High lumenal chloride in the lysosome is critical for lysosome function

[Editors’ note: the author responses to the first round of peer review follow.]

Reviewer #1:

Quoting a review from Jentsch (2013), exact measurements of lysosomal [Cl−] have not been reported due to the lack of suitable Cl− sensors for the high [Cl−] at low pH. However, from measurements with cells treated with low Cl− and from model calculations, lysosomal [Cl−]lumen is greater than 80 mM. 2010 data strongly point to a crucial role of Cl− transport in organellar physiology that "goes beyond merely providing the electrical shunt for proton pumping by the V-ATPase." Despite maintaining lysosomal conductance and normal lysosomal pH, Clcn7unc/unc mice showed lysosomal storage disease like mice lacking ClC-7. Although various proteins are known to be regulated by Cl−, the mechanism by which (mainly luminal) Cl− affects membrane traffic and organellar function has remained elusive; the 2010 analysis of Clcn5unc and Clcn7unc mice has suggested that luminal anion concentration is important all along the endosomal-lysosomal pathway.

Here, the authors use a novel, DNA based chloride sensor to try more accurately to determine lysosomal chloride levels. Like the previous work from the Jentsch lab, the findings using C. elegans lysosomes indicate a high level of lysosomal chloride. Chloride ion levels are decreased under conditions of lysosomal dysregulation due to mutations in genes that lead to lysosomal storage disorders. Not clear are the mechanisms underlying these observations.

Recently, the lysosome has been shown to represent a significant calcium store, and in worms, CUP-5 is the MCOLN-1 homolog that is important for lysosomal calcium levels and functions. If chloride is not the counterion for protons, it may be important for calcium homeostasis. Given that it is not clear how chloride influences lysosome function in disease states or in normal cells, it would be very important for these authors to clarify the connection between chloride accumulation defects and calcium uptake and regulation. Without this, the findings seem somewhat anecdotal, despite highlighting the recently uncovered importance of chloride ions in lysosome function. No doubt this is an interesting area for further investigation.

We now include additional experiments that address how low chloride in the lysosome could mechanistically connect to lysosomal storage disease. We pursued two hypotheses. One, suggested by this reviewer, was that low chloride could impede lysosomal Ca²⁺ accumulation, which is known to compromise lysosome function (1–3). We also explored whether chloride could directly impact the proper function of specific lysosomal enzymes (1).

Our studies revealed both mechanisms to be operational. Briefly, selectively inhibiting chloride channels with NPPB resulted in reducing lysosomal Ca²⁺ (Figure 5B-D). Further, the lysosomal enzymes cathepsin C and aryl sulfatase B showed reduced enzymatic activity upon reducing lysosomal Cl^- with NPPB (Figure 5E-G).

The results of these experiments are now presented as Figure 5 of the revised manuscript, along with the associated discussion in the subsection “High chloride regulates lysosome function in multiple ways”.

Reviewer #2:

[…] 1) Cl^- channels are known to exhibit poor anion selectivity. What is the anion selectivity for Clensor? When lysosomal [Cl^-] is reduced in some LSD cells, assuming that lysosome lumen is still electroneutral and iso-osmotic, what are substituted anions? Phosphate is probably an obvious candidate. Therefore, it would be helpful if the authors can show the effects of NO3(-) and PO4(3-) in Clensor in vitro.

We thank the reviewer for this suggestion. Figure 1—figure supplement 3 shows the selectivity of Clensor to various anions in the form of their sodium salts. This reveals that the sensitivity of Clensor to diverse biologically abundant anions including NO₃^- and PO₄^3- is negligible. This is attributed to the fact that Clensor's chloride-sensing module, BAC, undergoes collisional quenching selectively with halide ions because of their well-matched HOMO-LUMO energies (2,3). However, chloride is the most abundant physiological anion (4) hence BAC acts as a chloride sensor in biological systems.

2) Lysosomal delivery of Clensor might be slowed down in LSD cells, compared with wild-type (WT) cells. Could trafficking defects contribute to the observed reduction in lysosomal [Cl^-]? Given the 40 mM difference in late endosomal vs. lysosomal [Cl^-], it is plausible that a block in endosome maturation might affect the [Cl^-] measurement. I would assume that the calibration curves and maximal signals are identical for WT and LSD cells. Is that the case?

The reviewer's point is well taken. Before any Cl^- measurements were made in any mutant C. elegans, we performed colocalization studies of Clensor with LMP-1::GFP transgenic worms, where the relevant lysosomal storage disorder gene was knocked down by RNAi. These revealed ~80% colocalization at t = 60 min post injecting Clensor in all mutant backgrounds studied here, indicating that trafficking of the probe was not affected. Further, in the context of clh-6 worms, though there is an imbalance in chloride concentrations, lumenal pH of these compartments matches that of wildtype lysosomes suggesting that these compartments are indeed likely to be lysosomes. Several other mutants e.g., ncr-1, aman and manba showed Clensor accumulated in grossly misshapen and/or enlarged LMP1::GFP positive compartments, further reinforcing that the compartments are indeed lysosomes swollen due to the accumulated material.

In murine and human macrophages, we pre-labelled lysosomes with TMR-Dextran (0.5 mg/mL) using literature protocols (5), treat cells with U18666A and Conduritol β-epoxide (CBE) to induce Niemann Pick C and Gaucher's cellular phenotype, only then pulse Clensor for 30 min, chased for 60 min, and scored for co-localization with TMR-Dextran. Amitriptyline (AH) and NPPB were added 30 min after pulsing and chasing of Clensor to lysosomes. We observed that Clensor colocalized with lysosomes under each of these conditions, indicating that they do not suffer trafficking defects in these cell culture models of lysosomal storage diseases. This data is now presented as Figure 4—figure supplement 5.

Reviewer #3:

The work of Krishnan and co-workers describes the use of previously published fluorescent sensors for measuring lysosomal pH and Cl levels. Elegantly, the work was performed in circulating nematode macrophages as well as cultured mammalian cells. This is a very exciting application and the authors observe that a lack of chloride levels correlates with a loss of degrading capacity of the lysosome. The authors follow the hypothesis that the drop-in chloride content is responsible for driving lysosomal function. They tested model conditions for lysosomal storage diseases in C. elegans and found indeed reduced chloride levels under such conditions. However, this is not a prove that the chloride ion levels are instrumental. The question remains if chloride levels are the key component in lysosomal dysfunction or a by-stander effect.

Please see response to reviewer #1, point #1. In a nutshell, this is not a by-stander effect.

The authors argue that CF patients show lysosomal distress symptoms at the molecular level (changes in enzyme activities) similar to Niemann-Pick patients. What the authors do not consider in their argumentation is that CF patients share very little of the NPC phenotype. There is no early neurodegeneration in CF nor a liver accumulation of lipids such as cholesterol or sphingosine. As a result, the conclusions driven by the present study do not hold.

Actually, we never implied any connection between Niemann Pick C with CF. We only cited published literature that correlated reduced arylsulfatase activity and acid sphingomyelinase activity in cells derived from patients with CF (12,13). These enzymes are defective in mucopolysaccharidoses VI and Niemann Pick A/B diseases(14). These are distinct diseases from Niemann Pick C disease.

However, given the extensive new data that we added, we have dropped mention of CF as it can prove distracting to the reader.

Down to the bare bones, the study reveals very interesting data regarding the chloride ion levels in lysosomes but lacks any mechanistic insight. Unfortunately, there is no consideration (or even mentioning) of the calcium levels in the lysosome that have received quite some attention in the regulation of lysosomal function and signaling lately (for instance, see Medina et al. Nature Cell Biology 17, 288-299 (2015)). Chloride might change driving forces for lysosomal im- or export but a direct switching function through changes in protein conformation as is known for calcium ions would be a large surprise.

Please see response to reviewer #1, point #1. Briefly, lysosomal Ca²⁺ is indeed depleted when lysosomal chloride is reduced. However, the functions of specific lysosomal enzymes such as cathepsin C and aryl sulfatase B, is also affected. There is in vitro evidence that high Cl^- is necessary for the activity of these enzymes (15–19). Data to this effect is now presented as Figure 5 in the revised main manuscript.

For publication in eLife, I would expect a more profound mechanistic insight of what chloride ions are doing in lysosomes. So far, the authors present highly interesting observations in very relevant cells followed by a conclusion section that is in my opinion not coherent with the observed phenotype (in CF). To make this work suitable for eLife, I would expect measuring and/or manipulating calcium levels under conditions where lysosomal chloride levels are high or low, respectively. In addition, the authors could manipulate chloride levels in lysosomes acutely and observe effects spontaneously.

We have addressed this comment. Please see response to reviewer #1, point #1.