TRPML1 positions lysosomes and regulates actin-membrane linkers in astrocyte processes

Reviewer #1 (Public review):

Summary:

Late endosomes and lysosomes (LEL) are dynamic organelles with critical roles in cell physiology via transport of cargos to various destinations, degrading cargos, and as calcium stores. The latter is a less studied function of LELs, and virtually nothing is known about LEL function and transport in astrocytic processes. This manuscript investigates the dynamics of LELs in astrocyte processes co-cultured with neurons and finds that the lysosomal calcium channel Trpml1 regulates their positioning near astrocytic specializations (PAPs) downstream of synaptic activity.

Strengths:

Rigorous and well-controlled study of an understudied area of cellular neuroscience, namely regulation of organelle transport in astrocytes to shape synaptic environment and functionality.

Weaknesses:

Some of the same mechanistic links have been probed in neurons and other cell types, but astrocyte cell biology is still less extensively studied, making this an important contribution. Currently, only cultured astrocytes are being investigated.

Reviewer #2 (Public review):

Summary:

Many ion channels/transporters in endosomes and lysosomes remain poorly understood. Even though the functional importance of endo-lysosomes in astrocytes has been recently recognized in many neurological diseases including lysosomal storage disorders and neurodegenerative diseases, the basic biology has not been much studied. In this manuscript, Spivey et al. showed how one of the key ion channels i.e., TRPML1, regulates endosomes and lysosomes (LELs) in astrocytes -which are also not much investigated in the field, compared to neurons- and its activity may further affect the structure of astrocytes and synaptic activity of neighboring neurons. The authors elegantly use multiple controls from agonists, antagonists, and TRPML1 knockdown to corroborate the results. The data are very strong and well supported. I believe that revising a few parts of the manuscript will greatly augment the significance of this work.

Strength:

The rigor of the study and data using various controls. The quality of the data is also very impressive.

Weakness:

A limitation of the study is that the mechanistic experiments rely primarily on overexpression or genetic knockdown of TRPML1 approaches, both of which alter TRPML1 abundance on endolysosomal membranes and potentially introduce artifacts related to protein level, affecting luminal ion homeostasis. While the overall conclusions are convincing, validation in a genetic MCOLN1 knockout mouse model would provide more definite evidence for the proposed mechanism.

Reviewer #3 (Public review):

Summary:

In their manuscript, the authors assess how TRPML1 influences lysosome trafficking and function in astrocytes using a neuron-astrocyte coculture system. Specifically, it was found that activation of TRPML1 reduces LEL motility in astrocyte branches while TRPML1 increases it, and a model was proposed in which TRPML1 coordinates lysosomal positioning along astrocyte branches, enabling LELs to locally regulate actin-membrane linkers that influence PAP (peripheral astrocyte processes) structure and plasticity. The authors primarily use pharmacology to buttress their findings.

Strengths:

TRPML1 is currently under investigation as a potential therapeutic target for lysosomal storage disorders and neurodegenerative diseases such as AD and PD. The manuscript is hence timely and important as the crosstalk between glia cells and neurons is likewise increasingly recognized as disease-relevant.

Weaknesses:

Unfortunately, experiments performed to investigate the role of TRPML1 are based purely on pharmacological tools. No bona fide KO data are available. At least for some experiments, this should be done. MLIV iPSC lines are available.

If no KO controls are being provided, at least the authors shall use ML1-SA1 (EVP-169) as an agonist instead of ML-SA1, because ML-SA1 activates all three TRPML channels, which would be a major problem for this study. Likewise, the ML-SI3 antagonist also has effects on other TRPMLs. EDME is a blocker with higher specificity for TRPML1.

When using pharmacological tools, the original papers relating to these tools may be cited. For example:

- For ML-SA1 https://www.nature.com/articles/ncomms1735 and for MK6-83 https://www.nature.com/articles/ncomms5681

- ML-SI3 as a blocker for TRPML1 seems problematic: https://pubmed.ncbi.nlm.nih.gov/33187805/ ; an alternative may be: https://www.nature.com/articles/s41598-021-87817-4

No reference is made to other important regulators of lysosomal cation homeostasis such as TPC1 or TPC2, two other Ca2+/Na+ permeable cation channels, or other TRPML channels. The authors should provide some data supporting or excluding a role of these channels.