Y530F alone duplicated these effects. Inhibition of Src with siRNA, PP2 or pcDNA3/flag-SrcK298M abolished these effects. The pulmonary microvascular endothelial cells (PMVECs) isolated from receptor for AGEs (RAGE)-knockout mice decreased the phosphorylation of Src and attenuated the barrier dysfunction after AGE-treatment. In vivo study showed that the exudation of dextran from mesenteric venules was increased in AGE-treated mouse. This was attenuated in RAGE knockout or PP2-pretreated mice. Up-regulation of Src activity induced the phosphorylation of moesin, as well as activation and dissociation of VE-cadherin, while down-regulation of Src abolished these effects. FAK was also proved to interact with Src in HUVECs stimulated with AGEs. Our studies demonstrated that Src plays a critical role in AGE-induced microvascular hyperpermeability by phosphorylating moesin, VE-cadherin, and FAK respectively.The disruption of microvascular barrier in response to advanced glycation end products (AGEs) stimulation contributes to vasculopathy associated with diabetes mellitus. Here, to study the role of Src and its association with moesin, VE-cadherin and focal adhesion kinase (FAK) in AGE-induced vascular hyperpermeability, we verified that AGE induced phosphorylation of Src, causing increased permeability in HUVECs. Cells over-expressed Src displayed a higher permeability after AGE treatment, accompanied with more obvious F-actin rearrangement. Activation of Src with pcDNA3/flag-Src Microvascular barrier dysfunction and endothelial hyperpermeability are the crucial events in the development of inflammatory diseases, such as trauma, ischemia-reperfusion injury, arteriosclerosis, and especially, diabetes mellitus (DM). Vascular endothelial cells lining the intima of the blood vessels to form a semi-permeable barrier are the bases of microvascular barrier function. The disrupted barrier in response to a variety of stimuli causes endothelial hyperpermeability, exudation of vascular contents and inflammatory factors, transmigration of inflammatory cells, resulting in tissue edema and organ dysfunction. Advanced glycation end products (AGEs) are a group of compounds produced by the non-enzymatic glycation or glycoxidation of proteins, lipids, and nucleic acids, and play a crucial role in the pathogenesis of diabetic microangiopathy and macrovasculopathy. It is reported that AGEs accumulate in the tissue and plasma during aging, while markedly increase in patients with diabetes2356Src family kinases (SFKs) are the largest family of non-receptor tyrosine kinase, consisting of nine structurally related proteins, Src, Blk, Fyn, Yes, Lyn, Lck, Hck, Fgr and Yrk. These proteins share four Src homology (SH) domains involved in catalytic activity, protein-protein interaction, and cell membrane binding911Src is one of the most widely studied members in SFKs. Inhibition of Src has been reported to abolish the increases in albumin permeability caused by C5a-activated neutrophils, which indicates its significance in vascular hyper-permeabilityWe have previously reported that AGEs exerted dose- and time-dependent effects on monolayer permeability of human dermal microvascular endothelial (HMVECs)Once activated through phosphorylation at Tyr 419, the tyrosine kinase of Src can phosphorylate many proteins involved in the regulation of endothelial permeability. We asked whether Src activation was involved in the signal events evoked by AGEs in HUVECs. Cells were incubated with 100 μg/mL of AGE-BSA for different duration and phosphorylation of Src was determined by western blotting. The results showed that phosphorylation of Src 419 was rapidly enhanced at 10 min, reached a peak at 60 min, and then returned to baseline level at 120 min, while phosphorylation of Src 530 showed the contrary tendency during 120 min period. Incubation with BSA (Bovine Serum Albumin) alone had no effect on Src 419 phosphorylation . Src actTo further clarify the role of Src in AGE-induced endothelial barrier disruption, HUVECs were pretreated with a pan inhibitor of Src, PP2, for 90 min before stimulation with AGE-BSA. We revealed that PP2 significantly attenuated AGE-induced endothelial hyperpermeability , indicatTo investigate whether Src activation influence the cytoskeleton arrangement, F-actin morphology was revealed by rhodamine-phalloidin staining . We founTo investigate whether RAGE is required for Src activation in AGE-induced endothelial dysfunction, pulmonary microvascular endothelial cells (PMVECs) isolated from wild type and RAGE knockout mice were used. Our data showed that the elevated endothelial permeability by AGEs was completely abolished by RAGE knockout . MoreoveThe role of Src and RAGE in microvascular permeability was investigated in AGE-treated wild type (WT) and RAGE knockout mice. We revealed no exudation of FITC-dextran out of the venules in WT mice without AGE treatment. AGE-treated WT mice showed a significant increase in the exudation of FITC-dextran in the extravascular space. This exudation was markedly reduced by a pan Src inhibitor, PP2, and completely abated in RAGE knockout mice . These fSince moesin regulates endothelial permeability via modulating equilibrium between contractile forces generated by the endothelial cytoskeleton and adhesive forces produced at inter-endothelial junctions and cell-matrix attachment, here we tried to find out whether Src signaling influenced moesin phosphorylation. Consistent with our previous findingsGiven that Src acted as an upstream of VE-cadherin and caused the disruption of endothelial barrier in response to permeability-increasing agents such as bradykinin and histamineTo explore whether FAK was the downstream target of Src in AGE-treated HUVECs, cells were transfected with Src siRNA or pretreated with PP2 before the application of AGE-BSA. We found that AGE-BSA treatment induced a significant increase in FAK phosphorylation and this increase was inhibited by PP2 or Src siRNA . These fWe next investigated whether the phosphorylation of FAK played a role in the elevated endothelial permeability induced by AGEs. Cells were treated with FAK inhibitor PF573228 for 60 min before the application of AGE-BSA, and endothelial monolayer permeability was evaluated by TER and Pd. The results showed that inhibition of FAK significantly attenuated AGE-induced hyperpermeability of monolayer endothelial cells . The ovein vivo.Several studies have reported that AGEs increase vascular endothelial monolayer permeability and microvascular permeability. The mechanisms underlying these responses were complex and seemed to depend on the origins of cells and organs181920212223Activation of Src involves dephosphorylation of Y530 and autophosphorylation of the Y419, and the phosphorylation of Src at Y419 can be used as markers for activated SrcIn this study, AGEs were associated with RAGE to activate Src through a time- and dose-dependent phosphoarylation at Tyr419 in HUVECs, and in primary isolated PMVECs. RAGE knockout completely abated AGE-induced Src phosphorylation in PMVECs, consistent with the observation that the RAGE-specific antibody blocked the activation of Src kinase in vascular smooth muscle cellset al. has demonstrated that TNF-α can phosphorylate threonine 558 of moesin and enhance the permeability in pulmonary microvascular endothelial cells558, which leads to the disruption of endothelial barrier7T558A, while exaggerated by the active mutant pcDNA3/HA-moesinT558D, which mimicked the AGE-evoked endothelial responsesIt is important to note that the tyrosine kinase Src works as a signaling hub where various signaling pathways diverge. Although Src can phosphorylate many proteins implicated in endothelial hyperpermeability, this study focused on the phosphorylation of moesin, VE-cadherin and FAK. Moesin is the main ERM molecule expressed in endothelium30The intercellular adherens junctional protein, VE-cadherin, is phosphorylated, internalized and ubiquitinated in response to permeability-increasing agents such as bradykinin and histamine37382O2 or thrombin434442The role of FAK in the modulation of endothelial permeability has been extensively explored. Evidences show that FAK is auto-phosphorylated at tyrosine 397 after binding to integrin and provides binding sites for phosphorylation at 576, 577, 861 and 925 for several upstream signaling molecules including Src41This study did not rule out the possibility of cross-talks between moesin, VE-cadherin, and FAK signaling. For example, VEGF can promote the binding of FERM domain of FAK to the cytoplasmic tail of VE-cadherin, which facilitates the phosphorylation of beta-catenin at tyrosine-142 (Y142) and subsequently leads to the dissociation of VE-cadherin-beta-catenin complex and the breakdown of EC junctions467Taken together, this study demonstrated an involvement of Src in AGE-induced endothelial hyperpermeability. Since Src localized at the node which linked upstream AGEs/RAGE signaling and the three downstream pathways, VE-cadherin, moesin and FAK, one can assume that Src may be an appropriate target for the prevention and therapy of AGEs associated microvasculopathy.HUVECs were obtained from Sciencell. The sequences of oligonucleotide for c-Src and control siRNA were synthesized by GenePharma . The siRNA-targeted sequences were as follows: control siRNA, AATTCTCCGAACGTGTCACGT; Src siRNA, TGTTCGGAGGCTTCAACTCCT. The Src overexpressing plasmid (pcDNA3/flag-Src) and the mutants at Lys298  and at Tyr530  were synthesized by Genechem company . Antibody against Src , p-SrcY416 , p-moesin Thr558 , moesin , p-VE-cadherin Y658 , VE-cadherin , p-FAK Y925, and FAK were used. Src inhibitor PP2 (Cat# 529573) was acquired from Merck ; FAK inhibitor PF573228 (PZ0117) was purchased from Sigma .5/mL were seeded onto upper chamber of a trans-well with pore size of 0.4 μm. When endothelial monolayer grew confluent, TER was measured. The mean value of TER was expressed in the common unit (Ω cm2) after subtraction of the value of a blank cell-free filter. The relative changes of TER to baseline value were calculated by the formula:TER of HUVECs monolayer was measured using EVOMTER = TER of experimental wells/baseline TER of experimental wells - 1.2), V is the volume of the bottom chamber, [L] is the dextran concentration in upper chamber.Cells were grown onto transwell membrane till confluent and the tracer FITC-labeled dextran (1 mg/mL) was added to the top chamber for 45 minutes. Then the concentration of dextran in upper bottom chamber was determined with a HTS 7000 microplate reader. The permeability of endothelial monolayer were evaluated by the permeability coefficient of dextran calculated as follows: Pd = [A]/t × 1/A × V/[L], where [A] is the dextran concentration in bottom chamber, t refers to time in seconds, A indicates the area of the membrane  supplemented with protease and phosphatase inhibitors. Protein samples were subjected to SDS-PAGE separation, and then transferred to polyvinylidenedifluoride (PVDF) membranes. After blocked with 5% BSA, the membranes were incubated respectively with primary antibody for Src, p-Src, moesin, p-moesin, VE-cadherin, p-VE, FAK, or p-FAK at 1:1000 at 4 °C overnight, washed three times with each time for 10 min, and incubated with respective secondary antibody at room temperature for 1 h. After washed three times for 10 min each time, protein bands were visualized with chemiluminescence and densitometric analysis was operated by an imaging station.Total proteins were prepared using the lysis buffer . 48 h to 72 h after transfection, cells were treated with or without AGEs for 1 h. Then cells were lysed and subjected to Western blot.When HUVECs cultured in DMEM/F12 without antibiotics grew to 30%–50% confluence, they were transfected with siRNA (20 nmol/L) using siRNA-MateTMkit. HUVECs of 70%–80% confluence were transfected with plasmids using LipoFilterTM Liposomal Transfection Reagent acquired from HANBIO . Briefly, for a 6-well format, 0.4 μg DNA and 12 μl LipoFilterTM was incubated in 250 μl DMEM. The DNA complexes were added to the plates and incubated for 48 h–72 h, followed by stimulation with AGEs.The transfection of plasmid was carried out by LipoFilterECs were plated in microwells and cultured to confluence. Treated with indicated stimulation, sequentially, cells were washed in PBS three times with each time for 2 min, fixed in 4% formaldehyde for 10 min and permeabilized with 0.5% Triton X-100 at 4 °C for 15 min. Cells were washed three times, blocked in 5% BSA for 1 h, and incubated with VE-cadherin (1:200) at 4 °C overnight. After washed in PBS, cells were stained with FITC-conjugated secondary antibody (1:50). For F-actin staining, cells were incubated with rhodamine-phalloidin (2000 U/L) at room temperature for 1 h. Afterwards, cells were washed three times in PBS and detected by a Zeiss LSM780 laser confocal scanning microscope .The 18–20 gram male C57 mice used were purchased from the Laboratory Animal Center of Southern Medical University. RAGE knockout mice were kindly provided by Kanazawa University, Japan. The protocol using mice was approved by the Animal Care Committee of the Southern Medical University of China and in strict accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Mice were injected intraperitoneally with BSA or AGE-BSA at 10 mg/kg for consecutive seven days. For PP2 treatment group, PP2 (2 mg/kg) was given intraperitoneally 20 min before the daily AGE-BSA injection, and for control group, equal volume of DMSO was administrated. For the assay of microvascular exudation of FITC-dextran, mice were anesthetized with an intramuscular injection of 13.3% ethyl carbamate plus 0.5% chloralose (0.65 ml/kg) before all surgical manipulations and their carotid veins were cannulated. Sequentially, mice were placed on a Plexiglas platform mounted to an intravital upright microscope and a midline laparotomy was performed, then the mesenteric venules were selected for measurement. The FITC-dextran tracer was administrated via carotid veins at 100 mg/kg in 1 mL of APSS solution, followed by continuous infusion of dextran at 0.15 mg/kg/min to maintain its concentration. The excitation wave used to detect fluorescence was 488 nm and the emission wave was 525 nm. Changes in integrated optical intensity were calculated as follows: ΔI = 1 − (Ii − Io)/Ii, where ΔI indicates the changes in light intensity, Ii is the light intensity inside the vessel, while Io is the light intensity outside the vessel.7 total cells. Mix well and incubate for 15 min in the refrigerator (2 °C–8 °C). Place column in the magnetic field of a suitable MACS Separator. Apply cell suspension onto the column and collect unlabeled cells that pass through the column and repeat I for several times. Finally, remove column from the separator and place it on a suitable collection tube, flushing out the magnetically labeled cells into it. Purity of mouse PMVECs were evaluated by factor VIII labeling.Primary PMVECs were isolated using CD31 MicroBeads mouse according to the manufacture’s protocols provided by Miltenyi Biotec . Briefly, lungs from wild type and RAGE knockout mice were excised, sliced, digested in collagenase type I, and then filtered through 70 μm nylon filters. Centrifuge cell suspension at 300 × g for 10 minutes. Move out supernatant and add 10 μl of CD31 microbeads per 10All data were expressed as means ± SE from more than three independent experiments and analyzed using SPSS 16.0 software. Statistical comparisons were performed using one-way ANOVA and P < 0.05 was considered statistically significant.How to cite this article: Zhang, W. et al. Role of Src in Vascular Hyperpermeability Induced by Advanced Glycation End Products. Sci. Rep.5, 14090; doi: 10.1038/srep14090 (2015).


Levodopa (L-DOPA)-induced dyskinesias (LIDs) represent the major side effect in Parkinson’s disease (PD) therapy. Leucine-rich repeat kinase 2 (LRRK2) mutations account for up to 13 % of familial cases of PD. LRRK2 N-terminal domain encompasses several serine residues that undergo phosphorylation influencing LRRK2 function. This work aims at investigating whether LRRK2 phosphorylation/function may be involved in the molecular pathways downstream D1 dopamine receptor leading to LIDs. Here we show that LRRK2 phosphorylation level at serine 935 correlates with LIDs induction and that inhibition of LRRK2 induces a significant increase in the dyskinetic score in L-DOPA treated parkinsonian animals. Our findings support a close link between LRKK2 functional state and L-DOPA-induced abnormal motor behaviour and highlight that LRRK2 phosphorylation level may be implicated in LIDs, calling for novel therapeutic strategies. Parkinson’s disease (PD) is a neurodegenerative disorder characterized by the progressive loss of dopaminergic neurons from the substantia nigra pars compacta, resulting in a decrease of dopamine (DA) levels in the striatum. Up-to-date, DA replacement with L-3,4-dihydroxyphenylalanine methyl ester hydrochloride (L-DOPA) is the most effective therapeutic approach for PD. Unfortunately, as the disease progresses, L-DOPA administration is responsible for severe side effects known as L-DOPA-induced dyskinesias (LIDs) . The mosyclic adenosine monophosphate-dependent protein kinase (PKA) [Leucine-rich repeat kinase 2 (LRRK2) mutations account for up to 13 % of familial PD cases compatible with dominant inheritance , 5 and hse (PKA) . Accumulse (PKA) , and, atse (PKA) . Notablyse (PKA) . In thisGiven the tight functional correlation between LRRK2 and crucial LIDs molecular players, we measured LRRK2 expression and phosphorylation levels in the striatum from control, 6-OHDA and L-DOPA-treated non dyskinetic and dyskinetic rats. Interestingly, while LRRK2 total expression was unaltered in the different experimental groups, LRRK2 phosphorylation at Ser-935  were maintained on a 12-h light/dark cycle at 22 °C with food and water ad libitum. Procedures were carried out in accordance with the current European Law (Directive 2010/63/EU).The primary antibodies used in this study are rabbit anti-LRRK2 1:500 MJFF C41-2, rabbit anti-LRRK2 P-Ser-935 UDD2 10(12) , mouse anti-actin 1:1000, mouse anti-FLAG 1:1000, mouse anti myc 1:1000, mouse anti-synaptophysin 1:1000 .n = 100; 125–175 g) underwent a single stereotaxic injection of 6-OHDA  in the left medial forebrain bundle  as previously described [Rats  for 14 days. L-DOPA-induced abnormal involuntary movements (AIMs) were evaluated on days 4, 7, 10 and 14 of L-DOPA administration using a highly validated rat AIMs scale , 26. In n = 7) and Inhibitor III , or 0.5 % DMSO (n = 7) in the ispilateral striatum . Untreated fully lesioned animals displaying stable and severe dyskinesia  were included in the study as positive control. L-DOPA treatment was continued for 4 days after injection of LRRK2 inhibitors. To evaluate the effects of LRRK2 inhibition on dyskinesia, AIMs assessment on IN-1/InhibIII/DMSO-treated rats was carried out before the surgery (−18 h), on the day of the surgery and after 2 days .At day 15 of L-DOPA administration, fully lesioned rats showing a low dyskinetic behaviour underwent a single stereotaxic injection of 2 LRRK2 inhibitors IN-1 .post hoc test as indicated in the text.All data are expressed as mean ± SEM. Significance of the differences was analysed by unpaired two-tailed Student’s t test or ANOVA followed by Dunn’s and Bonferroni Not applicable.Not applicable.https://osf.io/u4nzd/.The datasets supporting the conclusions of this article are available in the Open Science Framework repository at


The cell's fate is largely governed by the dynamics of cellular events, brought about by the interplay of various proteins, in which post-translational modifications (PTMs) play a key role. Therefore, a better understanding of post-translational modification is of utmost importance to a biochemist. Over the years much work in this regard has been achieved through mass spectrometry, however, the “on-off” nature of PTMs, which accounts for cell's dynamics, has posed a serious challenge in quantitating them, especially with regards to measuring the occupancy of PTMs on a particular site. Protein phosphorylation has served as the best model for which measurement of occupancy has been attempted by employing stable isotope labeling and label-free approaches. But the reliability of these methods has mainly been limited to the relative quantitation between samples. Although the fractional occupancy/absolute quantitation of phosphorylation has been determined by grossly applying these methods, but overall they fall short of testability. Moreover, the field demands a broad approach which can even encompass a more challenging task of absolute quantitation of Cys-based PTMs.For quantitation, differential labeling of samples with stable isotopes or in the label-free approach the peak intensity of a peptide ion or identification frequency of the peptides of a particular protein is used for measurement. Stable isotope labeling is achieved through metabolic labeling in cell culture or by chemical labeling in post-metabolic stage. For the metabolic labeling of proteins, the cell cultures are grown in the presence of naturally abundant amino acids (light version), and amino acids  labeled with heavy isotopes , this method is known as stable isotope labeling with amino acids in cell culture (SILAC). The ratio of the ion intensities of the peptide (light vs. heavy) in the MS scan is used for quantitation (Ong et al., per se. These standards are the pre-selected synthetic peptides from the protein of interest that gets labeled with an isotope. A known amount of the isotope labeled peptides are mixed with the sample and then introduced into the mass spectrometer. Absolute abundance of the PTM can be obtained by measuring the relative abundance of the modified and unmodified peptides and comparing it with the amount of standard used. Notably, stable isotope labeling and synthetic peptide standards were used in absolute quantitation of phosphorylation on Ser1526 in human separase (Stemmann et al., Stable isotope labeling for absolute quantitation requires internal standards that are used to measure protein abundance Interestingly, an integrated phosphatase treatment and stable isotope labeling approach was adapted for determining the absolute phosphorylation occupancy in the yeast proteome. This approach did not make use of any standard, instead equal amounts of protein lysates after protease-digestion were either mock- or phosphatase treated, differentially labeled with stable isotope and subsequently mixed. The absolute phosphorylation site occupancy was represented by the single ratio between the non-phosphorylated peptides with and without phosphatase treatment (Wu et al., per se is very low. Thus, looking at the fast pace of the field and the continuous out-flux of ideas from the think-tank, the doorway for site occupancy measurement of Cys-based PTMs is already open but to come up with a foolproof method, would require some sound strategizing.With the present day, state-of-the-art technology and improved methodology we have made significant progress in characterizing phosphorylation and the databank is increasing every day. Nonetheless, another segment of the field; Cys-based PTMs considerably lags behind! The mechanistic insight of Cys-based modifications, in particular, S-nitrosylation and S-palmitoylation, is ever growing, and their involvement in regulating various cellular events is quite plausible. There are few remarkable methods developed over the years to identify the Cys-sites of these modifications with mass spectrometry, like the biotin-switch technique and SNO-Rac for S-nitrosylation (Jaffrey et al., The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Mer and Flt3 receptor tyrosine kinases have been implicated as therapeutic targets in acute myeloid leukemia (AML). In this manuscript we describe UNC1666, a novel ATP-competitive small molecule tyrosine kinase inhibitor, which potently diminishes Mer and Flt3 phosphorylation in AML. Treatment with UNC1666 mediated biochemical and functional effects in AML cell lines expressing Mer or Flt3 internal tandem duplication (ITD), including decreased phosphorylation of Mer, Flt3 and downstream effectors Stat, Akt and Erk, induction of apoptosis in up to 98% of cells, and reduction of colony formation by greater than 90%, compared to treatment with vehicle. These effects were dose-dependent, with inhibition of downstream signaling and functional effects correlating with the degree of Mer or Flt3 kinase inhibition. Treatment of primary AML patient samples expressing Mer and/or Flt3-ITD with UNC1666 also inhibited Mer and Flt3 intracellular signaling, induced apoptosis, and inhibited colony formation. In summary, UNC1666 is a novel potent small molecule tyrosine kinase inhibitor that decreases oncogenic signaling and myeloblast survival, thereby validating dual Mer/Flt3 inhibition as an attractive treatment strategy for AML. Though the treatment of acute myeloid leukemia (AML) has significantly improved over the past 30 years, average five-year survival rates are still less than 60% for pediatric patients and are progressively worse for older adult and elderly patients , 2. The The Mer receptor tyrosine kinase is abnormally expressed on 80–100% of AML patient samples, whereas little or no Mer is expressed in normal bone marrow precursor cells . Mer actFLT3-ITD mutant allele relative to the wild type allele (> 0.4) has been associated with a markedly poor progression-free survival [Internal tandem duplication (ITD) mutations of the FMS-like tyrosine kinase 3 (Flt3) receptor, found in ~20–30% of adult AML patient samples and ~15% of pediatric AML samples, is associated with poor prognosis in both patient populations –15. Specsurvival . Constitsurvival , 18. Givsurvival , 20. Intsurvival , such thsurvival . Issues Given the high prevalence of expression of both Mer and Flt3 and the data indicating their oncogenic roles in AML, targeting them together is an attractive therapeutic strategy, and could potentially result in better outcomes in high-risk patients, reduced need for chemotherapy in low-risk patients, or a therapeutic option for those who cannot tolerate high-intensity chemotherapy. We have developed UNC1666, a novel Mer and Flt3 targeted small molecule tyrosine kinase inhibitor with therapeutic potential in AML. In this publication, we show that this ATP-binding site competitive small molecule potently and selectively inhibits Mer and Flt3 kinase activation and downstream signal transduction resulting in growth inhibition and apoptosis of AML cell lines and primary patient myeloblasts.in vivo pharmacokinetic properties made UNC1062 unsuitable for in vivo studies. To develop further Mer inhibitors, a new pyrrolopyrimidine scaffold with better solubility was introduced using a structure-based design approach [i) proved this compound to be more potent and selective for Mer  compared to previously described Mer inhibitors [50 0.69 nM; Ki 0.67 nM) equipotently in enzymatic MCE assays. A comprehensive protein kinase profiling panel provided by Carna Biosciences was used to assess off-target kinase inhibition mediated by UNC1666 at a concentration of 46 nM, more than 50-fold higher than its MCE IC50 values against Mer and Flt3 (50 0.57 nM)  . Further50 560 nM) and Flt3 (MCE IC50 220 nM) relative to UNC1666.The small molecule UNC1653 Figure  has a pyhighAR”) and MOLM-13 , which express little (“dim”) and no Mer respectively, and have been used extensively as models of leukemia with Flt3-ITD mutations.We have previously described survival and proliferation signaling resulting from Mer tyrosine kinase activity in AML, including increased activation of Erk1/2, Akt and Stat6 . SimilarUsing these cell lines, we confirmed that short-term treatment with UNC1666 reduced Mer and Flt3 phosphorylation in a dose dependent manner, compared to vehicle alone (DMSO) or negative control UNC1653 Figure . UNC1666To investigate whether inhibition of other kinases might contribute to effects mediated by UNC1666, we also assessed expression of the Trk kinases as well as Tyro-3 and Axl. The cells lines used for these studies express very little or no Tyro-3, Axl or Trk protein .We then evaluated downstream Erk, Akt and Stat signaling after a two hour treatment with UNC1666. There was a consistent dose-dependent abrogation of Erk1/2, Akt and Stat6 phosphorylation in Mer expressing cell lines treated with UNC1666 compared to vehicle or UNC1653 Figure . The decp < 0.001). Importantly, apoptosis was induced at the same concentrations of UNC1666 required for inhibition of Mer . Again, induction of apoptosis correlated with inhibition of Flt3 . Cells were counted after six days of culture (Day 6) to assess rebound growth  over six days, whereas those treated with 100 nM UNC1666 only increased 2.5-fold (to 0.3 × 105 cells/ml) (p < 0.001). The MV4;11 cell line treated with vehicle demonstrated an average 67-fold increase (from 1.5 × 104 to 10 × 105 viable cells/ml) whereas cells treated with 50 nM UNC1666 only increased 13-fold (to 2.0 × 105 viable cells/ml) (p < 0.001).Malignant cells have previously demonstrated rebound growth after removal of TKIs in some clinical trials . To evalh Figure . Although Figure , these ch Figure . Samplesin vivo, AML cell lines were plated in equal number in soft agar and treated with UNC1666, negative control UNC1653, or vehicle in the overlaying medium. Vehicle treatment was assessed in duplicate and data from samples treated with UNC1666 or UNC1653 were normalized to the mean vehicle colony number for each experiment. Treatment with UNC1666 significantly decreased colony formation compared to cells treated with medium containing vehicle or UNC1653 .To assess the effect of UNC1666 in a longer-term assay which might more closely approximate the three-dimensional environment encountered 3 Figure . The KasFLT3-ITD alleles were detected using standard clinical molecular techniques . All samples except #123009 expressed Flt3-ITD mutations, though #11612 and #41206 were noted to have a low allelic ratio. Since there is a correlation between high allelic ratio of the Flt3-ITD (> 0.4) and poor prognosis in patients [highAR”) or low (“Flt3-ITDlowAR”) Flt3 allelic ratio. None of the patient samples demonstrate detectable levels of Trk proteins  is > 90% (one differing residue). In contrast, the sequence identity for the full Mer kinase domain compared to the full kinase domain of Flt3 is ~30%. UNC1666 exhibits selectivity for Mer and Flt3 over other kinases tested, with greater than 50-fold selectivity over closely related and structurally similar kinases Tyro-3 and Axl, co-members with Mer in the TAM family.The unacceptable cure rate and toxic consequences of intensive chemotherapy support the development of novel targeted therapies to improve long-term clinical outcomes in AML. Our previous work demonstrated the upregulation of the Mer tyrosine kinase in nearly all adult and pediatric patient samples collected at diagnosis or relapse. In contrast, normal bone marrow progenitors express very little Mer , 9. We pWe have found that Mer and Flt3-ITD can be co-expressed on AML patient samples, though a larger cohort of patient samples is needed to determine the frequency of dual Mer expression and Flt3-ITD mutation. Currently, little is known about the physical association of Mer and Flt3 receptors on leukemic blasts. Though studies to assess the physical association of Mer and Flt3 have not been reported, co-localization of Flt-3 and Axl, a member of the TAM kinase family along with Mer has been demonstrated . Based oGiven that Flt3-ITD is a known oncogenic target in AML and Mer is also known to play essential oncogenic roles in AML cells, combined inhibition of Mer and Flt3 is likely to be an effective strategy with broad applicability. Thus, we tested the efficacy of our dual Mer/Flt3 TKI in AML cell lines and patient samples. The MCE enzymatic assay data indicate that UNC1666 inhibits Mer and Flt3 with nearly equal potency. However, in cell-based assays, UNC1666 was more effective at reducing Flt3 phosphorylation compared to Mer phosphorylation. Inhibition of Mer and Flt3 phosphorylation in cell based assays correlated with functional effects mediated by UNC1666 in cell lines expressing Mer or Flt3-ITD.in vitro therapeutic intervention. Given the heterogeneity of downstream effector phosphorylation status in a given AML cell line or patient sample, blockade of both Mer and Flt3 signaling may affect independent downstream pathways and/or may function redundantly to more effectively inhibit shared pathways. Targeting these mutual pathways had a profound functional effect in AML patient samples that co-expressed Mer and Flt3, including decreased ability to evade apoptosis and decreased colony formation. Each of these effects occurred at concentrations between 5–100 nM, and correlated well with inhibition of Mer and Flt3 phosphorylation in cell-based assays. The observed correlations between inhibition of Mer or Flt3 and functional anti-leukemia effects mediated by UNC1666 in AML cells suggest that these effects are a result of on-target inhibition of Mer and Flt3, rather than non-specific inhibition of other kinases.Previously, we demonstrated signaling downstream of Mer through the pro-survival and anti-apoptotic Erk1/2, Akt and Stat6 pathways in AML. Other groups have shown similar downstream effectors of Flt3-ITD including Erk1/2, Akt and Stat5. Though there are have been additional effectors known downstream of both Mer and Flt3 in various cell types, we chose to analyze these molecules which function to promote cancer cell growth and prevent apoptosis and have well-characterized roles in leukemogenesis. In addition, other groups have noted heterogeneity in the phosphorylation status of all three of these molecules (in both Mer and/or Flt3-ITD dependent cell lines) depending on the cell line, tumor type, assay, and/or The studies presented here extend the action of UNC1666 to primary patient AML blasts. Interestingly, the anti-proliferative effects in patient samples were even more dramatic than in cell lines with near complete induction of apoptosis and growth inhibition at 50 nM. The patient sample conditions  suggest that Mer inhibition is likely to be effective even in the bone marrow microenvironment. The bone marrow niche provides a potential rich source of paracrine Gas6 , 29, a Min vivo bioavailability and pharmacokinetics as compared to earlier Mer inhibitors [in vitro work with primary patient samples demonstrated a dramatic effect at concentrations well within the range of what is clinically achievable using orally bioavailable TKIs, we anticipate that improved analogues will have similar results both in vitro and in vivo.Initially, UNC1666 was designed to have greater solubility to improve hibitors , 30. Unfin vivo bioavailability and a short half-life (data not shown), it was not possible to assess the toxicity profile associated with dual Mer/Flt3 inhibition in mice. As previously described, Flt3 inhibitors such as AC-220 are known have side effects including myelosuppression, though this is variable and is not present with some newer generation Flt3 inhibitors [Because UNC1666 has poor hibitors  due to ihibitors , 9. Prevhibitors , indicatDespite our finding that the majority of Mer-expressing cell lines have at least some degree of response to treatment with UNC1666 in apoptosis and colony formation assays, the level of Mer protein does not correlate with sensitivity to UNC1666. This observation has recently led to initiation of studies to investigate the characteristics of AML cells that might predict sensitivity to Mer inhibition. Based on previous studies indicating a prognostic role for Gas6 in AML, we hypothesize that paracrine or autocrine expression of Gas6 (or other Mer ligands) by the leukemia cell itself or by the supportive marrow stroma , 33, couin vivo experimentation with more bioavailable compounds that share UNC1666′s improved potency. These data also suggest a potential role for dual Mer/Flt3 inhibition in the treatment of human AML, where there is a true need for targeted agents to treat patients more effectively.In summary, Mer and Flt3 inhibition was effective at profoundly diminishing survival of AML blasts in both cell lines and primary patient samples. UNC1666 decreased Mer and Flt3 phosphorylation/activation and abrogated downstream signaling through the Stat, Akt and Erk pathways. The dose required for these signaling effects correlated well with induction of apoptosis and decreased colony formation. Prolonged effects were even noted on seemingly viable cell populations in replating assays, a promising indication for future in vivo bioavailability for use in the treatment of human disease, though it is not clear if these cancers would equally benefit from the dual Mer/Flt3 inhibition. The data presented here are likely to lead to a multitude of additional studies of our Mer/Flt3 inhibitors exemplified here by UNC1666, including evaluation of potential synergistic combination therapy, assessment in murine xenograft mice using patient derived xenografts, and ultimately to phase I clinical trials. The hypothesis that Mer is providing the neoplastic cell with a survival advantage and the observation that Mer inhibition sensitizes cancer cells to chemotherapy [Interestingly, several additional cancers are known to aberrantly express Mer, including acute lymphoblastic leukemia and melanoma, and inhibition of Mer in these cancers has demonstrated significant effects on malignant cell survival and apoptosis , 34. Theotherapy  suggest in vitro studies, compounds were dissolved in dimethyl sulfoxide . DMSO equivalent to the 300 nM UNC1666 treatment was used as a vehicle control.UNC1666 and UNC1653  were prepared as previously described , 30. KinCell lines MV4;11, NOMO-1, KG-1a, NB-4, and HEL were obtained from the German Collection of Microorganisms and Cell Culture (DSMZ); Kasumi-1 and U937 were obtained from the American Type Culture Collection (ATCC); MOLM-13 was a gift from Robert Arceci (Johns Hopkins); HS-27 was a gift from Kathrin Bernt (University of Colorado). AML cell lines were maintained in RPMI medium (HyClone) supplemented with 10% FBS and penicillin/streptomycin (cRPMI). Cell line identities were confirmed using short tandem repeat microsatellite loci analysis. HS-27 stromal cells were maintained in DMEM with 10% FBS and penicillin/streptomycin prior to co-culture. De-identified cord blood samples and primary patient myeloblasts apheresed from peripheral blood were obtained from the University of Colorado after written informed consent in accordance with the Declaration of Helsinki. Experiments conformed to regulatory standards as approved by the Colorado Multiple Institutional Review Board. AML patient samples were maintained as previously described , with HSThree million cells per condition were cultured in 24-well plates and treated with UNC1666, UNC1653, or vehicle for two hours. Whole cell lysates were prepared and proteins were resolved on Tris-Glycine SDS-PAGE gels (Invitrogen) and transferred onto nitrocellulose membranes. Membranes were blocked in tris-buffered saline with 0.1% Tween-20 containing 5% bovine serum albumin (when probing for Flt3) or 5% milk . For immunoprecipitation assays, cells were plated and treated as above, then treated with pervanadate for 10 minutes prior to cell lysis to stabilize the phosphorylation status. With the use of pervandate, it was not necessary to stimulate cells with exogenous Mer or Flt3 ligands  to detect phosphorylated Mer and Flt3 proteins. Anti-Mer antibody or anti-Flt3 and rec-Protein G-sepharose beads (Invitrogen) were added and lysates were incubated overnight on a rocking platform. Beads were washed and eluted proteins were resolved as above. Immunoprecipitate membranes were probed with an anti-phospho-Mer or anti-phospho-Flt3 antibody. After visualization, membranes were stripped and reprobed for total protein. The following antibodies were used according to manufacturer recommendations: anti-Mer ; anti-phospho-Mer (Phosphosolutions Inc.) , 39; ant5 cells, and 1 × 106 AML patient blasts were co-cultured with 3.5 × 105 HS27 cells per condition in 24 well plates, and treated for 72 hours. Harvested cells were washed, resuspended in PBS containing 1 μM YO-PRO®-1 iodide (Invitrogen) and 1.5 μM propidium iodide (PI) (Invitrogen), and incubated on ice for 15–20 minutes. Fluorescence was detected and analyzed using a FC500 flow cytometer with CXP data analysis software (Beckman Coulter). For cell cycle analyses, cells were fixed with 100% ethanol after treatment for 72 hours with UNC1666 or controls, stained with PI overnight, and analyzed on a Gallios flow cytometer. Flow cytometry data were analyzed using Modfit Cell Cycle Analysis software (Verity Software House). In replating assays, cells were similarly plated and treated, then harvested cells were washed twice, and 1.5 × 104 viable cells/ml were cultured in cRPMI for six days. Six days after replating, viable myeloblast count was determined by trypan blue exclusion using a Cedex XS Analyzer (Roche).To assess apoptosis, AML cell lines were cultured at 3 × 106 cells/ml in MethoCult H4434 Classic Methylcellulose-Based Medium with Recombinant Cytokines for Human Cells (StemCell Technologies) containing UNC1666 or vehicle in triplicate and colonies were grown for 10 days. Human mononuclear cells were isolated from umbilical cord blood samples using Ficoll-Paque PLUS . Cells were grown in serum-free IMDM (HyClone) media containing BIT 9500 Serum Substitute (StemCell Technologies), lipoprotein lipase (Millipore), and 2-mercaptoethanol (Sigma) for one hour, then plated in Methocult H4434 methylcellulose containing UNC1666 or vehicle at 2 × 106 cells/mL in triplicate and colonies were grown for 14 days. Cell line and patient sample colonies were counted using a GelCount colony counter (Oxford Optronix) and cord blood colonies were manually counted in a blinded, non-biased manner.Cell lines were plated at a density of 1000 cells/ml in 0.35% agar over 0.5% agar base layer. Agar was overlaid with cRPMI containing UNC1666, UNC1653, or vehicle. Colonies were grown for 14  or 21 days (Kasumi-1) prior to staining with 1 mg/ml nitrotetrazolium blue (Sigma-Aldrich). Treatment-containing medium was renewed twice weekly. Patient samples were plated at a density of 1 × 10p < 0.05.Statistical analyses were performed using GraphPad Prism software (v6.02), comparing UNC1666 and vehicle-treated samples. Cell cycle and replating assays were analyzed using two-way ANOVA. All other data were analyzed using one-way ANOVA. All statistics were corrected using Bonferroni's multiple comparisons test. Results were considered significant when


Egg belongs to the list of ingredients that must be labeled because of its frequent involvement in food allergies especially in childhood. Several thermal processes are commonly applied to egg and it is known that some egg-allergic people can tolerate well-cooked egg. Food allergens are characterized by their ability to elicit IgE antibodies (sensitization) in susceptible individuals and to display several IgE-binding epitopes. Allergenic potential influenced by different structural levels  can be greatly impacted by all modifications of these structures due to processing. Our work aimed to analyze the impact of pasteurization and boiling of egg and its fractions on in vivo reactivity and its relation to IgE-binding profiles to egg allergens/fractions in a cohort of children allergic to egg.Allergic children  were recruited, all had positive skin prick-tests (SPT) to raw whole egg and egg white and 80% to raw egg yolk. Reactivity to pasteurized and boiled fractions was evaluated by SPT. Sera samples taken at inclusion were used to draw profiles of IgE-reactivity against known allergens and proteins of egg white and against fractions of egg yolk by ELISA. Relative STP data and IgE-binding profiles were analyzed by hierarchical classification to evidence typical profiles of reactivity in this cohort.Pasteurization had very limited impact on structure of egg and only 10% and 5% of children STP became negative with pasteurized  whole egg or egg white. Boiling  greatly changed structure of egg products and had a higher effect than pasteurization since 55%, 27% and 67% of children SPT remained positive to boiled egg and yolk and white fractions. Two typical STP behaviors were evidenced by classification in this cohort. IgE-binding profiles largely differed among children but three typical profiles where extracted from data. Contingency analysis revealed links between typical STP and IgE-binding profiles with possible explanation of tolerance to cooked egg.


Adult hippocampal neurogenesis provides the dentate gyrus with heterogeneous populations of granule cells (GC) originated at different times. The contribution of these cells to information encoding is under current investigation. Here, we show that incoming spike trains activate different populations of GC determined by the stimulation frequency and GC age. Immature GC respond to a wider range of stimulus frequencies, whereas mature GC are less responsive at high frequencies. This difference is dictated by feedforward inhibition, which restricts mature GC activation. Yet, the stronger inhibition of mature GC results in a higher temporal fidelity compared to that of immature GC. Thus, hippocampal inputs activate two populations of neurons with variable frequency filters: immature cells, with wide‐range responses, that are reliable transmitters of the incoming frequency, and mature neurons, with narrow frequency response, that are precise at informing the beginning of the stimulus, but with a sparse activity.DOI:http://dx.doi.org/10.7554/eLife.08764.001 A number of cell types in the body are capable of dividing to produce two new cells. These are used either to replace damaged or worn out tissue, or to satisfy a need for additional cells. By contrast, the vast majority of the billions of neurons in the brain are produced before birth, and only a handful of brain regions retain the ability to generate new neurons throughout life.One of these brain regions is the hippocampus, which has roles in memory and navigation. The zone of the hippocampus that receives signals from other parts of the brain—known as the dentate gyrus—contains cells called granule cells that are still able to divide in adult animals. Newly formed cells mature over the course of a few weeks, but whether immature and mature cells make different contributions to processing the information that enters the hippocampus is unclear.By stimulating slices of mouse hippocampus using sequences of electrical pulses similar to those that occur naturally in the brain, Pardi et al. have shown that the granule cells' responses vary with age. Both mature and 4-week-old granule cells responded more strongly when the electrical pulses were applied at a slower rate. However, the immature cells could also respond to pulses applied at a faster rate more reliably than their mature counterparts.In contrast, mature cells signaled the arrival of the first pulse in a sequence more precisely than immature cells, with the majority of mature granule cells firing within the first three milliseconds of receiving a pulse. Additional experiments revealed that these differences arise because mature cells are more easily prevented from firing by inhibitory neurons that contact them, particularly in the presence of rapid sequences of pulses.By using immature and mature granule cells to encode different aspects of an incoming stimulus, the hippocampus may thus be able to maximize its ability to process information. These results raise questions regarding how parallel processing by immature and mature granule cells could affect the processing in CA3, the area that receives the information sent by these cells.DOI:http://dx.doi.org/10.7554/eLife.08764.002 The dentate gyrus (DG) is the main entrance of information to the hippocampus . ActivitAs activity arrives to the DG, it does not only activate GC but also inhibitory circuits . The intIn this work, we have addressed these questions focusing on the understanding of how excitation and inhibition interact to determine the response of GC. We found that due to a weaker influence of feedforward inhibitory circuits, immature neurons can respond to a wider range of frequencies than mature neurons, however, they are less time locked to the incoming stimuli. Mature neurons on the contrary show a higher temporal fidelity but are less effective in their responses to frequency. We suggest that immature and mature GC reflect complementary aspects of incoming activity.We investigated how immature GC process afferent activity from entorhinal inputs arriving at different frequencies and how they compare to mature GC in the adult mice hippocampus. We selected 4-week-old neurons as immature GC because this is the earliest stage at which adult-born GC can be reliably activated by an excitatory drive , exhibitslope) was chosen for all experiments (To investigate how 4-week-old (4wpiGC) and mature (matGC) GC respond to conditions of activity that resemble those occurring during hippocampus-dependent behaviors , we measeriments . Five treriments .Immature GC activate with a higher probability than mature GC at all frequencies, yet both GC populations showed decreased responsiveness for trains delivered at increasing frequency . ActivatThe ability of GC to spike at a given frequency reflects its ability to re-transmit time information to their target neurons. To analyze this aspect, we calculated the efficacy to reproduce each frequency. This was computed as the average of the probability of occurrence of action potentials in the same frequency range as that given by the stimulus. The results show that 4wpiGC have higher efficacy than matGC at all frequencies . This obThe decreased activation with frequency could simply be due to intrinsic physiological restrictions of GC or instead to characteristics of the activated circuit. Therefore, we studied the intrinsic capacity of 4wpiGC and matGC to fire action potentials at 20 and 40 Hz in response to injection of current pulses. Whole-cell current-clamp recordings were obtained from 4wpiGC and matGC, and trains of square current pulses were injected at increasing frequencies. All recorded GC started showing failures at frequencies near 100 Hz. Importantly, both 4wpiGC and matGC consistently followed 20 and 40 Hz . These rTo understand the mechanisms underlying the differential filtering found in matGC vs 4wpiGC, we studied the individual circuit components. Activation of mPP axons not only produces monosynaptic glutamatergic excitation of GC but also recruits feedforward GABAergic inhibitory circuits, which can modulate neuronal firing in response to afferent inputs . Thus, wActivation of matGC is strongly controlled by inhibition, since their activity significantly increases with PTX at every frequency . InteresTo study how the recruited inhibition affects the activation profiles of 4wpiGC and matGC, we investigated the precise contribution of excitatory and inhibitory components that control the activation of GC at different frequencies. We performed whole-cell voltage-clamp recordings to measure excitatory and inhibitory responses elicited at each stimulation frequency at a fix intensity as wherein before. Activation of mPP produced excitatory postsynaptic currents (EPSC) and inhibitory postsynaptic currents (IPSC), indicating that glutamatergic entorhinal axons directly activate immature and mature GC and also recruit inhibitory interneurons that synapse onto both populations . In ordeStimulation of the mPP evokes slower IPSC in 4wpiGC than in matGC , and as To make quantitative comparisons between the currents evoked in 4wpiGC and matGC at different frequencies, we calculated the mean peak EPSG, and the mean IPSG value at the peak EPSG, for the 10 pulses at each frequency. Thus, we obtained a single value of the EPSG and a single value of the IPSG, for 4wpiGC and matGC at each frequency. These data show that the evoked inhibition in both populations of GC increases with frequency. Excitation, on the contrary, slightly decreases . This slThe different rising kinetics of the recruited inhibition between matGC and 4wpiGC determines different time windows in which inhibition restricts excitation. To better understand the extent to which this difference affects activation as a function of frequency, we calculated the residual inhibition at different periods corresponding to the studied frequencies, and added the recruited inhibition observed on average at 1 Hz on top . The resOverall, these results show that the higher activation levels of 4wpiGC are due to a higher balance between the excitation and inhibition, which is mainly generated by a smaller inhibition at the time around spike generation.2/3) that reduces release probability in mossy fiber terminals . Thus, activation in response to stimulation of the mPP at the intensities used in the experiments (50% fEPSPslope) was well above threshold and unaltered by the application of DCG4  . ImmaturIn this work, we reveal that mPP stimulation with trains of stimulus at physiologically relevant frequencies evoke higher levels of activation in immature adult born hippocampal neurons, such that they are more reliable in transmitting different frequencies. The results also demonstrate that the activation differences between immature and mature GC are governed by the recruitment of feedforward inhibitory circuits. The small and slow inhibition, that allows immature neurons to be good transmitters of frequency, together with the weak excitation they receive, generates a worse temporal fidelity in those cells compared to mature neurons, that are time locked to the stimulus. Thus, activity arriving to the hippocampus at different frequencies could be reflected by the activation of graded populations of neurons, immature GC that are reliable transmitters of the incoming frequency, and mature GC that are precise at informing the beginning of the stimulus, but with sparse activity.Numerous studies have shown that brain activity occurs with certain temporal patterns and proposed that the timing of the signals is itself a code . ExtraceLocal inhibitory circuits are primarily responsible for differences in activation generated by trains of stimuli. The large increase in inhibition at high frequencies determines very low levels of activation. Thus, due to inhibitory circuits, the activity arriving to DG at different frequencies can be differentially decoded by mature and immature GC.We observed that at low frequencies, feedforward inhibition plays an important role in controlling activation whereas feedback inhibition is very low. As frequency increases, feedback inhibition also increases and adds to the control of activity at high frequencies.Despite the difference in intrinsic properties, activation differences between immature and mature GC are entirely generated by inhibition. The relation between recruited excitation and inhibition is crucial to determine GC activation . TherefoAdditionally, the growth of inhibitory currents in GC at high frequencies could be the result of specific recruitment of different types of interneurons affecting equally both GC. A recently published study in mature GC shows that dendritic inhibition is preferentially recruited with high frequency stimulation and grows along the train of pulses. Instead perisomatic inhibition is recruited at low and high frequencies, and decreases within a train . In the Our results show, in addition, that spikes in mature neurons are more time-locked to the incoming input than immature neurons, partly due to inhibition, but also probably to the stronger excitatory synapses they receive, as has been suggested. In vivo experiments, recording in the DG in animals with reduced neurogenesis show that they have reduced number of neurons with spiking broadly tuned to theta cycle, and most cells show a narrow tuning to a specific phase of theta, suggesting that immature cells could presumably contribute to the broadly tuned population. This notion may have functional implications in the influence that mature and immature neurons could have on CA3 .sparse code, in which information is encoded at any instant by the spiking of a small subset of neurons within the population, in general with low mean firing rates and high selectivity, that could increase information storage. And a dense code, in which most neurons are active at any moment and information is encoded by variations in firing rate. Our results suggest that mature and immature GC could represent these two different codes coexisting in the same structure and presumably serving at different functions. While mature GC could specifically represent a particular input, immature GC, with a higher ability to drive CA3, could be generating networks in CA3 that would presumably represent a particular experience occurring while they are immature.It has been proposed that there are two coding strategies in the nervous system , a spars5 particles/μl.A replication-deficient retroviral vector based on the Moloney murine leukemia virus was used to express RFP or GFP under a CAG promoter . RetroviFemale C57Bl/6J mice 6–7 weeks of age were housed at 4 mice per cage, with two running wheels. Running wheel housing started 2–4 days before surgery and continued until the day of slice preparation. For surgery, mice were anesthetized , and virus (1 µl at 0.125 µl/min) was infused into the dorsal area of the right DG using sterile microcapillary calibrated pipettes and stereotaxic references . Animals were killed for acute slice preparation 4 weeks after the surgery.PvalbCre;CAGFloxStopTom (PV-Tom) mice, B6;129P2-Pvalbtm1(cre)Arbr/J (PVcre) mice (B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J (Ai14) conditional reporter mice. Animals heterozygous for Cre and tomato were used.To generate re) mice  were croExperimental protocols were approved by the Institutional Animal Care and Use Committee Leloir Foundation  according to the Principles for Biomedical Research involving animals of the Council for International Organizations for Medical Sciences and provisions stated in the Guide for the Care and Use of Laboratory Animals.−, 2.5 KCl, 2.0 NaH2PO4, 25 NaHCO3, 0.5 CaCl2, 7 MgCl2, 20 dextrose, 1.3 Na+-ascorbate, 3.1 Na+-pyruvate, and 4 kynurenic acid. The right hippocampus was dissected and slices (400 µm thick) were cut transversally to the longitudinal axis in a vibratome and transferred to a chamber containing artificial cerebrospinal fluid : 125 NaCl, 2.5 KCl, 2.3 NaH2PO4, 25 NaHCO3, 2 CaCl2, 1.3 MgCl2, 1.3 Na+-ascorbate, 3.1 Na+-pyruvate, and 10 dextrose (315 mOsm). Slices were bubbled with 95% O2/5% CO2 and maintained at 30°C for >1 hr before experiments started. Salts were acquired from Sigma-Aldrich .Mice were anesthetized and decapitated at 4 weeks post injection (wpi). Brains were removed into a chilled solution containing (mM) 110 choline-Cl− neurons localized in the outer third of the GCL : 120 potassium gluconate, 4 MgCl2, 10 HEPES buffer, 0.1 EGTA, , 5 NaCl, 20 KCl, 4 ATP-tris, 0.3 GTP-tris, and 10 phosphocreatine . Loose-patch recordings were performed with ACSF-filled patch pipettes (5–6 MΩ). Field recordings were performed using patch pipettes (5 MΩ) filled with 3 M NaCl. Recordings were obtained using Axopatch 200B and Multiclamp 700B amplifiers, , digitized, and acquired at 20 KHz onto a personal computer using the pClamp10 software. Membrane capacitance and input resistance were obtained from current traces evoked by a hyperpolarizing step of 10 mV. Series resistance was typically 10–20 MΩ, and experiments were discarded if higher than 30 MΩ.Recorded neurons were visually identified by fluorescence and infrared DIC videomicroscopy. The mature neuronal population encompassed RFP the GCL . Whole-cslope, slope elicited at any given stimulus intensity was normalized to the fEPSPslope evoked at a stimulus intensity that evokes a maximal pop spike   blocked >70% of IPSCs. EPSCs were isolated by voltage clamping GC at the reversal potential of the IPSC measured for each individual neuron (∼60 mV). In turn, disynaptic IPSCs were recorded at the reversal potential of the EPSC (∼0 mV). When present, direct monosynaptic IPSC recorded in KYN  was subtracted from the IPSC. Synaptic excitatory and inhibitory conductances were computed as the EPSC or IPSC divided by the driving force at which the synaptic currents were recorded. 2-amino-5-phosphonovaleric acid  was perfused during recordings to block NMDA currents. We ruled out a possible influence of NMDA receptor-mediated currents on the activation of GC by recording spiking in the presence of AP5. No differences were found between spiking in ACSF and after application of AP5 in the bath (data not shown).Efficacy was computed as the average of the probability of occurrence of action potentials in the same frequency range as that given by the stimulus. Thus, a granule cell that responds with five consecutive action potentials in response to the train of 10 pulses, has an efficacy = 4/9 , while a GC with five discontinuous action potential firing, (without presenting two continuous) has an efficacy of 0/9. Efficacy was calculated for matGC and 4wpiGC .To dissect feedforward and feedback inhibitory components, after registering the total inhibition evoked by the stimulation of the mPP, mossy fiber synapses were blocked with DCG4  . In thist-test, Wilcoxon signed-rank test, two-sample F-test for equal variances, one-way or two-way ANOVA, and Bonferroni's post-hoc test, as indicated in the figure legends.Significant differences were assessed by two-tailed  review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent  The reviewers were wondering how much the observed frequency-dependent recruitment of GCs depends on the intrinsic properties of young vs. mature cells. This aspect should be addressed in the Discussion section.2) The reviewers ask why inhibition fails to influence the activity and the definition of the temporal window for action potential generation in young cells. The reviewers would have expected that although weak, it should have some effect on the activity of the young cells. Is inhibition indeed as weak as shown in the study or is the applied protocol insufficient to observe functional consequences?3) Concerns related to the normalization paradigms were raised and should be addressed by the authors in the revised manuscript.4) The relationship between excitation and inhibition seems to be similar between both young and mature GCs. A better presentation of the interaction between excitation and inhibition in 5) The data related to the effects of DCGIV are interesting, however, the authors should discuss the mild but existing (and published) blocking effects of DCGIV on perforant path-mediated inputs onto GCs.6) Since there seems to be some overlap in the results between previous papers from the same and other groups, the novel aspects of the study should be emphasized and better discussed in the revised manuscript.6) The influence of running wheels on the obtained data remained unclear and should be addressed in the Discussion. Please see below the original major criticisms of the reviewers:Reviewer 1:1) The authors argue that young and mature GCs have both the intrinsic ability to discharge at high frequency on the basis of square current injections in the soma. However, this is a different situation than stimulating PP inputs. Young GCs have higher input resistances, slower membrane time constants than mature ones. Thus, their responsiveness to input frequency should be different. Indeed, young GCs have been demonstrated to be more responsive/excitable than mature ones due to lower activation threshold by the authors in Marin-Burgin, Science 2012. Please revise your statement or alternatively discuss the influence of intrinsic properties on the results.2) In the subsection “Inhibitory control of activity in mature and immature GC”: please mention that the broader window of excitation of young GCs is not only dependent on the weaker inhibition but also on the intrinsic properties such as slower membrane time constant of young GCs.3) It remains a bit unclear why the authors differentiate between residual and recruited inhibition because at the end they sum both and compare them with synaptic excitation. The authors argue that a differentiation between residual and induced inhibition needs to be performed because of different multiple-pulse dynamics of synaptic inhibition. However, the authors do not provide information on the extent of these differences and how they may potentially impact total inhibition and thus discharge activity.4) Which IN types may contribute to the feedforward inhibition? The authors argue that PV-INs are recruited by PP stimulation. However, the time course of inhibition is slower than for feedback inhibition. This stays in contrast to the perisomatic location of PV-IN synapses. Therefore, the authors should discuss more precisely which IN types could potentially contribute to FF inhibition in this circuitry.Reviewer 2:1) The authors conclude that immature GCs have a wider time window in which excitation can elicit spikes without being affected by inhibition (subsection “Inhibitory control of activity in mature and immature GC”) but it is not clear why the inhibitory conductance fails to provide functional inhibition. Either there is interesting biology that renders immature GCs insensitive to inhibitory conductances, or there is a less interesting  possibility that the stimulus paradigm is insufficient to detect the functional consequence of the inhibitory conductance. Even the relatively small conductance shown in 2) In light of the above concern, the authors should carefully consider the use of their stimulus normalization paradigm. This procedure was apparently developed for CA1, where most cells can be driven to spike whereas in the DG this appears not to be the case (the authors suggest that only ∼ 50% of cells spike with inhibition intact). Results from this group's previous study  shows the non-linear relationship between stimulus intensity and % of spiking cells, so it is hard to understand how the pSpike  provides a sensitive measure of the number of active perforant path fibers. The normalization paradigm shown in 3) The main conclusion is that inhibition dictates the frequency-dependent firing characteristics of mature and 4 wpi cells. But this is not immediately obvious from the data in In addition, it appears that the synaptic currents in 4) The dissection of feed-forward and feedback components of inhibition is potentially very interesting, but I am not convinced by the method since it relies entirely on the selective blockade of mossy fiber synapses by DCG-IV. In fact, 1 micromolar DCG-IV is well known to block medial peforant path evoked EPSCs i.e. . Is therReviewer 3:1) It is not clear how the use of running wheels in all the experiments affects the interpretation of the data. There is growing evidence that suggests that running changes rates of maturation of DGCs and afferent connectivity . Since one of the aims of this study is to probe differences between immature adult-born DGCs and mature DGCs, it is important that the authors directly address whether running has an effect on activation of 4 weeks old adult-born DGCs following mPP stimulation at different frequency-trains (by comparing with a non-running group).2) The notion that intrinsic properties, but local circuitry underlying inhibition and excitations, determines recruitment of immature and mature DGCs in response to PP stimulation is not new and has been addressed previously in Dieni et al., JNSc 2013. In this study, the authors used a dual pathway stimulation protocol (MML and OML) and found that ratio of inhibitory and excitatory drive onto DGCs is influenced by stimulus frequency and dictates spiking probability. The relevance of the main findings of this study should be integrated into the discussion of results here.3) The extent of mossy cell contribution to activation of DGCs following mPP stimulation in slices is not clear. Does stimulation of EC Layer II inputs to DG differentially activate 4 week vs. mature DGCs at 20 Hz? This caveat should be discussed. Reviewer 1:1) The authors argue that young and mature GCs have both the intrinsic ability to discharge at high frequency on the basis of square current injections in the soma. However, this is a different situation than stimulating PP inputs. Young GCs have higher input resistances, slower membrane time constants than mature ones. Thus, their responsiveness to input frequency should be different. Indeed, young GCs have been demonstrated to be more responsive/excitable than mature ones due to lower activation threshold by the authors inMarin-Burgin, Science 2012. Please revise your statement or alternatively discuss the influence of intrinsic properties on the results.The reviewer is correct in regard to the higher input resistance of immature GC. A direct consequence of this property is that they are more efficient at translating synaptic currents into membrane depolarization, even though they receive a weaker excitatory drive (due to the reduced number of contacts) as shown in We added some clarifications in the text to further emphasize this idea in the Results and Discussion sections.2) In the subsection “Inhibitory control of activity in mature and immature GC”: please mention that the broader window of excitation of young GCs is not only dependent on the weaker inhibition but also on the intrinsic properties such as slower membrane time constant of young GCs.As we stated in the previous point, the main difference in activation between young and mature GC is due to the inhibitory network rather than to intrinsic properties (as shown in 3) It remains a bit unclear why the authors differentiate between residual and recruited inhibition because at the end they sum both and compare them with synaptic excitation. The authors argue that a differentiation between residual and induced inhibition needs to be performed because of different multiple-pulse dynamics of synaptic inhibition. However, the authors do not provide information on the extent of these differences and how they may potentially impact total inhibition and thus discharge activity.We thank the reviewer for the observation. We agree that it was not very clear on the text the necessity to differentiate between recruited and residual inhibition. We changed the subsection “Synaptic mechanisms underlying frequency dependence and responsiveness of immature and mature GC“ to better explain how the different rising kinetics of the recruited inhibition between matGC and 4wpiGC determines a different time window in which inhibition restricts excitation.4) Which IN types may contribute to the feedforward inhibition? The authors argue that PV-INs are recruited by PP stimulation. However, the time course of inhibition is slower than for feedback inhibition. This stays in contrast to the perisomatic location of PV-IN synapses. Therefore, the authors should discuss more precisely which IN types could potentially contribute to FF inhibition in this circuitry.” to discuss this topic.We think that the slow FF inhibition we observe in 4wpiGC corresponds to perisomatic slower IPSC because in a previous study we directly stimulated perisomatic inhibition and found postsynaptic currents with slower kinetics in immature GC . The synaptic mechanism underlying the slower inhibition in 4wpiGC would require further investigation, but we hypothesize that perisomatic inhibition is incipient at this neuronal age and could, thus, be immature and physiologically different. We think that differences could still reside on PV+ interneuron contacts. However, we would need to investigate in detail that synapse, by doing pair recordings from PV neurons onto immature and mature GC. Those experiments are in our plans but will take time, presumably for a future study. We expanded the subsection “Inhibitory circuits in the control of activityReviewer 2:1) The authors conclude that immature GCs have a wider time window in which excitation can elicit spikes without being affected by inhibition (subsection “Inhibitory control of activity in mature and immature GC”) but it is not clear why the inhibitory conductance fails to provide functional inhibition. Either there is interesting biology that renders immature GCs insensitive to inhibitory conductances, or there is a less interesting  possibility that the stimulus paradigm is insufficient to detect the functional consequence of the inhibitory conductance. Even the relatively small conductance shown inin 4wpi cells should provide some degree of inhibition at 1 and 10 Hz.Based on the data shown here  and on o2) In light of the above concern, the authors should carefully consider the use of their stimulus normalization paradigm. This procedure was apparently developed for CA1, where most cells can be driven to spike whereas in the DG this appears not to be the case (the authors suggest that only ∼ 50% of cells spike with inhibition intact). Results from this group's previous study  shows the non-linear relationship between stimulus intensity and % of spiking cells, so it is hard to understand how the pSpike  provides a sensitive measure of the number of active perforant path fibers. The normalization paradigm shown inalso does not show that the pSpike is maximal (panel B), and there is a very poor fit of the fEPSP data (panel C). It is not clear why the normalization procedure is required for these experiments, but if it is, the authors should confirm that the fEPSP is linearly related to number of active PP fibers (that could be assayed by the fiber volley) and that their measure of input strength is unaffected by blockade of inhibition. It is important to have similar stimulation parameters to compare the effect of GABAergic inhibition shown inwith the results of. An alternative approach would be to compare the effect of PTX on spiking in each cell to allow a within-cell comparison, which would obviate the need for the normalization paradigm.A) We use normalization for important reasons. Due to variability the way the slice is cut, in the location of the stimulation electrode , the excitability of a particular slice, it is really impossible to compare across slices using stimulation intensity. We added an example of how different slices present different input output curves of activation in B) We agree that a better measure of the input is the fiber volley. Unfortunately it is not as reliable as the fEPSP in the recordings, in the sense that the signal is not always clearly present since it can be hidden by the stimulation artifact. For that reason we use the fEPSP as an indirect measure of the input. We do that because the fEPSP increases linearly with the fiber volley along a wide range (in the recordings where it was possible to measure it). We have changed the example for one that has incorporated the fiber volley and added an inset panel in C) We always measure the fEPSP in absence of PTX at the beginning of the experiment to have the same input activated in both control and PTX conditions. We did some experiments in the same cells and results were consistent with the data of the pooled slices.We have clarified the normalization protocol in the Results and in Materials and methods .3) The main conclusion is that inhibition dictates the frequency-dependent firing characteristics of mature and 4 wpi cells. But this is not immediately obvious from the data in, since the frequency-dependent patterns of excitation and inhibition shown in 3D and E appear to be very similar in mature and 4wpi GCs (despite their different spiking patterns). This could be an issue of presentation – perhaps illustrating frequency-dependent differences in the E/I ratio between mature and 4 wpi cells within the same graph would make this point more clear.In addition, it appears that the synaptic currents inwere recorded in AP5 but the spiking data was not. Since the NMDAR component could become a source of depolarization during high frequency stimulation, it should be included in the synaptic analysis or the authors should rule it out by demonstrating a lack of effect of AP5 on spiking.”.A) To better illustrate the frequency-dependent differences in the E/I ratio between mature and 4 wpi cells we added a panel in B) We were also concerned about the effect of AP5 on spiking. We had previously performed experiments controlling for the effect of AP5 on spiking and found no effect (we did not include those experiments in the previous version of the manuscript). We have now mentioned that observation in the Methods, subsection “Evoked postsynaptic currents and conductances”.4) The dissection of feed-forward and feedback components of inhibition is potentially very interesting, but I am not convinced by the method since it relies entirely on the selective blockade of mossy fiber synapses by DCG-IV. In fact, 1 micromolar DCG-IV is well known to block medial peforant path evoked EPSCs (i.e.and many subsequent studies). The authors should repeat this critical control experiment using EPSCs rather than spiking, since supra-threshold excitation could potentially mask a suppression of MPP synaptic strength that would confound all the analysis shown in. Furthermore, since GC spiking presumably is required for feedback inhibition, it is difficult to understand how the feedback IPSG is greatest during 40 Hz stimulation, which is the frequency when GCs spike the fewest action potentials (comparewith). Is there a frequency-dependent recruitment of feedback interneurons? Showing the FB-IPSC during the train might be useful in this regard.A) We were aware that DCG4 affects mPP pathway (we added a reference of that in the text now) and for that reason we could not do experiments of the effect of that drug on GC spiking. We used DCG4 only to study feed-forward inhibition, which is also recruited by the mPP pathway. However, inhibitory interneurons tend to display a very low threshold to respond to afferent inputs. Therefore, we reasoned that presumably at the stimulation intensity used in all experiments (50% fEPSP), interneurons would be above threshold even though the drug increased their spiking threshold. Thus, FF-IPSC recorded in GC would not be affected by DCG4. To test this idea we did experiments checking the effect of DCG4 on PV interneurons activation upon stimulation of the mPP . Indeed,B) Regarding feedback inhibition, we added a supplementary figure  showing Reviewer 3:1) It is not clear how the use of running wheels in all the experiments affects the interpretation of the data. There is growing evidence that suggests that running changes rates of maturation of DGCs and afferent connectivity . Since one of the aims of this study is to probe differences between immature adult-born DGCs and mature DGCs, it is important that the authors directly address whether running has an effect on activation of 4 weeks old adult-born DGCs following mPP stimulation at different frequency-trains (by comparing with a non-running group).This point is worth clarifying. First, we consider running wheel housing a condition that is slightly closer to the wild environment that normal caging, so animals can voluntarily run. In addition, we use running wheels to increase neurogenesis at the time of viral transduction, which in turn increases the density of labeled cells, as shown in several previous papers from van Praag 1999 onwards. In our labs, we have routinely used wheels for that purpose  The notion that intrinsic properties, but local circuitry underlying inhibition and excitations, determines recruitment of immature and mature DGCs in response to PP stimulation is not new and has been addressed previously in Dieni et al.,JNSc 2013. In this study, the authors used a dual pathway stimulation protocol (MML and OML) and found that ratio of inhibitory and excitatory drive onto DGCs is influenced by stimulus frequency and dictates spiking probability. The relevance of the main findings of this study should be integrated into the discussion of results here.We agree that the findings of that study are relevant to ours and we apologize for the missing reference; we have added it in the Introduction and Discussion.3) The extent of mossy cell contribution to activation of DGCs following mPP stimulation in slices is not clear. Does stimulation of EC Layer II inputs to DG differentially activate 4 week vs. mature DGCs at 20 Hz? This caveat should be discussed.Indeed mossy cells could contribute to excitation and inhibition that GC receive, but we did not address this issue in our work. Nevertheless, we have added a line in the Discussion regarding this topic together with a reference: “Also, there could be a differential contribution of mossy cells, which can affect inhibition and excitation on GC ”.


Ambra1, after exposure to a hypoxia mimetic, cobalt chloride (CoCl2). We observed that, upon CoCl2 administration, activation of the apoptotic machinery was concomitant with down-regulation of the pro-autophagic factor Ambra1, without affecting transcription. Additionally, co-treating the cells with the caspase inhibitor z-VAD-FMK did not restore Ambra1 protein levels, this implying the involvement of other regulatory mechanisms. Partial re-localization of Ambra1 mRNA to non-translating fractions and cytoplasmic P-bodies was further detected. Thus, in this pseudohypoxic context, Ambra1 mRNA translocation to P-bodies and translational suppression correlated with increased cell death.Hypoxia has been associated with several pathological conditions ranging from stroke to cancer. This condition results in the activation of autophagy, a cyto-protective response involving the formation of double-membraned structures, the autophagosomes, in the cytoplasm. In this study, we investigated the cellular mechanisms regulating the autophagy gene  Interestingly, no significant changes in Ambra1 mRNA levels were observed at 18h, the time-point at which protein levels decreased  was unchanged in control and pseudohypoxic conditions  to non-translating fraction upon pseudohypoxia, resulting in the decrease of Ambra1 protein levels. Since un-translating mRNAs are often stored in cytoplasmic P-bodies, we analyzed the localization of Ambra1 mRNA in the cytoplasm before and after exposure to hypoxic conditions. Even though Ambra1 mRNA is stable in stress conditions, our data suggest that, at least in part, it is sequestered and stabilized in P-bodies, thus becoming inaccessible to the protein translation machinery. This sequestration may then result in a decrease in protein levels, in time with activation of cell death and suppression of the pro-survival pathway of autophagy. By contrast, Beclin 1 mRNA does not relocate to P-bodies at all in similar conditions, indicating that the mechanism here described does not concern all autophagy regulators. Although in general the decay of mRNAs is a consequence of prolonged stress conditions that lead to apoptosis, it is plausible that the maintenance of a pool of Ambra1 mRNA may represent an important reserve for the cell in case normal conditions are restored. Indeed, this would allow Ambra1 prompt translation.Our data implies the involvement of a translational control in this phenomenon. To test this hypothesis, polysome profile analysis was performed and we observed translocation of Ambra1 mRNA moves to P-bodies, it certainly needs further investigation. There is evidence for interaction of P-bodies with actin filaments and/or microtubule structures [Ambra1 mRNA with or without CoCl2 treatment and its effect on cell death and survival in hypoxic conditions. Furthermore, since the co-localization of Ambra1 mRNA and P-bodies after CoCl2 treatment is only partial, it would be interesting to know if this messenger can also localize in other specific foci, such as stress granules.As for the mechanism by which ructures ,22. Alsoructures . TherefoS1 Fig2 500 μM for 8 hours in presence or not of chloroquine 20 μM (CHQ) for the last hour of treatment.Representative images of HeLa cells not treated (NT) ore treated with CoCl(EPS)Click here for additional data file.S2 FigAmbra1 mRNA (red) and Ge-1 protein (green) co-localization after 18 hr treatment with CoCl2. Blue: Nuclear staining with DAPI. (B) total Ambra1-positive dots were counted in treated and untreated cells: the graph shows that their total number is unchanged.(A) The image represents 3D-analysis of (EPS)Click here for additional data file.S3 Fig2.  Ge-1 protein;  Beclin 1 mRNA;  Merge image. Scale bars: 10 μmLeft panels (A-C) represents control cells, whilst right panels (D-F) shows HeLa cells treated for 18 hr with 500 μM CoCl(EPS)Click here for additional data file.


Stroke immediately sets into motion sustained excitotoxicity and calcium dysregulation, causing aberrant activity in neuronal nitric oxide synthase (nNOS) and an imbalance in the levels of nitric oxide (NO). Drugs targeting nNOS-originated toxicity may therefore reduce stroke-induced damage. Recently, we observed that a redox-modulating agent of the NO metabolome, S-nitrosoglutathione (GSNO), confers neurovascular protection by reducing the levels of peroxynitrite, a product of aberrant NOS activity. We therefore investigated whether GSNO-mediated neuroprotection and improved neurological functions depend on blocking nNOS/peroxynitrite-associated injurious mechanisms using a rat model of cerebral ischemia reperfusion (IR).1412 of nNOS. GSNO treatment of IR animals decreased IR-activated nNOS activity and neuronal peroxynitrite levels by reducing nNOS phosphorylation at Ser1412. The Ser1412 phosphorylation is associated with increased nNOS activity. Supporting the notion that nNOS activity and peroxynitrite are deleterious following IR, inhibition of nNOS by its inhibitor 7-nitroindazole or reducing peroxynitrite by its scavenger FeTPPS decreased IR injury. GSNO also decreased the activation of AMP Kinase (AMPK) and its upstream kinase LKB1, both of which were activated in IR brain. AMPK has been implicated in nNOS activation via Ser1412 phosphorylation. To determine whether AMPK activation is deleterious in the acute phase of IR, we treated animals after IR with AICAR (an AMPK activator) and compound c (an AMPK inhibitor). While AICAR potentiated, compound c reduced the IR injury.IR increased the activity of nNOS, the levels of neuronal peroxynitrite and phosphorylation at SerTaken together, these results indicate an injurious nNOS/peroxynitrite/AMPK cycle following stroke, and GSNO treatment of IR inhibits this vicious cycle, resulting in neuroprotection and improved neurological function. GSNO is a natural component of the human body, and its exogenous administration to humans is not associated with any known side effects. Currently, the FDA-approved thrombolytic therapy suffers from a lack of neuronal protective activity. Because GSNO provides neuroprotection by ameliorating stroke’s initial and causative injuries, it is a candidate of translational value for stroke therapy. Ischemic stroke  and transient ischemic attack (TIA) obstruct oxygen and nutrient supply to the brain. Instantaneously, the obstruction of blood supply causes necrotic neuronal death in the injury core; however, even greater apoptotic neuronal damage is caused by nitroxidative stress and neuroinflammation in the cortical penumbra and hippocampal areas . NeuronanNOS contributes approximately 90% to NOS activity in normal rodents . Of the 331; however, immediately following stroke injury, nNOS is activated by NMDA receptor-mediated excitotoxicity and a sustained calcium influx through site-specific phosphorylation (Ser1412) and denitrosylation (Cys331). In such an environment, nNOS-derived NO is converted to peroxynitrite by an instantaneous diffusion-limited reaction with superoxide -1H-imidazole-4-carboxamide (AICAR) and 5,10,15,20-tetrakis(4-sulfonatophenyl) porphyrinato iron (III) (FeTPPS) were obtained from Abcam  and Calbiochem, , respectively. All other chemicals and reagents used were purchased from Sigma-Aldrich , unless stated otherwise.GSNO was purchased from World Precision Instruments  and dorsomorphin dihydrochloride (Compound c) from Tocris Bioscience . 5-Amino-1- arginine  to L- citrulline in the presence or absence of the competitive NOS inhibitor L-NAME using an NOS assay kit from Cayman Chemicals  and following the instructions described therein. Total cNOS activity was determined by subtracting calcium independent NOS activity. The nNOS inhibitor, 7-NI (50 mg/kg), and the NOS inhibitor, L-NAME (30 mg/kg), were used to differentiate between endothelial nitric oxide synthase and nNOS activity [NOS activity in the penumbra brain area was determined by the conversion of L-[4,5-activity . The actN-(1-naphthyl)-ethylene-diamine dihydrochloride and yields a reddish-violet diazo dye whose absorbance was measured in the visible range at 540 nm. The assay was performed as described by the manufacturer.The levels of NO (as nitrite) in the penumbral brain area were determined using a Roche’s test “Nitric oxide colorimetric assay” kit. The estimation was based on the following reaction: nitrites + sulfanilamide + t test as appropriate or one-way ANOVA with Tukey post hoc for three or more groups. Values were expressed as mean ± standard deviation (SD). A p value less than 0.05 was considered statistically significant.Statistical analysis was performed using Graph pad prism 5.01 software . StatistIncreased nNOS activity and the participation of nNOS-derived peroxynitrite in stroke injury have been reported . HoweverTo examine whether GSNO-mediated decreased levels of peroxynitrite in IR brain correlate with neuroprotection, we evaluated neuronal degeneration using Nissl Figure a, axonal1412) [1412). We observed IR-induced increased levels (p < 0.001) of phosphorylated Ser1412 at 1 h of reperfusion  . In such1412) . Peroxyn412 . In n Figure a, b, res7-NI, a selective nNOS inhibitor, is reported to reduce infarct volume in IR models , 40. How1412 and thus its increased activity is regulated by several kinases, including Akt and AMPK. While Akt is inhibited [nNOS activation occurs via phosphorylation of Sernhibited , AMPK isnhibited . Peroxyn1412), leading to peroxynitrite formation. We observed that the AMPK inhibitor Comp c as well as the peroxynitrite scavenger FeTPPS-treated animals (at 1 h after IR) had significantly reduced (p < 0.001) infarct volume (Figure AMPK activation in the acute phase of IR is deleterious, and these injurious effects are linked with increased peroxynitrite levels , 41. Pere Figure a and impe Figure b. In cone Figure a, b. Howe Figure a, b.FiguStroke is one of the most disabling conditions in the United States. The most severe injury after ischemia or reperfusion happens within a couple of hours of initial stroke, leading to the wisdom in stroke care that “time is brain” . This coNeuroimaging data in patients who have stroke or transient ischemic attack show that the territory of the middle cerebral artery branches suffers from multiple infarcts more frequently than other areas . Inflamm1412, thus maintaining its own formation and causing sustained neuronal cell death. In this study, we tested whether GSNO down regulates aberrant nNOS activity by reducing the levels of peroxynitrite through reversing peroxynitrite-induced phosphorylation/dephosphoryaltion mechanisms as elucidated in Figure Early neuronal cell death in IR is caused by nNOS activation and peroxynitrite formation . Peroxyn2+ influx, leading to secondary modification of nNOS, such as denitrosylation of Cys331, and phosphorylation of Ser1412 [1412 [1412 as early as 1 h following IR (Figure 1412 (Figure nNOS activation requires NMDA-receptor-mediated Ca Ser1412 . Several12 [1412 , 50–54, 1412 of nNOS, thus increasing nNOS activity. Unlike AMPK, Akt is inhibited following IR in a time dependent manner, likely by peroxynitrite [172 by its upstream kinase LKB1. Peroxynitrite has been reported to be potent activator of LKB1/AMPK [Decreased activation of AMPK and reduced IR injury in nNOS KO mice indicate that AMPK activation is dependent on nNOS activity . AMPK acynitrite . Our datynitrite . On the ynitrite , 58; addynitrite . It is hynitrite , 41. IR-KB1/AMPK . In suchOur data on nNOS and AMPK are consistent with the notion that sustained activation of AMPK results in detrimental effects in stroke , 41. AMPIn the present study, we have shown that the nNOS/peroxynitrite/AMPK vicious cycle is an attractive therapeutic target for stroke therapy. The study provides evidence that GSNO-mediated mechanisms of phosphorylation and dephosphorylation of nNOS and AMPK, in inhibiting aberrant nNOS and thus reducing peroxynitrite, protected against neuronal loss and aided in functional recovery following IR Figure . GSNO is