Despite the ubiquitous expression of NMNAT1, patients do not manifest pathologies other than retinal degeneration. Here we demonstrate that widespread NMNAT1 depletion in adult mice mirrors the human pathology, with selective loss of photoreceptors highlighting the exquisite vulnerability of these cells to NMNAT1 loss. Conditional deletion demonstrates that NMNAT1 is required within the photoreceptor. Mechanistically, loss of NMNAT1 activates the NADase SARM1, the central executioner of axon degeneration, to trigger photoreceptor death and vision loss. Hence, the essential function of NMNAT1 in photoreceptors is to inhibit SARM1, highlighting an unexpected shared mechanism between axonal degeneration and photoreceptor neurodegeneration. These results define a novel SARM1-dependent photoreceptor cell death pathway and identifies SARM1 as a therapeutic candidate for retinopathies.Leber congenital amaurosis type nine is an autosomal recessive retinopathy caused by mutations of the NAD LCA is the most common cause of blindness in children and about 70% of LCA cases are associated with mutations in genes related to the visual cycle, cGMP production, ciliogenesis, or transcription. Recently, more than thirty mutations in the nuclear NAD9 (LCA9) . Despitel stress . While i in mice , it is nNmnat1 mutations (V9M and D243G) exhibit severe retinal degeneration while the most common LCA9 mutation (E257K), which is not fully penetrant  robustly protects against ischemic and glaucomatous loss of the axon and soma , while cenetrant , inducesenetrant . In retienetrant . The reqse light .+ metabolism. Indeed, many of the enzymes involved in photoreceptor function are dependent on NAD+ as a cofactor, and for some of these proteins mutations in their corresponding genes lead to blindness. These include variants in the NAD+ or NADPH dependent retinal dehydrogenases like RDH12 that cause LCA13  that is the central executioner of axon degeneration , a disorder associated with severe, early-onset retinal degeneration and vision loss  and CAG-CreERT2, which expresses a tamoxifen-activated Cre recombinase from the ubiquitous actin promoter. We chose a conditional approach because NMNAT1 knockout embryos are lethal (fl/fl: CAG-CreERT2Nmnat1 and control mice with tamoxifen. We first used RT-PCR to measure Nmnat1 mRNA in the retina at 21 days after tamoxifen and found that it was significantly decreased in NMNAT1 cKO (fl/fl: CAG-CreERT2Nmnat1 + tamoxifen) compared with wild-type (WT) mice  mice . To inve of NAD+ . We next vessels  arrow in vessels . Quantit vessels . Hence, To gain insights into the temporal aspects of the retinal degenerative process, we analyzed retinal morphology at seven time points after tamoxifen administration. The loss of nuclei in the ONL layers were evident at 25 days post tamoxifen injection and robust retinal thinning was evident at 33 days post tamoxifen injection . We measfl/fl: CAG-CreERT2Nmnat1 treated with tamoxifen, untreated fl/fl: CAG-CreERT2Nmnat1 or fl/flNmnat1 treated with tamoxifen. In mutant animals in which Nmnat1 was excised, we observed a complete loss of both scotopic (rod-driven responses) and photopic (cone-driven responses) responses, indicating the loss of Nmnat1 in mature retina causes severe photoreceptor dysfunction . We examined three cohorts of mice: function . This isout mice . While pIn addition to NMNAT1, mammalian cells encode two other NMNAT isoforms; NMNAT2 that is localized in the Golgi and cytosol, and NMNAT3 that is localized inside the mitochondria. Since the loss of NMNAT1 induced retinal degeneration, we wished to determine the role of NMNAT2 and 3 in the retinal structure/function. A previous study showed that NMNAT2 knockout mice are perinatally lethal and have truncated optic nerves as well as peripheral axon degeneration . We coulCAG-CreERT2. We therefore generated mice lacking NMNAT1 specifically in rod photoreceptors by crossing the fl/flNmnat1 mice with Rhodopsin-Cre (Rho-Cre) mice (fl/fl:Rho-CreNmnat1 mice at 6-weeks-of-age. Similar to previous findings using Crx-Cre that expresses Cre recombinase in developing photoreceptors as early as E11 (fl/fl:Rho-CreNmnat1 retina (fl/fl:Rho-CreNmnat1 mice (Identifying the cells that are vulnerable to NMNAT1 loss is key to understanding LCA9 pathogenesis. The severe loss of the ONL nuclei  induced re) mice . We analy as E11 , histoloy as E11 . The quae retina  as well e retina . ConsistCre mice . In addiCre mice  that is Cre mice .OPN1LW-Cre. We crossed fl/flNmnat1 mice with OPN1LW-Cre mice in which Cre recombinase expression is driven by the human red/green pigment (OPN1LW) promoter starting at P10  in the contralateral eye. We confirmed the expression of NMNAT1-HA in a subset of outer nuclear cells and a minor population of inner nuclear cells  containing the photoreceptor-specific human rhodopsin kinase (hGRK1) promoter driving GFP . Virus war cells . Mice thar cells . There war cells . Hence, + loss and cellular degeneration in the retina. To test this hypothesis, we crossed fl/fl:CAG-CreERT2Nmnat1 mice with SARM1 knockout mice (fl/fl: CAG-CreERT2:Sarm1-/-Nmnat1 mice. Nmnat1 was excised in these mice via tamoxifen administration at 2 months of age (NMNAT1 cKO: SARM1 KO). First, we assessed SARM1 activation via measurement of cADPR, a product of the SARM1 NAD+ cleavage enzyme and a biomarker of SARM1 activity as well as NAD+  that is localized to photoreceptor cells are associated with LCA13. Combined deletion of retinal dehydrogenases, RDH12 and RDH8, results in mouse retinal degeneration , which is the rate limiting enzyme for GTP synthesis and, in turn, is required for cGMP production. cGMP is indispensable for the regulation of photoreceptor membrane potential and calcium concentration upon light stimulation. IMPDH1 mutations cause both a dominant form of retinitis pigmentosa (RP10) and LCA11. These results highlight the central role of NAD+ metabolism in the photoreceptor.Retinal NADe models . Reducedneration . NAD+ is+ synthesis. Despite the broad functions of nuclear NAD+ in all cell types, the sole consequence of LCA9-associated NMNAT1 mutations is retinal dysfunction/degeneration without systemic abnormalities. Previous studies, and our results, show early loss of photoreceptor cells in NMNAT1-deficient retina  is a naturally occurring, non-pathogenic virus used in gene therapy studies to restore structure and function to diseased cells. Recently, the U.S. FDA approved an AAV-RPE65 vector as a therapeutic reagent for LCA2 and other biallelic RPE65 mutation associated retinal dystrophies . Theoret+ plays such a central role in photoreceptors, the identification of the NAD+ biosynthetic enzyme NMNAT1 as the cause of LCA9 suggests that photoreceptor degeneration in LCA9 is due the reduction in NAD+ synthesis. Surprisingly, we demonstrate here that this is not the essential function for NMNAT1 in photoreceptors. Instead, NMNAT1 is required to restrain the activity of the prodegenerative NADase SARM1. When NMNAT1 is deleted from SARM1 KO photoreceptors, the photoreceptors do not die and but instead maintain their physiological function, demonstrating that these cells do not require NMNAT1 as long as SARM1 is not present. This finding is perfectly analogous to the relationship between NMNAT2 and SARM1 in the axon. NMNAT2 KO mice are perinatal lethal and have dramatic axonal defects, but NMNAT2, SARM1 double KO mice are viable and have a normal lifespan  which have FRT sites flanking promoterless LacZ-neomycin phosphotransferase gene (beta Geo) expression cassette located between exon 2 and 3 together with loxP sites flanking exon three was obtained from EUCOMM (NMNATtm1a(EUCOMM)Wtsi, RRID:MGI:5782147). This mouse expresses functionally null truncated NMNAT1 (exon 1 and 2) fused to beta Geo. FRTgeo;loxPNmnat1 +/ heterozygote mice were viable and fertile however, in consistent with former results, no whole body knockout  was born  have two loxP sites flanking the third exon. Then fl/+Nmnat1  mice were crossed with mice expressing inducible Cre recombinase under actin promoter  and fl/fl: CAG-CreERT2Nmnat1 mice were generated. All genotypes were confirmed by genomic PCR. NMNAT1 whole body knockout mice were generated by injecting 100 μg/g 4-hydroxytamoxifen (Sigma) into 6 to 8 weeks old fl/fl: CAG-CreERT2Nmnat1 with IP for total 10 days with 2 days rest after first 5 days injection. The last day of injection was counted as day 0 after tamoxifen injection. Mice expressing Cre recombinase (CAG-CreERT2) were obtained from The Jackson Laboratory. To generate mice lacking Nmnat1 specifically from rod photoreceptors, we crossed fl/flNmnat1 mice with mice carrying a copy of the Rhodopsin- iCre75 transgene, in which Cre recombinase expression is driven by the rhodopsin promoter starting postnatally at P7, which were provided by Dr. Ching-Kang Jason Chen , which were provided by Dr. Yun Le (tm1(KOMP)Mbp, Knockout Mouse Project (KOMP)) in our facility and crossed with C57BL/6 J mice for at least five generations.Animal studies were carried out under approved protocols from animal studies committee at Washington University. NMNAT1 mutant mice (was born . Next Nmenotypes . The resson Chen . To gene. Yun Le . SARM1 k. Yun Le . NMNAT3 CVCL_0045) by CaPO4 transfection. Cells were harvested and lysed by successive freeze thaw cycles. Virus within the lysate was purified by discontinuous iodixanol step gradients followed by further purification via column chromatography on a 5 ml HiTrap Q sepharose column using a Pharmacia AKTA FPLC system . Vectors were then concentrated and buffer exchanged into Alcon BSS  supplemented with Tween 20 (0.014%). Virus was titered by qPCR relative to a standard and stored at −80C° as previously described (Plasmids containing the photoreceptor-specific human rhodopsin kinase (hGRK1) promoter up stream of either GFP or HA-tagged NMNAT1 were packaged in AAV8(Y733F) capsid. The detailed methodology of vector production and purification has been previously described . Brieflyescribed . HEK293 Mice were anesthetized with a mixture of ketamine (70–80 mg/kg) and xylazine (15 mg/kg) injected intraperitoneally. The pupil was dilated with 1% tropicamide and topical anesthesia  was also applied to the eye. A self-sealing scleral incision was made by using the tip of a 31 G needle with the bevel pointed down. Then a 33G needle on a Hamilton syringe was inserted into the scleral incision and 1 µl of AAV containing solutions were injected in the subretinal space inducing a transient retinal detachment. The needle was slowly removed to prevent reflux and an ophthalmic ointment of neomycin/polymyxin B sulfate/bacitracin zinc was applied to the injected eye.Digital color fundus photography was performed using the Micron III retinal imaging system (Phoenix Research Laboratories). Prior to fundus imaging, mice were anesthetized with an intraperitoneal injection of 86.9 mg/kg ketamine and 10 mg/kg xylazine and administered 1.0% tropicamide eye drops (Bausch and Lomb) to dilate the pupils.ERG was performed as previously described  by using2. On the day of preparation, Trizol was directly added to the frozen retina and tissues were homogenized with Polytron and RNA was extracted using Trizol (Thermo Fisher Scientific) and chloroform (Sigma) phase separation. Quantitative RT-PCR reaction was performed with primers  with reaction mixture  and monitored with Prism 7900HT (ABI) and analyzed with delta-CT method.Mice were euthanized and eyeballs were enucleated and retinas were dissected and immediately freeze in liquid NAB_1549585) and secondary antibody Jackson Immuno Research Laboratories, AlexaFluo@568, 111-585-003 were used to visualize the HA-tagged NMNAT1. Primary antibody against rhodopsin  was used to identify the photoreceptor outer segment. Slides were analyzed under the microscope  after the nuclear staining with DAPI and mounting . For X-Gal staining, retina were dissected and fixed in the cold fixation buffer  for 1 hr, wash with detergent rinse , and incubated with X-Gal solution  for 10 hr in the dark at room temperature. Tissues were rinsed with PBS then fix with 4% PFA for 1 hr then paraffin sections were prepared and the sections were analyzed under the microscope .Mice were euthanized and eyeballs were enucleated and fixed in 4% formalin for 8 hr then washed with PBS and then embedded in paraffin. The thickness of the retinal layers or outer nuclear layers was measured using HE stained sections and plotted against the distance from the optic nerve head. The numbers of nuclei in the outer nuclear layer were analyzed using HE stained retinal sections. Outer nuclear layer was visually determined and the number of nucleus in each layer was counted and normalized by the length parallel to each layer of the retina. Data were expressed relative to the total number of nuclei in the WT. For immunostaining of the HA epitope tag, paraffin embedded eye sections were deparaffinized and treated with formic acid (70% in water) for 15 min at room temperature. Sections were rinsed and treated with blocking solution (goat IgG). Primary antibody against HA  and then centrifuged . Clear supernatant was transferred to new tube containing 100 μl chloroform and vigorously shake then centrifuged . The chloroform extraction was repeated three times. Clear aqueous phase (120 μl) was transferred to new tube and then lyophilized and stored at −80°C until measurement. Lyophilized samples were reconstituted with 60 µl of 5 mM ammonium formate (Sigma) and centrifuged at 12,000 x g for 10 min. Cleared supernatant was transferred to the sample tray. Serial dilutions of standards for each metabolite in 5 mM ammonium formate were used for calibration. Liquid chromatography was performed by HPLC  with Atlantis T3   with standard curves and normalized by the protein amount in the sample.Mice were euthanized and eyeballs were enucleated and retinas were dissected and immediately freeze in liquid N Waters) . For ste Waters) . The metSample number (n) was defined as a number of mice or replicates and indicated in the figure legend. Data comparisons were performed using Mann-Whitney U test, Kruskal-Wallis test, one-way ANOVA, or two-way ANOVA using R. F and P values for ANOVA were reported for each comparison in corresponding figure legends. For multiple comparisons, Holm-Bonferroni multiple comparison for one-way-ANOVA and Tukey post-hoc test for two-way-ANOVA were used. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:[Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]eLife. Your article has been reviewed by a Senior Editor, a Reviewing Editor, and three reviewers. The following individuals involved in review of your submission have agreed to reveal their identity: Rui Chen (Reviewer #3).Thank you for submitting your work entitled "SARM1 depletion rescues NMNAT1 dependent photoreceptor cell death and retinal degeneration" for consideration by eLife at this time.Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in Your paper on the underpinnings of Leber congenital amaurosis type 9 (LCA9), an early onset retinal neurodegenerative disease due to loss of function mutations in the nuclear NAD+ synthesis enzyme NMNAT1, was reviewed by three reviewers and me. Your study, involving a careful series of loss- and gain-of-function studies on NMNAT1 using conditional or tissue-specific NMNAT1 knockout mice, presents a new angle on mechanism of this disease in that you connect photoreceptor degeneration with the long known link between loss of NMNAT and activation of SARM1, the "executioner" of axon degeneration.eLife guidelines for proper rigor stand for addressing the sample size in your work.As you can see in the attached reviews, there are a number of concerns raised by the reviewers. Most notably, reviewers #1 and #2 are concerned about low sample sizes for your statistical analyses. In the consultation session with the reviewers, reviewer #3 agrees that in many cases, the number of mice is low, but finds this acceptable given the large number of different conditional knock out mice reported in this paper and that actCre, iCre, HGRPCre, and Vmd2Cre show consistent results. However, the Another concern is on the value of the therapeutic experiments on partial rescue by AAV-NMNAT1: You show proof-of-principle that AAV supplementation with NMNAT1 may help, but you would need to address timing in testing with regard to disease onset in order to make claims about its efficacy as a therapeutic. The therapeutic approach could be downplayed and placed with a more cogent explication of the pros and cons in the Discussion section.Finally, reviewer #1 cites a recent paper on a Sarm1 KO promoting rod and cone photoreceptor cell survival in a model of retinal degeneration . This paper does offset the novelty of your study, but your study, if amended, has the potential to elucidate additional mechanistic aspects.While it may be difficult to perform more experiments to increase your n in this time of lab closing and ramp-up, at the very least you may be able to address other major issues on your quantitative analysis and minor points  for which you may already have data.Reviewer #1:The study by Sasaki et al., examine the role of NMNAT1 and SARM1 in retinal degeneration.In my view there are two aspects to the study, the first is more pre-clinical and the second explore the basis for retinal degeneration in Nmnat1 mutations. I personally find the paper somehow disappointing on both.Following extensive characterization of the Nmnat1 cKO, the authors turn to rescue experiments by AAV infection to "test the therapeutic potential of AAV-mediated genereplacement" as they claim. There are two problems with these experiments:1) The authors use the conditional KO and ablate Nmnat1 two months after birth, but this a genetic disease, the complete KO is much more appropriate.2) The AAV is injected before the ablation of Nmnat1 (Results section), this does not mimic any therapeutic approach.The rescue is not impressive, I would like to see the wild type in the graphs of Figure 6C-E.There is not much new on basis for retinal degeneration here as well, which pool of Nmnat1 is the protecting, nucleus or cilia is highly interesting but this is not studied. What is the type of cell death? The authors point that it is apoptotic (Results section), but Sarm induce non-apoptotic cell death.Data rigorouslyIn some cases, the N is too low see Figure 1C and Figure 7F-H, this leads to conflicting results is there NAD+ depletion in the Nmnat1 cKO? In Figure 7F there is in Figure 1C maybe with more samples. The authors claim that SARM1 is not protecting through elevation of NAD+ levels, but according to Figure 7F this is exactly what is happing in the Double KO.Current literatureeLife to decide if this is affecting the novelty of this study.Recent paper demonstrated that sarm1 KO promotes rod and cone photoreceptor cell survival in a model of retinal degeneration . It is up to Reviewer #2:Mutations in the NAD+ synthesis enzyme NMNAT1 are associated with Leber congenital amaurosis type 9 (LCA9). However, little is known about the role of NMNAT1 in the retina and in regards to retinal degeneration. The authors examine retina morphology and physiology in mice after knockout: ubiquitous, in the rods, cones or RPE cells. They find that retinal degeneration occurs strongly with the loss of NMNAT1 in the rod cells, and that AAV supplementation with NMNAT1, or SARM1 knockout, protects against the retinal degenerative phenotype. This work has high significance and the authors utilize multiple model systems to support their hypothesis. The possible targeting of SARM1 as a therapeutic is highly intriguing. However, there is some clarification needed in the manuscript and low sample sizes are concerning to fully support the suggested results.1) The authors make a point of discussing the vascular phenotype after tamoxifen induced knockout of NMNAT1 by a ubiquitous cre-driver. However, there is no mention of this vascular phenotype in any of their conditional model systems, and it is not a phenotype typically associated with LCA. The authors should mention this within their discussion and touch upon the potential reasons for lack of this phenotype when loss of NMNAT1 occurs in the rods, cones, or RPE cells alone as opposed to ubiquitously lost within the retina.2) As the rod and cone conditional loss of NMNAT1 displayed different phenotypic outcomes, it would be interesting to see staining of the rod and cone photoreceptors in each model system to see if they were preferentially affected. The authors provide H and E images within the manuscript and should have sections from their mutant mouse lines available.3) The authors mention that NMNAT2 interacts with SARM1 and that NMNAT1 may be compensating for this action in the photoreceptor cell. Can the authors examine NMNAT2 levels (via western blot or immunostaining) after tamoxifen-induced loss of NMNAT1? It would be interesting to see if NMNAT2 levels change in the retina, and if NMNAT1 levels are altered in other retinal cells when lost in their conditional knockout models.4) It would be interesting to see if NMNAT1 levels are altered in other retinal cells when lost in their conditional knockout models. Can the authors examine NMNAT1 levels (via western blot or immunostaining) in these mice?5) LCA is an early-onset retinal degenerative disease. The conditional knockout models that were used in this study did not express cre until after one week of age in the mouse. The authors should include a small discussion on this point in their paper.6) AAV restoration of NMNAT1 was provided before disease onset, and not delivered after tamoxifen injection. It still supports the author's hypothesis that NMNAT1 plays a key role in retinal degeneration, but the authors over-state the therapeutic effect in their paper. It would also be interesting to test the effects of the AAV on the retinal cells and not just the photoreceptors, the authors could include that in their discussion. They should make sure that they do not over-state the therapeutic benefit of the AAV injections.7) The sample sizes provided in the figure legends do not designate whether they refer to the number of mice or number of retinas/eyes examined. Please correct.8) The photopic ERG results in the RPE conditional knockout are lower than wild-type controls without any error bars rising above WT levels. This is one of the cases where the authors note an N = 2 for statistical analysis. The authors should consider whether an increased sample size or a later timepoint tested might provide a significant ERG phenotype in this mouse line, and that their conclusion that the RPE does not play a role may be incorrect.9) There appear to be error bars missing from some of the ERG figures. Please check and make sure all error bars are present in the graphical results.10) The authors should mention sample sizes for the histological analyses (H and E staining) in the figure legend or Results section. It would also be helpful to discuss whether the degeneration phenotype was variable between mice or followed a similar degeneration pattern in all mice in each of their conditional knockout models.Reviewer #3:In this study, the authors have generated multiple knock out mice lines to systematically investigate the function of Nmnat1 in the retina. Compared to previously published studies of Nmnat1 knock out mice, this study combines cell type-specific knock out and rescue experiment to illustrate that Nmnat1 play important function primarily in photoreceptor cells, not RPEs. In addition, as the Sram1 mutant rescues the Nmnat1 knock out phenotype along with increasing NMN level and cADPR expression in the Nmnat1 knock out, the author provides convincing evidence that degeneration induced in Nmnat1 mutation is mediated through activation of Sram1. The overall data quality is high and the result is convincing. My specific comments and suggestions are:1) As the thickness of ONL and INL change across the retina, the reduction can be better measured by measuring the thickness at multiple positions across the retina and representing the results in a spider graph.2) Relative minor degeneration of INL neurons is observed. I am wondering if all cell types in the INL are affected. Staining of the cell types in INL can be conducted to better characterize the degeneration.3) It seems that there is a theme that NMNAT1 is required only for retinal neurons. If this is the hypothesis, I am wondering why RGCs are not affected in the mutant.4) The authors should fix the gene nomenclature convention. For mouse, the gene symbols are italicized, with only the first letter in upper-case.eLife. Your article has been reviewed by Huda Zoghbi as the Senior Editor, a Reviewing Editor, and three reviewers. The reviewers have opted to remain anonymous.Thank you for submitting your article "SARM1 depletion rescues NMNAT1 dependent photoreceptor cell death and retinal degeneration" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife but that some conclusions require a modest amount of additional new data, as they do with your paper, we are asking that the manuscript be revised to either limit claims to those supported by data in hand, or to explicitly state that the relevant conclusions require additional supporting data.We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, when editors judge that a submitted work as a whole belongs in eLife, either of which would be linked to the original paper.Our expectation is that the authors will eventually carry out the additional experiments and report on how they affect the relevant conclusions either in a preprint on bioRxiv or medRxiv, or if appropriate, as a Research Advance in Summary:Your study examines mutations in the NAD+ synthesis enzyme NMNAT1 that are associated with Leber congenital amaurosis type 9 (LCA9). You follow the role of NMNAT1 in the retina with regards to retinal degeneration, determining retinal morphology and physiology in mice after ubiquitous, rod- and cone-specific knockout. You show that retinal degeneration occurs with the loss of NMNAT1 in rod cells, and that a loss of SARM1, usually studied in the context of axon degeneration, protects against the retinal degenerative phenotype. This work points to an important pathway underlying retinal degeneration and possible targeting of SARM1 as a therapeutic. The reviewers have appreciated your revisions, in particular, the increase in n, and the deletion of the pre-clinical portion of the manuscript. Your study provides insight into the basis for retinal degeneration in mutation of Nmnat1. There are a few remaining amendments requested by the reviewers.Essential revisions:You provide AAV restoration of NMNAT1 before disease onset in your mouse model. Although you acknowledge that you did not test the AAV supplementation after disease onset at a clinically relevant time point, you indicate in both the Results section and Discussion section that AAV gene therapy had a significant rescue of the ERG, implying that the b-wave was also rescued. However, the scotopic b-wave was not significantly different between the treated and control eyes, nor was the photopic ERG, beyond the ERG a-wave response. Please correct these statements.The level of NMN, NAD+ and cADPR is measured in the whole retina. Given that severe degeneration of photoreceptor cell is observed at the time of measurement, it is difficult to tell how much of the change is due to cell degeneration and cell type composition change. To this end, while it is clear that survival of photoreceptor cell depends on NMNAT1, you might check if subtle defects are observed in other cell types in the retina since NMNAT1 is ubiquitously expressed in all retinal cell types, by staining with cell type specific markers.eLife. Your revised article has been evaluated by Huda Zoghbi (Senior Editor) and a Reviewing Editor.Thank you for resubmitting your work entitled "SARM1 depletion rescues NMNAT1 dependent photoreceptor cell death and retinal degeneration" for further consideration by The study is excellent and the manuscript has been improved, but there is one remaining issue that needs to be addressed before acceptance, as outlined below:Please clearly state in the Materials and methods section, that the HEK293 ATCC CRL-1573 cell lines were used two years ago for viral production and thus could not be authenticated now. [Editors’ note: the authors resubmitted a revised version of the paper for consideration. What follows is the authors’ response to the first round of review.]Reviewer #1:There are two problems with these experiments:1) The authors use the conditional KO and ablate Nmnat1 two months after birth, but this a genetic disease, the complete KO is much more appropriate.The NMNAT1 whole body knockout mouse is embryonic lethal and so we could not perform the suggested experiment. Instead, we performed a conditional deletion in adult mice and found that NMNAT1 is required for the maintenance of photoreceptors and also performed both rod and cone specific knockouts that occur much earlier, at about one week postnatally. Despite the very different pattern and timing of knockouts, we got quite consistent results from these experiments, suggesting that our findings reflect a fundamental role for NMNAT1 in photoreceptors.2) The AAV is injected before the ablation of Nmnat1 (Results section), this does not mimic any therapeutic approach.The rescue is not impressive, I would like to see the wild type in the graphs of Figure 6C-E.We agree that this approach does not mimic gene therapy, and in response to this comment and the editor’s comments we have dramatically toned down any discussion of this as a model of therapy. We have moved these rescue data from the main figures to the supplemental data and, instead of discussing this as a candidate therapy, now emphasize that this is a genetic rescue experiment useful for demonstrating that NMNAT1 is required in photoreceptor cells. We now explicitly describe the limitations of our experiment as a model of gene therapy.There is not much new on basis for retinal degeneration here as well, which pool of Nmnat1 is the protecting, nucleus or cilia is highly interesting but this is not studied. What is the type of cell death? The authors point that it is apoptotic (Results section), but Sarm induce non-apoptotic cell death.We respectfully disagree with the reviewer. Our finding that loss of NMNAT1 induces retinal degeneration via activation of SARM1 is novel and quite unexpected. In the retina field the belief based on published literature was that NMNAT1 was required for photoreceptor survival due to its role in NAD biosynthesis to supply NAD+ to photoreceptors.Here, we show that its essential function in photoreceptor survival is to inhibit SARM1. In the axon degeneration field, it is well known that NMNAT2 inhibits SARM1 activity in the axon, but no one had previously suggested that NMNAT1 would play an analogous role in a neuronal cell body. Our findings are surprising, not intuitive, and important for both our understanding of retinal neurodegeneration and axon degeneration.Reviewer #2:[…] However, there is some clarification needed in the manuscript and low sample sizes are concerning to fully support the suggested results.1) The authors make a point of discussing the vascular phenotype after tamoxifen induced knockout of NMNAT1 by a ubiquitous cre-driver. However, there is no mention of this vascular phenotype in any of their conditional model systems, and it is not a phenotype typically associated with LCA. The authors should mention this within their discussion and touch upon the potential reasons for lack of this phenotype when loss of NMNAT1 occurs in the rods, cones, or RPE cells alone as opposed to ubiquitously lost within the retina.We agree that these data were ancillary to the point of the manuscript and so we removed the angiogram data.2) As the rod and cone conditional loss of NMNAT1 displayed different phenotypic outcomes, it would be interesting to see staining of the rod and cone photoreceptors in each model system to see if they were preferentially affected. The authors provide H and E images within the manuscript and should have sections from their mutant mouse lines available.We believe that rods and cones are preferentially affected in the corresponding KO mice for a number of reasons. First, both the Rod and HGRP Cre driver mice have been previously characterized and show specificity. Second, both our anatomical and ERG data support the selective nature of the KOs. For example, the contributions of rods and cones to the various components of the ERG are well known in the literature, and our results are consistent with selective disruption of rods and cones in the respective KO lines.3) The authors mention that NMNAT2 interacts with SARM1 and that NMNAT1 may be compensating for this action in the photoreceptor cell. Can the authors examine NMNAT2 levels (via western blot or immunostaining) after tamoxifen-induced loss of NMNAT1? It would be interesting to see if NMNAT2 levels change in the retina, and if NMNAT1 levels are altered in other retinal cells when lost in their conditional knockout models.4) It would be interesting to see if NMNAT1 levels are altered in other retinal cells when lost in their conditional knockout models. Can the authors examine NMNAT1 levels (via western blot or immunostaining) in these mice?Despite extensive efforts on our part, we have not found reliable antibodies for NMNAT1 or NMNAT2 for immunocytochemical analysis. We would point out, however, that even if there were a compensatory increase in NMNAT2 levels in photoreceptors after NMNAT1 depletion, this is not sufficient to block SARM1 activation.5) LCA is an early-onset retinal degenerative disease. The conditional knockout models that were used in this study did not express cre until after one week of age in the mouse. The authors should include a small discussion on this point in their paper.We added this discussion point as requested.6) AAV restoration of NMNAT1 was provided before disease onset, and not delivered after tamoxifen injection. It still supports the author's hypothesis that NMNAT1 plays a key role in retinal degeneration, but the authors over-state the therapeutic effect in their paper. It would also be interesting to test the effects of the AAV on the retinal cells and not just the photoreceptors, the authors could include that in their discussion. They should make sure that they do not over-state the therapeutic benefit of the AAV injections.We appreciate the comment and agree. As such, we moved these data to the supplement and have dramatically toned down any claims about therapy, focusing instead on the study as a proof of genetic rescue.7) The sample sizes provided in the figure legends do not designate whether they refer to the number of mice or number of retinas/eyes examined. Please correct.The sample number is the number of mice and this is now stated clearly in the methods and figure legends. We also now provide all source data that shows color coded data for individual mice.8) The photopic ERG results in the RPE conditional knockout are lower than wild-type controls without any error bars rising above WT levels. This is one of the cases where the authors note an N = 2 for statistical analysis. The authors should consider whether an increased sample size or a later timepoint tested might provide a significant ERG phenotype in this mouse line, and that their conclusion that the RPE does not play a role may be incorrect.Because we would not be able to generate more mice in a timely manner, we have removed these data from the paper. As this was a negative result and we have positive results with two other tissue specific Cre lines, we feel that this has no significant impact on the paper.9) There appear to be error bars missing from some of the ERG figures. Please check and make sure all error bars are present in the graphical results.All of the error bars were included, but for some data the error bars were smaller than the size of symbols. We have now increased the size of the error bars to ensure that they are visible for all figures.10) The authors should mention sample sizes for the histological analyses (H and E staining) in the figure legend or Results section. It would also be helpful to discuss whether the degeneration phenotype was variable between mice or followed a similar degeneration pattern in all mice in each of their conditional knockout models.As shown in the Figure 3B and its legends, we analyzed the conditional knockout retinal structure using 3 mice at each of 7 different time points  and obtained the retinal degeneration time course. All mice analyzed showed a consistent retinal degeneration time course.Reviewer #3:1) As the thickness of ONL and INL change across the retina, the reduction can be better measured by measuring the thickness at multiple positions across the retina and representing the results in a spider graph.As requested, we now provide a spider graph showing retinal and ONL thickness and agree that this is an excellent way to represent the data. We also provide representative whole eye images that clearly show the consistent loss of ONL and INL nucleus across the entire retina.2) Relative minor degeneration of INL neurons is observed. I am wondering if all cell types in the INL are affected. Staining of the cell types in INL can be conducted to better characterize the degeneration.While this is an interesting question, we think that this is beyond the scope of this paper which is focused on photoreceptor degeneration and function.3) It seems that there is a theme that NMNAT1 is required only for retinal neurons. If this is the hypothesis, I am wondering why RGCs are not affected in the mutant.While we do not know the answer, we speculate that NMNAT2 is likely a major NMNAT in RGCs and so the loss of NMNAT1 is not sufficient to activate SARM1. Unfortunately, there are no available NMNAT2 conditional knockout mice or antibodies to test retinal expression of NMNAT2. This will be an interesting future project.4) The authors should fix the gene nomenclature convention. For mouse, the gene symbols are italicized, with only the first letter in upper-case.We fixed the nomenclature as requested.[Editors’ note: what follows is the authors’ response to the second round of review.]Essential revisions:You provide AAV restoration of NMNAT1 before disease onset in your mouse model. Although you acknowledge that you did not test the AAV supplementation after disease onset at a clinically relevant time point, you indicate in both the Results section and Discussion section that AAV gene therapy had a significant rescue of the ERG, implying that the b-wave was also rescued. However, the scotopic b-wave was not significantly different between the treated and control eyes, nor was the photopic ERG, beyond the ERG a-wave response. Please correct these statements.We agree that our statements are not sufficient to point out that ERG recovery is partial. We corrected the statements as below.1) Results sectionOriginal statement“we observed significantly increased scotopic a-wave amplitudes in AAV-NMNAT1 treated retinas compared with retinas injected with AAV-GFP  accompanied by a small increase in the scotopic and photopic b-wave amplitudes between AAV-NMNAT1 and AAV-GFP treated retinas .”Revised statement“we observed significantly increased scotopic a-wave amplitudes in AAV-NMNAT1 treated retinas compared with retinas injected with AAV-GFP . There were also small, but statistically insignificant, increases in the scotopic and photopic b-wave amplitudes between AAV-NMNAT1 and AAV-GFP treated retinas .”2) Discussion sectionOriginal statement“Delivery of AAV8(Y733F)-hGRK1-NMNAT1 significantly improved the retinal phenotype of mice deficient for NMNAT1, however the virus was delivered prior to deletion of NMNAT1.”Revised statement“Delivery of AAV8(Y733F)-hGRK1-NMNAT1 prior to deletion of NMNAT1 resulted in partial improvement of the retinal phenotype, in particular the scotopic a-wave, of mice deficient for NMNAT1.”The level of NMN, NAD+ and cADPR is measured in the whole retina. Given that severe degeneration of photoreceptor cell is observed at the time of measurement, it is difficult to tell how much of the change is due to cell degeneration and cell type composition change. To this end, while it is clear that survival of photoreceptor cell depends on NMNAT1, you might check if subtle defects are observed in other cell types in the retina since NMNAT1 is ubiquitously expressed in all retinal cell types, by staining with cell type specific markers.eLife or the equivalent in the future as suggested by the editors.We agree that the whole retinal metabolite changes could be due not only to photoreceptor degeneration but also to changes in cells other than photoreceptors. As the reviewer suggests, immunocytochemistry to shed light on the state of other cell types in the degenerating retina will provide valuable information. Because our main conclusions will not be affected by results of these experiments, we choose to provide these data as a Research Advance in [Editors’ note: what follows is the authors’ response to the second round of review.]The study is excellent and the manuscript has been improved, but there is one remaining issue that needs to be addressed before acceptance, as outlined below:Please clearly state in the Materials and methods section, that the HEK293 ATCC CRL-1573 cell lines were used two years ago for viral production and thus could not be authenticated now.Thank very much for the suggestion on the cell line authentication. We added the description “HEK293 cells were used more than two years ago for viral production and thus could not be authenticated now.” in the method section. We appreciate careful considerations on our manuscript.


A 58-year-old man presented with recurrence of chronic myeloid leukemia (CML) after complete molecular remission in the setting of non-compliance with imatinib. He was restarted on imatinib and was also noted to have IgG kappa monoclonal gammopathy of undetermined significance (MGUS). The patient re-achieved molecular remission after resumption of imatinib, but his MGUS progressed to smoldering myeloma and he was eventually diagnosed with multiple myeloma (MM) and initiated on treatment for MM with thalidomide, bortezomib and dexamethasone. He has responded well to treatment of the myeloma and continues concurrent maintenance imatinib treatment for CML and is being evaluated for bone marrow transplant. The association of two concurrent hematological malignancies, CML and MM, is very rare and has been infrequently reported in literature. The pathophysiology of this has not yet been fully understood. This case report reviews the various theories to explain this and discusses the potential challenges of simultaneous treatment of MM and CML. Multiple myeloma (MM) is a monoclonal disorder of plasma cells which have differentiated from lymphoid B cells. Therefore, the abnormal cell types in CML and MM are distinctly different. The instances of both MM and CML in the same patient occurring in a synchronous or metachronous manner are extremely rare. There are several factors that have been postulated to be related to this occurrence. These include age, gender, race, exposure to environmental carcinogens or radiation, epigenetic upregulation/downregulation, as a result of progression or treatment of one malignancy potentiating the development of malignant cells (which acquire antiapoptotic ability) and mechanisms to evade immune surveillance, chronic antigenic stimulation, genetic polymorphisms. At present there are multiple theories but insufficient data to make any definite conclusions about the mechanism of co-existence of CML and MM. With the advent of novel therapies and improving survival in patients with CML and MM, there is value in further investigation regarding the pathophysiology and clinical characteristics of such cases3. Additionally, the occurrence of more than one hematological malignancy in the same patient presents treatment challenges because it can lead to the possibilities of drug-drug interactions and medication toxicities. Analysis of the treatment protocols of these patients and their follow-up will be required to assess the risks and benefits of different treatment options. At this time, due to paucity of data, the management is tailored according to the patient’s individual risk factors and clinician’s judgement.Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder of pluripotent hematopoietic stem cells. It results from a translocation t  (q34q11) known as the Philadelphia chromosome creating a BCR-ABL fusion gene, which is transcribed into proteins with abnormal tyrosine kinase activity that drives abnormal white blood cell (WBC) proliferationThis is a rare case of the occurrence of IgG MM and CML in a single patient and reviews the management of these diseases.A 58-year-old man with history of CML presented in December 2015 for re-establishment of care after being lost to follow-up. He was first diagnosed in December 2007 with CML and was treated with imatinib (400mg daily). He achieved complete molecular remission but was unfortunately lost to follow-up after 2012. The patient stated that he had stopped taking imatinib in September 2015 due to family issues and stressors. When he presented in December 2015, he had no specific complaints. He was noted to have mild pallor on exam, but no lymphadenopathy or hepatosplenomegaly. Labs were significant for leukocytosis and blasts noted on peripheral smear. BCR-ABL FISH/PCR was also positive indicating relapse of CML and the transcripts at the time of diagnosis were typical. His peripheral smear showed only rare blasts (< 10 %) and the patient appeared to be in the chronic phase of CML. A bone marrow biopsy was not repeated at this time.The patient was re-initiated on treatment with imatinib 400mg daily. Within 2 weeks of resuming treatment he was noted to have an improvement in WBC count. However, he was incidentally detected to have an elevated total protein level. In view of this serum protein, electrophoresis was ordered and the patient was found to have an IgG kappa monoclonal gammopathy of undetermined significance (MGUS). This diagnosis was established because the total M protein spike was <3g/dL, bone marrow biopsy showed < 10% plasma cells and the patient had no evidence of end organ damage. Skeletal survey was done which showed no lytic lesions. Imatinib was continued for CML and he was monitored closely for progression of plasma cell dyscrasia.With regard to the patient’s CML, he achieved molecular remission in November 2016 with 3 log reduction in the 3 tested transcripts b2a2, b3a2, e1a2 and without detectable Philadelphia chromosome. However, he had a steady increase in the paraprotein level from December 2015 to April 2019 without any symptoms. He did not develop myeloma defining events such as hypercalcemia , renal insufficiency (creatinine clearance <40mL/min or serum creatinine >2mg/dL), anemia  or bony pain/lytic lesions . These are together referred to as the CRAB phenomenon. His serum free light chain ratio remained at <100mg/L.th rib, left and right ischium, and a lesion in the second lumbar (L2) vertebra. He also underwent a bone marrow biopsy in July 2019, which showed 50% plasma cells expressing CD 38, CD 138, dim/partial CD 117, CD 56 and kappa light chain restriction. No BCR ABL gene rearrangement was noted. Thus, it confirmed the diagnosis of MM.In May 2019, the patient had further rise in creatinine and paraprotein level and had an increase in serum free light chain ratio. Therefore, he underwent a PET CT in June 2019. This showed increased uptake in the left 4MM interphase fluorescence in situ hybridization (FISH) panel analysis of CD138+ enriched plasma cells was positive for three CCND1 signals consistent with trisomy 11 and for extra signals for chromosomes 7, 9 and 15. There were no cells with FGFR3-IGH, CCND1-IGH, or IGH-MAF fusions. Results for 1p/1q, 13q and TP53 were normal. Extra signals for probes targeting the chromosomes reported above suggest the presence of a hyperdiploid clone. Thus, the patient’s cytogenetic testing was negative for high risk factors.The trends of serum protein electrophoresis are seen in2) and dexamethasone (40mg/day once a week) in September 2019. This regimen was preferred over the VRd  due to the concern for increased risk of pancytopenia with concurrent use of imatinib and lenalidomide.The patient was started on treatment for ISS stage II standard risk myeloma with thalidomide (50mg/day daily for 21 days followed by 7 days off), weekly bortezomib  and resolved completely. Thus far he has completed 6 cycles of treatment for myeloma with concurrent imatinib and has been referred to a transplant center to assess his eligibility for the same.4.The occurrence of CML and MM together is very rare. There are multiple explanations that have been suggested for co-existence of more than one hematological malignancy. One of the theories is that there is a common progenitor stem cell. The Philadelphia chromosome is observed not only in granulocytes but also in cells of the monocytic, erythroid, megakaryocytic and lymphoid series. This finding supports the concept of a common pluripotent progenitor cell. The transformation to lymphoid cells in the blast phase of the CML also suggests a relationship between the myeloid and lymphoid lineageet al.5 that imatinib inhibits MM cell proliferationin vitro. On the contrary, it has been reported that imatinib has a stimulatory effect on MM cells through activation of Erk1 and Erk2 mitogen activated protein kinases (MAP kinases). There have also been reports of MM developing in non CML patients such as those with gastrointestinal stromal tumors treated with imatinib6 Carulliet al.7 looked at the possible interference of imatinib with plasma cell phenotype. Their study looked at 30 patients and found that 70% of these patients had an abnormal plasma cell phenotype, which they defined as plasma cells which lack CD 19. Some of these cells showed additional aberrations such as expression of CD 56. Although this cannot establish a causal relationship between imatinib and MM, it merits further investigation.The role of imatinib in promoting development of MM is debatable. There is some evidence from Pandiella7.Imatinib mesylate is a tyrosine kinase inhibitor, which is the standard of care for CML as a first line agent. It has activity against different genes involved in cellular transformation such as BCR-ABL1, c-KIT, PGFR-α and β and Jak 2. Imatinib in CML acts by competing with ATP to bind to the BCR ABL1 tyrosine kinase and thereby inhibiting the WBC proliferation that it effects8.Treatment of MM is based on whether the patient has standard or high-risk disease, which in turn is determined by cytogenetic analysis. High risk features constitute t , t , del17p13, t  and 1q gain. Additionally, patients should be assessed for eligibility to receive an autologous stem cell transplant. In standard risk patients such as ours the standard first line treatment is RVd regimen, which comprises lenalidomide, bortezomib and dexamethasone. Post induction therapy, if patients are eligible for hematopoietic cell transplant (HCT), they may choose either an early HCT or delayed HCT strategy. Autologous HCT is the mainstay, although allogenic HCT is still largely investigational. Post-transplant patients need maintenance therapy as well. Transplant in eligible patients receive either a two/three drug induction regimen followed by maintenance therapy. Melphalan, cyclophosphamide and thalidomide are also part of first line treatment options for myeloma. In patients with relapse, several of the newer agents are being used, which include the new proteasome inhibitor (carfilzomib), immunomodulatory drugs , inhibitors of NF-κB, MAPK and AKT, histone deacetylase inhibitors (like vorinostat and panobinostat), and monoclonal antibodies 9.Due to the rarity of coexistence of more than one hematological malignancy in the same patient, we do not have robust data on treatment regimens. Current treatment is based on factoring in the patient’s individual risk factors and the physician’s judgement and experience. Even though there is concern for imatinib being associated with MGUS and myeloma, co-administration of imatinib with myeloma treatment seems to be reasonable. Myeloma treatment both for high risk and standard risk group patients involves a proteasome inhibitor, such as bortezomib. Both bortezomib and imatinib are metabolized by microsomal enzyme CYP3A4. However, imatinib is a potent inhibitor of CYP3A4, while bortezomib is only a weaker inhibitor. Reduction of bortezomib dosing to once weekly instead of twice seems to be associated with less adverse effects when used in conjunction with imatinib10Additionally, bisphosphonates, which are used as supportive treatment in myeloma, have also been shown to inhibit CML cell lines and induce apoptosis synergistically with imatinib through the inhibition of prenylation of Ras and Ras-related proteinsThis case report presents an experience with a CML patient who over time progressed to develop the entire spectrum of myeloma, starting with MGUS followed by smoldering myeloma and ultimately MM requiring treatment. At this point it is difficult to say if this association is coincidental or related to some other mechanism and this is a subject that requires further research.The most essential take-home points from this case are as follows:1. MM can be diagnosed in patients with one of the following: >10% plasma cells in bone marrow OR biopsy proven bony/extramedullary plasmacytoma and any one of the CRAB phenomenon OR any of the following- >/= 60% plasma cells in bone marrow OR serum free light chain ratio >/= 100 OR >1 focal lesion on MRI studies.2. Both MGUS and smoldering myeloma are characterized by an absence of myeloma defining events. In MGUS: paraprotein level <3g/dL, bone marrow plasma cells <10%; while in smoldering myeloma: paraprotein level >3g/dL or urinary monoclonal protein >/= 500mg in 24 hours and/or clonal bone marrow plasma cells 10 to 60%3. Co-existence of CML and MM is very rare. Etiology is probably multifactorial but there is a possibility that this is because CML and MM share a common pluripotent progenitor stem cell which can differentiate into both lymphoid and myeloid lines.4. There is concern that the imatinib may lead to alteration of the plasma cell phenotype and it may be worthwhile to monitor serum electrophoresis and protein levels in patients who have received imatinib treatment.5. Co-administration of bortezomib and imatinib is feasible. Since CYP3A4 is important for metabolism of both drugs, administration of bortezomib as weekly instead of twice weekly may help to reduce adverse effects of bortezomib.All data underlying the results are available as part of the article and no additional source data are required.Written informed consent for the publication of the case report and any associated images was obtained from the patient. No further comments.Are enough details provided of any physical examination and diagnostic tests, treatment given and outcomes?YesIs the case presented with sufficient detail to be useful for other practitioners?PartlyIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment?YesIs the background of the case’s history and progression described in sufficient detail?YesReviewer Expertise:Myeloid leukemias, clinical trials, Genetic syndromes in leukemia.I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. This is an uncommon case presentation of two apparently unrelated hematopoietic malignancies.et al., NIH) Association of Multiple myeloma following CLL or other low grade B cell lymphoma may be related to terminal changes of B cell to Plasma cells . We suggested another potential theory of multiple simultaneous malignancies includes the sharing of a common malignant pluripotent progenitor stem cell which may allow further transformation of both lymphoid and myeloid differentiations. We had published a similar case in 2014 in a patient of CML with t9:22) along with JAK2 mutation and Myeloma  has been linked to increased cell survival, proliferation, and malignant transformation  and has been found in all hematopoietic cell lineages including erythropoietic cells, megakaryocytes, macrophages, and B-lymphocytes .Are enough details provided of any physical examination and diagnostic tests, treatment given and outcomes?YesIs the case presented with sufficient detail to be useful for other practitioners?PartlyIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment?YesIs the background of the case’s history and progression described in sufficient detail?YesReviewer Expertise:Leukemia, Lymphoma, Myeloma with molecular genetic applicationsI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. In this case report, the authors have discussed a patient with chronic myeloid leukemia and metachronous IgG multiple myeloma. The clinical presentation and disease course are well described and discussed. Please mention the transcripts at diagnosis .At CML disease relapse, please indicate if the patient was still in the chronic phase. Was there a bone marrow biopsy done at that time, if so what was the percentage of plasma cells. As the diagnosis of MGUS requires BMBx? Please indicate if the clonal plasma cells were <10% facilitating the diagnosis of MGUS.Please indicate if the x-axis in Figure 1-2 denotes months?It will be interesting to know the most common Grade >3 AEs experienced by the patient  and resolved completely. He has now completed 6 cycles of treatment for myeloma and is being evaluated for bone marrow transplant. He has had no other side effects.   Thanks for updating. Please make necessary inclusions of those comments in the manuscript.


Malaria transmission is dependent on the formation of gametocytes in the human blood. The sexual conversion rate, the proportion of asexual parasites that convert into gametocytes at each multiplication cycle, is variable and reflects the relative parasite investment between transmission and maintaining the infection. The impact of environmental factors such as drugs on sexual conversion rates is not well understood. We developed a robust assay using gametocyte-reporter parasite lines to accurately measure the impact of drugs on sexual conversion rates, independently from their gametocytocidal activity. We found that exposure to subcurative doses of the frontline antimalarial drug dihydroartemisinin (DHA) at the trophozoite stage resulted in a ~ fourfold increase in sexual conversion. In contrast, no increase was observed when ring stages were exposed or in cultures in which sexual conversion was stimulated by choline depletion. Our results reveal a complex relationship between antimalarial drugs and sexual conversion, with potential public health implications. Plasmodium falciparum is responsible for the most severe forms of human malaria. Repeated rounds of its ~48 hr intraerythrocytic asexual replication cycle result in an exponential increase in parasite numbers and are responsible for all clinical symptoms of malaria. At each round of replication, a small subset of parasites commits to differentiation into non-replicative sexual forms termed gametocytes, which are the only form transmissible to a mosquito vector. Sexual commitment is marked by epigenetic activation of the expression of the master regulator PfAP2-G, a transcription factor of the ApiAP2 family  are potent antimalarial drugs that rapidly kill asexual parasites. After activation by cleavage of their endoperoxide bond by hemoglobin degradation products, ARTs produce reactive oxygen species and free radicals that result in widespread damage in parasite proteins and lipids. However, because ARTs have a very short elimination half-life in the human circulation (~1–3 hr), their application as monotherapy was discontinued to avoid infection recrudescence and development of drug resistance. Artemisinin-based combination therapies (ACTs), consisting of ART and a long-acting partner drug, are the current frontline treatment for uncomplicated as well as severe malaria cases . ResistaTreatment with antimalarial drugs such as chloroquine (CQ) or sulfadoxine-pyrimethamine is usually associated with increased gametocytemia (density of gametocytes in the blood) on the days following drug administration, whereas treatment with ACTs results in reduced gametocytemia and transmission to mosquitoes . DespiteAlthough it has been proposed that the increase of gametocytemia observed after treatment with some drugs may reflect stimulation of sexual conversion, there is no direct linear relationship between conversion rates and the prevalence and density of circulating gametocytes . The dynP. falciparum in vitro cultures, yielding inconsistent results. While some studies reported increased sexual conversion upon exposure to specific doses of drugs such as CQ or ART , which allows accurate flow cytometry-based detection of very early gametocytes within a few hours after sexual conversion  gametocyte-specific promoter (NF54-10.3-Tom line)  . Using tom line) . All appom line) .In an additional set of experiments, we tested the impact of a DHA pulse at lower concentrations (≤5 nM) on sexual conversion . A 2 nM We also tested the impact on sexual conversion of higher DHA doses up to 30 nM, a concentration that kills ~90% of the parasites . In cholNF54-gexp02-Tom line produced in cultures treated with 5 nM DHA at the trophozoite stage (in the presence of choline) matured through stages I to V without any apparent morphological alteration. Furthermore, mature, stage V gametocytes were able to exflagellate after activation and to infect mosquitoes productively  at the early ring stage that reduced growth by <25%  did not cultures . This unnversion . In an athe drug . In thesthe drug .NF54-10.3-Tom reporter line and flow cytometry or light microscopy analysis of Giemsa-stained smears to measure sexual conversion by the SCC pathway. However, IFA analysis of this parasite line using anti-Pfs16 antibodies revealed an increase in the proportion of parasites expressing this endogenous protein upon DHA exposure  pathway , rather exposure . The sigIn these experiments, choline depletion did not increase sexual conversion via the SCC route . This reUsing the same drug pulse approach, we assessed whether CQ, a drug with a different mode of action than DHA , also stWe also tested the effect of an unrelated type of stress, a 3 hr heat shock at 41.5°C mimicking a malarial febrile episode, on sexual conversion. Exposure of choline-supplemented cultures at the trophozoite stage to heat shock, which reduced survival by ~40%, resulted in a ~ fourfold increase in sexual conversion and gametocytemia .pfap2-g (PF3D7_1222600), we analyzed the transcript levels for this gene after a DHA pulse, and also for one of its earliest known targets, gexp02 (pfap2-g and gexp02 relative to the serine-tRNA ligase (PF3D7_0717700) reference gene in choline-supplemented cultures, but not in choline-depleted cultures (pfap2-g or gexp02 (pfap2-g or gexp02 transcript levels against ubiquitin-conjugating enzyme (PF3D7_0812600) as a reference gene , DHA did not further stimulate sexual conversion at either stage. The accurate determination of the impact of DHA on sexual conversion rates at different stages was possible thanks to the development of an assay that uses a short drug pulse and reporter parasite lines that enable very early detection of gametocytes by flow cytometry. A limitation of our study is that we only used wild type parasite lines that do not carry mutations in the PfKelch13 protein associated with artemisinin resistance . Future The overall impact of a drug on the transmission potential of an infection depends on its effect on the sexual conversion rate, and on several other factors. In the case of ARTs, the stimulation of sexual commitment at the trophozoite stage may not result in an overall increase in transmission due to rapid clearance of asexual parasites, which prevents new rounds of gametocyte production, and to the activity of the drug against developing and mature gametocytes. Indeed, several studies have observed that treatment with drug combinations containing ARTs reduce gametocyte density and the duration of gametocyte carriage . NotwithP. falciparum exposed to low doses of DHA at the ring stage were also consistent with these prediction, as this resulted in moderately reduced sexual conversion rates. In contrast, experiments in which a pulse of DHA or CQ at a low dose was administered at the trophozoite stage showed the opposite trend, such that the subcurative treatment stimulated sexual conversion. This latter result is in line with some previous studies using P. falciparum . Whether the impact of stress on sexual conversion rates is positive (enhancement) or negative (reduction) also remains controversial . Evolutilciparum  or a murlciparum . A possifections . Thus, afections , or heatortholog .pfap2-g activation, which likely involves cellular components that are absent during the ring stage. Because stimulation of sexual conversion by choline depletion has been shown to involve GDV1  or by exposure to CQ shows a similar stage dependency, suggesting that the ring stage is largely insensitive to stimulation of sexual conversion. At the ring stage, some types of stress, such as exposure to DHA, may induce latency of a rather small fraction of the parasites as a means of population survival , rather lve GDV1 , which ilve GDV1 , which mlve GDV1 : the drulve GDV1  and heatlve GDV1 , and DHAlve GDV1 . FurtherP. falciparum sexual conversion rates that occur in response to a pulse of DHA. We demonstrate remarkable plasticity in sexual conversion rates, and a complex response that depends on the stage of the parasites at the time when they are exposed to the drug, the drug dose, and the metabolic state (presence or absence of choline). This complex scenario may explain the discrepant results obtained by previous studies. The assay that we have developed to test the impact of DHA on sexual conversion rates can be used to test the impact of any other drug or condition, as shown here for CQ and heat shock. Of note, the success of malaria elimination efforts largely depends on the ability to reduce or interrupt transmission. Although our results are not of immediate public health concern because the overall impact of treatment with ACTs is a reduction of the transmission potential, at least when compared with other drugs, the capacity of ARTs to induce sexual conversion must be taken into account. Otherwise, under certain conditions, treatment may result in an increase in transmission that could jeopardize efforts to eliminate malaria.Altogether, here we provide a detailed characterization of the changes in NF54-gexp02-Tom, E5-gexp02-Tom, and NF54-10.3-Tom were previously described and characterized , with B+ erythrocytes (3% hematocrit) and standard RPMI-HEPES parasite culture medium containing 0.5% Albumax and supplemented with 2 mM choline (Cultures were regularly maintained at 37°C under shaking (100 rpm) or static conditions in a hypoxic atmosphere (2% O choline . Erythro choline .Cultures for the production of mature gametocytes for mosquito infection were maintained in a semi-automated shaker incubator system as described . Fresh hNF54-10.3-Tom) or 0–10 hpi (NF54-gexp02-Tom) for exposure at the ring stage, or at 25–30 hpi for exposure at the trophozoite stage. After 3 hr, the drug was removed and fresh, pre-warmed culture medium was added. In some experiments, 200 nM DHA was maintained for 48 hr as a ‘kill’ control  cultures at ~1.5% parasitemia were split in two and one culture was maintained with a 2 mM choline supplement whereas the other had no choline added. Drug pulses with DHA (Sigma-Aldrich no. D7439) or CQ (Sigma-Aldrich no. C6628) were performed at 1–6 hpi  at the next cycle after drug exposure (measured at ~30–35 hpi or ~30–40 hpi) in treated cultures relative to control cultures, and expressed as percentage. Parasitemia was measured by flow cytometry (see below).NF54-gexp02-Tom and E5-gexp02-Tom lines, the sexual conversion rate was calculated as the sexual stage parasitemia divided by the total  parasitemia, and expressed as percentage. Asexual and sexual parasites were quantified by flow cytometry at ~30–35 hpi or ~30–40 hpi (D1) of the cycle after drug treatment, in the absence of chemicals that inhibit asexual replication. When using the NF54-10.3-Tom line, the sexual conversion rate was measured by dividing the gametocytemia on day 3 (D3) by the initial ring stage parasitemia on D0. In this case, cultures were treated with 50 mM N-acetyl-D-glucosamine  from D1 onwards to inhibit asexual replication. In experiments with the NF54-10.3-Tom line, gametocytemia was also measured on D0 to identify gametocytes already present in the culture at the beginning of the experiment, but it was found to be negligible. In any case, it was subtracted from D3 gametocytemia, such that only gametocytes newly formed during the assay were considered. Unless otherwise indicated, statistical analysis of differences in sexual conversion was performed using one-way ANOVA with Tukey HSD as the post hoc test. Variance was assumed to be homogenous because the sample size was the same for all groups, and they contain the same type of data.A schematic of our approach to determine sexual conversion rates is provided in Flow cytometry analysis to measure parasitemia at the cycle of drug exposure was performed using the nucleic acid stain SYTO 11 (0.016 μM) (Life Technologies no. S7573) and a BD FACSCalibur machine as previously described . To measImmunofluorescence assays (IFA) were performed as previously described . Brieflypfap2-g and gexp02 were normalized against the housekeeping genes serine-tRNA ligase and ubiquitin-conjugating enzyme. All qPCR primers used have been previously described (Trizol reagent (Invitrogen no. 15596026) was used to collect and preserve total RNA, followed by extraction using a protocol designed for samples with low RNA concentration . Brieflyescribed . StatistAnopheles stephensi mosquitoes using the standard membrane feeding assay (SMFA)  was added to the cultures for 3 hr, and 24 hr later  culture conditions were changed to medium with 10% human serum instead of Albumax and choline, and GlcNac (50 mM) was added to kill asexual stages. GlcNac was maintained for 4 d. Gametocyte cultures were followed during 9–13 d after DHA-treatment with medium changes twice a day, but without replenishing with fresh erythrocytes. At days 9–13, gametocyte development was analyzed in Giemsa stained blood smears and exflagellation was monitored after activation as described . Gametocy (SMFA) . Oocystsy (SMFA) . Statisty (SMFA)  was perf In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:Plasmodium falciparum sexual conversion. The study reveals that exposure to subcurative doses of dihydroartemisinin stimulates parasite sexual conversion, in a dose and stage-dependent manner. This work illustrates the complex interplay between drug exposure, parasite stage and metabolic conditions in the trade-off between asexual proliferation and formation of transmission forms.Transmission of malaria parasites from humans to mosquitoes depends on the formation of sexual blood stages, the gametocytes. Here, Portugaliza et al., capitalize on reporter parasite lines expressing fluorescence markers in early gametocytes to measure the effects of artemisinin, the front-line antimalarial drug, on Decision letter after peer review:Plasmodium falciparum" for consideration by eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Dominique Soldati-Favre as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Catherine Lavazec (Reviewer #3).Thank you for submitting your article "Artemisinin exposure at the trophozoite stage enhances sexual conversion in the malaria parasite The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife". Please let us know if you would like to pursue this option. As the editors have judged that your manuscript is of interest, but as described below that additional experiments are required before it is published, we would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). First, because many researchers have temporarily lost access to the labs, we will give authors as much time as they need to submit revised manuscripts. We are also offering, if you choose, to post the manuscript to bioRxiv  along with this decision letter and a formal designation that the manuscript is "in revision at Summary:Plasmodium falciparum.This is an interesting and well-presented study showing that exposure to subcurative doses of antimalarial drugs stimulates sexual conversion in the malaria parasite P. falciparum cultures. They found that exposure to subcurative doses of dihydroartemisinin at the trophozoite stage results in an increase in sexual conversion, mimicking depletion of choline in the culture medium. This effect is associated with an upregulation of AP2G, the master regulator of sexual conversion. Sexual conversion is also enhanced by exposure to another antimalarial drug, chloroquine, albeit to a lesser extent. In contrast, the ring stage appears to be insensitive to sexual conversion stimulation by artemisinin or choline depletion.The authors exploited recently published gametocyte-reporter parasite lines to measure the impact of drug treatment on sexual conversion in P. falciparum, so the conceptual novelty is somewhat limited. While the meaning of such a phenomenon in a clinical context remains unclear, the data are solid and this study provides a novel tool to investigate the signaling pathways leading to sexual conversion.Stress, including drug-induced stress, is known to stimulate sexual conversion in Essential revisions:1) Effect on ring stagesA main novel finding here is that ring stages of Pf are refractory to sexual conversion following drug and metabolic changes. DHA arrests ring stages before killing them, so DHA-treated rings might require a longer latent period before conversion to gametocyte. The authors should measure conversion at least 24-48hours after their measurement for DHA-treated trophozoites. In Figure 2, Did the authors test other concentrations of DHA on rings? Since they show that the DHA effect at the trophozoite stage is dose-dependent, one may expect that DHA affects the sexual conversion at the ring stage with concentrations higher than 10 nM.2) Assessment of sexual conversion.It is not clear how and when the total % of gametocytes was measured following drug treatments  and whether DHA increases both the proportion of early gametocyte and subsequent development into mature gametocyte after 10-14 days or if this increase was driven merely by a reduction in asexual forms or increased RBC lysis following drug exposure. In this study, and in the previous article by Portugaliza et al., (2019), the authors define the sexual conversion rate as the proportion of parasites that convert into sexual forms at each replicative cycle, which can be estimated as the gametocytemia divided by the initial parasitemia. This is indeed how the authors calculated the sexual conversion rate when using the NF54-10.3-Tom line (measured by dividing the gametocytemia on day 3 (D3) by the initial ring stage parasitemia on D0). However, when using the NF54-gexp02-Tom reporter line, the authors used another calculation   parasitemia, and expressed as percentage"). This corresponds to the proportion of gametocytes and not exactly to the sexual conversion rate, which is over-estimated when the number of asexual parasites is reduced due to the effects of the drug. Therefore, the axis label in graphs 1D, 2D, 3B, 4B, 5C, etc…should be changed to something like "proportion of gametocytes". The authors should consider using a more accurate calculation of the sexual conversion, as they did in their 2019 paper with the NF54-gexp02-Tom parasites or in this study with the NF54-10.3-Tom.3) A major question is whether DHA exposure induction of sexual conversion is via a choline-dependent or independent pathway. Choline depletion induces sexual conversion and may present as a confounding effect in DHA treated trophozoites since DHA alkylates lipids, especially in trophozoites. Choline measurements should have been done in the DHA or CQ treated parasites.4) It seems that only one single transmission experiment was performed (Supplementary figure 5), with a very small number of mosquitoes. The authors should repeat this experiment to strengthen the conclusion that ART enhances production of functional gametocytes. Summary:This is an interesting and well-presented study showing that exposure to subcurative doses of antimalarial drugs stimulates sexual conversion in the malaria parasite Plasmodium falciparum.The authors exploited recently published gametocyte-reporter parasite lines to measure the impact of drug treatment on sexual conversion in P. falciparum cultures. They found that exposure to subcurative doses of dihydroartemisinin at the trophozoite stage results in an increase in sexual conversion, mimicking depletion of choline in the culture medium. This effect is associated with an upregulation of AP2G, the master regulator of sexual conversion. Sexual conversion is also enhanced by exposure to another antimalarial drug, chloroquine, albeit to a lesser extent. In contrast, the ring stage appears to be insensitive to sexual conversion stimulation by artemisinin or choline depletion.Stress, including drug-induced stress, is known to stimulate sexual conversion in P. falciparum, so the conceptual novelty is somewhat limited. While the meaning of such a phenomenon in a clinical context remains unclear, the data are solid and this study provides a novel tool to investigate the signaling pathways leading to sexual conversion.P. falciparum, there is a paucity of studies providing unambiguous evidence for a specific effect of drugs on sexual conversion rates (as discussed in our Introduction and Discussion section). Thus, whether or not drugs can induce sexual conversion remains a highly controversial subject that we hope to settle with our approach and the data presented in this manuscript.We thank the reviewers for the careful review, for the important suggestions to improve the manuscript and for appreciating the interest and quality of our work. However, we would like to note that although stress in general and drug-related stress has indeed been frequently proposed to stimulate gametocyte production in Essential revisions:1) Effect on ring stagesA main novel finding here is that ring stages of Pf are refractory to sexual conversion following drug and metabolic changes. DHA arrests ring stages before killing them, so DHA-treated rings might require a longer latent period before conversion to gametocyte. The authors should measure conversion at least 24-48hours after their measurement for DHA-treated trophozoites. In Figure 2, Did the authors test other concentrations of DHA on rings? Since they show that the DHA effect at the trophozoite stage is dose-dependent, one may expect that DHA affects the sexual conversion at the ring stage with concentrations higher than 10 nM.We performed a new set of experiments in which parasites were exposed to different concentrations of DHA at the ring stage and sexual conversion was measured at a later time, as suggested by the reviewers. We measured gametocytemia by flow cytometry at ~24 and ~48 hpi of the next multiplication cycle. These experiments did not reveal any difference between sexual conversion rates determined at the two time points , indicating that the reduction in sexual conversion after DHA exposure at the ring stage is not attributable to a slowdown of sexual development or conversion in DHA-treated cultures. This is now discussed in the text . We also tested the effect of exposing parasites at the ring stage to higher DHA concentrations, as suggested by the reviewers, and found that sexual conversion rates progressively decrease with higher DHA concentration .2) Assessment of sexual conversion.It is not clear how and when the total % of gametocytes was measured following drug treatments  and whether DHA increases both the proportion of early gametocyte and subsequent development into mature gametocyte after 10-14 days or if this increase was driven merely by a reduction in asexual forms or increased RBC lysis following drug exposure. In this study, and in the previous article by Portugaliza et al., (2019), the authors define the sexual conversion rate as the proportion of parasites that convert into sexual forms at each replicative cycle, which can be estimated as the gametocytemia divided by the initial parasitemia. This is indeed how the authors calculated the sexual conversion rate when using the NF54-10.3-Tom line (measured by dividing the gametocytemia on day 3 (D3) by the initial ring stage parasitemia on D0). However, when using the NF54-gexp02-Tom reporter line, the authors used another calculation   parasitemia, and expressed as percentage"). This corresponds to the proportion of gametocytes and not exactly to the sexual conversion rate, which is over-estimated when the number of asexual parasites is reduced due to the effects of the drug. Therefore, the axis label in graphs 1D, 2D, 3B, 4B, 5C, etc…should be changed to something like "proportion of gametocytes". The authors should consider using a more accurate calculation of the sexual conversion, as they did in their 2019 paper with the NF54-gexp02-Tom parasites or in this study with the NF54-10.3-Tom.We agree with the reviewers that correct determination of sexual conversion rates is critical for this study. From the reviewers’ comments we feel that we did not explain our methods with sufficient clarity in the first version of the manuscript. Therefore, we have now added Figure 1—figure supplement 1 in which the different methods that we used to determine sexual conversion rates are described in detail.gexp02-Tom reporter lines is indeed equivalent to the “initial ring stage parasitemia on Day 0” used with other parasite lines . Gametocytemia was measured at different times depending on the marker used, but given that gametocytes are non-replicating, this does not affect the determination of sexual conversion rates. Using the gexp02-tdTom line we previously showed that gametocytemia is stable between 10-15 and 96-101 hpi, such that sexual conversion rates determined by analysis at these different times were almost identical .In all of our approaches and with all parasite lines, the determination involves calculating the proportion of Generation 1 parasites that develop into sexual forms (Generation 0 are the parasites that are exposed to the different conditions and Generation 1 are the parasites of the next cycle), as in our previous studies. Please note that the “total  parasitemia” used in experiments with the Regarding the question of how and when parasitemia and gametocytemia were determined in Figure 1E and Figure 3E, they were determined by flow cytometry in Generation 1. Values in panels E are from the same flow cytometry determination used to calculate conversion rates, which was performed at the time indicated in the schematic (panel A) included in each figure. To make this more clear, we have included this information in several figure legends  and in Results sectionsubsection “Exposure to DHA at the trophozoite stage enhances sexual conversion”. Figure 1E shows that the increase in the sexual conversion rate shown in Figure 1D does not result merely from a reduction in asexual forms, but rather from a net increase in gametocytemia, as explained in the text . No erythrocyte lysis was observed in any of our experiments at any of the drug concentrations tested.Furthermore, as part of the mosquito infection experiments , we measured gametocytemia on day ≥10, and found that the observed increase in sexual conversion rate and early gametocytemia in DHA-treated cultures (pulse at the trophozoite stage) results in an increase in gametocytemia measured at day ≥10 (mature gametocytes).We hope that the new figure and the changes in the figure legends and the text make our methods to determine the sexual conversion rate more clear, and the reviewers agree that the labeling of the axis is correct in all figures.3) A major question is whether DHA exposure induction of sexual conversion is via a choline-dependent or independent pathway. Choline depletion induces sexual conversion and may present as a confounding effect in DHA treated trophozoites since DHA alkylates lipids, especially in trophozoites. Choline measurements should have been done in the DHA or CQ treated parasites.We fully agree with the reviewers that understanding the mechanism of stimulation of sexual conversion by DHA and the potential involvement of LysoPC/ choline in this mechanism would be of great interest, but we consider that this is beyond the scope of this manuscript. In addition, addressing this molecular mechanism would involve using metabolomics techniques that are not established technologies in our laboratories . This would be a full new study in itself, because to address the mechanism meaningfully these experiments should be performed under different conditions and at multiple stages. We hope that the reviewers agree that the publication of our findings, including the first comprehensive characterization of the effect of DHA on sexual conversion, should not be delayed to perform experiments on the underlying mechanisms, which should be the subject of future investigations.However, we have performed a new set of experiments in which we found that a heat shock at the trophozoite stage also enhances sexual conversion. While we are aware that these experiments do not directly address the mechanism of induction of sexual conversion by DHA, these observations suggest that sexual conversion can be stimulated by types of stress that are not known or predicted to have an impact on choline levels. We have included these new results in the manuscript .4) It seems that only one single transmission experiment was performed , with a very small number of mosquitoes. The authors should repeat this experiment to strengthen the conclusion that ART enhances production of functional gametocytes.We performed new mosquito infection experiments that confirmed the idea that gametocytes produced from DHA-treated cultures are fully viable and infectious. In these experiments we observed an increase in the number of mature gametocytes and exflagellating centers in cultures treated with 5 nM DHA, following the increase in sexual conversion (relative to control cultures). In addition, when mosquitoes were fed with these cultures, an increase in the number of oocysts per mosquito and sporozoites per mosquito (relative to control cultures) was observed. The data from these additional experiments strongly suggests that exposure to low concentration DHA at the trophozoite stage enhances not only sexual conversion but also transmission to mosquitoes. The new results are presented in the new main Figure 2 and described in subsection “Exposure to DHA at the trophozoite stage enhances sexual conversion”.


Acute myocardial infarction (AMI) is a disease with high morbidity and mortality. Some new biomarkers can help us to improve the life quality and prognosis of AMI patients.  We therefore performed a systematic review and meta-analysis on the use of galectin-3  for assessing prognosis of AMI patients.  We searched Medline, Embase, Web of Science, Cochrane Library, SinoMed, China National Knowledge Infrastructure (CNKI), and Wanfang database up to June 2019. Trials included using galectin-3 to estimate prognosis in myocardial infarction (MI) patients. Z −0.22, 95% CI: −0.34, −0.09). The correlation between galectin-3 and infarct size was not significant in 119 patients . Higher galectin-3 was associated with increased all-cause mortality in 2343 AMI patients .  We identified 10 trails with a total of 2809 participants. The negative correlation between galectin-3 and left ventricular ejection fraction (LVEF) was significant in 505 AMI patients (Fisher's  The limited evidence suggests that galectin-3 is likely to predict the adverse outcomes in MI patients, but it is not significantly correlated with infarct size after MI. More high-quality trials with longer-term follow-up are still needed to confirm this finding. Coronary artery disease (CAD) is one of the most common types of disease around the world. Although state-of-the-art treatment and multiple biomarkers can be used for early diagnosis and estimation of prognosis in CAD patients , some evGal3 is a beta-galactoside binding lectin which can be produced by macrophages, vascular smooth muscle cells (VSMCs), and endothelium . Gal3 inThe clinical use of gal3 for heart failure (HF) has been widely explored, and it is useful for the diagnosis and accurate estimation of prognosis in HF patients . AlthougMyocardial infarction (MI) is one of the main causes that lead to HF, and some new biomarkers can help us to improve the diagnosis and prognosis of MI patients –17. ReceWe included randomized controlled trials, prospective cohort studies, or case-controlled studies that enrolled MI patients. The eligibility studies had to report the following results: (1) gal3 levels and clinical outcomes during follow-up and (2) the Pearson or Spearman correlation coefficient between gal3 and cardiac function (LVEF or infarct size). Echocardiography or cardiac magnetic resonance (CMR) imaging functional data were extracted; echocardiography data were preferentially used unless MRI data were available. The serum gal3 was measured with the ELISA; therefore, we excluded studies where the participants were chosen from community or gal3 was measured by using some other methods instead of ELISA.The following electronic databases were searched for the original review: PubMed, Web of Science, SinoMed, China National Knowledge Infrastructure (CNKI), and Wanfang database. Keywords included “galectin3,” “cardiovascular disease,” “coronary heart disease,” “acute coronary system,” and “myocardial infarction.” Medical subject headings (MeSH) or equivalent and text word terms were used. The publications written in English or Chinese were included in our search.Two review authors independently reviewed titles and abstracts of all studies identified by the electronic searches and retrieved potentially relevant studies. We then read the full text of relevant studies and excluded any study that did not satisfy the inclusion criteria. All disagreements were resolved by discussion with a third review author until a consensus was achieved. In case of missing or unclear data for our analysis, we contacted the corresponding authors to clarify or get the data that were missing.2 statistic). We used the random effects model (Mantel-Haenszel (M-H) method) to pool the data.In order to get the standard error (SE) depending on the value of the Pearson correlation coefficient, a Fisher transformation was used to convert each correlation coefficient into an approximately normal distribution. The inverse variance formula was used to calculate the data . For eacTwo authors independently assessed the risk of bias of included studies according to the Cochrane Handbook (Version 5.1.0) and rated each domain as having a low risk of bias, a high risk of bias, or an unclear risk of bias. Disagreements were settled by discussion or consulted a third review author.The database searches generated 419 (PubMed), 826 (Web of Science), and 158  hits and 406 after reduplication. Screening the titles and abstracts identified 35 papers for formal inclusion or exclusion, of which 10 papers met the inclusion criteria. Details of the screening process through the review are given in Eight studies recruited participants with MI . The pooled results of these studies are shown in Z −0.22, 95% CI: −0.34, −0.09) with a relatively high level of heterogeneity . We conducted subgroup analysis to explore the statistical heterogeneity.Seven studies provided the r value between gal3 and LVEF which were measured during AMI. The pooled results of these four studies revealed that the negative correlation between gal3 and LVEF was statistically significant , but there was a high level of heterogeneity . The forest plot between gal3 and LVEF is shown in Four studies (505 patients) provided the Z −0.31, 95% CI: −0.52, −0.09). Three studies (119 patients) reported the data of gal3 and LVEF which were measured at relatively long time after MI (Mayr 2012 4 months and Singsaas 2016 1 year and 4.4 years). There was a statistically significant negative correlation between gal3 and LVEF . The forest plot is shown in We also found that three studies (119 patients) evaluated the association between gal3 and infarct size . Although these three studies showed negative correlation between gal3 and infarct size, the overall effect was not significant , and there was a high level of heterogeneity . The forest plot is shown in MI is one of the common diseases with high morbidity and mortality. It was our interest to explore the effectiveness of gal3 to predict the mortality or HF onset after MI. We found that five studies (2343 patients) evaluated the association between gal3 and mortality or HF onset . Our pooled results indicated that higher gal3 was associated with increased all-cause mortality in MI patients .LVEF, frequently used for assessment of left ventricular, is an important parameter for predicting an unfavorable clinical outcome in AMI patients. After acute myocardial injury, LV remodeling is more or less happened, and a series of mechanical and neurohormonal factors may take part in this process that results in the progressive deteriorate of LVEF , leading2 = 53%, P=0.05) in this analysis. One of the main reasons was that the time of detecting LVEF and collecting the serum sample were not permanent. In Szadkowska's study, peripheral blood samples were collected within 36–60 h after acute MI, and LVEF was assessed on the 3rd–5th day of MI [The meta-analysis showed that gal3 significantly predicted the decline of LVEF, but there was a high level of heterogeneity . However, the results should be interpreted seriously because we just included 3 studies in this analysis. More importantly, the time of collecting the blood sample and performing cardiac magnetic resonance (CMR) imaging was different.Infarct size is a major parameter of cardiac function after MI , 34 and,MI, one subtype of CAD, is one of the major causes of HF characterized by high morbidity and mortality. It was our interest to explore the value of gal3 for predicting MACE and mortality in these patients. We found that three studies examined the association between gal3 level and death , 28, 29.Although we endeavored to search out all relevant studies, until now there are only a small number of trials included in this review; besides, the most of the studies in our review included relatively limited numbers of participants. Particularly, there was a variation in the status of participants between studies. Most of the studies included a varied range of follow-up time that might have a bearing on these results. In the future, we still need more studies with long follow-up time to further assess the value of gal3 in the aspect of prognosis of MI.The limited trial evidence suggests gal3 is statistically negatively correlated with LVEF in MI patients. More importantly, the available trials are supportive of favourable effects of gal3 on predicting mortality or LVEF after MI. However, in this field, there is still one large on-going trial in which gal3 concentration increased risk of all-cause mortality in MI patients during mid-term follow-up . High-quSerum gal3 has been regarded as a novel biomarker to predict long-term adverse outcomes in HF patients; however, the role of gal3 in diagnosis and assessing the prognosis of MI is still controversial. In our study, we found that gal3 was statistically correlated to the LVEF with a relatively high level of heterogeneity. In AMI patients, the negative correlation between gal3 and LVEF was statistically significant, but there was a high level of heterogeneity. After a relatively long time of MI, there was the statistically significant negative correlation between gal3 and LVEF, and there was a low level of heterogeneity. The negative correlation between gal3 and infarct size was not significant. There was a high level of heterogeneity. More importantly, our pooled results indicated that higher gal3 is related to the increased all-cause mortality in MI patients during the follow-up period between 30 days and 5.4 years. The limited evidence suggests that gal3 is likely to predict the adverse outcomes and LVEF in MI patients, but it is not significantly correlated with infarct size after MI. We are still looking forward to more and more reliable and persuasive clinical trials in the future.