Author Response
The following is the authors’ response to the original reviews.
Reviewer #1 (Public Review):
Summary:
Through an unbiased genomewide KO screen, the authors identified loss of DBT to suppress MG132-mediated death of cultured RPE cells. Further analyses suggested that DBT reduces ubiquitinated proteins by promoting autophagy. Mechanistic studies indicated that DBT loss promotes autophagy via AMPK and its downstream ULK and mTOR signaling. Furthermore, loss of DBT suppresses polyglutamine- or TDP-43-mediated cytotoxicity and/or neurodegeneration in fly models. Finally, the authors showed that DBT proteins are increased in ALS patient tissues, compared to non-neurological controls.
Strengths:
The idea is novel, the evidence is mostly convincing, and the data are clean. The findings have implications for human diseases.
Reply: We thank the reviewer for the supportive comments.
Weaknesses:
More experiments are needed to establish the connections between DBT and autophagy. The mechanistic studies are somewhat biased, and it's unclear whether the same mechanism (i.e., AMPK-->mTOR) can be applied to TDP-43-mediated neurodegeneration. Also, some data interpretation has to be more accurate.
Reply: We thank the reviewer for raising these questions, and we have provided additional evidence in the revised manuscript to support the model that DBTKO can enhance autophagy and induce resistance to TDP-43-associated toxicity. This is described in greater detail below.
(1) To provide further evidence for the connection between DBT and autophagy, we have introduced additional controls. For the additional controls, we have included the AMPK shRNA and drug treatment controls (Fig.4D, Fig.S4B), and these results suggest that reducing the AMPK level renders DBTKO cells sensitive to MG132 toxicity. We also added the TSC1 shRNA and mTOR agonist treatment controls (Fig.5E, Fig.S4G), and the results show that increasing mTOR levels also make the DBTKO cells sensitive to MG132.
(2) To further confirm the roles of AMPK and mTOR in DBTKO cells, we introduced the AMPK agonist (EX229) and mTOR inhibitors (RAD001 and AZD8055) in co-treatment experiments with MG132 and then measured cell survival (Fig.S4D, S4G). The results indicate that promoting AMPK activation or inhibiting mTOR can enhance cell resistance to MG132-induced toxicity.
(3) Additionally, we included the overexpression and rescue experiments for DBT and analyzed the AMPK-ULK1 signaling in WT RPE1 and DBTKO cells (Fig.S5D, S5E). The results indicate that the increase of DBT can significantly reduce the phosphorylation of AMPK/ULK1 and the levels of the autophagy marker LC3II. Together, these results suggest that DBT plays an important role in autophagy.
(4) We had shown in the original version of the manuscript that DBTKO renders cells more resistant to TDP-43-associated toxicity, similar to the tolerance of MG132-induced toxicity. Here we further show that expression of TDP-43M337V enhances the phosphorylation of AMPK in the DBTKO cells (Fig. S7A), similar to the effect of the MG132 treatment. These results suggest that the resistance of DBTKO cells to MG132 or TDP-43-assoicated toxicity shares a similar mechanism of activated the AMPK signaling.
Reviewer #2 (Public Review):
Summary:
Hwang, Ran-Der et al utilized a CRISPR-Cas9 knockout in human retinal pigment epithelium (RPE1) cells to evaluate for suppressors of toxicity by the proteasome inhibitor MG132 and identified that knockout of dihydrolipoamide branched chain transacylase E2 (DBT) suppressed cell death. They show that DBT knockout in RPE1 cells does not alter proteasome or autophagy function at baseline. However, with MG132 treatment, they show a reduction in ubiquitinated proteins but with no change in proteasome function. Instead, they show that DBT knockout cells treated with MG132 have improved autophagy flux compared to wildtype cells treated with MG132. They show that MG132 treatment decreases ATP/ADP ratios to a greater extent in DBT knockout cells, and in accordance causes activation of AMPK. They then show downstream altered autophagy signaling in DBT knockout cells treated with MG132 compared to wild-type cells treated with MG132. Then they express the ALS mutant TDP43 M337 or expanded polyglutamine repeats to model Huntington's disease and show that knockdown of DBT improves cell survival in RPE1 cells with improved autophagic flux. They also utilize a Drosophila model and show that utilizing either a RNAi or CRISPR-Cas9 knockout of DBT improves eye pigment in TDP43M337V and polyglutamine repeat-expressing transgenic flies. Finally, they show evidence for increased DBT in postmortem spinal cord tissue from patients with ALS via both immunoblotting and immunofluorescence.
Strengths:
This is a mechanistic and well-designed paper that identifies DBT as a novel regulator of proteotoxicity via activating autophagy in the setting of proteasome inhibition. Major strengths include careful delineation of a mechanistic pathway to define how DBT is protective. These conclusions are largely justified, but additional experiments and information would be useful to clarify and extend these conclusions.
Reply: We thank the reviewer for the supportive comments.
Weaknesses:
The large majority of the experiments are evaluating suppression of drug (MG132) toxicity in an in vitro epithelial cell line, so the generalizability to disease is unclear. Indeed, MG132 itself has been shown to modulate autophagy, and off-target effects of MG132 are not addressed. While this paper is strengthened by the inclusion of mouse-induced motor neurons, Drosophila models, and postmortem tissue, the putative mechanisms are minimally evaluated in these models.
Also, this effect is only seen with MG132 treatment, at a dose that causes markedly impaired cell survival. In this setting, it is certainly plausible that changes in autophagy could be the result of differences in cell survival, as opposed to an underlying mechanism for cell survival.
Additional controls would be useful to increase confidence that DBT knockdown is protective via modulation of autophagy.
While the authors report increased DBT in postmortem ALS tissue as suggestive that DBT may modulate proteotoxicity in neurodegeneration, this point would be better supported with the evaluation of overexpression of DBT in their model.
Reply: We appreciate the reviewer for raising these questions, and we have provided further evidence in the revised manuscript to support the proposed mechanism that DBTKO confers resistance to MG132-induced toxicity through activation of autophagy. This is discussed in greater detail below.
(1) To provide further mechanistic analysis, we have included additional controls for the analysis of AMPK signaling in Fig. 4D and Fig. S4B. These results demonstrate that using drugs or shRNAs to reduce AMPK activity can decrease DBTKO survival. We have also shown that that an increasing the AMPK activity with an activator enhances the survival of both WT and DBTKO cells under MG132 treatment (Fig. S4D), suggesting that DBTKO cells resist MG132-induced toxicity through the activation of AMPK signaling.
(2) We have included additional controls for the analysis of mTOR signaling in Fig. 5E and Fig. S4F. The results in Fig. 5E show that reducing TSC1 using shRNAs can decrease DBTKO survival. We also added the experiments with mTOR agonist MHY1485 as a control in Fig. S4F. These results indicate that mTOR activation can promote DBTKO cells' sensitivity to MG132 toxicity. To further confirm the importance of mTOR in DBTKO-mediated resistance to MG132 toxicity, we included the mTOR inhibitors RAD001 and AZD8055 in the co-treatment experiments with MG132, and then measured cell survival (Fig. S4G). The results show that both mTOR inhibitors can enhance cell resistance to MG132-induced toxicity (Fig. S4G). These findings suggest that mTOR inhibition is required for DBTKO-mediated cell survival under MG132 treatment.
(3) To further test the hypothesis that DBT knockdown is protective via modulation of autophagy, we have introduced the overexpression of DBT and the rescue of DBT in DBTKO cells to analyze the AMPK signaling that regulates autophagy (Fig. S5E). The results demonstrate that overexpression of DBT significantly reduced the phosphorylation of AMPK and ULK1 (Fig. S5E). In the rescue experiment, the results mirror those of the overexpression experiment, showing a significant reduction in the phosphorylation of AMPK and ULK1 (Fig. S5E). We also analyzed the autophagy marker LC3II in both the overexpression and rescue experiments, and the results indicate that increasing the DBT level specifically reduces the LC3II level (Fig. S5D). These results support the model that loss of DBT promotes the activation of autophagy.
(4) To test the hypothesis that DBT may modulate proteotoxicity in neurodegeneration, we included the studies with TDP-43M337V and found that the expression of the mutant TDP43 enhanced the phosphorylation of AMPK in the DBTKO cells (Fig. S7A), consistent with the observations made with MG-132 treatment. Together with other findings in the manuscript, these results indicate that DBTKO can sensitize the activation of the AMPK signaling and confer the resistance to TDP-43-associated toxicity.
