Neuroscience Research Group, Medical Research Institute, Faculty of Medicine, University of Antioquia (UdeA), SIU Medellin, Colombia.
National Center for Genome Sequencing, University of Antioquia (UdeA), SIU Medellin, Colombia.
Cytotherapy. 2018 Jan;20(1):45-61. doi: 10.1016/j.jcyt.2017.09.011. Epub 2017 Nov 2.
Stem cell transplantation is an excellent option for regenerative or replacement therapy. However, deleterious microenvironmental and endogenous factors (e.g., oxidative stress) compromise ongoing graft survival and longevity. Therefore, (transient or stable) genetically modified cells may be reasonably thought to resist oxidative stress-induced damage. Genetic engineering of mesenchymal stromal cells (MSCs) obtained from Wharton's jelly tissue may offer some therapeutic potential. PARKIN is a multifunctional ubiquitin ligase able to protect dopaminergic cells against stress-related signaling. We, therefore, evaluated the effect of the neurotoxicant 6-hydroxydopamine (6-OHDA) on regulated cell death signaling in MSCs and investigated whether overexpression of PARKIN in MSCs was capable of modulating the effect of 6-OHDA.
We transiently transfected Wharton's jelly-derived MSCs with an mCherry-PARKIN vector using the Lipofectamine LTX method. Naïve MSCs and MSCs overexpressing PARKIN were exposed to increasing concentrations of 6-OHDA. We used light and fluorescence microscopy, flow cytometry, immunocytochemistry staining, in-cell Western and Western blot analysis.
After 12-24 h of 6-OHDA exposure, we detected dichlorofluorescein (DCF)-positive cells (80%) indicative of reactive oxygen species (H2O2) production, reduced cell viability (40-50%), decreased mitochondrial membrane potential (ΔΨm, 35-45%), DNA fragmentation (18-30%), and G1-arrested cell cycle in the MSCs. 6-OHDA exposure increased the expression of the transcription factor c-JUN, increased the expression of the mitochondria maintenance Phosphatase and tensin homologue-induced putative kinase 1 (PINK1) protein and increased the expression of pro-apoptotic PUMA, caspase-3 and apoptosis-inducing factor (AIF). 6-OHDA exposure also significantly augmented the oxidation of the oxidative stress sensor, DJ-1. Overexpression of PARKIN in MSCs not only significantly reduced the expression of cell death and oxidative stress markers but also significantly reduced DCF-positive cells (50% reduction).
6-OHDA induced apoptosis in MSCs via generation of H2O2, activation of c-JUN and PUMA, mitochondrial depolarization and nuclei fragmentation. Our findings suggest that PARKIN protects MSCs against 6-OHDA toxicity by partly interacting with H2O2, reducing the expression of c-JUN, PUMA, AIF and caspase-3, and maintaining the mitochondrial ΔΨm.
干细胞移植是一种用于再生或替代治疗的极好选择。然而，有害的微环境和内源性因素（例如氧化应激）会损害正在进行的移植物存活和寿命。因此，（暂时或稳定）基因修饰细胞可能被认为能够抵抗氧化应激诱导的损伤。对来源于 Wharton 胶组织的间充质基质细胞（MSCs）进行基因工程改造可能具有一定的治疗潜力。PARKIN 是一种多功能泛素连接酶，能够保护多巴胺能细胞免受应激相关信号的影响。因此，我们评估了神经毒性 6-羟多巴胺（6-OHDA）对 MSCs 中调节性细胞死亡信号的影响，并研究了 PARKIN 在 MSCs 中的过表达是否能够调节 6-OHDA 的作用。
我们使用 Lipofectamine LTX 方法，通过瞬时转染 mCherry-PARKIN 载体对 Wharton 胶衍生的 MSCs 进行转染。未处理的 MSCs 和过表达 PARKIN 的 MSCs 暴露于递增浓度的 6-OHDA 下。我们使用荧光显微镜、流式细胞术、免疫细胞化学染色、细胞内 Western blot 和 Western blot 分析。
在 6-OHDA 暴露 12-24 小时后，我们检测到二氯荧光素（DCF）阳性细胞（80%），表明活性氧（H2O2）的产生、细胞活力降低（40-50%）、线粒体膜电位（ΔΨm，~35-45%）、DNA 片段化（18-30%）和 G1 期细胞周期阻滞。6-OHDA 暴露增加了转录因子 c-JUN 的表达，增加了线粒体维持磷酸酶和张力蛋白同源物诱导的潜在激酶 1（PINK1）蛋白的表达，并增加了促凋亡 PUMA、caspase-3 和凋亡诱导因子（AIF）的表达。6-OHDA 暴露还显著增加了氧化应激传感器 DJ-1 的氧化。在 MSCs 中转染 PARKIN 不仅显著降低了细胞死亡和氧化应激标志物的表达，而且还显著降低了 DCF 阳性细胞（减少约 50%）。
6-OHDA 通过生成 H2O2、激活 c-JUN 和 PUMA、线粒体去极化和核片段化诱导 MSCs 凋亡。我们的研究结果表明，PARKIN 通过与 H2O2 部分相互作用、降低 c-JUN、PUMA、AIF 和 caspase-3 的表达以及维持线粒体 ΔΨm，来保护 MSCs 免受 6-OHDA 毒性的影响。
