Department of Human Science and Promotion of quality of Life, San Raffaele Open University of Rome, Via di Val Cannuta, 247, 00166 Roma, Italy.
Section of Human Anatomy and Histology, Department of Biomedical and Biotechnological Sciences, University of Catania, Via S. Sofia, 87, 95123 Catania, Italy.
Int J Mol Sci. 2020 Apr 22;21(8):2943. doi: 10.3390/ijms21082943.
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease of complex etiology leading to motor neuron degeneration. Many gene alterations cause this pathology, including mutation in Cu, Zn superoxide dismutase (SOD1), which leads to its gain of function. Mutant SOD1 proteins are prone to aberrant misfolding and create aggregates that impair autophagy. The hypoxic stress is strictly linked to the disease progression since it induces uncontrolled autophagy activation and the consequent high rates of cell death. Previously, we showed that pituitary adenylate cyclase-activating polypeptide (PACAP) exerts neurotrophic activity in cultured mSOD1 motor neurons exposed to serum deprivation. To date, no studies have examined whether the protective effect of PACAP on mSOD1 cells exposed to hypoxic insult is mediated through the regulation of the autophagy process. In the present study, we used the neuroblastoma-spinal cord-34 (NSC-34) cell line, stably expressing human wild type or mutant SOD1 G93A, to represent a well characterized in vitro model of a familial form of ALS. These cells were exposed to 100-µM desferrioxamine mesylate salt for 24h, to mimic the hypoxic stress affecting motor neurons during the disease progression. Our results showed that PACAP treatment significantly reduced cell death and hypoxia-induced mSOD1 accumulation by modulating the autophagy process in G93A motor neurons, as revealed by the decreased LC3II and the increased p62 levels, two autophagy indicators. These results were also confirmed by evaluating the vacuole formation detected through light chain 3 (LC3) immunofluorescence. Furthermore, the PACAP effects on autophagy seem to be mediated through the activation of the MAPK/ERK signaling pathway. Overall, our data demonstrated that PACAP exerts an ameliorative effect on the mSOD1 motor neuron viability by modulating a hypoxia-induced autophagy process through activation of MAPK/ERK signaling cascade.
肌萎缩侧索硬化症（ALS）是一种复杂病因导致运动神经元退行性变的进行性神经退行性疾病。许多基因改变导致这种病理学，包括铜，锌超氧化物歧化酶（SOD1）的突变，导致其功能获得。突变的 SOD1 蛋白易于异常错误折叠并产生聚集体，从而损害自噬。缺氧应激与疾病进展密切相关，因为它诱导不受控制的自噬激活和随后的高细胞死亡率。先前，我们表明，垂体腺苷酸环化酶激活肽（PACAP）在暴露于血清剥夺的培养的 mSOD1 运动神经元中发挥神经营养活性。迄今为止，尚无研究检查 PACAP 是否对暴露于缺氧刺激的 mSOD1 细胞的保护作用是否通过调节自噬过程来介导。在本研究中，我们使用神经母细胞瘤-脊髓-34（NSC-34）细胞系，稳定表达人野生型或突变型 SOD1 G93A，代表一种家族性肌萎缩侧索硬化症的体外模型。这些细胞用 100-µM 去铁胺甲磺酸盐处理 24 小时，模拟疾病进展过程中影响运动神经元的缺氧应激。我们的结果表明，PACAP 通过调节 G93A 运动神经元中的自噬过程显著降低细胞死亡和缺氧诱导的 mSOD1 积累，这通过降低 LC3II 和增加 p62 水平（两种自噬指标）来揭示。通过评估通过轻链 3（LC3）免疫荧光检测到的空泡形成也证实了这些结果。此外，PACAP 对自噬的作用似乎是通过激活 MAPK/ERK 信号通路介导的。总的来说，我们的数据表明，PACAP 通过激活 MAPK/ERK 信号级联来调节缺氧诱导的自噬过程，对 mSOD1 运动神经元活力产生改善作用。
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