheral blood immune cells. These data are suggestive of potential therapeutic utility of PARP inhibitors in the sphere of rare demyelinating diseases, and in particular MLD.

Graphical Abstract

Graphical abstract. Proposed mechanism of action of Olaparib in sulfatide-treated astrocytes. Human astrocytes treated for 24 h with sulfatides increase PARP-1 expression and die. PARP-1 overexpression is modulated by Ca²⁺ release from the endoplasmic reticulum, thus enhancing intracellular Ca²⁺ concentration. PARP-1 inhibition with Olaparib reduces Ca²⁺ influx and cell death. Olaparib also decreases IL-6, IL-8, IL-17, and CX3CL1 release from sulfatide-stimulated astrocytes, suggesting that PARP-1 plays a role in dampening neuroinflammation in MLD. This is confirmed by the reduction of immune cell migration such as lymphocytes, NK cells, and T cells towards sulfatide-treated astrocytes. Moreover, mitochondrial stress and ROS production induced by sulfatides are rescued by PARP-1 inhibition. Future studies will focus on the signaling cascades triggered by PARP-1-mediated currents in reactive astrocytes and Olaparib as a potential therapeutic target for MLD.

中文翻译：

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阐明奥拉帕尼在脑硫脂诱导的人星形胶质细胞毒性和神经炎症中的治疗效用

异染性脑白质营养不良 (MLD) 是一种严重的脱髓鞘、常染色体隐性遗传性脑白质营养不良，无法治愈。该疾病的基础是芳基硫酸酯酶 A 基因 (ARSA) 突变，导致该溶酶体酶活性不足，从而导致脑内半乳糖神经酰胺-3-O-硫酸酯（硫苷脂）积聚。大脑中的大部分影响归因于少突胶质细胞中硫苷脂的积累及其细胞损伤。相比之下，对于星形胶质细胞中硫苷脂的毒性知之甚少。聚（ADP-核糖）聚合酶 (PARP) 抑制剂是一种抗癌疗法，已在许多神经退行性疾病和炎症性疾病的临床前模型中证明有效，但从未针对 MLD 进行过测试。在这里，我们检查了硫苷脂对人星形胶质细胞的毒性作用以及市售 PARP-1 抑制剂奥拉帕尼对这种细胞损伤的恢复。培养的人星形胶质细胞用浓度逐渐增加的脑硫苷脂 (5–100 μM) 处理，并加或不加奥拉帕尼 (100 nM)。使用细胞活力测定来确定奥拉帕尼是否可以解除脑硫苷脂诱导的毒性。还使用免疫荧光、钙 (Ca ²⁺ ) 成像、ROS 和线粒体损伤测定来探索硫苷脂和奥拉帕尼的作用。进行 ELISA 并测量外周血免疫细胞的趋化性，以检查奥拉帕尼对人星形胶质细胞硫苷脂诱导的炎症的影响。在这里，我们建立了硫苷脂诱导的星形胶质细胞毒性的浓度依赖性（24小时EC 50∼20 μM）模型_。我们的数据表明，硫苷脂诱导的星形胶质细胞毒性涉及（i）PARP-1激活，（ii）促炎细胞因子释放，以及（iii）增强外周血免疫细胞的化学吸引力。此外，这些脑硫苷脂诱导的作用被奥拉帕尼（IC ₅₀ ∼100 nM）减弱。此外，硫苷脂会导致人星形胶质细胞中ROS产生、线粒体应激和Ca ²⁺信号传导受损，这表明代谢发生了变化，并且奥拉帕尼（100 nM）治疗也可以缓解这些变化。我们的数据支持硫苷脂可以驱动星形胶质细胞死亡的假设，并证明奥拉帕尼可以抑制硫苷脂诱导的毒性的许多方面，包括线粒体应激、炎症反应以及人星形胶质细胞与外周血免疫细胞之间的通讯。这些数据表明 PARP 抑制剂在罕见脱髓鞘疾病（特别是 MLD）领域具有潜在的治疗效用。

图形概要

图形概要。奥拉帕尼在硫苷脂处理的星形胶质细胞中的拟议作用机制。用硫苷脂处理 24 小时的人星形胶质细胞会增加 PARP-1 的表达并死亡。PARP-1 过表达受内质网Ca ²⁺释放的调节，从而提高细胞内 Ca ²⁺浓度。Olaparib 抑制 PARP-1 可减少 Ca ²⁺流入和细胞死亡。奥拉帕尼还减少脑硫苷脂刺激的星形胶质细胞释放 IL-6、IL-8、IL-17 和 CX3CL1，表明 PARP-1 在抑制 MLD 中的神经炎症中发挥作用。免疫细胞（例如淋巴细胞、NK 细胞和 T 细胞）向硫苷脂处理的星形胶质细胞迁移的减少证实了这一点。此外，抑制 PARP-1 可以挽救硫苷脂诱导的线粒体应激和 ROS 产生。未来的研究将集中于反应性星形胶质细胞中 PARP-1 介导的电流触发的信号级联反应，以及奥拉帕尼作为 MLD 的潜在治疗靶点。