The role of lysosomal Ca2+ in the regulation of autophagy

The ER is the largest organelle found in the cell and acts as the largest Ca2+ store, with a [Ca2+]ER around 500 µM while the [Ca2+]cyt is around 100 nM. Various proteins, including the SERCA pump and the IP3 receptor, regulate the Ca2+ content of the ER. The high [Ca2+]ER is important for the role of Ca2+ as second messenger, but also for the proper functioning of the ER chaperones involved in the folding of newly synthesised proteins and in the protein quality control system. Recently it was shown that the lysosomes are Ca2+ sources with a [Ca2+]lys found to be around 500 µM as well. For Ca2+ import in the lysosomes, a role has been proposed for the Ca2+/H+ exchanger, while several Ca2+-release channels have been described including TRPML1 and TPC2.

Macroautophagy, or in short autophagy, is a process always present at basal levels in order to degrade and recycle old or malfunctioning proteins and/or organelles. Cargo is engulfed in an autophagosome which eventually fuses with a lysosome, where degradation takes place. Under stress conditions, autophagy can be upregulated in order to provide the cell with nutrients and energy. These conditions include e.g. nutrient deprivation, hypoxia, pathogen infection and ER stress.

Ca2+ has an important role in autophagy, and can act both in an inhibitory and a stimulatory way. While a moderate Ca2+ transfer into the mitochondria is necessary for proper functioning of the Krebs cycle and energy production and consequently inhibits autophagy. Furthermore, various proteins involved in autophagy regulation are dependent on cytosolic Ca2+. Finally, release of lysosomal Ca2+ has a role in e.g. autophagosome-lysosome fusion, and e.g. starvation-, rapamycin- and resveratrol-induced autophagy are known to be dependent on Ca2+.

The ER is also important for protein and lipid synthesis. However, when the synthesis and folding of proteins is impaired, un- or misfolded proteins accumulate in the ER lumen. In order to be able to alleviate and counteract this so-called ER stress, the ER contains sensors that can activate the UPR. The UPR consists of three branches, i.e. the IRE1α, the ATF6 and the PERK branches, that together aim to promote cell survival by reducing general protein synthesis and activating autophagy. When this is not sufficient to resolve the ER stress, however, the cell will go into apoptosis.

Here, we investigated the role of Ca2+ in ER stress-induced autophagy using the L-proline analogue AZC. We found that treatment of HeLa cells for 3-9 h with 5 mM AZC activated both the ATF6 and PERK, but not the IRE1α, arm of the UPR. This treatment also resulted in the upregulation of the ER chaperone BiP but the expression levels of ERp57 and ERp72 were not affected. Furthermore, we demonstrated that AZC induces LC3 lipidation within 6 h of treatment, and that this was dependent on PERK activation. Additionally, both PERK activation and LC3 lipidation depended on cytosolic Ca2+. Apart from this, we found that 6 h pre-treatment with AZC did not affect the ER Ca2+ content, although the rise in cytosolic Ca2+ when ER Ca2+ release was induced appeared lower. Interestingly, the rate by which the cytosol was cleared from Ca2+ released from the ER was increased, explaining the lower detected cytosolic Ca2+ rise. The increased clearance rate did not depend on the expression levels of the main Ca2+ transporters IP3R, SERCA, PMCA or MCU, and was not related to a changed Ca2+ uptake in the mitochondria.

Secondly, we investigated the role of the lysosomal Ca2+ channel TPRML1 in autophagy using its agonist MK6-83. We found that 3 h treatment with 10 µM MK6-83 led to a strong increase in LC3-II independently from mTOR inhibition, and we could attribute this to a specific effect of MK6-83 on TRPML1. Interestingly, the LC3 lipidation did not occur when MK6-83 was applied in combination with starvation, while pharmacological induction of autophagy using torin1 or resveratrol did not interfere with MK6-83-induced LC3 lipidation. Furthermore, the decrease of phospho-TFEB levels upon MK6-83 treatment was abolished when MK6-83 was used in combination with starvation, suggesting a possible relation between mTOR inactivity, TFEB activation and LC3 lipidation.

In short, we found that Ca2+ is essential for AZC-induced activation of the PERK branch of the UPR and subsequent LC3 lipidation, and that AZC affects cytosolic Ca2+ clearance after emptying the ER Ca2+ store. Furthermore, we found that TRPML1 strongly induces LC3 lipidation independently from mTOR and possibly dependent on TFEB activation. This was however inhibited by starvation, possibly due to a negative feedback mechanism reactivating mTORC1.

In time, understanding the role of Ca2+ in the UPR and autophagy may contribute to improved treatment strategies for e.g. Alzheimer’s disease and mucolipidosis type IV.