Functional contribution of the transcription factor ATF4 to the pathogenesis of amyotrophic lateral sclerosis
Authors: Estefanía Lopez
Claudio Hetz
Soledad Matus
Melissa Nassif
Vicente Valenzuela
Journal: PLoS ONE (2013)
Discipline: Multidisciplinary
Keywords: Endoplasmic reticulum stress; ATF4; amyotrophic lateral sclerosis; SOD1(G86R); apoptosis
Affiliations: Neurounion Biomedical Foundation, Santiago, Chile. 

Abstract:  Endoplasmic reticulum (ER) stress represents an early pathological event in amyotrophic lateral sclerosis (ALS). ATF4 is a key ER stress transcription factor that plays a role in both adaptation to stress and the activation of apoptosis. Here we investigated the contribution of ATF4 to ALS. ATF4 deficiency reduced the rate of birth of SOD1(G86R) transgenic mice. The fraction of ATF4(-/-)-SOD1(G85R) transgenic mice that were born are more resistant to develop ALS, leading to delayed disease onset and prolonged life span. ATF4 deficiency completely attenuated the induction of pro-apoptotic genes, including BIM and CHOP, and also led to quantitative changes in the ER protein homeostasis network. Unexpectedly, ATF4 deficiency enhanced mutant SOD1 aggregation at the end stage of the disease. Studies in the motoneuron cell line NSC34 demonstrated that knocking down ATF4 enhances mutant SOD1 aggregation possibly due to alteration in the redox status of the cell. Our results support a functional role of ATF4 in ALS, offering a novel target for disease intervention. 

Retracted: No

PubPeer comments:
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 I marked the splices event mentioned in post #1 above. Moreover, the bands of LC3 and HSP90 in Fig. 3 seem identical to the first 4 linesof Fig. S3. The last 2 lines of Fig. S3 are not continous to the previous 4 lines of the same membrane. The HSP90 band in Fig. S3 are also strangly misalligned with the above bands suggesting that they might be from different gels. ![file] , 
 We thank users for noticing these issues. These experiments were performed by the first author Dr. Soledad Matus (postdoctoral fellow at that time) with a research assistant. Regarding comments to figure 3A, **the splicing of the bands was declared on the figure legend. **Then, all data was quantified and presented in panel B of the same figure. Importantly, we realized that we did state that each western blot has individual loading controls since we used acrylamide gels with different concentrations and could only blot a few proteins per membrane. A representative loading control was shown. Here we provide the experiments indicating the individual bands that were selected based on the quantification (to show band intensities closer to the average signal). In Figure 1, red boxes show the bands selected in the figure. Notice that not all western blots were loaded in the same order or do not contain the same number of animals. The whole experiment involved running two blots to analyze the full panel of animals. We apologize for these omissions in the legends. Regrettably, when we review this data, we realized that there was a mistake in selecting the loading control bands, and they only corresponded for one animal group. However, because the loading control between this group was similar, the conclusions of the experiments are not altered by this mistake. We apologize for this practice since it was performed to better fit with the global quantification of animals to the representative protein band. We plan to submit a correction to PloS One to provide data from the full experiment.u00a0 ![file] **Figure 1:** Western blots of Figure 3A Plos One. For LC3B detection, extracts from non-treated and Tunicamicyn (2,5ug m O.N) treated cells were included as negative (-) and positive controls (+).u00a0 For comments on figure 4B, the bands were indeed spliced together, obtained from the same film, and this was not stated in the figure legend. A few years ago, the computer of Dr. Matus was stolen, and we lost many of the backup information of this article. We were able to recover independent experiments from the backups of a research assistant showing virtually the same result. Here we include two examples of the experiments performed as repeats that were performed many times showing the same results (Figure 2). ![file] **Figure 2:** Left image, western blot of NSC34 cells stably transduced with lentiviral vectors expressing shRNA against IRE1a u00a0(I),u00a0XBP1 (X), ATF6 (6), ATF4 (4) mRNA or Mock (empty vector). Cells were transiently transfected with expression vectors for mutant SOD1G85R fused to EGFP. After 72 hrs., mutant SOD1 aggregation was monitored by western blot using anti-GFP antibody. In this experiment, the effect Betamercaptoethanol (b) was included. Right image, the effects of ATF4 knockdown (A) was evaluated in cells transfected with expression vectors for wild type (WT) and mutant SOD1G85R fused to EGFP in cells transduced with a lentiviral vector expressing shRNA against ATF4 (A) or luciferase (L) as control. ![file] Regarding comment of figure S3, the aim of the experiment was to include the positive control needed for the same experiment presented in Figure 3A. The image was simplified for clarity (Figure 3). In figure legends is stated that this figure was composed. We apologize if this was not clear enough to indicate that splicing of bands was used to generate this figure. ![file] **Figure 3:** Complete experiment that generated figure S3. Regarding comments of figure 5C, this image was not spliced. We are not able to provide original films because they were lost, but here we provide other repeats of the experiment showing exactly the same result (Figure 4). ![file] **Figure 4:** Western blot of NSC34 transiently transfected with the expression vector for mutant SOD1G85R fused to EGFP. After 72 hrs., mutant SOD1 aggregation was monitored by Western blot using anti-GFP antibody. In this experiment, the effect of N-acetylcysteine (NAC), Trolox, Arginine (Arg) and Cysteine (Cis) was tested. HSP90 was used as loading control. ,