Vervliet Tim, Pintelon Isabel, Welkenhuyzen Kirsten, Bootman Martin D, Bannai Hiroko, Mikoshiba Katsuhiko, Martinet Wim, Nadif Kasri Nael, Parys Jan B, Bultynck Geert
KU Leuven, Laboratory of Molecular and Cellular Signaling, Department of Cellular and Molecular Medicine, B-3000 Leuven, Belgium.
University of Antwerp, Laboratory of Cell Biology and Histology, Department of Veterinary Sciences, 2610 Antwerp, Belgium.
Biochem Pharmacol. 2017 May 15;132:133-142. doi: 10.1016/j.bcp.2017.03.011. Epub 2017 Mar 18.
The inositol 1,4,5-trisphosphate receptors (IPRs) and intracellular Ca signaling are critically involved in regulating different steps of autophagy, a lysosomal degradation pathway. The ryanodine receptors (RyR), intracellular Ca-release channels mainly expressed in excitable cell types including muscle and neurons, have however not yet been extensively studied in relation to autophagy. Yet, aberrant expression and excessive activity of RyRs in these tissues has been implicated in the onset of several diseases including Alzheimer's disease, where impaired autophagy regulation contributes to the pathology. In this study, we determined whether pharmacological RyR inhibition could modulate autophagic flux in ectopic RyR-expressing models, like HEK293 cells and in cell types that endogenously express RyRs, like C2C12 myoblasts and primary hippocampal neurons. Importantly, RyR3 overexpression in HEK293 cells impaired the autophagic flux. Conversely, in all cell models tested, pharmacological inhibition of endogenous or ectopically expressed RyRs, using dantrolene or ryanodine, augmented autophagic flux by increasing lysosomal turn-over (number of autophagosomes and autolysosomes measured as mCherry-LC3 punctae/cell increased from 70.37±7.81 in control HEK RyR3 cells to 111.18±7.72 and 98.14±7.31 after dantrolene and ryanodine treatments, respectively). Moreover, in differentiated C2C12 cells, transmission electron microscopy demonstrated that dantrolene treatment decreased the number of early autophagic vacuoles from 5.9±2.97 to 1.8±1.03 per cellular cross section. The modulation of the autophagic flux could be linked to the functional inhibition of RyR channels as both RyR inhibitors efficiently diminished the number of cells showing spontaneous RyR3 activity in the HEK293 cell model (from 41.14%±2.12 in control cells to 18.70%±2.25 and 9.74%±2.67 after dantrolene and ryanodine treatments, respectively). In conclusion, basal RyR-mediated Ca-release events suppress autophagic flux at the level of the lysosomes.
肌醇1,4,5 - 三磷酸受体（IPRs）和细胞内钙信号传导在调节自噬（一种溶酶体降解途径）的不同步骤中起着关键作用。然而，主要在包括肌肉和神经元在内的可兴奋细胞类型中表达的细胞内钙释放通道——兰尼碱受体（RyR），尚未在自噬方面得到广泛研究。然而，这些组织中RyRs的异常表达和过度活性与包括阿尔茨海默病在内的几种疾病的发病有关，其中自噬调节受损是病理的一个因素。在本研究中，我们确定了药理学上抑制RyR是否能调节异位表达RyR的模型（如HEK293细胞）以及内源性表达RyRs的细胞类型（如C2C12成肌细胞和原代海马神经元）中的自噬通量。重要的是，HEK293细胞中RyR3的过表达损害了自噬通量。相反，在所有测试的细胞模型中，使用丹曲林或兰尼碱对内源性或异位表达的RyRs进行药理学抑制，通过增加溶酶体周转率来增强自噬通量（自噬体和自溶酶体的数量以mCherry - LC3斑点/细胞来衡量，在对照HEK RyR3细胞中为70.37±7.81，在丹曲林和兰尼碱处理后分别增加到111.18±7.72和98.14±7.31）。此外，在分化的C2C12细胞中，透射电子显微镜显示丹曲林处理使每个细胞横截面的早期自噬泡数量从5.9±2.97减少到1.8±1.03。自噬通量的调节可能与RyR通道的功能抑制有关，因为两种RyR抑制剂都有效地减少了HEK293细胞模型中显示自发RyR3活性的细胞数量（对照细胞中为41.14%±2.12，在丹曲林和兰尼碱处理后分别降至18.70%±2.25和9.74%±2.67）。总之，基础的RyR介导的钙释放事件在溶酶体水平抑制自噬通量。
