Gene transfer of master autophagy regulator TFEB results in clearance of toxic protein and correction of hepatic disease in alpha-1-anti-trypsin deficiency

Alpha-1-anti-trypsin deficiency is the most common genetic cause of liver disease in children and liver transplantation is currently the only available treatment. Enhancement of liver autophagy increases degradation of mutant, hepatotoxic alpha-1-anti-trypsin (ATZ). We investigated the therapeutic potential of liver-directed gene transfer of transcription factor EB (TFEB), a master gene that regulates lysosomal function and autophagy, in PiZ transgenic mice, recapitulating the human hepatic disease. Hepatocyte TFEB gene transfer resulted in dramatic reduction of hepatic ATZ, liver apoptosis and fibrosis, which are key features of alpha-1-anti-trypsin deficiency. Correction of the liver phenotype resulted from increased ATZ polymer degradation mediated by enhancement of autophagy flux and reduced ATZ monomer by decreased hepatic NFκB activation and IL-6 that drives ATZ gene expression. In conclusion, TFEB gene transfer is a novel strategy for treatment of liver disease of alpha-1-anti-trypsin deficiency. This study may pave the way towards applications of TFEB gene transfer for treatment of a wide spectrum of human disorders due to intracellular accumulation of toxic proteins.

Thank you for the submission of your manuscript to EMBO Molecular Medicine. We have now heard back from the three Reviewers whom we asked to evaluate your manuscript.
All Reviewers are quite positive and find the topic of the study important. They do, however raise a number of concerns about the interpretation of the results and important technical issues, which should be convincingly addressed with further experimentation where necessary, in a revision of the current manuscript. Furthermore, as you will see from the enclosed reports, Reviewers 1 and 3 both express concern on the mouse embryo fibroblast experiments in Figure 1 with respect to the observed increase in mutant ATT protein secretion under TFEB overexpression.
I should remind you that it is EMBO Molecular Medicine policy to allow a single round of revision only and that, therefore, acceptance or rejection of the manuscript will depend on the completeness of your responses included in the next, final version of the manuscript.
Revised manuscripts should be submitted within three months of a request for revision; they will otherwise be treated as new submissions, except under exceptional circumstances in which a short extension is obtained from the editor. I look forward to seeing a revised form of your manuscript as soon as possible. ***** Reviewer's comments ***** Referee #1 (General Remarks): Reviewer comments: The manuscript by Pastore et al., describes an interesting gene transfer strategy for the treatment of AAT-deficiency. The strategy consists in the overexpression of the TFEB transcription factor in the liver that increases autophagy and lysosome biogenesis in the hepatocytes reducing the accumulation of the hepatotoxic ATZ protein (AAT mutant protein). The therapeutic effect is clear and has been very well demonstrated by the authors in a relevant animal model of AAT deficiency. Importantly constitutive expression of TFEB is not toxic and the reduction of ATZ expression levels reduces the risk of HCC development. One interesting aspect of this strategy is that it will be valid for patients with different genetic deficiency leading to AAT accumulation. Furthermore this therapeutic strategy could be apply to other disease associated to the intracellular accumulation of misfolded proteins.
However, some authors have previously reported the use of molecules to increase autophagy for the treatment of AAT deficiency reducing the novelty of the approach presented in this work.
The paper has some general and some specific flaws.
1. The quality of figures 1A and 1B should be improved since it is not clear which are the ATZ bands in each case.
2. The authors are using a long term expression vector, toxicity analysis should be also performed at later time points after vector administration.
3. Since the reduction of intracellular ATZ is associated with an increase of ATZ secretion in which percentage the reduction of ATZ after TFEB expression is due to autophagy or to the increase of protein secretion? Why TFEB expression increase ATZ secretion? Carbazepine (CBZ) induces ATZ reduction by activation of autophagy but also by proteasome degradation; did the author check if the proteasome play a role on ATZ reduction after TFEB expression?
4. It has been reported that Autophagy abrogation increase ATZ secretion so it will be expected that autophagy activation will reduce ATZ secretion, the authors should comment on this.
5. Does TFEB expression result in ATZ secretion to the circulation? 6. CBZ induce an increase in the number of autophagosomes, however, TFEB expression decrease the number of autophagosomes, the explanation given by the authors is that this is due to fusion of the autophagosomes to the lysosomes, meaning that once the autophagomes are fused to the lysosomes they cannot be detected? 7. It will be interesting to see if TFEB expression induces ATG7, ATG12 and LC3 expression in the liver.
8. The authors should analyse if TFEB expression affects the secretion of AAT and depending on the results they should comment on how lung pathology associated with AAT-deficiency will be affected.
9. Indicate in Fig 2D that the graph represents the fold increase in TFEB expression.

Referee #2 (General Remarks):
The manuscript by Pastore, et al., attempts to confirm the hypothesis that augmentation of autophagy will increase the degradation of Z-mutant alpha-1 antitrypsin (ATZ) both in cell lines and in the transgenic ATZ over-expressing mouse model.
The basis for the experiments is augmentation of transcription factor EB (TFEB). This is done in cell lines by transfection, which are then assessed by pulse-chase labeling, both in transient transfections and stable cell lines.
Subsequent experiments utilized helper-dependendent Ad vectors (HDAd) to sugment TFEB in the transgenic mouse model. The key finding that Z-AAT accumulation in the liver was reduced was demonstrated convincingly. Other experiments demonstrated a decrease in both monomeric and polymeric ATZ. The changes in ATZ accumulation were correlated with a number of other key findings including an improvment in mitochondrial ultrastructure and a demonstration of autophagy markers being increased.
Finally, there is evidence presented that the HDAd-TFEB augmentation actually decreased the amount of AAT mRNA. This reinforces a concept presented as a model in Figure 8, that the cellular response to the mutant AAT results in increased IL-6-mediated up-regulation of AAT expression.

Referee #3 (Comments on Novelty/Model System):
This is a nice straightforward study that uses a robust model of A1AT liver disease to show that gene trasfer of TFEB prevents mutant A1AT accumulaton, liver injury, and liver fibrosis. The results are very good but expected.

Referee #3 (General Remarks):
This is a nice straightforward study that uses a robust model of AAT liver disease to show that gene trasfer of TFEB prevents mutant AAT accumulaton, liver injury, and liver fibrosis.
1. The MEF data in figure 1 is the least convincing. Why is there increased secretion of A1AT with TFEB treatment? The pulse chase is also difficult to interpret. Why not use primary cultures of hepatocytes from the A1AT Z mouse to demonstrate the effects? 2. Figure 2 is very impressive, but would benefit from a higher magnification of 2A in order to clearly visualize the PAS positive inclusion bodies.
3. Figure 4. The HDAd-TFEB is on different gels than the controls so that comparisons are tenuous. The whole experiment should be run on one gel. Dear Editor, Thank you for considering our manuscript entitled "Gene transfer of master autophagy regulator TFEB results in clearance of toxic protein and correction of hepatic disease in alpha-1antitrypsin deficiency" by Pastore et al. for publication in EMBO Molecular Medicine. We appreciated the thoughtful comments of the reviewers and we included the information pertinent to address their concerns. Enclosed please find a revision that responds to the reviewers' comments and concerns. We have highlighted in red the changes we made in the revised manuscript.
Please see below for a detailed response to each reviewer's comments. We have done our best to respond to the concerns of reviewer #1 and #3 (reviewer #2 did not had specific critiques) and have addressed them in point-by-point fashion.

Reviewer #1 comments: The manuscript by Pastore et al., describes an interesting gene transfer strategy for the treatment of AAT-deficiency. The strategy consists in the overexpression of the TFEB transcription factor in the liver that increases autophagy and lysosome biogenesis in the hepatocytes reducing the accumulation of the hepatotoxic ATZ protein (AAT mutant protein). The therapeutic effect is clear and has been very well demonstrated by the authors in a relevant animal model of AAT deficiency. Importantly constitutive expression of TFEB is not toxic and the reduction of ATZ expression levels reduces the risk of HCC development. One interesting aspect of this strategy is that it will be valid for patients with different genetic deficiency leading to AAT accumulation.
Furthermore this therapeutic strategy could be apply to other disease associated to the intracellular accumulation of misfolded proteins. However, some authors have previously reported the use of molecules to increase autophagy for the treatment of AAT deficiency reducing the novelty of the approach presented in this work.
The paper has some general and some specific flaws.

The quality of figures 1A and 1B should be improved since it is not clear which are the ATZ bands in each case.
Author's reply: We have repeated the in vitro studies and we are now presenting images of improved quality that show ATZ bands more clearly (revised Fig. 1).

The authors are using a long term expression vector, toxicity analysis should be also performed at later time points after vector administration.
Author's reply: We have evaluated toxicity also at later time points (up to 6 months postinjection) in the PiZ mice injected with HDAd-TFEB vector and in controls ( Supplementary  Figures 4 and 5).

Since the reduction of intracellular ATZ is associated with an increase of ATZ secretion in which percentage the reduction of ATZ after TFEB expression is due to autophagy or to the increase of protein secretion? Why TFEB expression increase ATZ secretion? Carbazepine (CBZ) induces ATZ reduction by activation of autophagy but also by proteasome degradation; did the author check if the proteasome play a role on ATZ reduction after TFEB expression?
Author's reply: We have repeated the pulse and chase experiments and performed a careful determination of the ATZ band in the media that was not previously performed. Besides the reduction of intracellular ATZ, upon quantification in media we observe a decreased ATZ secretion in cells transfected with TFEB. We are providing these new data on ATZ secretion in the revised Fig. 1 (please see panels A and C of revised Fig. 1). We treated MEFs with MG132 proteasome inhibitor and we observed in TFEB transfected cells a similar reduction in intracellular ATZ compared to untreated cells. Therefore, TFEB does activate proteasome degradation of ATZ in MEF. This new data has been included as panel E in revised Fig. 1.

It has been reported that Autophagy abrogation increase ATZ secretion so it will be
expected that autophagy activation will reduce ATZ secretion, the authors should comment on this.
Author's reply: We indeed observed a reduction of both intracellular and extracellular ATZ (please see panels A and C of revised Fig. 1). The results of our studies are now consistent with the reported role of autophagy on ATZ secretion.

Does TFEB expression result in ATZ secretion to the circulation?
Author's reply: Consistent with the reduction in monomeric ATZ (Fig. 5), we observed a reduction in serum ATZ over time in HDAd-TFEB injected mice. These data have been added as Fig. 3.
6. CBZ induce an increase in the number of autophagosomes, however, TFEB expression decrease the number of autophagosomes, the explanation given by the authors is that this is due to fusion of the autophagosomes to the lysosomes, meaning that once the autophagomes are fused to the lysosomes they cannot be detected?
Author's reply: The reviewer is correct. Upon fusion with the lysosome, autophagosome loses rapidly its specific molecular markers (such as LC3) and its identity as an organelle. Consistent with previously published observations (Teckman et al. Am J Physiol Gastrointest Liver Physiol 279:G961-G974, 2000), we found an increase in autophagosome number in PiZ mice compared to wild-type mice (Fig. 4F). As the reviewer pointed out, HDAd-TFEB injected PiZ mice exhibited less autophagosomes than control PiZ mice, likely because their fusion with lysosomes is activated by TFEB, as previously reported (Settembre et al., Science 332:1429-1433, 2011.
7. It will be interesting to see if TFEB expression induces ATG7, ATG12 and LC3 expression in the liver.
show that samples were run all together. However, for consistency with other experiments presented in the manuscript, we would prefer to show 3 out of the 5 samples.
Thank you again for your encouraging response and we hope the revised version of this manuscript may now be suitable for publication in a future issue of EMBO Molecular Medicine. 2nd Editorial Decision 11 December 2012 Thank you for the submission of your revised manuscript to EMBO Molecular Medicine. We have now received the enclosed report from the Reviewer 1 who was asked to re-assess it. As you will see the Reviewer is generally satisfied but raises a specific concern based on your revised version, with respect to the data on ATZ secretion.
I acknowledge that you mention and briefly discuss the discrepancy in your point-by-point response, but I must agree with the Reviewer that the new data require a better explanation.
Please submit your revised manuscript within two weeks and possibly before the holiday break.
I look forward to reading a new revised version of your manuscript as soon as possible.