Basal ryanodine receptor activity suppresses autophagic flux
Basal ryanodine receptor activity suppresses autophagic flux
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DOI：
10.1016/j.bcp.2017.03.011
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发表时间：
2017-05
影响因子：
5.8
通讯作者：
T. Vervliet;I. Pintelon;K. Welkenhuyzen;M. Bootman;H. Bannai;K. Mikoshiba;W. Martinet;N. N. Kasri-N.;J. Parys;G. Bultynck
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文献类型：
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作者：
T. Vervliet;I. Pintelon;K. Welkenhuyzen;M. Bootman;H. Bannai;K. Mikoshiba;W. Martinet;N. N. Kasri-N.;J. Parys;G. Bultynck
肌醇1，4，5-三磷酸受体(IP3Rs)和细胞内钙信号在调节溶酶体降解途径--自噬的不同步骤中起着关键作用。Ryanodine受体(RyR)是一种细胞内钙释放通道，主要表达于可兴奋的细胞类型，包括肌肉和神经元，然而，与自噬有关的研究尚未广泛。然而，这些组织中RyRs的异常表达和过度活性已与包括阿尔茨海默病在内的几种疾病的发生有关，自噬调节受损参与了该病的病理。在这项研究中，我们确定了药物抑制RyR是否可以调节异位RyR表达模型中的自噬通量，如HEK293细胞和内源性表达RyR的细胞类型，如C2C12成肌细胞和原代海马神经元。重要的是，RyR3在HEK293细胞中的过表达削弱了自噬通量。相反，在所测试的所有细胞模型中，使用丹曲林或兰诺定药物抑制内源性或异位表达的RyRs，通过增加溶酶体翻转来增加自噬通量(自噬小体和自溶酶体的数量以mCherry-LC3点/细胞衡量，分别从对照HEK RyR3细胞的70.37%±77.81增加到丹曲林和兰诺定处理后的111.18±77.72和98.14%±77.31)。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早期自噬空泡的数量从每细胞横截面的5.9±2.97个减少到1.8个±1.03个。自噬通量的调节可能与RyR通道的功能抑制有关，因为在HEK293细胞模型中，两种RyR抑制剂都有效地减少了显示自发RyR3活动的细胞数量(丹曲林和兰诺定处理后，分别从对照细胞的41.14%±22.12%和18.70%±22.25%和9.74%±2.67%)。总之，基础RyR介导的钙释放事件在溶酶体水平上抑制了自噬通量。
The inositol 1,4,5-trisphosphate receptors (IP3Rs) and intracellular Ca2+signaling are critically involved in regulating different steps of autophagy, a lysosomal degradation pathway. The ryanodine receptors (RyR), intracellular Ca2+-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 Ca2+-release events suppress autophagic flux at the level of the lysosomes.