In this study, the authors report on the morphological and functional alterations of lysosomes and late endosomes in an in vitro model of neuronal aging. Between 21 and 28 DIV somatic and neuritic endolysosomes become enlarged, less acidic, less degradative yet they show a higher level of cathepsin D and a higher proportion of late endosomes (positive for CI-M6PR). The proximal-to-distal gradient of endolysosomes along neurites is also more shallow with in vitro aging, reflecting a shift in the antero-/retrograde balance of their motility in neurites. Interestingly, similar findings were made in aged mouse brains (18-mo hippocampal CA1 region and prefrontal cortex), showing the relevance of the in vitro aging model findings. In the last part, the authors attempt to relate these changes with the loss of excitatory synapses between 21 and 28 DIV using drug treatments (TRPML1 agonist or chloroquine overnight).

The study is interesting, carefully designed and executed. Data are accurately described. Overall, the study provides compelling evidence for the reported endolysosomal changes. The last part on the causal relationship between these changes and synapse loss in the in vitro model is less convincing because the drugs used have pleiotropic effects. The induction of lysosomal Ca2+ efflux by TRPML1 agonists induces multiple effects, including the stimulation of lysosome fusion with other membranes and the activation of transcription factors for lysosomal biogenesis. Moreover, the molecular basis for the reported effect on lysosome acidification is unclear. Similarly, the neutralization of acidic organelles by chloroquine induce multiple effects.

I also have 2 points about the lysosomal pH assay. First, it would be useful to quantitate the pH shift of aged lysosomes using a ratiometric pH probe, for instance Oregon Green dextran. Second, in my experience, the yield of Lysotracker fixation is very poor. Imaging this dye immediately after mounting does not solve this pitfall. I would suggest to restrict this assay to live neuron imaging.

Minor points:
1) The designation of 21-DIV/28-DIV neurons as ‘mature’/’aged’ should be defined on p. 5

2) p. 6, line 1, ‘… indicating that ELs are positioned more distally in aged neurons…”: the neurite/soma ratio does not seem higher at 28 DIV than 21 DIV.

3) p. 6, last paragraph, ‘… suggesting a change in Cat D lifetime’: why do the authors favor slower degradation over increased expression?

4) p. 8, line 4, ‘suggesting an enlargement of distal ELs with aging’: the enlargement occurs in fact in the 3 neuritic regions (Fig. 2C)

5) p. 8, 2nd paragraph, ‘Interestingly, inhibition of synaptic activity increases…’ (missing word)

6) ‘accumulate/ing DQ-BSA…’ (p. 8, 3rd paragraph; p.9 , last paragraph) is an improper term. The DQ-BSA signal reflects the dequenching (through degradation) of this substrate, not its accumulation

7) p. 11, lines 1-3, ‘… LAMP1 was enriched in the synaptosomal fraction of the aged brain’: the enrichment of LAMP1 in P3 fractions with aging is not significant in Fig. 4I.

8) The text could be more concise.

The authors addressed most reviewers' concerns. Although some questions remain (quantitation of lysosomal pH changes; mechanism of reported ML-SA1 effect), the new data significantly strengthen the manuscript.