Biochemistry Department, Federal University of Santa Catarina, 88040-900 Florianópolis, SC, Brazil.
Cellular Neurobiology Laboratory, Salk Institute for Biological Studies, CA 92037 La Jolla, USA.
Oxid Med Cell Longev. 2019 Mar 27;2019:2715810. doi: 10.1155/2019/2715810. eCollection 2019.
Hyperosmotic stress is an important pathophysiologic condition in diabetes, severe trauma, dehydration, infection, and ischemia. Furthermore, brain neuronal cells face hyperosmotic stress in ageing and Alzheimer's disease. Despite the enormous importance of knowing the homeostatic mechanisms underlying the responses of nerve cells to hyperosmotic stress, this topic has been underrepresented in the literature. Recent evidence points to autophagy induction as a hallmark of hyperosmotic stress, which has been proposed to be controlled by mTOR inhibition as a consequence of AMPK activation. We previously showed that methylglyoxal induced a decrease in the antioxidant proteins thioredoxin 1 (Trx1) and glyoxalase 2 (Glo2), which was mediated by AMPK-dependent autophagy. Thus, we hypothesized that hyperosmotic stress would have the same effect.
HT22 hippocampal nerve cells were treated with NaCl (37, 75, or 150 mM), and the activation of the AMPK/mTOR pathway was investigated, as well as the levels of Trx1 and Glo2. To determine if autophagy was involved, the inhibitors bafilomycin (Baf) and chloroquine (CQ), as well as ATG5 siRNA, were used. To test for AMPK involvement, AMPK-deficient mouse embryonic fibroblasts (MEFs) were used.
Hyperosmotic stress induced a clear increase in autophagy, which was demonstrated by a decrease in p62 and an increase in LC3 lipidation. AMPK phosphorylation, linked to a decrease in mTOR and S6 ribosomal protein phosphorylation, was also observed. Deletion of AMPK in MEFs did not prevent autophagy induction by hyperosmotic stress, as detected by decreased p62 and increased LC3 II, or mTOR inhibition, inferred by decreased phosphorylation of P70 S6 kinase and S6 ribosomal protein. These data indicating that AMPK was not involved in autophagy activation by hyperosmotic stress were supported by a decrease in p-ULK1, an AMPK phosphorylation site. Trx1 and Glo2 levels were decreased at 6 and 18 h after treatment with 150 mM NaCl. However, this decrease in Trx1 and Glo2 in HT22 cells was not prevented by autophagy inhibition by Baf, CQ, or ATG5 siRNA. AMPK-deficient MEFs under hyperosmotic stress presented the same Trx1 and Glo2 decrease as wild-type cells.
Hyperosmotic stress induced AMPK activation, but this was not responsible for its effects on mTOR activity or autophagy induction. Moreover, the decrease in Trx1 and Glo2 induced by hyperosmotic stress was independent of both autophagy and AMPK activation.
高渗应激是糖尿病、严重创伤、脱水、感染和缺血等重要的病理生理条件。此外，在衰老和阿尔茨海默病中，脑神经元细胞也面临高渗应激。尽管了解神经细胞对高渗应激反应的稳态机制具有重要意义，但这一主题在文献中却很少被提及。最近的证据表明，自噬诱导是高渗应激的标志之一，据推测，这是由于 AMPK 激活导致 mTOR 抑制的结果。我们之前曾表明，甲基乙二醛诱导抗氧化蛋白硫氧还蛋白 1（Trx1）和甘油醛 3-磷酸脱氢酶 2（Glo2）的减少，这是由 AMPK 依赖性自噬介导的。因此，我们假设高渗应激会产生相同的效果。
用 NaCl（37、75 或 150 mM）处理 HT22 海马神经细胞，研究 AMPK/mTOR 通路的激活情况，以及 Trx1 和 Glo2 的水平。为了确定自噬是否参与其中，使用了自噬抑制剂巴弗霉素（Baf）和氯喹（CQ）以及 ATG5 siRNA。为了检测 AMPK 的参与，使用了 AMPK 缺陷型小鼠胚胎成纤维细胞（MEFs）。
高渗应激明显诱导自噬增加，这通过 p62 减少和 LC3 脂质化增加来证明。还观察到 AMPK 磷酸化，与 mTOR 和 S6 核糖体蛋白磷酸化减少有关。高渗应激诱导的自噬，如 MEFs 中 AMPK 缺失所检测到的 p62 减少和 LC3 II 增加，或 mTOR 抑制，由 P70 S6 激酶和 S6 核糖体蛋白磷酸化减少推断，并不受 AMPK 缺失的影响。这些表明 AMPK 不参与高渗应激诱导的自噬激活的数据得到了 p-ULK1（AMPK 磷酸化位点）减少的支持。用 150 mM NaCl 处理 6 和 18 小时后，Trx1 和 Glo2 的水平下降。然而，在 HT22 细胞中，Baf、CQ 或 ATG5 siRNA 抑制自噬并不能防止 Trx1 和 Glo2 的减少。在高渗应激下，AMPK 缺陷型 MEFs 表现出与野生型细胞相同的 Trx1 和 Glo2 减少。
高渗应激诱导 AMPK 激活，但这与 mTOR 活性或自噬诱导无关。此外，高渗应激诱导的 Trx1 和 Glo2 减少与自噬和 AMPK 激活均无关。
