Modulation of intracellular calcium homeostasis blocks autophagosome formation

Abstract

Cellular stress responses often involve elevation of cytosolic calcium levels, and this has been suggested to stimulate autophagy. Here, however, we demonstrated that agents that alter intracellular calcium ion homeostasis and induce ER stress-the calcium ionophore A23187 and the sarco/endoplasmic reticulum Ca (2+)-ATPase inhibitor thapsigargin (TG)-potently inhibit autophagy. This anti-autophagic effect occurred under both nutrient-rich and amino acid starvation conditions, and was reflected by a strong reduction in autophagic degradation of long-lived proteins. Furthermore, we found that the calcium-modulating agents inhibited autophagosome biogenesis at a step after the acquisition of WIPI1, but prior to the closure of the autophagosome. The latter was evident from the virtually complete inability of A23187- or TG-treated cells to sequester cytosolic lactate dehydrogenase. Moreover, we observed a decrease in both the number and size of starvation-induced EGFP-LC3 puncta as well as reduced numbers of mRFP-LC3 puncta in a tandem fluorescent mRFP-EGFP-LC3 cell line. The anti-autophagic effect of A23187 and TG was independent of ER stress, as chemical or siRNA-mediated inhibition of the unfolded protein response did not alter the ability of the calcium modulators to block autophagy. Finally, and remarkably, we found that the anti-autophagic activity of the calcium modulators did not require sustained or bulk changes in cytosolic calcium levels. In conclusion, we propose that local perturbations in intracellular calcium levels can exert inhibitory effects on autophagy at the stage of autophagosome expansion and closure.

Defects in calcium homeostasis and mitochondria can be reversed in Pompe disease

“…A number of studies suggested that a rise in cytosolic Ca 2C is a powerful inducer of autophagy, [68][69][70][71] although other reports do not support this finding. 72,73 In our study, the normalization of Ca 2C levels by verapamil did not have any appreciable effect on autophagy.…”

Unbiased Cell-based Screening in a Neuronal Cell Model of Batten Disease Highlights an Interaction between Ca2+ Homeostasis, Autophagy, and CLN3 Protein Function

“…Rab7 is known to be a key regulator of autophagosomal maturation, and thapsigargin appears to specifically disrupt a step upstream of Rab7 association with the autophagosome (32,37). These facts, along with the evidence that endogenous CLN3 may be present in a Rab7-Rab7-interacting lysosomal protein complex (39) prompted us to assess Rab7 association with autophagosomes in our mouse cerebellar cell system as well.…”

“…It has been proposed that thapsigargin disrupts autophagosome maturation by interfering with Rab7-mediated autophagosomal-lysosomal fusion (32), perhaps even upstream of Rab7, inhibiting closure of the pre-autophagosomal structure (37). Interestingly, CLN3 has also been proposed to mediate Rab7 function, and overexpressed CLN3 protein has been reported to co-immunoprecipitate with a Rab7-interacting lysosomal protein complex (39).…”

“…1D). Notably, thapsigargin doses in this range have previously been shown in healthy cells to reduce but not completely abolish SERCA activity (37). The effect of low dose thapsigargin treatment on the CbCln3 ⌬ex7/8/⌬ex7/8 cells was also observed by immunoblot analysis, where 100 nM thapsigargin treatment increased lipidated GFP-LC3-II levels in the CbCln3 ⌬ex7/8/⌬ex7/8 cells, but GFP-LC3-II levels were not significantly altered by 100 nM thapsigargin in the wild type cells (Fig.…”

Autophagy of cytoplasmic bulk cargo does not require LC3

“…To test if this conclusion could be extended to other cell types and to conditions without cycloheximide, macroautophagic cargo (LDH) sequestration was measured in LNCaP prostate carcinoma cells, 8 a cell type that (unlike hepatocytes) allows genetic knockdown experiments to be performed. We found that the LC3 family members expressed in these cells (LC3A1, LC3A2, LC3B1) could be collectively knocked down by a combination of siRNAs.…”