Basal ryanodine receptor activity suppresses autophagic flux.
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.
KU Leuven, Laboratory of Molecular and Cellular Signaling, Department of Cellular and Molecular Medicine, B-3000 Leuven, Belgium.
Tim
T
Vervliet
University of Antwerp, Laboratory of Cell Biology and Histology, Department of Veterinary Sciences, 2610 Antwerp, Belgium.
Isabel
I
Pintelon
KU Leuven, Laboratory of Molecular and Cellular Signaling, Department of Cellular and Molecular Medicine, B-3000 Leuven, Belgium.
Kirsten
K
Welkenhuyzen
The Open University, School of Life, Health and Chemical Sciences, Walton Hall, Milton Keynes MK7 6AA, United Kingdom.
Martin D
MD
Bootman
RIKEN Brain Science Institute (BSI), Laboratory for Developmental Neurobiology, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
Hiroko
H
Bannai
RIKEN Brain Science Institute (BSI), Laboratory for Developmental Neurobiology, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
Katsuhiko
K
Mikoshiba
University of Antwerp, Laboratory of Physiopharmacology, Department of Pharmaceutical Sciences, B-2610 Antwerp, Belgium.
Wim
W
Martinet
Radboud University Medical Center, Donders Institute for Brain, Cognition and Behaviour, Department of Cognitive Neuroscience, Department of Human Genetics, 6500HB Nijmegen, The Netherlands.
Nael
N
Nadif Kasri
KU Leuven, Laboratory of Molecular and Cellular Signaling, Department of Cellular and Molecular Medicine, B-3000 Leuven, Belgium.
Jan B
JB
Parys
KU Leuven, Laboratory of Molecular and Cellular Signaling, Department of Cellular and Molecular Medicine, B-3000 Leuven, Belgium. Electronic address: geert.bultynck@kuleuven.be.
Geert
G
Bultynck
D000818:Animals; D001343:Autophagy; D002478:Cells, Cultured; D003620:Dantrolene / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D051381:Rats; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism*
D019837:Ryanodine Receptor Calcium Release Channel; D003620:Dantrolene
Alzheimer’s disease; Autophagy; Bafilomycin A1 (PubChem CID: 6436223); Ca(2+); Dantrolene; Dantrolene (PubChem CID: 6604100); Lysosome; Ryanodine (PubChem CID: 11317883); Ryanodine receptor