Drosophila SARM1 regulatory domain complexed with this activator, the vacor metabolite VMN, which as the most potent activator yet known is likely to support drug development for human SARM1 and NMNAT2 disorders. This study indicates the mechanism of neurotoxicity and pesticide action by vacor, raises important questions about other pyridines in wider use today, provides important new tools for drug discovery, and demonstrates that removing SARM1 can robustly block programmed axon death induced by toxicity as well as genetic mutation.Axon loss underlies symptom onset and progression in many neurodegenerative disorders. Axon degeneration in injury and disease is promoted by activation of the NAD-consuming enzyme SARM1. Here, we report a novel activator of SARM1, a metabolite of the pesticide and neurotoxin vacor. Removal of SARM1 completely rescues mouse neurons from vacor-induced neuron and axon death in vitro and in vivo. We present the crystal structure of the  Sarm1 deletion confers robust axon protection, and even lifelong protection against lethality caused by NMNAT2 deficiency  plays a central, pro-degenerative role in programmed axon death  . This axficiency , SARM1 hIn the past 2 years, there has been intense focus on structure-activity studies of SARM1 to help drive drug development. Recent structural data revealed the existence of an allosteric pocket in SARM1 armadillo-repeat (ARM) domain which is important for the regulation of SARM1 activity. NMN binds to this pocket, leading to SARM1 activation . NAD Ji can alsoSarm1-/- neurons and their axons are completely resistant. We unexpectedly find that vacor metabolite VMN directly binds to and activates SARM1, causing neuronal death. This study elucidates the mechanism of action of vacor likely underlying its lethal neurotoxic effect in humans. Vacor use is banned, but structurally related pyridines are widely-used and present in drugs, pesticides and food, raising important questions about their potential toxicity and SARM1 as a mediator of environmental neurotoxicity. Our findings also provide important new tools for drug discovery. To our knowledge, VMN is the most potent activator of SARM1 reported so far, and our structural data reveal essential information on SARM1 regulation which will aid drug development to block SARM1 activation.Here, we have investigated whether vacor, a disused pesticide and powerful neurotoxin associated with human peripheral and central nervous system disorders  and axonIntracellular conversion of vacor into its metabolites has been suggested to contribute to its toxicity . We firs-/-Sarm1 DRG and SCG neurites  injection  of vacor-/-Sarm1 neurons  and inhibition (Ki)  .While NMN activates SARM1  and promARM recombinantly, we used the ARM domain of Drosophila SARM1 , produced in E. coli, to investigate if VMN interacts directly with dSARM1ARM. Isothermal titration calorimetry (ITC) analysis showed that VMN bound to dSARM1ARM with a Kd value of 2.83 ± 0.16 μM (1:1 molar ratio) (ARM (1.69 Å resolution). dSARM1ARM contains eight tandem armadillo repeats (ARM1-8) stacking into an unusually compact right-handed superhelix  . To furtperhelix . The boum ARM1-5 . The intm ARM1-5 ; and eigRM1-5 (2 . The hydRM1-5 (2 .ARM than NMN (Kd = 6.39 ± 0.04 μM), whose own interaction with the ARM domain was recently reported  structures of ligand-free and NAD-bound hSARM1, representing the inactive state of the protein (ARM due to the rotation of its nicotinic acid (NA) portion towards the N-terminus of the protein, and is therefore unable to form any interactions with the C-terminal region of the protein , thus preventing NAD from inducing a more compact conformation in the ARM domain , R437 (human R157), and K476 (human K193), because these residues are highly conserved and specifically interact with VMN through their side chains. We introduced the corresponding W103A, R157A, and K193R mutations into human SARM1 and exogenously expressed them in  neurons . Unlike  neurons . We pick neurons . Crucial neurons .ARM structures and the differences from the ligand-free and NAD-bound hSARM1ARM structures provide the explanation for the mechanism of how these ligands target the same allosteric site, but regulate SARM1 activity differently. The data not only provide support for a key physiological role of NMN in the regulation of SARM1 activity and axon degeneration, but given VMN’s greater potency than NMN, we anticipate that VMN and vacor will be important tools to support drug discovery in several ways. The identification of key residues in the ARM domain involved in the potent activation of SARM1 by VMN should facilitate rational drug design; further understanding why VMN activates SARM1 more potently than NMN could lead to the development of ligands with higher-affinity and selectivity to lock SARM1 in its inactive state. It will be important to expand also on VMN inhibition functions. Finally, we have also recently reported that recombinant hSARM1 uses vacor for base exchange to produce VAD  Act under Project Licences PPL P98A03BF9 and PP1824519, and in accordance with the Association for Research in Vision and Ophthalmology’s Statement for the Use of Animals in Ophthalmic and Visual Research.IMSR_JAX:000664), Sarm1-/- (RRID:MGI:3765957) and SWld (RRID:MGI:4438866) E13.5-E14.5 mouse embryos and SCG ganglia were dissected from postnatal day 0–2 mouse pups. Littermates from Sarm1+/- crosses were used when possible, as indicated in figure legends. Explants were cultured in 35 mm tissue culture dishes pre-coated with poly-L-lysine  and laminin  in Dulbecco’s Modified Eagle’s Medium  with 1 % penicillin/streptomycin, 50 ng/ml 2.5 S NGF  and 2 % B27 (Gibco). Four µM aphidicolin (Merck) was used to reduce proliferation and viability of small numbers of non-neuronal cells (Sarm1-/- SCG ganglia were incubated in 0.025 % trypsin (Merck) in PBS (without CaCl2 and MgCl2) (Merck) for 30 min, followed by incubation with 0.2 % collagenase type II (Gibco) in PBS for 20 min. Ganglia were then gently dissociated using a pipette. Dissociated neurons were plated in a poly-L-lysine and laminin-coated area of ibidi μ-dishes (Thistle Scientific) for microinjection experiments. Dissociated cultures were maintained as explant cultures, except that B27 was replaced with 10 % fetal bovine serum (Merck) and 2.5 S NGF was lowered to 30 ng/ml. Culture media was replenished every 3 days. For most experiments, neurites were allowed to extend for 7 days before treatment.DRG ganglia were dissected from C57BL/6J , and VMN or vehicle (H2O) just prior to imaging (time 0 hr). When used, FK866  and NAM (Merck) were added at the same time as vacor. For neurite outgrowth and long-term survival assays, multiple doses of vacor or vehicle were added by replacing media with fresh media containing the drugs at the timepoints indicated in the figure. The drug concentrations used are indicated in the figures and figure legends. Vacor was dissolved in H2O with 4 % 1 N HCl or DMSO; quantitation of the dissolved stock was performed spectrophotometrically (ɛ340nm 17.8 mM–1cm–1).For most experiments, DRG and SCG neurons were treated at day in vitro (DIV) 7 with vacor (Greyhound Chromatography) or vehicle (HPhase contrast images were acquired on a DMi8 upright fluorescence microscope (Leica microsystems) coupled to a monochrome digital camera (Hamamatsu C4742-95). The objectives used were NPLAN 5 X/0.12 for neurite outgrowth assays and HCXPL 20 X/0.40 CORR for neurite degeneration assays. Radial outgrowth was determined by taking the average of two measurements of representative neurite outgrowth for each ganglion at DIV2-3-5-7. Measurements were made from overlapping images of the total neurite outgrowth. For neurite degeneration assays, the degeneration index was determined using a Fiji plugin . For eacSarm1-/- SCG neurons were microinjected using a Zeiss Axiovert S100 microscope with an Eppendorf FemtoJet microinjector and Eppendorf TransferMan micromanipulator. Plasmids were diluted in 0.5 X PBS (without CaCl2and MgCl2) and filtered using a Spin-X filter (Costar). The mix was injected directly into the nuclei of SCG neurons using Eppendorf Femtotips . Cell suspension was pipetted into each side of the upper channel of the microfluidic device. On DIV7, a difference of 100 μl of media between chambers was introduced and drugs were added to the compartment with the lower hydrostatic pressure. To calculate the % of viable neurons, 1 μg/ml propidium iodide (PI) (Thermo Fisher Scientific) was added to the media 15 min before drug addition. Phase contrast and fluorescence microscopy images were acquired on a DMi8 upright fluorescence microscope (Leica microsystems) coupled to a monochrome digital camera (Hamamatsu C4742-95). The objective used was HCXPL 20 X/0.40 CORR. For each experiment, the degeneration index was calculated from the average of two distal fields of neurites per condition, whereas the % of viable neurons remaining relative to the first time point was calculated from the average of three fields of cell bodies (staining positive for PI) per condition; the total number of experiments is indicated in the figure legends.IMSR_JAX:000664), or -/-Sarm1 (RRID:MGI:3765957) mice were intravitreally injected with either 2 μL vacor, DMSO (vehicle) or PBS. Injections were performed as previously described . Images were captured, blindly, from eight regions per retina using a 20 X objective, and automatically quantified with the Fiji plugin ‘Simple RGC’  fractions were washed in ice-cold PBS and rapidly frozen in dry ice and stored at −80 °C until processed. Tissues were ground in liquid N2 and extracted in HClO4 by sonication, followed by neutralisation with K2CO3. NMN and NAD were subsequently analysed by spectrofluorometric HPLC analysis after derivatisation with acetophenone .VMN and VAD were synthesised as previously reported  with minCVCL_1926) and had been authenticated by STR profiling. Mycoplasma contamination was not detected. Recombinant, full-length C-terminal Flag-tagged wild-type and K193R human SARM1 (hSARM1-Flag) were expressed in HEK293T cells and purified by immunoprecipitation. HEK cells at 50–70% confluence were transfected with wild-type and K193R hSARM1-Flag expression construct (pLVX-IRES-ZsGreen vector backbone) using Lipofecatmine 2000 (Thermo Fisher Scientific). To boost wild-type and K193R hSARM1-Flag expression, media were supplemented with 2 mM nicotinamide riboside (prepared from Tru Niagen capsules by dissolving the contents and passing through a 0.22 µm filter) at the time of transfection. After 24 hr, cells were collected and washed in cold PBS and lysed for 10 min with trituration by pipetting and repeated vortexing in ice-cold KHM buffer  with cOmplete, Mini, EDTA-free protease inhibitor cocktail (Roche). After centrifugation for 5 min at 3000 rpm in a chilled microfuge, the supernatant was collected and diluted to 1 µg/µl in KHM buffer after determination of its protein concentration by the Pierce BCA assay (Thermo Fisher Scientific). For immunoprecipitation, 1 ml of extract was incubated overnight at 4 °C with rotation with 20 µg/ml anti-FLAG M2 antibody  and 50 µl Pierce magnetic protein A/G beads (Thermo Fisher Scientific) pre-washed with KHM buffer. Beads were collected on a magnetic rack and washed 3 x with 500 µl KHM buffer and 1 x with PBS (with protease inhibitors) and then resuspended in PBS containing 1 mg/ml BSA. hSARM1-Flag concentration in the bead suspension was determined relative to an hSARM1 standard (purified from insect cells) by immunoblotting using a rabbit polyclonal antibody raised against the SAM domains of human SARM1 (both kindly provided by AstraZeneca).Human embryonic kidney (HEK) 293T cells  were obtained from the American Type Culture Collection  HPLC   in buffer HEPES/NaOH 50 mM, pH 7.5, and 250 μM NAD. NMN, VMN, and VAD were added at the concentrations indicated in the figures. Reactions were initiated by adding NAD, incubated at 25 °C in a water-bath, stopped at appropriate times by HClO, cADPR)  or under, cADPR) . The pro, cADPR) . One uni, cADPR)  and thenVmax) represents the rate in the absence of the effector  corrected by the two parameters between parentheses, the first for activation and the second for inhibition, taking into account opposite and independent contributions exerted by the single but dual effector I. X is the ‘relative activity fold activation‘ factor at saturating concentrations of I, Y is the residual activity fraction at saturating concentration of I, aK is the affinity constant of I for the activation site, iK is the affinity constant of I for the inhibition site, and n is the Hill coefficient indicating cooperativity (if ≠ 1).that represents a Michaelis-Menten equation adapted to be valid for readily reversible effectors, that is, including activators and inhibitors binding via distinct sites to one protein target. Maximum velocity (ARM (residues 315–678) was cloned into the pMCSG7 expression vector at the SspI site, using ligation-independent cloning (ARM mutants were generated using the Q5 Site-Direct Mutagenesis Kit (New England BioLabs). Plasmids were transformed into BL21 (DE3) cells. Large-scale expression of dSARM1ARM and its mutants was performed using the auto-induction method . PMSF (phenylmethanesulfony fluoride) was added to the cell resuspension at 1 mM concentration. Cell resuspension from 1 l of culture was sonicated at the amplitude of 40 % for 120 s (10 s on and 20 s off). Lysed cells were centrifuged at 15300× g for 40 min at 4 °C. The supernatant was loaded onto the HisTrap HP 5 ml Ni column, followed by washing using 10 column volumes of lysis buffer. The bound protein was eluted using elution buffer , and incubated with TEV (tobacco etch virus) protease to remove the N-terminal 6× histidine tag in the SnakeSkin Dialysis Tubing, 3.5 K MWCO (Thermo Fisher Scientific), dialysed against the buffer containing 20 mM HEPES (pH 8.0), 300 mM NaCl and 1 mM DTT at 4 °C overnight. The cleaved protein was reloaded onto the Ni column and flow-through was collected, concentrated to 10 ml and injected onto the Superdex 75 HiLoad 26/600 column  equilibrated with gel-filtration buffer (10 mM HEPES pH 8.0 and 150 mM NaCl). The pure target protein, as confirmed by SDS-PAGE analysis and mass spectrometry, was concentrated to 10 mg/ml and stored at –80 °C.Codon-optimised cDNA of dSARM1 cloning . Plasmidn method . In brieARM (10 mg/ml) and VMN were incubated at 1: 10 molar ratio at 4 °C overnight. Hanging drops, containing 2 μl of protein complex and 2 μl of well solution , were equilibrated against 500 μl of well solution at 20 °C. Crystals with irregular shapes were observed after 3–5 days. In situ partial proteolysis happened during the process of crystallisation, as the analysis of the crystals via SDS-PAGE analysis and mass spectrometry indicated that the crystallised protein only spanned residues 370–678, rather than residues 315–678. Crystals cryo-protected in paratone-N diffracted to 1.7–2.3 Å resolution at the Australian Synchrotron MX2 beamline. X-ray diffraction data was collected at the wavelength of 0.95372 Å. Raw data was processed using XDS (RRID:SCR_015652)  (ARM (PDB: 7LCZ) (SCR_014224) (SCR_014222) (SCR_000305) (Schrodinger), PDBsum (RRID:SCR_006511) (SCR_015749) (ARM (RMSD = 0.3 Å over 296 Cα atoms) in the asymmetric unit.Protein crystallisation, diffraction data collection and structural determination dSARM1_015652) ; data re_007255) . Phases B: 7LCZ)  as a sea_014224) . The mod_014222)  and Phen_014222) , and ana_006511)  and PISA_015749) . The cryd) were obtained after fitting the integrated and normalised data to a single-site binding model using NanoAnalyze (TA Instruments).ITC experiments were performed in duplicate on Nano ITC (TA Instruments). All proteins and compounds were dissolved in a buffer containing 10 mM HEPES (pH 8.0) and 150 mM NaCl. The baseline was equilibrated for 600 s before the first injection. VMN (0.6 mM) was titrated as 20–25 injections of 1.96 μL every 200 s, into 45 μM protein. The heat change was recorded by injection over time and the binding isotherms were generated as a function of molar ratio of the protein solution. The dissociation constants  as a loading control. Quantification of band intensity was determined by densitometry using Fiji (RRID:SCR_002285).Following treatment with vacor, DRG ganglia were separated from their neurites with a scalpel. Neurites and ganglia were collected, washed in ice-cold PBS containing protease inhibitors (Roche), and lysed directly in 15 µl 2 x Laemmli buffer containing 10 % 2-mercaptoethanol (Merck). Samples were loaded on a 4-to-20% SDS polyacrylamide gel (Bio-Rad). Membranes were blocked for 3 hr in 5 % milk in TBS  plus 0.05 % Tween-20 (Merck) (TBST), incubated overnight with primary antibody in 5 % milk in TBST at 4 °C and subsequently washed in TBST and incubated for 1 hr at room temperature with HRP-linked secondary antibody (Bio-Rad) in 5 % milk in TBST. Membranes were washed, treated with SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific) and imaged with Uvitec Alliance imaging system. The following primary antibodies were used: mouse monoclonal anti-SARM1  (1:5000)SCR_002798). The appropriate tests used and the n numbers of each individual experiment are described in the figure legends. A p-value < 0.05 was considered significant.Statistical testing of data was performed using Prism  feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Suzanne Pfeffer as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Marc Freeman (Reviewer #2); Mohanish Deshmukh (Reviewer #3).Thank you for submitting your article "Neurotoxin-mediated potent activation of the axon degeneration regulator SARM1" for consideration by The reviewers have discussed their reviews with one another, in consultation with the Reviewing Editor. Among the comments, the overall enthusiasm is high and the results of vacor and its metabolite VMN as an activator of Sarm were considered to be well done and novel. The significance of the results and the structural data was also strong. Several revisions were recommended to improve the clarity and to extend the findings.Essential revisions:1) VMN is an activator of Sarm at low concentration and an inhibitor at high concentrations. Is VMN's inhibitory activity working on the active site? Is there any additional evidence for how VMN inhibits Sarm either through the active site or through an allosteric site. Two of the three reviewers requested further insight into this question and felt the answer would give mechanistic insight into a new activator and inhibitor of SARM.2) Do high VMN concentrations (or vacor) inhibit axotomy induced axon degeneration? Any data to address this question should be included.3) Some reorganization of the manuscript would be helpful. Many of the supplemental figures contains key data. They should be included with the four figures presently in the submission. The Introduction is dated and does not include consideration of the allosteric pocket.Reviewer #1 (Recommendations for the authors):1) Is the supplement to figure 1 the authors use compartment chambers to show that vacor can trigger local degeneration of cell bodies and axons. They state that with cell body application "vacor-induced cell death causes eventual secondary neurite degeneration." However, in their data, cell body application of vacor causes more rapid and robust distal axon degeneration than does direct application of vacor to the distal axons. This is not consistent with the "eventual neurite degeneration" and suggests a different and more active mechanism of degeneration in that case. Is the axon degeneration in the distal compartment still Sarm dependent? This should at a minimum be discussed, and now that inhibitors of Sarm are available could be experimentally addressable.2) The authors report that VMN not only activates Sarm, but also inhibits Sarm. The authors claims that this inhibitory function "could reveal critical information on how Sarm activity is regulated," yet they do not attempt to understand the mechanism of inhibition. Is the inhibitory role of VMN working through the same allosteric pocket? Is it acting on the catalytic site in the enzyme? With the nice biochemical system the authors have established this seems easily addressable and would be an important addition to a manuscript describing how VMN regulates Sarm activity.3) The introduction in this manuscript is quite similar to the version published on Bioarchive about a year ago. In that year, there has been tremendous progress in understanding the mechanism of Sarm activation, with the publication of four papers describing the structure of allosteric ligands binding to Sarm and the definition of the roles of NMN and NAD in Sarm activation/inhibition. The introduction should be updated in order to better place the current work in context.4) There are only four figures but many supplemental figures that are quite important. The paper would be much easier to follow if it were re-organized into a more traditional 7-8 figure format.5) The final sentence in the abstract claims that the study shows that Sarm can "permanently block programmed axon death induced by toxicity." This is overstated, as the authors only show Sarm knockout protects for three days after a single vacor injection, and in vitro after two doses separated by two days. This does not speak to whether the protection is permanent, since this would require studies of long-term toxin exposure.6) The manuscript would benefit from a more comprehensive analysis of how this work relates to prior work published by these authors. For example, the point mutants in the binding pocket of Sarm were previously analyzed by these authors for their role in NMN binding and Sarm activation. This should be mentioned and the findings compared. Similarly, there is a comparison of the crystal structure for NMN and NAD, but not for their recently described structure of Sarm bound to NAMN.7) The authors show very convincingly that Sarm activation leads to loss of retinal ganglion cells. A couple of previous studies using different injury models have come to different conclusions about whether Sarm causes RGC death. It would be interesting to comment on how the author's current data relates to those prior findings in Sarm knockouts.Reviewer #2 (Recommendations for the authors):8) The abstract claims that the structure of VMN bound to the Sarm regulatory domain will "facilitate drug development for some human disorders including ALS and some polyneuropathies." Since this binding pocket has already been well defined in previous studies, these is too grand of a claim for the abstract, which should focus more on what the manuscript has actually demonstrated. Commentary on hopes for improved drug development would be more appropriate for the Discussion section.None. This is a rigorous and complete study.Reviewer #3 (Recommendations for the authors):The experiments very well done and the results are interesting. The use of multiple models both in vitro and in vivo as well as the biochemical and structural studies are a strength of this study. Essential revisions:1) VMN is an activator of Sarm at low concentration and an inhibitor at high concentrations. Is VMN's inhibitory activity working on the active site? Is there any additional evidence for how VMN inhibits Sarm either through the active site or through an allosteric site. Two of the three reviewers requested further insight into this question and felt the answer would give mechanistic insight into a new activator and inhibitor of SARM.We appreciate these comments. We agree that VMN-mediated inhibition of SARM1 is interesting and understanding how this inhibition works is certainly one of our future plans. We have started working towards this and our preliminary data suggest that the mechanism of inhibition might not simply be explained by VMN binding to the active or allosteric site. Therefore, unravelling how VMN inhibits SARM1 is likely to be a fairly substantial undertaking at this stage, which we feel goes beyond the scope of the current study. We prefer to keep the focus at present on the toxic effect of vacor and its metabolite VMN, along with the many exciting implications of this including drug development and the potential for activation of SARM1 by other environmental toxins.2) Do high VMN concentrations (or vacor) inhibit axotomy induced axon degeneration? Any data to address this question should be included.We thank the reviewers for these interesting suggestions; there are, however, some experimental limitations which need to be considered. For instance, vacor is insoluble in culture media at concentrations higher than 100µM, limiting the range of concentrations that can be tested. We have treated axotomised neurites with 100µM vacor; as expected, we did not see protection as vacor is normally very toxic at this concentration.Similarly, it is unclear how much VMN actually enters the cell, considering that it is a charged molecule. 500µM VMN (the concentration used in this study) is the first concentration that causes toxicity in neurons. This is substantially higher than the first toxic concentration of vacor (50µM), suggesting that VMN cell-permeability is limited. Therefore, reaching high enough concentrations of VMN in neurons to confer protection is challenging.We would be happy to share preliminary data on these points for revision purposes, but we would prefer not to add these to the manuscript as interpretation of results in this instance is subject to many caveats.3) Some reorganization of the manuscript would be helpful. Many of the supplemental figures contains key data. They should be included with the four figures presently in the submission. The Introduction is dated and does not include consideration of the allosteric pocket.We appreciate this feedback and have made the following changes to address reviewers’ comments:– We have changed the format of the paper into a more traditional 7 figure paper. Some important data that were in supplementary have now been moved to the main text. Specifically, Figure 1 —figure supplement 1 is now Figure 1. We have added a comparison with the structure of SARM1 bound to NaMN. All structural data are now in Figure 6. Finally, we have moved all data on SARM1 mutants in figure 7.– We have added a paragraph in the introduction to include the latest work in the field; .– We have made a number on minor changes in response to reviewers’ comments, detailed below. We have used the word tracked changes function to highlight changes to the text.Reviewer #1 (Recommendations for the authors):1) Is the supplement to figure 1 the authors use compartment chambers to show that vacor can trigger local degeneration of cell bodies and axons. They state that with cell body application "vacor-induced cell death causes eventual secondary neurite degeneration." However, in their data, cell body application of vacor causes more rapid and robust distal axon degeneration than does direct application of vacor to the distal axons. This is not consistent with the "eventual neurite degeneration" and suggests a different and more active mechanism of degeneration in that case. Is the axon degeneration in the distal compartment still Sarm dependent? This should at a minimum be discussed, and now that inhibitors of Sarm are available could be experimentally addressable.We thank the reviewer for pointing this out. We have discussed this important point in the main text; .2) The authors report that VMN not only activates Sarm, but also inhibits Sarm. The authors claims that this inhibitory function "could reveal critical information on how Sarm activity is regulated," yet they do not attempt to understand the mechanism of inhibition. Is the inhibitory role of VMN working through the same allosteric pocket? Is it acting on the catalytic site in the enzyme? With the nice biochemical system the authors have established this seems easily addressable and would be an important addition to a manuscript describing how VMN regulates Sarm activity.We appreciate these comments. We agree that VMN-mediated inhibition of SARM1 is interesting and understanding how this inhibition works is certainly one of our future plans. We have started working towards this and our preliminary data suggest that the mechanism of inhibition might not simply be explained by VMN binding to the active or allosteric site. Therefore, unravelling how VMN inhibits SARM1 is likely to be a fairly substantial undertaking at this stage, which we feel goes beyond the scope of the current study. We prefer to keep the focus at present on the toxic effect of vacor and its metabolite VMN, along with the many exciting implications of this including drug development and the potential for activation of SARM1 by other environmental toxins.3) The introduction in this manuscript is quite similar to the version published on Bioarchive about a year ago. In that year, there has been tremendous progress in understanding the mechanism of Sarm activation, with the publication of four papers describing the structure of allosteric ligands binding to Sarm and the definition of the roles of NMN and NAD in Sarm activation/inhibition. The introduction should be updated in order to better place the current work in context.We thank the reviewer for the suggestion. We have now updated the introduction to reflect recent developments in the field; .4) There are only four figures but many supplemental figures that are quite important. The paper would be much easier to follow if it were re-organized into a more traditional 7-8 figure format.We have now reorganised the manuscript into a more traditional 7 figure format. Specifically, Figure 1 —figure supplement 1 is now Figure 1. All structural data are now in Figure 6. Finally, we have moved all data on SARM1 mutants which were previously in supplementary material in figure 7.5) The final sentence in the abstract claims that the study shows that Sarm can "permanently block programmed axon death induced by toxicity." This is overstated, as the authors only show Sarm knockout protects for three days after a single vacor injection, and in vitro after two doses separated by two days. This does not speak to whether the protection is permanent, since this would require studies of long-term toxin exposure.We thank the reviewer for pointing this out. We have changed the word “permanently” with “robustly” in the abstract.6) The manuscript would benefit from a more comprehensive analysis of how this work relates to prior work published by these authors. For example, the point mutants in the binding pocket of Sarm were previously analyzed by these authors for their role in NMN binding and Sarm activation. This should be mentioned and the findings compared. Similarly, there is a comparison of the crystal structure for NMN and NAD, but not for their recently described structure of Sarm bound to NAMN.We thank the reviewer for this suggestion. We have now added the comparison with the structure of SARM1 bound to NaMN , and discussed this in the text; .7) The authors show very convincingly that Sarm activation leads to loss of retinal ganglion cells. A couple of previous studies using different injury models have come to different conclusions about whether Sarm causes RGC death. It would be interesting to comment on how the author's current data relates to those prior findings in Sarm knockouts.We have now included a sentence in the discussion on this interesting point; .8) The abstract claims that the structure of VMN bound to the Sarm regulatory domain will "facilitate drug development for some human disorders including ALS and some polyneuropathies." Since this binding pocket has already been well defined in previous studies, these is too grand of a claim for the abstract, which should focus more on what the manuscript has actually demonstrated. Commentary on hopes for improved drug development would be more appropriate for the Discussion section.We thank the reviewer for this suggestion and we have now made changes to the abstract in line with the reviewer’s suggestion; .


Assessment of screw pattern, implant density (ID), and optimization of 3D correction through computer-based biomechanical models.To investigate how screw pattern and ID affect intraoperative 3D correction of thoracic curves in adolescent idiopathic scoliosis, and how different correction objectives impact the optimal screw pattern.Screw pattern, ID, correction objectives and surgical strategies for posterior fusion of AIS are highly variable among experienced surgeons. The “optimal” instrumentation remains not well defined.en bloc derotation, and compression/distraction as primary correction maneuvers. 3D correction descriptors were quantified in the coronal, sagittal and transverse planes. An objective function weighting the contribution of intraoperative 3D correction and mobility allowed rating of the outcomes of the virtual surgeries. Based on surgeon-dependent correction objectives, the optimal result among the simulated constructs was identified.10 patient-specific multibody models of representative adolescent idiopathic scoliosis Lenke 1A cases were built and used to compare alternative virtual correction surgeries. Five screw patterns and IDs  were simulated, considering concave rod rotation, Low-density (ID ≤ 1.4) constructs provided equivalent 3D correction compared to higher (ID ≥ 1.8) densities (average differences ranging between 2° and 3°). The optimal screw pattern varied from case to case, falling within the low-density screw category in 14% of considered scenarios, 73% in the mid-density (1.4 < ID < 1.8) and 13% in the high-density. The optimal screw pattern was unique in five cases; multiple optima were found in other cases depending on the considered correction objectives.Low-density screw patterns provided equivalent intraoperative 3D correction to higher-density patterns. Simulated surgeon’s choice of correction objectives had the greatest impact on the selection of the optimal construct for 3D correction, while screw density and ID had a limited impact.N/A. The treatment of adolescent idiopathic scoliosis (AIS) through posterior instrumented fusion has significantly evolved in the last decades. Since the introduction of pedicle screws, correction techniques have continuously evolved, allowing powerful control on 3D spine shape through a variable number of anchoring points . Screw p3D correction objectives are defined as the choice to target one or more specific descriptors of spinal deformity on different anatomical planes  using specific maneuvers. Surgeon preference regarding correction objectives are believed to be responsible for the high variability among surgeons and may affect the final outcome , 19, 20.en bloc derotation , 2820, 2Assumptions in the modeling may set some limitations on the results of this study. To limit the number of tested independent variables, the same number of fused levels, fixed screw patterns/ID and correction techniques were simulated for all cases. While, in reality, surgeons may adjust the surgical technique depending on the available anchoring points, curve flexibility and span a wider range of possibilities , 11, 26.The objective function here considered was limited to one key metric per plane describing the intraoperative condition, but it may be further expanded to include other spine descriptor, as well as other non-geometrical parameters , which have already been reported in dedicated studies , 25, 26.To conclude, this biomechanical comparative study demonstrates that screw patterns with low (ID ≤ 1.4 screws/level) and high (ID ≥ 1.8) implant densities provide equivalent intraoperative correction in the three anatomical planes in a representative cohort of ten thoracic AIS curves. Moreover, the identification of the optimal screw pattern for every curve depends on surgeons’ preference regarding specific correction objectives.The proposed patient-specific approach represents a promising tool to assess and optimize the surgery planning of complex spinal deformities including surgeon-dependent correction objectives.


Aspergillus nidulans global phosphoproteome during mycelial growth and show that under these conditions, H2O2 induces major changes in protein phosphorylation. Among the 1964 phosphoproteins we identified, H2O2 induced the phosphorylation of 131 proteins at one or more sites as well as the dephosphorylation of a larger set of proteins. A detailed analysis of these phosphoproteins shows that H2O2 affected the phosphorylation of critical regulatory nodes of phosphoinositide, MAPK, and TOR signaling as well as the phosphorylation of multiple proteins involved in the regulation of gene expression, primary and secondary metabolism, and development. Our results provide a novel and extensive protein phosphorylation landscape in A. nidulans, indicating that H2O2 induces a shift in general metabolism from anabolic to catabolic, and the activation of multiple stress survival pathways. Our results expand the significance of H2O2 in eukaryotic cell signaling.Reactive oxygen species (ROS) regulate several aspects of cell physiology in filamentous fungi including the antioxidant response and development. However, little is known about the signaling pathways involved in these processes. Here, we report  Among these, superoxide is produced during respiration and by dedicated enzymes like NADPH oxidases [2O2 is generated by spontaneous or superoxide oxidase-mediated dismutation of superoxide. H2O2, considered as the most important ROS in redox biology [2O2 concentrations [Aerobic organisms generate reactive oxygen species (ROS) during normal metabolism. ROS are Ooxidases , while ctrations . For mantrations . Howevertrations ,6, planttrations  and animtrations  signal ttrations ,10.Neurospora crassa, the differentiation of asexual spores involves three morphogenetic transitions, each preceded by an hyperoxidant state that is characterized by the increased production of ROS [N. crassa [A. nidulans [Podospora anserina [Sordaria macrospora [A. nidulans, ROS also regulate important cellular processes such as mitochondrial division [The importance of ROS in cell differentiation has been documented in several species of filamentous fungi. In n of ROS , the pron of ROS ,13,14,15. crassa ,17, A. nnidulans , Podospoanserina , Sordaricrospora , and othdivision ,22 and tdivision ,26,27,282O2 are mediated by the oxidation of critical cysteine or methionine residues in specific proteins [2O2 can impact protein phosphorylation [2O2 can also regulate protein phosphorylation. This is the case or Aurora kinase A, in which the oxidation of a cysteine (C290) prevents the essential phosphorylation of adjacent T288, in its activation segment. This cysteine, conserved in a group of human CAMK, AGC, and AGC-like kinases, is also important to regulate the activity of fission yeast Srk1 and Pka1 kinases [Many of the direct biological effects of Hproteins . Indeed,rylation ,31. In a kinases .A. nidulans and other fungi, H2O2 induces the phosphorylation and nuclear localization of the Hog1/p38 orthologous stress-activated MAPKs SakA and MpkC [2O2 and both proteins are important for proper catalase catB gene induction [2O2 also induces the interaction of SakA and MpkC with several proteins including the SrkA kinase. In response to H2O2, SrkA is translocated to the nucleus in a SakA-dependent process [2O2 resistance, H2O2-mediated induction of catB and other genes, and for proper development. NapA also regulates the expression of multiple genes during asexual development and regulates carbon utilization [2O2 activates the SakA pathway, which is highly conserved and critical for stress survival in eukaryotes, shows the importance of H2O2 in cell signaling. However, the general knowledge about the pathways involved in ROS perception and ROS signal transduction in filamentous fungi is still very limited.In and MpkC ,25, and and MpkC . Both Sanduction . SakA an process . In addi process  and NapA process ,27 are nlization ,27. The A. nidulans, global phosphoproteomic analysis has been used to study MpkA functions in cell wall stress [Phosphorylation is the most ubiquitous posttranslational modification found in proteins , and by l stress , PKA-depl stress , and genl stress .2O2 signaling in A. nidulans. At the same time, we present an extensive review of the signal transduction and metabolic pathways that were affected in response to H2O2. We found that H2O2 has major effects on the phosphorylation of phosphoinositide, MAPK, TOR, and other kinase signaling networks that control growth, stress responses, and development. Moreover, the effects of H2O2 in critical metabolic enzymes indicate that a major change in metabolism, from anabolic to catabolic, is induced by H2O2. Our results provide a set of specific predictions on how ROS affect specific signal transduction pathways that can be experimentally tested.We carried out a global phosphoproteomic analysis to identify the proteins and pathways potentially involved in HAspergillus nidulans strain CLK43 (pabaA1 yA2 veA1) was used as the wild type strain to compare with previous results [2O2 effects on protein phosphorylation, liquid cultures were treated as reported [A. nidulans mycelia was grown in liquid culture for 12 h and then treated or not with 10 mM H2O2 during 10 min. After this, triplicate samples from each condition were frozen with liquid nitrogen and processed for phosphoproteomic analysis. results . It was  results . To detereported . Brieflyw/v) DTT. The final pellet was washed twice with cold acetone, followed by a final wash with 80% cold acetone. Precipitated proteins were solubilized in 50 mM Tris-HCl buffer pH 8 with 6 M urea. Protein concentration was determined using the Bradford Protein Assay  and a nano spectrophotometer .Frozen mycelia was ground to a fine powder in a precooled mortar using liquid nitrogen. The resulting powder was resuspended in 50 mM Tris-HCl buffer pH 7.4 containing 6 M urea, 5 mM DTT, and protease inhibitor  and phosphatase inhibitor  cocktails. Samples were kept on ice in 50 mL conic tubes and homogenized for 5 min with a Tissue Tearor homogenizer . Subsequently, the extracts were transferred to a Branson 2200 sonicator  and sonicated for 5 min. The protein extract was clarified by centrifugation at 4000 rpm for 20 min and 4 °C. Soluble proteins were precipitated as reported [2 Phosphopeptide Enrichment Kit , following the manufacturer’s instructions.An aliquot of 3 mg of protein from each sample was reduced with 10 mM DTT at 37 °C for 1 h. Subsequently, iodoacetamide was added to a final concentration of 15 mM and samples were incubated at room temperature for 30 min in the dark. The samples were diluted with three volumes of 50 mM Tris-HCl buffer pH 8. Trypsin  was added in a ratio of 1:60 (trypsin: protein) and samples were incubated at 37 °C for 18 h. The resulting peptides were desalted by solid phase extraction with HyperSep SPE columns . Phosphopeptides were enriched using the High-Select™ TiOLC–MS/MS analysis was carried at the Institut de Recherches Cliniques de Montreal (IRCM), Canada. Prior to LC-MS/MS, protein digests were re-solubilized in 1% acetonitrile/1% formic acid and loaded onto a Self-Pack C18 column installed in the Easy-nLC 1200 system . The buffers used for chromatography were 0.2% formic acid (buffer A) and 90% acetonitrile/0.2% formic acid (buffer B). Peptides were eluted with a two-slope gradient at a flow rate of 250 nL/min. The LC system was coupled to a Orbitrap Fusion Tribrid™ mass spectrometer  through a Nanospray Flex Ion Source. Peptide ions were fragmented in the HCD collision cell and analyzed in the linear ion trap with a target value of 1e4 and a normalized collision energy of 29 V. MS3 scanning was performed upon detection of a neutral loss of phosphoric acid  in MS2 scans. Target ions selected for fragmentation were dynamically excluded for 30 s after two MS2 events.Aspergillus nidulans protein database (version 4 November 2019). The peak list files were generated with Proteome Discoverer (v2.3) using the following parameters: minimum mass set to 500 Da and maximum mass set to 6000 Da. Mascot was searched with a fragment ion mass tolerance of 0.60 Da and a parent ion tolerance of 10.0 PPM. Cysteine carbamidomethylation was specified in Mascot as a fixed modification. Methionine oxidation and serine, threonine, and tyrosine phosphorylation were specified as variable modifications.Protein database searching was performed with Mascot 2.5  against the UniProt Scaffold  was used to validate MS/MS based peptide and protein identifications. The search was performed against a decoy database. Peptide identifications were accepted if they could be established at greater than 90.0% probability by the Peptide Prophet algorithm . ProteinFunctional enrichment analysis was performed using NeVOmics, bioinformatic tool based in Python with R packages . STRING 2O2, we grew A. nidulans mycelia in liquid culture for 12 h, which corresponds to a phase of active hyphal growth [2O2 for 10 more min. Triplicate biological samples from each condition were processed for phosphoproteomic analysis. These conditions were based on previous results showing that this H2O2 treatment induces the phosphorylation of the stress MAPK SakA [catB gene expression and catalase B activity [2O2 concentration used in these experiments was relatively high. However, the actual H2O2 intracellular concentration must be much lower, considering that H2O2 is efficiently decomposed by the activity of the catalases [2O2 concentration has been estimated in blood plasma [2O2  are involved in the response to other types of physiological stresses such as nutrient starvation.To determine phosphoproteomic changes in response to Hl growth , and theAPK SakA ,25 and iAPK SakA , and othAPK SakA  as well activity ,27,28,33activity , the H2Oatalases ,50, peroatalases ,27 expreatalases . As a red plasma ,53. More2O2 identified a total of 1964 proteins phosphorylated at one or more sites , which correspond to the regulation of biological processes, protein transport, and organelle organization [We carried out a Gene Ontology  and KEGGnization . RegardiA. nidulans phosphoproteome corresponds to proteins involved in cell signal transduction.Overall, these results indicate that under growing conditions, a large proportion of 2O2 induces phosphorylation changes in specific signal transduction pathways. MAP kinases are regulated by phosphorylation at both the threonine and tyrosine of the conserved TXY motif within the activation loop [2O2 treatment induce phosphorylation of the MAPK SakA TGY activation loop at T and Y residues [2O2, it became phosphorylated at the T171 and Y173 residues, and a novel phosphosite was detected at T176 in the presence of H2O2 , pheromone (Fus3), and cell-wall integrity (Slt1) MAPK pathways and identified their phosphorylation changes in response to H2O2  might be responsible for SakA and MpkC T176 phosphorylation. Consistent with this, CaMKII is required for H2O2-induced phosphorylation of p38 in endothelial cells [2O2-specific phosphorylation, suggesting that they might be involved in H2O2 sensing and be potentially linked to the SakA/MpkC pathway  promotes its nuclear exclusion, resulting in cell cycle arrest [2O2-specific. Srk1 T463 corresponds to SakA T413, which NetPhos identifies as a putative substrate of SakA orthologous kinase p38. This supports the role of SakA in SrkA phosphorylation in response to H2O2 and suggests that SrkA integrates inputs from additional H2O2-sensitive phosphorylation pathways. A single H2O2-specific phosphosite was detected in phosphatase NimT, the ortholog of S. pombe Cdc25, while 14-3-3 protein AN5744 single phosphosite S211 was dephosphorylated in H2O2. By analogy with S. pombe osmotic response, we propose that A. nidulans H2O2 response involves the sequential phosphorylation of SakA, SrkA, and NimT  [2O2, and Networkin predicts Ste20 and Pkc1 as the kinases responsible for S137 and S138 phosphorylation, respectively. Notably, previous studies in yeast have shown that Ste20 kinase phosphorylates HTB2 S10 during H2O2-induced apoptosis. Moreover, phosphonull HTB2 S10A mutants are resistant to H2O2-induced apoptosis, while phosphomimetic HTB2 S10E mutants induce apoptosis [2O2 as well as its possible regulation by kinases SakA, SrkA, Ste20, and PKC.SakA and SrkA physically interact with other proteins including histone H2B  . We detepoptosis . These rA. nidulans, interacts with SakA and SrkA in response to H2O2 [2O2-specific. According to NetPhos, FabM T368 would be phosphorylated by p38, supporting a role for SakA and/or MpkC in FabM phosphorylation. In addition to FabM, the phosphorylation of other putative mRNA-binding proteins was affected by H2O2 (2O2. Pbp1 interacts with FabM homolog Pab1 to regulate poly(A) tail synthesis [Ashbya gossypii Whi3 regulates cell cycle and cell polarity by forming condensate-like structures, and the phosphorylation of individual residues provides specificity to different condensates [2O2, AN7700 was found dephosphorylated at two sites and phosphorylated at T216. These results suggest that by regulating the phosphorylation of these RNA-binding proteins through SakA/MpkC and/or other kinase pathways, H2O2 might regulate the formation of stress granules and other ribonucleoprotein condensates involved in growth and stress responses. In addition, H2O2 can regulate liquid–liquid phase separation by mediating the reversible oxidation of proteins like Pbp1, as it occurs in S. cerevisiae [The poly-A mRNA binding protein FabM, involved in asexual development  and stre to H2O2 . Three p by H2O2 , suggestynthesis , and alsynthesis , is simidensates . In H2O2revisiae .2O2 [S. pombe SakA homolog Sty1/Spc1 phosphorylates AtfA homolog Atf1 at the serine in PLSP (S172) motif, and this regulates its stability and function [2O2 treatment (T159) and two (S132 and S136) being H2O2-specific  shows constitutive physical interaction with SakA [S. pombe phosphatase pyp1, involved in the negative regulation of the stress MAPK Sty1/Spc1 [2O2 treatment  kinase activity and mitotic exit [2O2 and one (S151) H2O2-specific. PsrA is required for conidia germination on glucose media [S. cerevisiae PsrA homolog Psr2 inhibits TORC1 signaling through its interaction with Whi2 (see TOR section). In H2O2, putative alkaline phosphatase Pho8/AN10563 was dephosphorylated at S150 and phosphorylated on S14. S. cerevisiae Pho8 is involved in the regulation of autophagy [2O2 not only affected DipA phosphorylation, but also the phosphorylation of its substrate DenA . These results suggest that H2O2 regulates both proteolysis and peroxisome dynamics by regulating the activation of DipA. Notably, H2O2 also differentially affected the phosphorylation of both PP2A (protein phosphatase 2A) complex regulatory subunits [The phosphatases responsible for SakA dephosphorylation are not known in ith SakA  and is tty1/Spc1 . We detereatment . This sus lethal . In addiphatases . Five phf stress . Ten photic exit . Phosphase media . S. cereutophagy . Phosphautophagy . Moreoveutophagy . H2O2 nosubunits . The PP22O2 activates the SakA/MpkC pathway and support its role in the phosphorylation of AtfA, SrkA, NimT, FabM, and histone H2B. They also suggest a connection between this pathway and CAM-II kinases CmkA and CmkC in H2O2 sensing. Moreover, H2O2 also regulates the phosphorylation of several mRNA-binding proteins and phosphatases, potentially related to this and other MAPK pathways.In summary, our results show that HA. nidulans regulates not only development, but also secondary metabolism and has been involved in cell wall and oxidative stress responses [A. nidulans contains G-alpha subunits FadA, GanA, and GanB. FadA and GanB signaling is mediated by the cAMP/PKA pathway to regulate development and secondary metabolism [2O2  and Y184 (with and without H2O2). This indicates that MpkB is activated by H2O2, as phosphorylation of both residues in the TEY activation loop is required for activity in all members of this MAPK family [The pheromone pathway in esponses . It is cesponses , all of esponses ,83. G-presponses . A. nidutabolism ,86 and ctabolism , while Gism [2O2 . As showunctions . We confK family .A. nidulans STRIPAK complex, composed of StrA, SipA, SipB, SipC, SipD, SipE, and SipF subunits, is required for MpkB phosphorylation and regulates resistance to oxidative stress, development, and secondary metabolism [2O2-specific phosphosites  complex regulates many cellular processes including signal transduction and development . A. nidutabolism . We detephosites . Notablyory role . This suA. nidulans and other fungi [A. flavus, VosA [2O2-specific phosphosites in VeA (S374) and VosA (T247) and two invariable phosphosites in LaeA (S39 and S70) and SteA (S550 and T552). In addition, several enzymes directly involved in the biosynthesis of secondary metabolites such as fatty acid synthases (FAS) and polyketide synthases (PKS) were detected as phosphoproteins  in ene brlA ,105 and and stuA ,106,107.nd dopey  are requoprotein .A. nidulans as it is required for light-mediated inhibition of sexual development and for mycotoxin production in a VeA-dependent fashion. ImeB contains a TXY motif (TTY) of the type found in MAP kinases, and the substitution of any of these three amino acids produced inactive versions of this kinase [Ustilago maydis [2O2. Notably, we found all three TTY residues phosphorylated; H2O2 induced the dephosphorylation of T211 and the phosphorylation of T210, while Y212 phosphorylation was not affected by H2O2. This suggests that H2O2 regulates sexual development and secondary metabolism by activating MpkB and by modulating ImeB activity, possibly through the differential phosphorylation of its TTY motif.The ImeB kinase, conserved form fungi to animals, is related to MpkB function in s kinase . Moreoveo maydis . We deteA. nidulans, MpkA is the MAPK generally associated with cell wall maintenance and repair pathways, which are similar in yeast [A. nidulans [The fungal cell wall, composed mainly of chitin and glucans, needs to be remodeled during polar growth, cell differentiation, and in response to environmental stress. Although SakA, MpkC , and Mpkin yeast  and A. nnidulans . In thesnidulans . Rho1 isnidulans  for a renidulans . As discnidulans .2O2 P2 producing kinase Mss4 [2O2. All these phosphosites (amino acids S65–S284) are concentrated in the N-terminal portion of the protein including N-terminal and kinase domains. Part of the N. crassa MSS-4 N-terminal domain is critical for its correct membrane association [S. cerevisiae Mss4 hyperphosphorylation results in increased plasma membrane association and a higher production of PtdInsP2 [2O2-induced phosphorylation changes in thee A. nidulans Sst4 and Mss4 homologs result in the activation of the phosphoinositide signaling pathway, leading to the activation of both the RhoA-MpkA and TORC2 signaling pathways (see phosphoinositide/TOR section). Along this line, the phosphorylation of A. nidulans phosphoinositide-dependent kinase PkhA  was also heavily affected by H2O2, which induced the dephosphorylation of eight residues and the phosphorylation of T207 (S. cerevisiae Pkh2 (see TOR section), the second one being involved in Pkc1 phosphorylation and MAPK activation [2O2-specific. Fourteen phosphosites were detected in MAPKKK BckA; seven dephosphorylated and one phosphorylated in H2O2. Somewhat unexpectedly, MkkA six phosphosites were found to be dephosphorylated in H2O2. Nevertheless, the MAPK MpkA was detected active (phosphorylated at T100 and Y102) with and without H2O2. Notably, we detected additional phosphosites Y98 and T105, which in H2O2 were dephosphorylated and phosphorylated, respectively. A recent report shows that yeast MpkA homolog Slt2 contains two phosphosites outside the TEY motif, one of which (T195) is also critical for the activity of this MAPK [2O2-induced phosphorylation of MpkA at T105 contributes to its activation. Downstream of MpkA, the transcription factor RlmA contained two invariant and one (T315) H2O2-specific phosphosites.As shown in 66 MssA, , the ortase Mss4 , containociation , while SsP2 . Therefo of T207 , Table 3tivation , and alshis MAPK . As MpkA2O2 activates phosphoinositide signaling (discussed in more detail in the next section) with the consequent activation of RhoA and the downstream CWI pathway to regulate cell wall polymer biosynthesis, actin organization, exocytosis, and the cell cycle.In summary, our results are consistent with a model in which HA. nidulans, Cdc42 homologue ModA plays a major role in polarity establishment [S. cerevisiae, the active GTP-bound conformation of Cdc42 binds to and activates PAK kinases Cla4 and Ste20 [A. fumigatus [Cdc42 GTPases are conserved regulators of cell polarity, cell cycle, and morphogenesis in eukaryotic cells ,123,124.lishment . In S. cnd Ste20 ,123, andnd Ste20 ,126. In umigatus . Other cumigatus . Bud3, aumigatus . Bem2 anumigatus . Bem3 coumigatus .2O2 , while MsbA single phosphosite was dephosphorylated in H2O2. Two H2O2-specific phosphosites  were detected in Cla4 and, notably, 17 phosphosites were detected in Cdc24 (AN5592); three dephosphorylated and three phosphorylated in H2O2. Eighteen phosphosites were found in Bud3 (AN0113); six dephosphorylated and six phosphorylated in H2O2. Twelve phosphosites were detected in Bem1 scaffold protein homolog BemA; four absent in H2O2 and one H2O2-specific. These results suggest that ShoA, MsbA, and ModA integrate different environmental inputs including ROS signals to regulate actin and septin cytoskeletons and polarized secretion via their downstream effectors including PAKs Cla4 and Ste20, MpkB, and possibly SakA MAPKs.While we did not detect Cdc42 (ModA) as a phosphoprotein, the phosphorylation pattern of critical regulators of its activity was notably affected by H2O2 , Table 4S. cerevisiae by Mss4 phosphatidylinositol 4-phosphate 5-kinase or by Tep1, a homologue of mammalian PTEN phosphatase [S. pombe results in the accumulation of its substrate PI P3 [2+ release from intracellular stores [2+ signaling.The phosphoinositide family (PIs) includes seven types of phosphorylated phosphatidylinositol, localized in different organelle membranes, which function as lipid signaling molecules by recruiting and/or activating diverse effector proteins. Although PI(5)P and PIP2 have not been detected in fungi, PIP2 has been involved in the regulation of the actin cytoskeleton, endocytosis, and Rho-Pkc1 signaling cascade in yeast . The levsphatase . By cata3,4,5)P3 . The hydr stores , therefo2O2 induced major changes in the phosphorylation of phosphoinositide kinases Stt4 and Mss4  and D , and phosphatidylinositol phosphatases AN7745, AN8053, and AN0925 P3 levels [As indicated previously (RhoA and MpkA section), our results show that Hand Mss4 , Table 5 by H2O2 ,137. In  AN10780 . This su3 levels .2O2 affected the phosphorylation of multiple proteins containing FYVE P2. The MCC/eisosome is considered as a membrane tension sensor complex and has also been involved in morphogenesis, cell wall integrity, and environmental stress resistance (see [2O2. PilA contained a unique H2O2-specific phosphosite (S60), while the Nce102 single phosphosite (S170) was dephosphorylated in H2O2. Pil1 phosphorylation has been associated with eisosome stability. However, is not clear yet whether such phosphorylation increases or decreases eisosome stability under different growth conditions [Candida albicans [In addition to regulating the MpkA MAPK pathway, phosphoinositides are critical to recruit and/or activate diverse effector proteins, mainly through FYVE, PH, or PX domains including PAK kinase Cla4, Rho1 GEF Rom2, Cdc42 GAP Bem3 , and TORing FYVE , PH  is a conserved kinase of the phosphoinositide 3-kinase (PI3K)-related kinase family that actually functions as a serine/threonine kinase. It plays a crucial role in the integration of diverse environmental cues such as nutritional status and stress to regulate cell growth in eukaryotes. The TOR kinase forms multi-subunit TOR complex 1 (TORC1) and TOR complex 2 (TORC2), which show different substrate specificities. These complexes control kinases of the AGC family, which play essential roles in growth regulation. However, for any activity at all, these AGC kinases must be phosphorylated on a threonine conserved in their activation loop (T-loop) by the 3-phosphoinositide dependent kinase PDK1 . PDK1, iactivity . In contmodeling .2O2 affected the phosphorylation patterns of components of both TOR complexes as well as the phosphorylation of some downstream AGC kinases  and its phosphorylation at S985. AN4639 is the ortholog of S. cerevisiae TORC1-specific subunit Kog1 (RAPTOR in humans). Likewise, H2O2 induced the dephosphorylation of Lst8 ortholog AN1335 at S267 and its phosphorylation at S273. S. cerevisiae Lst8 associates with both TORC1 and TORC2 complexes. Consistent with this, mutation of LST8 produces effects similar to the ones produced by rapamycin, which inhibits TORC1 but not TORC2 as well as cell wall defects mediated by TORC2 [S. cerevisiae Sch9 AGC kinase is phosphorylated by Tor1 under nitrogen rich conditions and required for TORC-mediated regulation of ribosome biogenesis and entry into G0 phase. A. nidulans Sch9 ortholog SchA plays redundant functions with protein kinase A (PkaA) during asexual spore germination and participates in carbon catabolite repression [A. fumigatus, schA gene expression is dependent on SakA MAPK and ΔschA mutants show increased SakA phosphorylation, suggesting a link between TORC1 and the SakA pathway [2O2.We found that H kinases , Table 9by TORC2 . Downstrpression . In A. f pathway . Of six S. cerevisiae both, TORC1 inactivation and phosphatase Nem1 activation result in dephosphorylation and activation of the lipin phosphatidate phosphatase Pah1, which is necessary for autophagy  was found phosphorylated at S53 and dephosphorylated a two phosphosites in H2O2, suggesting that PkaR might play a role in Maf1 phosphorylation and the oxidative stress response. In agreement with this, conidia from pkaR null mutants are sensitive to H2O2 in A. fumigatus [S. cerevisiae, we detected that Maf1 ortholog AN7681 was highly phosphorylated (15 phosphosites) during growth (in the absence of H2O2) and dephosphorylated at six residues in the presence of H2O2. Phosphatase A7750, a potential candidate to dephosphorylate Maf1 ortholog AN7681, was detected with ten total phosphosites, from which two were dephosphorylated in H2O2 and one was H2O2-specific . In contrast, Rts1 ortholog ParA (AN9467) was dephosphorylated at three phosphosites and phosphorylated on only one site (S147) in H2O2  and Pkc1 [S. cerevisiae TORC2 complex consists of specific subunits Tsc11 , Avo1 (SIN1 ortholog), and TOR common subunit Lst8. We detected three phosphosites in the Tsc11 ortholog AN10756, one of them phosphorylated (Y108) only in H2O2.The TORC2 complex plays a critical role for the survival of human cells to H3 levels . RICTOR 3 levels ,158. In and Pkc1 . The S. A. nidulans Avo1 (AN6304), and Ypk1  orthologs were all dephosphorylated in the presence of H2O2  by PDK kinases Pkh1/2 is essential for its activity ,160. Our of H2O2 . NotablyA. nidulans PkhA  and Ypk1 (PkcB) orthologs are both essential kinases. The deletion of pkhA and pkcB results in cells that initiate irregular growth before arresting as microcolonies, consistent with the idea that PkcB is regulated by PkhA [2O2 and one (S207) H2O2-specific. Since PkcB essential T-loop phosphorylation was lost in H2O2, these results suggest that H2O2 induces a transient inactivation of both PhkA and PkcB. This is consistent with the fact that FpkA, another potential PkcB substrate [2O2. Moreover, flipase DnfA, another potential FpkA substrate, was also dephosphorylated at two sites (S78 and T258) and phosphorylated at one (S50) in H2O2. Both kinases had additional phosphosites, which did not change in the presence of H2O2  in H2O2 and seven  H2O2-specific. Adenylate cyclase CyaA [2O2. The phosphodiesterase PdeA (AN0829) had a single H2O2-specific phosphosite (S325) and notably, putative kinase AN7572, the ortholog of S. cerevisiae Rim15 [2O2.Although the cAMP-PKA pathway is often related to glucose sensing, the starvation for other essential nutrients such as nitrogen results in the downregulation of PKA targets . This mimponents . The phoase CyaA  containeae Rim15 , containS. cerevisiae, TOR1C inhibition with rapamycin induces a global pattern of gene expression similar to those induced by nitrogen starvation [2O2 and the activation of amino acid catabolism, gluconeogenesis, and the pentose phosphate pathway. These responses are consistent with the inactivation of TORC1, the activation of TORC2, and a downregulation of the cAMP-PKA pathways by H2O2.In summary, TORC1 promotes anabolic processes including protein synthesis, glucose metabolism, and nucleotide and lipid biosynthesis. In arvation . As disc2O2. Previous work has shown that oxidative stress can affect nuclear transport [2O2. Protein AN3910 had the higher phosphosite number in this category, with 20 total phosphosites; 12 dephosphorylated in H2O2, one present only in H2O2, and seven that remained unchanged. AN3910 orthologs in S. pombe and S. cerevisiae are nuclear inner membrane proteins related to subtelomeric heterochromatin organization. Twelve phosphosites were detected in A. nidulans Nup159 (AN2086) with three unique to H2O2-treated samples. Nup159 is a nuclear pore protein similar to yeast Nup159 and human protein Nup214 [2O2-specific. Interestingly, it has been reported that oxidative stress induces the phosphorylation of human Nup159 and SonB homologs, affecting the exportation of nuclear proteins [The phosphorylation of many proteins involved in transcriptional regulation including proteins involved in nuclear-cytoplasmic transport  and chroransport ,167,168.ransport . Using Gn Nup214 . In addin Nup214 , one of proteins .2O2 induced phosphorylation changes in many other transcription factors (2O2-specific phosphorylation of Hsf1 (T449) and Nmy1 (S304)   at motifA. nidulans and other fungi. In A. nidulans, Yap1 homolog NapA is required for the induction of catalase CatB in response to H2O2 and for resistance to H2O2 and other types of oxidative stress as well as for the regulation of multiple genes during asexual spore formation [2O2 [2O2 sensing by S. cerevisiae Yap1 and its homologs involves the oxidation of specific cysteines, which results in its nuclear accumulation and transcriptional activation. However, the phosphorylation of oxidized nuclear Yap1 has also been reported [C. albicans, a correlation between the phosphorylation of oxidized Cap1 and Cap1-dependent gene expression has been reported [2O2 and one (T316) H2O2-specific, predicted as a GSK3 substrate.We examined the phosphorylation of YapA and SrrA , as thesormation ,27. Likeion [2O2 . H2O2 sereported , and in reported . NeverthS. cerevisiae response regulator Skn7. Like Skn7 [2O2-specific fashion, potentially by kinase Cdk5. The phosphorylation changes of other transcription factors involved in nitrogen metabolism are discussed in SrrA is the homolog of ike Skn7 , SrrA isike Skn7 . In contike Skn7 . Althougike Skn7 . In this2O2 are AN4894, AN2771, AN5048, AN0091, AN5836, and AN3688  and N. crassa (S477) and is phosphorylated (S439) in S. pombe [2O2-specific phosphosite (S331) was also detected in AN5048. AN5048 is similar to Yox1, which interacts with Mcm1 and ECB proteins to regulate cell cycle in S. cerevisiae. Yox1 is also a substrate of the NimX orthologous kinase Cdc28, which we found phosphorylated at two sites, one of them (T14) detected only in H2O2. AN2771, a putative transcription factor similar to S. cerevisiae RBA50 was phosphorylated at S143 only in the presence of H2O2, possibly by SakA/MpkC, according to NetPhos  was phosphorylated at T247, while the only two phosphosites detected in putative transporter CrnA (AN1008) were dephosphorylated in H2O2. H2O2-specific phosphosites were also detected in nitrogen-related transcription factors AN1927 (S174), AN4489 (S12), and UaY (S922). AN1927 is regulated by nitrate limitation [S. pombe [S. cerevisiae DAL81, a positive regulator of genes involved in multiple nitrogen degradation pathways [2O2-induced phosphorylation of these transcription factors results in their activation and the consequent induction of amino acid and purine degradation pathways.Under our experimental conditions, nitrate has to be transformed to the preferred nitrogen sources of glutamate and glutamine through a pathway that consumes both energy and reducing power. The process involves the transport of nitrate to the cell by transporters NtrA/NtrB, its subsequent conversion to nitrite by nitrate reductase NiaD, and then to ammonia by nitrite reductase NiiA. Ammonia is then transformed to glutamate and glutamine by NADP-glutamate dehydrogenase GdhA and glutamine synthase (GS), respectively. The transcriptional regulation of the nitrate assimilatory pathway is carried out by the nitrate-specific activator NirA and by AreA . As show cytosol . This opmitation  and its S. pombe . AN4489 pathways . UaY is pathways . We propgdhA gene is a highly regulated process. It requires AreA [2O2-specific phosphosites. It is unknown if such H2O2-induced phosphorylation results in the activation or inactivation of these transcription factors. However, it should be considered that NADP-glutamate dehydrogenase is itself irreversible, inactivated by ROS [2O2-specific phosphosite, and this residue is conserved in fungal and human enzymes. In H2O2, nitrate reductase NiaD (AN1006) N-terminus was dephosphorylated, while putative nitrite reductase NiiA (AN1007) acquired three phosphosites. A consistent hypothesis is that high H2O2 levels result in the inactivation of the nitrogen assimilation pathway at both transcriptional and posttranslational levels, perhaps as a transitory mechanism to prevent the futile consumption of energy and reducing power in non-growing conditions, under which amino acid and fatty acid catabolism would be favored. The fact that NAD-glutamate synthase (GOGAT) and catabolic NAD-glutamate dehydrogenase GdhB [2O2  , another S phase . Dia2 is S phase . By anal2O2. S. cerevisiae ortholog Otu1, involved in ER-associated degradation of substrates poly-ubiquitinated by Hrd1, has been detected as phosphorylated at S134 [AN3449, a deubiquitinase of the OTU family, was phosphorylated  only in H at S134 . Althoug at S134 .A. nidulans. Its stability depends on its phosphorylation status and its localization in either the nucleus or cytoplasm. DenA phosphorylation at S243 and S245 stabilizes the protein during growth and initial asexual development. Later in development, DenA phosphorylation at S253 destabilizes the protein [2O2. As indicated before, phosphatase DipA, which controls this phosphorylation-dependent stability of DenA [2O2  was detected in AN6547, the ortholog of the S. cerevisiae Pre5 20S proteasomal subunit. The phosphorylation of different proteosome subunits has been shown to result in increased activity [The SumO protease UlpB is required for the production of sexual spores, resistance to DNA damaging compounds, and the production of the secondary metabolite sterigmatocystin . We deteactivity .2O2-specific phosphosites. AcrB, which interacts genetically with the ubiquitination pathway [2O2. CreD, containing arrestin domains and PY motifs, is the ortholog of S. cerevisiae Rod1 [2O2, we detected that two out of six CreD phosphosites were removed and two were present only in this condition.Two proteins involved in the regulation of carbon source utilization, AcrB (AN3597) and CreD (AN4170), contained H pathway , containiae Rod1 . Rod1 iniae Rod1 . In H2O22O2 would result in major metabolic changes including the accumulation of α-ketoglutarate and the induction of gluconeogenesis. α-ketoglutarate has direct  and indirect  antioxidant properties [2O2 [2O2 induced changes in the phosphorylation status of PPP enzymes (2O2-specific phosphosite (S418)  . This enrevisiae  and its revisiae . AN1015,2O2 S. cerevisiae Uga2 succinate semialdehyde dehydrogenase ortholog AN3829 was phosphorylated at T261 and T262, while alanine transaminase Alt1 ortholog AN1923 was phosphorylated on S72. In contrast, putative GABA transporter AN8990 was dephosphorylated at S23 and S531  shunt, a conserved metabolic route that bypasses two steps of the Krebs cycle and generates NAD(P)H, has been involved in stress response and ROS mitigation, as GABA accumulates under different stress conditions ,213,214.and S531 . These r2O2 induces phosphorylation changes in multiple regulatory proteins. However, we found that H2O2 also induced phosphorylation changes in proteins directly involved in ROS metabolism , and PrxA unique phosphosite (S30) was dephosphorylated in H2O2. TpxA and TpxB play minor functions in H2O2 detoxification and TpxA elimination results in decreased asexual sporulation [As shown before, Htabolism , as is tposition . CatC sh in rice . Peroxiraperones . We deterulation .A. nidulans NoxA and its regulatory subunit NoxR (AN6046) are essential for sexual development and partially required for asexual sporulation [2O2. In N. crassa, the NOX-1 catalytic subunit is localized in the vacuolar system and the plasma membrane, and NOX-1 and NoxR ortholog NOR-1 colocalize only in discrete vesicular structures [2O2 suggests that ROS produced in one cell compartment  might affect the production of ROS by NOX enzymes in a different cell compartment .On the other hand, NADPH oxidases (NOX) are enzymes that produce ROS in a regulated manner and its regulation by ROS is not known in fungi . A. nidurulation ,217. Noxrulation , which irulation ,220. We 2O2 in vitro [A. nidulans OXR1 , containing the conserved cysteine, had four phosphosites  clustered at the C-terminus of the TLDc domain, all of which were dephosphorylated in the presence of H2O2. S315 and T319 are conserved in the Aspergilli, suggesting that their dephosphorylation in H2O2 might be important for OxrA function. Three H2O2-specific phosphosites were detected in AN1100, a hypothetical 5-oxoprolinase potentially involved in glutathione biosynthesis.OXR1 (oxidant resistant) is a protein conserved in eukaryotes and whose depletion from fungi to humans results in increased sensitivity to oxidative stress . Althougin vitro . The TLDin vitro  and is t2O2 has profound regulatory effects in cell physiology by affecting signal transduction, nitrogen and carbon metabolism, and gene expression. H2O2 induced phosphorylation changes in several proteins involved in cell cycle arrest including NimT, Cdc42 regulators, PP2A phosphatase, SconB, HulE, and Aurora kinase, which indicates that this is an important component of the response to H2O2. We propose that a large part of H2O2-induced changes in MAPK and TOR signaling, and the crosstalk between these pathways is mediated by its effects on phosphoinositide metabolism. We have provided a set of specific predictions on how ROS affect specific pathways, which can be valuable in designing future experiments.Our results indicate that H


Fetal alcohol syndrome (FAS) is characterized by disrupted fetal brain development and postnatal cognitive impairment. The targets of alcohol are diverse, and it is not clear whether there are common underlying molecular mechanisms producing these disruptions. Prior work established that acute ethanol exposure causes a transient increase in tyrosine phosphorylation of multiple proteins in cultured embryonic cortical cells. In this study, we show that a similar tyrosine phosphorylation transient occurs in the fetal brain after maternal dosing with ethanol. Using phospho-specific antibodies and immunohistochemistry, we mapped regions of highest tyrosine phosphorylation in the fetal cerebral cortex and found that areas of dendritic and axonal growth showed elevated tyrosine phosphorylation 10 min after maternal ethanol exposure. These were also areas of Src expression and Src family kinase (SFK) activation loop phosphorylation (pY416) expression. Importantly, maternal pretreatment with the SFK inhibitor dasatinib completely prevents both the pY416 increase and the tyrosine phosphorylation response. The phosphorylation response was observed in the perisomatic region and neurites of immature migrating and differentiating primary neurons. Importantly, the initial phosphotyrosine transient (~ 30 min) targets both Src and Dab1, two critical elements in Reelin signaling, a pathway required for normal cortical development. This initial phosphorylation response is followed by sustained reduction in Ser3 phosphorylation of n-cofilin, a critical actin severing protein and an identified downstream effector of Reelin signaling. This biochemical disruption is associated with sustained reduction of F-actin content and disrupted Golgi apparatus morphology in developing cortical neurons. The finding outlines a model in which the initial activation of SFKs by ethanol has the potential to disrupt multiple developmentally important signaling systems for several hours after maternal exposure.The online version contains supplementary material available at 10.1007/s12035-021-02467-x. The CDC estimates that 0.2–1.5 per 1000 live births are children with FASD, a syndrome characterized by disrupted fetal brain development and postnatal intellectual disability (ID) –6. HowevThis study extends our recent finding that ethanol exposure leads to SFK activation, inappropriate tyrosine phosphorylation in cultured primary neurons . One conIn prior studies, we observed similarities between ethanol-exposed and Reelin signaling–deficient embryonic neurons including disrupted cellular orientation, primary dendrite numbers, and Golgi compaction , 16. BecIn this study, we show that a similar disruption of SFK-dependent phosphorylation occurs in the fetal brain after maternal ethanol exposure. The robust nature of the ethanol-induced tyrosine phosphorylation response allowed us to use immunohistochemistry (IHC) to map changes of phosphorylated tyrosine content in fetal brain sections. The areas of maximal change include the marginal zone (MZ) where Reelin-dependent dendritogenesis occurs and the intermediate zone (IZ) that contains the forming white matter tracts. These areas also correlate with areas of high Src protein expression and Src activation . We find that maternal ethanol exposure first causes aberrant Src activation and Dab1 phosphorylation followed by sustained n-cofilin dephosphorylation and Golgi apparatus disruption. This suggests that maternal ethanol exposure may significantly impair SFK and Reelin signaling in the fetal brain.All animal procedures were performed in accordance with the Institutional Animal Care and Use Committee of SUNY Upstate Medical University. C57BL/6J mice (Jackson Laboratory) were used in this study. The day of plug discovery is considered embryonic day 0 (E0). Pregnant C57BL/6J were housed in a temperature-controlled room with a 12:12-h light-dark cycle. Intraperitoneal injections of pregnant dams were performed on E15 with either control phosphate-buffered saline (PBS) or PBS/ethanol to achieve 4 g ethanol/kg bodyweight, equivalent to 0.5% v/v. In some experiments, the Src family kinase inhibitor dasatinib (20 mg/kg bodyweight) was injected 30 min prior to ethanol or control injections. Animals were sacrificed 10, 30, 120, and 240 min after injection. Individual embryonic brains were rapidly isolated. Half of the brain was drop fixed in a 4% paraformaldehyde (PFA) PBS solution and then cryoprotected in 30% sucrose before cryostat sectioning for later histological analysis. The dorsal cortex of the other half brain was dissected for subsequent western blot analysis.Tail blood was collected in heparin coated tubes 10 min prior to the ethanol injections to establish a baseline, and then blood was taken at 10 min, 30 min, 1 h, 2 h, and 4 h after injection. Amniotic fluid was collected immediately after the injected dam had been sacrificed and the uterus exposed. A microsyringe was used to puncture the uterine wall and amniotic sac. A small volume of amniotic fluid was then withdrawn from the amniotic sac for subsequent analysis. The maternal blood and fetal amniotic fluid samples were then centrifuged at 2000 rpm for 15 min and stored at – 80 °C prior to analysis with an Analox Alcohol Analyzer .The 30 μm coronal cryosections were applied to glass slides and allowed to dry at room temperature overnight. Tissue sections were washed in phosphate-buffered saline (PBS) for 15 min and then blocked in 5% donkey serum, 1% BSA, and 0.2% Triton X-100 for 1 h at room temperature and incubated with primary antibody at 4 °C overnight. Mouse anti-pY99 , rabbit anti-MAP2 , rabbit anti-pY416 , rabbit anti-pan Src antibody , rabbit anti-Dcx  or H2O.Cortical tissue (E15) was dissociated using 0.25% trypsin at 37 °C for 20 min. A 10:10 buffer  was used to neutralize trypsin. Neurons were resuspended in Neurobasal medium supplemented with 2% B27, 1% GlutaMAX, and 1% penicillin/streptomycin  and plated onto PDL-coated glass coverslips in 24-well plates. After 3 day in vitro (DIV), cultures were exposed to ethanol  or PBS and then fixed for subsequent immunocytochemistry. For western blot analysis, cortical cultures were plated on PDL-coated 12-well tissue culture plates at a final concentration of 2 × 103VO4; 1 mM NaF) on DIV 3. All samples were loaded onto 10% SDS-PAGE gels and transferred to 0.45 μm PVDF Immobilon®-FL membranes (EMD Millipore). After incubation with Odyssey blocking buffer (LI-COR Biosciences) for 1 h at room temperature, the membranes were incubated with the following primary antibodies overnight at 4 °C. The primary antibodies were anti-pY99 , anti-pY416 , anti-pan Src antibody , anti-Dab1 , anti-p-Cofilin , anti-Cofilin  and anti-GAPDH  were used as loading controls. Appropriate secondary antibodies IRDye® 800CW and IRDye® 680RD (LI-COR Biosciences) were used, and membranes were scanned using the Odyssey® CLx system (LI-COR Biosciences).Both adult and E15 embryonic dorsal cortex and primary cortical neuron lysates were collected in radio immunoprecipitation assay (RIPA) buffer  with protease inhibitors  and phosphatase inhibitors (1 mM Na2/CO2) at 37 °C for 48 h before ethanol or control treatments. Explants from both control and ethanol groups were drop fixed in 4% PFA for subsequent histological analysis.Explant preparation and ex utero electroporation were performed on E13 embryos using 0.8 mg/ml of pCAG-GFP construct . Hemisph14Qlu/J transgenic mouse that expresses dsRed fluorescent protein from a Doublecortin (Dcx) promoter element 21] and c+ cells were infrequently detected in either condition, and no difference in positive cells was identified between conditions 4 h after exposure  levels were observed in fetal cortical lysates at 10 min after maternal exposure and persisted for at least 30 min before returning to baseline by 4 h Fig.  A and B.ure Fig. . This fiTo determine whether the in vivo fetal cortical response depended on SFK activation, we pretreated pregnant dams with dasatinib (BMS-354825). Dasatinib is an FDA-approved inhibitor of SFKs, with additional inhibition of BCR-ABL  28], an, an28], We used anti-phosphotyrosine (pY99) as well as anti-microtubule-associated protein 2 (MAP2) IHC to determine the spatial and temporal patterns of increased tyrosine phosphorylation in the developing cortex. Drop-fixed hemi-brains were cryosectioned and immunostained for subsequent confocal microscopy. Ethanol caused prominent pY99 signal increases in the apical area of the ventricular zone (VZ), where neural precursors are localized, the intermediate zone (IZ) that contains migrating neurons and developing axonal tracts, the subplate (SP) and marginal zone (MZ) that contain transient populations of neurons critical for early cortical patterning Fig. . ImportaIn our previous study, 10 min of ethanol exposure caused an increase in activation loop phosphorylation of Src but not the related kinase Fyn, indicating that Src activity is involved in the response . To dete+ cells are found at different stages of migration and development across the cerebral wall. PBS or ethanol (400 mg/dL) was added to the culture medium for 10 min, and explants were immediately drop fixed for subsequent histological analyses. Consistent with our findings from in utero exposed embryos, ethanol-treated explants showed significantly more pY99 signal in the VZ, IZ, and MZ compared to control-treated explants  or an equivalent volume of water. Consistent with our prior findings from primary cultures derived from SW embryos 37] and cIn this study, we provide evidence that maternal exposure to ethanol leads to rapid activation of Src family kinases and an elevation of tyrosine phosphorylation in multiple fetal cortical regions and cell types. Importantly, this brief phosphorylation increase is followed by an extended period of at least 2 h in which the Reelin signaling effector n-cofilin is dephosphorylated and F-actin content across the cerebral wall is reduced. In addition, we observed disrupted Golgi apparatus morphology that is similar to that observed with deficiency in Reelin signaling. These findings suggest that ethanol exposure may selectively impact SFK-dependent signaling pathways in the fetal cortex after maternal ethanol exposure.Our current findings both in vitro and in vivo outline a model in which transient SFK-activation by ethanol initiates a sequence of events that potential could impact signaling for extended periods of time. During the first phase of the ethanol response, Src is activated causing the tyrosine phosphorylation of its own activation loop, and Dab1, an adaptor protein essential for Reelin signaling. Our western blot analyses reveal that activation of SFKs by ethanol causes non-specific phosphorylation of multiple other protein species besides Dab1 at ~ 80 kD and Src itself at ~ 57 kD and this response is distinct from the relatively specific activation of Src and Dab1 by Reelin , 44. Altreeler cortical phenotype and profound layering and dendritic disruptions during early cortical development [SFKs are essential components of several developmentally important signaling pathways  46] [47 [4746] , mice sielopment , 49. ThiThe elevation of phosphotyrosine content by ethanol exposure is relatively brief (5–30 min) and is followed by a tyrosine dephosphorylation phase when total phosphotyrosine levels drop to baseline. During the latter period in vitro, application of Reelin protein could not initiate Reelin signaling, and no increase in Dab1 phosphorylation could be elicited . During Similarly, we observed disrupted Golgi morphology in the developing apical dendrite after maternal ethanol exposure which paralleled the morphological disruptions observed with Reelin-signaling disruptions , 42. As Disruption of SFK signaling may also impact neurite growth in other ways besides altering Reelin signaling  45] [46 4645] . Src has5 . Src hThe mechanism of ethanol-dependent Src activation is not clear: Ethanol activates a closely related SFK (Fyn) by inhibiting a regulatory STEP  , 69. Howhttps://gp3.mpg.de) shows that these phosphatases are expressed in the developing mouse cortex at E14.5, raising the possibility that their dysregulation may contribute to the later stage of the acute ethanol response. Alternatively, n-cofilin dephosphorylation may be due to the inhibition of kinases such as LIMK1 which phosphorylate n-cofilin at Ser3 [Altered phosphatase activity functioning downstream of Src may be involved in the n-cofilin Ser3 dephosphorylation that is sustained for hours after initial ethanol exposure. While phosphatases are generally thought to be constitutively active and not highly regulated, regulatory mechanisms have been identified . For exa at Ser3 . N-cofil at Ser3  and to m at Ser3 . UnderstEthanol exposure causes broad disruptions to the developing nervous system –6, and eSupplementary Figure. Time course of maternal blood ethanol concentrations (BEC) and fetal amniotic fluid concentrations (AEC) over 4 h following ethanol exposure at E15 (PNG 747 kb)High Resolution Image (TIF 277 kb)


These actions are associated with increased concentration of chloride ions in immature neurons [(Cl−)i] that determines the depolarizing strength of ion currents mediated by GABAA receptors, a ligand-gated Cl− permeable ion channel. During neuron maturation the (Cl−)i progressively decreases leading to weakening of GABA induced depolarization and enforcing GABA function as principal inhibitory neurotransmitter. A neuron restricted potassium-chloride co-transporter KCC2 is a key molecule governing Cl− extrusion and determining the resting level of (Cl−)i in developing and mature mammalian neurons. Among factors controlling the functioning of KCC2 and the maturation of inhibitory circuits, is Smoothened (Smo), the transducer in the receptor complex of the developmental protein Sonic Hedgehog (Shh). Too much or too little Shh-Smo action will have mirror effects on KCC2 stability at the neuron membrane, the GABA inhibitory strength, and ultimately on the newborn susceptibility to neurodevelopmental disorders. Both canonical and non-canonical Shh-Smo signal transduction pathways contribute to the regulation of KCC2 and GABAergic synaptic activity. In this review, we discuss the recent findings of the action of Shh-Smo signaling pathways on chloride ions homeostasis through the control of KCC2 membrane trafficking, and consequently on inhibitory neurotransmission and network activity during postnatal development.In the early stages of the central nervous system growth and development,  A ionotropic receptor channels (GABAAR). The inhibitory strength of GABAAR transmission is dependent on intracellular neuronal chloride concentration [(Cl−)i], that is relatively high (15–25 mM) in immature neurons and decreases progressively to 4–6 mM in mature neurons. This developmental change of (Cl−)i is determined primarily by activity of two cation-chloride cotransporters sodium-potassium-chloride cotransporter type 1 (NKCC1) and potassium-chloride cotransporter type 2 (KCC2)  synaptic transmission and chloride flux through GABAShh is a secreted glycoprotein preserved through the evolution and mostly known for its morphogenetic role during early phases of central nervous system development such as neural cell proliferation, neural progenitor cell fate, or neuronal differentiation . In the A) or repressor (Gli3R) forms of Glioma-associated (Gli) gene transcription factors  oscillations apparent at the neuronal primary cilium synchronous with Ca2+ waves in the neuronal soma. The Shh-induced increase in Ca2+ spike activity depends on both intracellular Ca2+ stores and extracellular Ca2+ influx through voltage dependent calcium channels and transient receptor potential 1 channel  that occur during the first postnatal weeks  and disaactivity . The subs effect . This reist SAG;  on theirist SAG; . The int channel . Moreove channel . These r channel . Other Sactivity . In thisactivity . Other sactivity . Althougactivity . In lung−)i, brought about by the increased contribution of the Cl− extruder KCC2 . On the other hand, an alteration of the Shh-Smo pathway has also been identified in TLE (−)i was not evaluated in the latter works, together above studies indicate the potential contribution of the Shh-Smo pathway to Cl− disruption in TLE patients.The postnatal maturation of GABAergic inhibitory transmission is linked to the developmental sequence of GABA shift from depolarizing to inhibitory actions which results from a developmental decrease in . Analysis of the phosphorylation state of Ser940 and Thr1007 residues of KCC2 revealed a strong and significant increase of Ser940 phosphorylation in neurons expressing Smo-CA, and significant decrease of Ser940 phosphorylation in neurons with Smo-ΔN  Activation of Smo downstream pathway with Smo-CA construct increases phosphorylation of KCC2 at Ser940 and consequently the stability of the co-transporter at the cell surface and its activity, thereby decreasing the intracellular chloride concentration and rendering GABA hyperpolarizing ; (2) Com as BDNF . As ment as BDNF . In line as BDNF  or down- as BDNF ; (3) A t as BDNF . Cholest as BDNF .In this review, we highlight the significant potential role of Shh-Smo signaling in chloride homeostasis through the control of KCC2 cell surface expression and activity, which in turn fine-tune the strength of inhibitory synaptic transmission. A weak or an absence of Shh-Smo signaling delayed the GABA polarity shift, leading to an unbalanced excitation to inhibition ratio. A similar role of Shh-smo signaling has been shown on astrocytes in mature cortex . The conThese open new research avenues on the interplay between Shh and GABA in neurodevelopmental disorders like Autism Spectrum Disorders (ASD), Down Syndrome and, epilepsy . As promBOX 1: Phosphorylation-dependent control of KCC2.932, Thr934, Ser937, Ser940, Thr906, Thr1007) results in change of the ion-transport activity of KCC2 measured in heterologous expression system (940 and Thr906 plus Thr1007) were extensively studied for their role in native neuronal environment both in-vivo and in-vitro. Briefly, the protein kinase C (pkC) phosphorylation of Ser940 leads to stabilization of KCC2 on neuron’s surface leading to enhancement of ion-transport activity, whereas Ser940 dephosphorylation is associated with transporter internalization and reduction of the KCC2 activity  including four members (WNK1–4) and the downstream SPAK/OSR1  pathway , the National Center for Scientific Research (CNRS), the A*MIDEX project (ANR-11-IDEX-0001-02) funded by the “Investissements d’Avenir” French Government Program, managed by the Agence Nationale de la Recherche. IM was supported by French Foundation for Epilepsy Research. MH was supported by a Doctoral fellowship from the French Ministry of higher Education and Research.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.