High basal autophagic activity in the brain revealed by systemic quantitative analysis using GFP-LC3-RFP mice

Reviewer #1 (Public review):

Summary:

The authors develop a GFP-LC3-RFP autophagy reporter under the control of the Rosa26 locus to measure autophagic flux in mouse embryos as well as adult tissues. While image quantification is consistently used, the authors also develop a semi-high-throughput assay for measuring autophagic flux using a microplate reader. Additionally, the authors cross these mice with a Cre-inducible Atg5-deletion mouse model, allowing the investigation of how autophagy flux is affected upon loss of Atg5. With this model, they demonstrate that loss of Atg5 leads to an increased ratio of GFP/RFP intensity in multiple tissues, including the brain, revealing that the brain undergoes basal autophagy. They further go on to show that the increase in GFP/RFP intensity upon Atg5 loss is greater in adult tissues compared to their embryonic counterparts. The development of an animal model, along with quantitative tools to measure the model, will have a high impact on the field. However, the analyses from the data presented do not fully justify the conclusions.

Strengths:

(1) A mouse model to better measure autophagy.

(2) The plate-reader-based method to quantify autophagy across tissues.

(3) Assessment of autophagy in many different tissues.

(4) Crossing the reporter mouse with the Atg5f/f mouse to assess basal autophagy.

Weaknesses:

(1) While the tool is of high impact, there is little new biological or mechanistic insight provided in these studies.

(2) The quantification and normalization method is unclear, making it difficult to compare across tissues accurately.

(3) Differential expression across cell types is not well documented or taken into account for comparisons.

(4) There is no consideration for sex as a biological variable.

Reviewer #2 (Public review):

Summary:

The aim of the authors was to measure starvation-induced and basal autophagy in vivo across several tissues and developmental stages. For this, they developed a novel mouse model expressing the GFP-LC3-RFP reporter. They also aimed to provide a more high-throughput method for autophagy flux measurements than assessment by imaging and developed an assay based on a microplate reader.

Strengths:

(1) Good validation of the mouse model. The knock-in strategy is well explained and illustrated.

(2) The model has potential to be applied to a wide range of research questions. The Cre-dependent expression allows for customization of KO timing, which will be beneficial in developmental studies.

(3) The authors presented consistent findings using two different methods to quantify autophagy, strengthening the robustness of their results.

(4) The authors demonstrated the validity of the high-throughput method (microplate reader).

Weaknesses:

(1) The comparison of neuronal populations in different areas of the brain is not ideal. In the cerebellum, Purkinje cells were chosen, which are rare and not representative of this tissue, as well as functionally very different from the neurons in the hippocampus and cortex that they were compared to.

(2) The explanation of the GFP-LC3-RFP construct and specifically if/how autophagosome formation can be measured and distinguished from flux could be clearer.

Conclusion:

The work presented is thorough, and the authors achieved their goals for this study. The effort used to further investigate unexpectedly high basal levels of autophagy in the brain is well appreciated and adds value to this paper. The conclusions of the authors are mostly very well supported by the data provided. The well-structured description of the results, along with clear figures, allows the reader to comprehend the authors' reasoning in reaching their conclusions.

The presented mouse model has great potential for a lasting positive impact on the research field of in vivo study of autophagy. The method of utilizing a microplate reader will also benefit future research where semi-high throughput is an advantage. Together, the information provided in this study not only presents new methodology that will allow the investigation of new research questions, but also provides novel information about in vivo autophagy flux at the selected developmental stages that opens up new follow-up research questions.