Lysosomal adaptation: How cells respond to lysosomotropic compounds

Abstract

Lysosomes are acidic organelles essential for degradation and cellular homoeostasis and recently lysosomes have been shown as signaling hub to respond to the intra and extracellular changes (e.g. amino acid availability). Compounds including pharmaceutical drugs that are basic and lipophilic will become sequestered inside lysosomes (lysosomotropic). How cells respond to the lysosomal stress associated with lysosomotropism is not well characterized. Our goal is to assess the lysosomal changes and identify the signaling pathways that involve in the lysosomal changes. Eight chemically diverse lysosomotropic drugs from different therapeutic areas were subjected to the evaluation using the human adult retinal pigmented epithelium cell line, ARPE-19. All lysosomotropic drugs tested triggered lysosomal activation demonstrated by increased lysosotracker red (LTR) and lysosensor green staining, increased cathepsin activity, and increased LAMP2 staining. However, tested lysosomotropic drugs also prompted lysosomal dysfunction exemplified by intracellular and extracellular substrate accumulation including phospholipid, SQSTM1/p62, GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) and opsin. Lysosomal activation observed was likely attributed to lysosomal dysfunction, leading to compensatory responses including nuclear translocation of transcriptional factors TFEB, TFE3 and MITF. The adaptive changes are protective to the cells under lysosomal stress. Mechanistic studies implicate calcium and mTORC1 modulation involvement in the adaptive changes. These results indicate that lysosomotropic compounds could evoke a compensatory lysosomal biogenic response but with the ultimate consequence of lysosomal functional impairment. This work also highlights a pathway of response to lysosomal stress and evidences the role of TFEB, TFE3 and MITF in the stress response.

Chloroquine inhibits autophagic flux by decreasing autophagosome-lysosome fusion

“…In line with these observations, we did not find a decrease in LysoTracker Red-positive puncta upon CQ treatment as previously shown [29,[55][56][57]. Our results and other recent studies [30,58,59], indicate that CQ does not substantially decrease lysosomal acidity, and the lysosomes retain their capacity to degrade delivered material. Although we cannot exclude that CQ treatment has a different effect on lysosomal pH depending on the cell type, one has to be careful in interpreting signal intensities of LysoTracker Red.…”

“…Therefore, part of the discrepancy on CQ effects on lysosomal pH might be attributed to how the pH was estimated. Moreover, CQ may induce a temporal elevation of lysosomal pH [30,61], which is of a transient nature and is followed by a stable, lasting re-acidification of the lysosomes [30]. The kinetics of this transient phase may also differ from cell type to cell type.…”

“…Because there are discrepancies in the literature about whether or not CQ raises the lysosomal pH [29,30], and our data indicated that a concentration of 100 µM had no major effect ( Figure 1(C,D) and S1(B)), we assessed the lysosomal acidity in U2OS cells exposed to increasing concentrations of CQ using the LysoTracker Red dye (Figure S1(C)). Surprisingly, LysoTracker Red-positive puncta were present in cells treated with CQ at concentrations ranging from 25 to 200 µM (Figure S1(C)), but also at concentrations of 400 and 800 µM when cells started to display clear signs of stress (data not shown).…”

A lysosome‐plasma membrane‐sphingolipid axis linking lysosomal storage to cell growth arrest

“…Lysosomal accumulation of uncatabolized material is a hallmark of a large number of diseases, including lysosomal storage disorders, Alzheimer's disease, Parkinson's disease, and brain aging (5)(6)(7). Moreover, it has been recently demonstrated that diverse lysosomotropic drugs accumulate within the lysosomes, affecting their function (8,9). However, the molecular mechanism responsible for the onset of cell damage upon lysosomal engulfment remains unknown to date; this represents the main challenge in the field.…”

Correction of Niemann-Pick type C1 disease with the histone deacetylase inhibitor valproic acid

“…CQ is a well-established lysosomotropic compound, which, in its free base form, can easily traverse the lipid bilayer of the lysosome and become ionized in its lumen, thereby reducing its permeability (120). The accumulation of CQ in the lumen of the lysosome serves as a sink for free protons culminating in an increase in the lysosomal pH (121), an effect which works to normalize the hyperacidification of the lysosome in response to HDACi treatment.…”