Webinar: Autophagy Webinar with Patrice Codogno  

Follow-Up Q&A with Dr. Codogno

Which factors are necessary for autophagosome-lysosome fusion? Is it possible that autophagosome formation is induced but fusion is not taking place due to lack of acidification?

There are multiple molecular actors that govern autophagosome lysosome fusion. For specific details, I recommend reading recent reviews on this topic (Nakamura and Yoshimori 2017 J Cell Sci and Reggiori and Ungermann 2017 J Mol Biol). The process involves Rab GTPases (e.g., Rab 7), SNAREs including Syntaxin 17, ESCRT proteins, TECPR1, PLEKHM1 and ATG14. ATG14 is a protein required for the autophagosome formation in a complex with Beclin 1 and Vps34 (or class III phosphatidylinositol kinase).
In the absence of lysosomal acidification, the fusion of autophagosomes with lysosomes can take place.  For example, lysosomotropic agents (e.g., chloroquine) do not block fusion but bafilomycin A1, which inhibits vacuolar ATPase, blocks fusion between autophagosomes and lysosomes. This effect is not due to the lack of lysosome acidification but rather due to the inhibitory effect of bafilomycin A1 on the Ca²⁺ pump SERCA (Mauvezin et al 2015 Nat Commun).

Do you find there is any role for the extracellular glycocalyx (seen in endothelial i.e.) in autophagy?

That’s an excellent question which is currently under investigation. Glycocalyx is a potential candidate to mediate the effect between shear stress and autophagy in endothelial cells.

In the biology of aging, what is the balance between autophagy vs increase in protein production?

During aging in most organisms there is a slow-down of protein synthesis without major changes in the fidelity of translation. In fact, the general process of protein homeostasis or proteostasis is altered including the capacity of protein folding and degradative pathways such as autophagy. Restoring proteostasis, for instance autophagy capacities, is beneficial for longevity. In regard to autophagy, many studies in multiple organisms have shown that restoring autophagy increases longevity and decreases diseases associated with aging.

Is there any contribution of late endosome-er contact in autophagosome formation?

Late endosomes can fuse with autophagosomes to form a compartment called an amphisome. This compartment is acidic and degradative. In the final degradative step of autophagy, amphisomes can fuse with lysosomes. Early endosomes and recycling endosomes contribute to autophagosome formation. For example, ATG9 and ATG16L1 are transported from the plasma membrane through the endocytic compartments to contribute to the early stage of autophagosome formation.

Are autophagosome membranes definitely derived from PM membranes?

The plasma membrane contributes to the formation of autophagosomes but the autophagosome is not built up at the plasma membrane. ATG9 and ATG16L1 are transported from the plasma membrane via the endocytic pathway to the site of autophagosome biogenesis. The phagophore emanates from the ER during starvation. The plasma membrane contributes to autophagosome biogenesis via the recruitment of ATG (9 and 16) and through ER-plasma membrane contact sites.

Does ATG5 have any non-autophagy related roles in lysosome/ endosome functions?

ATG5 can in fact modulate the late stage of autophagy. The primary role of the ATG12-ATG5 conjugate is to control autophagosome formation with ATG16L1. ATG5 also interacts with TECPR1, a protein required for autophagosome maturation (fusion with lysosome). In this way ATG5 conjugated to ATG12 can modulate the maturation of autophagosomes. Interestingly, the binding of ATG5 to ATG16L1 and TECPR1 are mutually exclusive.

Does the size/volume of endothelial cells change in response to low or high shear stress?

In contrast to kidney epithelial cells, shear stress (high or low) does not affect endothelial cell size. Interestingly, autophagy is important for the alignment of endothelial cells in the direction of the flux and in response to shear stress.

Is it possible that long lasting stress could lead to autophagy and could also cause transformation into cancer cells?

Your question raises an important point regarding the role of autophagy in cancer. I would say that generally speaking autophagy is protective in the early stage of cancer and tumor supportive in the late stage of cancer. However, this rule is far from universal. For example, autophagy has been shown to contribute to early stage colon cancer. Thus, the relationship between long lasting stress, autophagy and tumor development should be analyzed case by case.

Could you comment on how chemical modulation of autophagy benefits diseases differently? For example, if a drug molecule acts as a stimulator of autophagy, this is often considered a poor response because it may result in the development of drug resistance in cancer cells. Thus, a cancer drug should be an autophagy inhibitor, however, an autophagy stimulating drug may be good for Alzheimer’s disease (and atherosclerosis).

The beneficial effect of chemical modulation of autophagy is of course dependent on the role of autophagy in disease. Stimulation of autophagy has been shown to be beneficial in many pathologies, neurodegenerative disease, and atherosclerosis because the autophagic flux is impaired or blocked in these diseases. In many types of tumors autophagy is a mechanism associated with drug resistance. In this setting blocking autophagy is an adjuvant to cancer treatment to ameliorate the efficiency of cancer therapy. Several clinical trials are ongoing in this area.

Due to the effect of 3-AMA on class I PI3K and its positive effect on autophagy, would you recommend this molecule as a specific inhibitor of autophagy?

While long-term treatment with 3-methyladenine can inhibit class I PI3K (see Wu et al 2010 J Biol Chem), this treatment can also have a seemingly paradoxical stimulatory effect on autophagy. More broadly, we have to be careful about pharmacological inhibition of autophagy in terms of dose response and kinetics. The use of these drugs whenever possible should be complemented with genetic approaches to invalidate the expression of ATG. Here again care should be taken because ATG can also be involved in non-autophagy processes (see the autophagy guidelines Klionsky et al 2016 Autophagy).

Do you think LC3B and p62 are the best specific markers of autophagy?

To have a more complete answer to this question you can consult the autophagy guidelines (Klionsky et al 2016 Autophagy). So far LC3B and p62 are used as markers of the autophagic pathway. Of course, if you want to study the early autophagy stage, you can use other markers such as the recruitment of WIPI2, ATG9 and some others. Keep in mind that LC3B-II formation can also be involved in conjugation not related to autophagy and that the level of p62 mRNA can be influenced by different stress situations. Mizushima and colleagues reported that in some cell lines the level of p62 mRNA can be increased by the production of amino acids during autophagy. During our study on autophagy and shear stress we observed that the p62 mRNA levels were increased by shear stress. For this reason, we choose not to use the amount of p62 as a readout to analyze the autophagic flux under shear stress. LC3B and p62 are reliable markers of autophagy but you must be careful when using these markers. The gold standard to detect autophagic structures is EM observation and this technique, whenever possible, should be coupled to LC3B and p62 based readouts.

Have you correlated ER stress with autophagy?

In the studies presented during the webinar we did not investigate the effect of shear stress on ER stress. It is known that ER stress triggers autophagy, in general.

Is the regulation of autophagy different in fast vs/ slow proliferating cells?

Your question is important. However, the answer to this question depends on the stimuli for autophagy induction. I can say that slow proliferating cells are more dependent on autophagy than fast proliferating cells, in order to maintain homeostasis of the intracellular milieu. Fast proliferating cells can dilute protein aggregates and damaged organelles in daughter cells.

What is the effect on autophagy during p62 inhibition in different cell types?

p62 is engaged in the selective form of autophagy. Its inhibition can cause accumulation of damaged structures. However, the consequence of p62 inhibition can be cell type specific.