Department of Clinical Chemistry and Biochemistry, Military-Medical Faculty, Medical University of Lodz, Lodz, Poland.
Department of Biochemistry and Molecular Biology, Hollings Cancer Center, Medical University of South Carolina, Charleston, United States.
Curr Alzheimer Res. 2019;16(3):209-218. doi: 10.2174/1567205016666190228121157.
There is a body of evidence that neurodegenerative disease entities are directly correlated with the perturbations on the molecular level. Hence, the ER stress-mediated Unfolded Protein Response (UPR) is activated resulting in PERK-dependent phosphorylation of the Eukaryotic initiation factor 2 (eIF2α). Thus, the levels of ATF4 and CHOP proteins are significantly increased, which subsequently switches the pro-adaptive branch of the UPR into the pro-apoptotic directly leading to neuronal loss and initiation of the neurodegenerative process. The aim of the presented study was the evaluation of the biological activity of highly specific, small-molecule inhibitors of the PERKdependent UPR signaling pathway.
The study was conducted on rat astrocytic DI TNC1 cell line. The level of p-eIF2α was measured by Western blot technique, the cytotoxicity of the investigated compound was assessed by the MTT assay and using the FITC-conjugated Annexin V (Annexin V-FITC) to indicate apoptosis and propidium iodide (PI) to indicate necrosis. The effect of tested compound on cell cycle progression was measured by flow cytometry, where the PI-labelled nuclei were analysed for DNA content.
As a result one of the investigated compound LDN-0060609 triggers a significant inhibition of the eIF2α phosphorylation in DI TNC1 cell line. Moreover, we showed that compound LDN-0060609 is non-cytotoxic and has no effect on cell cycle progression.
In conclusion, LDN-0060609 may constitute a novel, targeted treatment approach against neurodegenerative diseases, including Alzheimer's disease (AD), where pathogenesis and progression are closely associated with the overactivation of the PERK-dependent UPR signaling pathway.
有大量证据表明，神经退行性疾病实体与分子水平上的干扰直接相关。因此，内质网应激介导的未折叠蛋白反应（UPR）被激活，导致 PERK 依赖性磷酸化真核起始因子 2（eIF2α）。因此，ATF4 和 CHOP 蛋白的水平显著增加，这随后将 UPR 的促适应性分支直接切换为促凋亡，导致神经元丢失和神经退行性过程的启动。本研究的目的是评估 PERK 依赖性 UPR 信号通路的高度特异性小分子抑制剂的生物学活性。
该研究在大鼠星形胶质细胞 DI TNC1 细胞系上进行。通过 Western blot 技术测量 p-eIF2α 的水平，通过 MTT 测定和 FITC 缀合的 Annexin V（Annexin V-FITC）来指示细胞凋亡和碘化丙啶（PI）来指示细胞坏死来评估研究化合物的细胞毒性。通过流式细胞术测量测试化合物对细胞周期进程的影响，其中对 PI 标记的细胞核进行 DNA 含量分析。
结果表明，研究的一种化合物 LDN-0060609 可显著抑制 DI TNC1 细胞系中 eIF2α 的磷酸化。此外，我们表明化合物 LDN-0060609 无细胞毒性，对细胞周期进程没有影响。
总之，LDN-0060609 可能构成一种针对神经退行性疾病的新型靶向治疗方法，包括阿尔茨海默病（AD），其发病机制和进展与 PERK 依赖性 UPR 信号通路的过度激活密切相关。
