Autophagy-Dependent Secretion: Crosstalk between Autophagy and Exosome Biogenesis

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this review, the authors summarize recent studies on the interaction between autophagy and exosome formation, noting that some topics are not fully covered. I recommend a minor revision before acceptance, including the following points:

1.     Many studies have shown that autophagy can increase the lysosomal degradation of exosomes. However, under certain conditions, such as nutrient deprivation or rapamycin treatment, autophagy activation appears to increase exosome production (line 377-383). The authors should address this conflict and provide a more comprehensive discussion in the review. Additionally, referencing more recent studies on this topic, particularly those examining the role of growth factors (doi.org/10.1080/15548627.2019.1596479) and stress (doi.org/10.1002/advs.202302622).

 2.     It is strongly recommended to include figures or tables in the section discussing the significance of amphisomes, to enhance the clarity and impact of the presentation.

Author Response

We greatly appreciate your constructive feedback and valuable suggestions on our manuscript. In accordance with your recommendations, we have conducted a revision to provide a more thorough discussion on the dual roles of autophagy in both the lysosomal degradation and the production of exosomes. We have also expanded our review to include recent studies on the role of growth factors and stress in this context.

1. Regarding the apparent conflict in the role of autophagy in exosome dynamics, we have added a detailed examination of the conditions under which autophagy promotes the lysosomal degradation of exosomes versus when it enhances exosome release. Our revised text now includes a nuanced discussion on the activation of autophagy under different stressors and growth conditions, referencing the studies you highlighted (Lines 379-407). This addition elucidates the context-dependent nature of autophagy's influence on exosome biogenesis and release, providing insights into the underlying mechanisms and physiological relevance of these processes.

2. To address the second point, we have integrated figure 2  that illustrate the beneficial or detrimental effects of amphisomes. These visual aids are designed to enhance the reader's understanding of the complex interplay between autophagy and exosome formation and to underscore the importance of amphisomes in cellular communication and waste management.

We believe that these revisions have significantly strengthened the manuscript, clarifying the intricate relationship between autophagy and exosome dynamics, and providing a comprehensive overview that is both informative and accessible to our readers.

Thank you for the opportunity to revise our work.

Reviewer 2 Report

Comments and Suggestions for Authors

This review describes the interplay involving mechanisms ruling autophagy and exosome biogenesis/release. The paper is interesting and well written, only some refinements are necessary.

1)     Page 1 lines 42-43 “and have been identified as having significant potential for therapeutic and diagnostic applications.” reference(s) is (are) missing.

2)     Page 2 lines 64-66 “Supporting this concept is the presence of amphisomes, which blend the characteristics and functionalities of both vesicle types and contribute to cellular secretion machinery.” reference(s) is (are) missing.

3)     Page 2-3 lines 93-98 “Apart from these degradative functions, autophagy also engages in non-degradative processes, such as secretory autophagy. This unique pathway diverges from the conven tional route, enabling cells to release proteins and other substances into the extracellular environment instead of directing them towards lysosomal degradation. This secretion extends autophagy's role beyond intracellular turnover to include significant involvement in cellular secretion and distribution of materials” reference(s) is (are) missing.

4)     Page 3 lines 133-135 “Secretory autophagy was first identified through research aimed at investigating the extracellular release mechanisms of leaderless Proteins, which lack an N-terminal signal sequence and are therefore incapable of entering the Endoplasmic Reticulum (ER).” reference(s) is (are) missing.

5)     Page 3 lines 143-150 “The process of secretory autophagy can be conceptually divided into three primary stages: 1- cargo recruitment to the forming autophagosome, for instance, through cargo receptors like TRIM16; 2- transport of the receptor-cargo complex to the autophagosome membrane via the R-SNARE protein SEC22B; 3- internalization of the cargo protein into the autophagosome, followed by fusion with the plasma membrane. This fusion is facilitated by a combination of SNARE proteins, specifically Sec22 on the autophagosomes, SNAP23 and SNAP29 and their cognate partners STX3 and STX4 on plasma membrane. This ultimately leads to the secretion of cargo proteins into the extracellular milieu.” reference(s) is (are) missing.

6)     Page 4 line 180 “The TRIM protein family, encompassing approximately 80 members,” reference(s) is (are) missing.

7)     Page 5 Figure 1, please indicate the software used to produce the artwork.

8)     Page 8 lines 361-365 “Toll-like receptors (TLRs) are critical components of the innate immune system, playing a central role in the recognition of various pathogens, including bacteria, viruses, and fungi. They are known to be involved in the regulation of autophagy. TLR2/6 is a heterodimeric receptor that, upon binding with lipoproteins, triggers a signaling pathway resulting in the induction of autophagy.” reference(s) is (are) missing.

9)     Page 9 line 437 “PMID: 1575680; PMID: 9008703”, if these are references, please add them in the bibliography and correct the reference enumeration in the text.

10)  Page 9 lines 511-513 “Varizella Zoster, in addition to being able to accumulate in host cell amphisomes[82], uses amphisomes for transportation to the cell surface[82], apparently for subsequent release to the outside” the same reference is repeated twice in the same sentence and some spaces are missing.

11)  Paragraph 3.5.4. please add spaces wherever required.

12)  Page 17 lines 843-847 “The E2 subunit of the pyruvate dehydrogenase complex (PDCE2) is localized on the inner mitochondrial membrane and is a part of the enzyme complex responsible for the conversion of pyruvate to acetyl-CoA in the mitochondrial respiration process. In damaged and stressed cells, PDCE2 can be translocated from the mitochondria to the cell surface and in some cases may act as an autoantigen.” reference(s) is (are) missing.

13)  Page 17 line 857-858 “PACSINs regulate intracellular vesicle trafficking, cytoskeletal rearrangement, caveolar biogenesis, neuronal development, and cell migration.” reference(s) is (are) missing.

14)  Page 18 lines 879-883 “MVB-mediated exosomal secretion is known to be an essential regulator of healthy reproduction, protecting high quality spermatozoa and eliminating feeble and defective spermatozoa. These exosomes interact with germ cells to maintain the homeostasis of spermatogenesis. The exosomal cargo proteins act together to ensure proper sperm motility, metabolism, oxidation-reduction, acrosome reaction, capacitation, and fertilization.”, lines 892-894 S-phase kinase-associated protein 1 (SKP1) is the assembly factor of SKP1–CUL1–F-box protein complexes, also known as CUL1-RING ubiquitin ligases, a family of E3 ligases found in all eukaryotes, which mediates the proteasomal degradation of hundreds of cellular regulatory proteins” and lines 908-917 “The cGAS-STING pathway plays an important role in innate immunity, serving as a sentinel for detecting cytosolic DNA—an indicator of viral infections or genomic instability. Within this pathway, the cyclic GMP-AMP synthase (cGAS) acts as a DNA sensor. Upon binding to cytosolic DNA, cGAS catalyzes the synthesis of cyclic GMP-AMP, which then binds to the stimulator of interferon genes (STING) transmembrane protein residing in the endoplasmic reticulum (ER) membrane. This binding triggers a conformational change in STING, leading to its activation. Active STING, in turn, recruits and activates the TANK-binding kinase 1 (TBK1) and the transcription factor interferon regulatory factor 3 (IRF3). This cascade ultimately culminates in the production of type I interferons (IFNs) and various proinflammatory cytokines, setting in motion an antiviral immune response.” References are missing.

15)  In the text many extended explanations of acronyms are put in parentheses, see for example page 3 line 109 “ULK1 (unc-51 like autophagy activating kinase 1)”. Please kindly explain all the acronyms in the text putting the acronym in parentheses after the extended explanation, as indicated in the guidelines https://www.mdpi.com/journal/cimb/instructions

16)  Please check typos like the missing space in line 449 page 10 “fusion[72], [73].” and the double punctuation at page 13 line 627 “health. [107].” throughout the text.

Author Response

Thank you for the detailed review and constructive feedback on our manuscript. We have taken all your comments into consideration and have made the necessary refinements.

1. We have inserted the appropriate references on page 1, lines 43-44, to substantiate the therapeutic and diagnostic potential of exosomes.

2. Additional references have been included on page 2, lines 64-66, to support the discussion on amphisomes.

3. On pages 2-3, lines 93-98, we have now cited sources that detail the non-degradative roles of autophagy, such as in secretory pathways.

4. Relevant literature has been referenced on page 3, lines 133-135, to elucidate the extracellular release mechanisms of leaderless proteins.

5. We have addressed the requirement for citations on page 3, lines 143-150, by providing references that discuss the primary stages of secretory autophagy.

6. On page 4, line 181, references regarding the TRIM protein family have been added.

7. The software utilized for the creation of Figure 1 on page 5 has now been indicated in the figure legend.

8. We have included references on page 8, lines 361-365, to back the role of TLRs in autophagy regulation.

9. The PMIDs mentioned on page 9, line 460, have been correctly integrated into the bibliography, with the in-text reference numbers duly adjusted.

10. Redundancies and spacing issues noted on page 9, lines 511-513, have been rectified.

11. All required spaces have been added in Paragraph 3.5.4.

12. Missing references for the information on PDCE2 have been incorporated on page 17, lines 872-874.

13. A citation for the role of PACSINs, mentioned on page 17, line 887, has been included.

14. For the sections on MVB-mediated exosomal secretion in reproduction (page 19, lines 909-914), the role of SKP1 (page 19, line 925), and the cGAS-STING pathway (page 19, line 943), the required references have been added.

15. We have ensured that acronyms are now explained in accordance with the journal's guidelines.

16. A meticulous review of the manuscript has been conducted to correct any typographical errors, including those related to spacing and punctuation.

We trust that these revisions have satisfactorily addressed your concerns and have enriched the manuscript. We are hopeful that the improvements made will facilitate the manuscript's publication.