{"paper_id":"0caa5e12-8248-45d4-99f8-60fc9d9e8983","body_text":"1 \n \nValidating FOXO4 as a therapeutic target for protecting against ischemia-reperfusion-\ncaused neuronal injury  \n \n \n \nYasin Asadi, Frahad Gorjipour, Anand Chakroborty, Rozenn K. Moundounga, Abena Dwamena, \nErin Gilstrap, and Hongmin Wang* \n \n \nDepartment of Pharmacology and Neuroscience, Garrison Institute on Aging, and Center of \nExcellence for Translational Neuroscience and Therapeutics,  Texas Tech University Health \nSciences Center (TTUHSC), School of Medicine, Lubbock, Texas 79430-6592, USA.  \n \n \n \n \n*Corresponding author:  \nHongmin Wang, Ph.D., Professor, Department of Pharmacology and Neuroscience, Texas Tech \nUniversity Health Sciences Center (TTUHSC), School of Medicine, Lubbock, Texas 79430 -\n6592, USA. Tel: +18067437089, Email: Hongmin.Wang@ttuhsc.edu \n \n \n \n \n \n \n \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted March 14, 2025. ; https://doi.org/10.1101/2025.03.13.643180doi: bioRxiv preprint \n\n2 \n \nAbstract: \nBackground: Previous data suggest that in the conditions of I/R in mice and the oxygen-glucose \ndeprivation (OGD) in cell cultures, FOXO4 facilitates inflammation and oxidative stress in non-\nbrain tissues, indicating that downregulation of FOXO4 may be neuroprotective in I/R-induced \ninjury in the brain. However, this possibility has not been tested in the cerebral I/R condition. \nMethod: FOXO4 knockout (KO) and wild-type (WT) primary neuronal cultures were treated \nwith an oxidative stress inducer, menadione (MD), or OGD, and then cell viability was assessed \nvia ATP and MTT assays. The KO and WT mice at 2-3 months were subjected to one-hour (h) \ntransient middle cerebral artery occlusion (tMCAO). Mice were sacrificed after 24 h for TTC \nstaining or after 48 h for immunohistochemical staining. Alternatively, animals were allowed to \nsurvive 1-10 days after tMCAO to test their functional recovery. Furthermore, using a structure-\nbased approach combined with cell-based assays, we screened FOXO4 inhibitors and identified \nactinomycin D (ActD) as a potent FOXO4 inhibitor. We also tested the therapeutic role of ActD \nin both in vitro and in vivo models of ischemic stroke.  \nResult: KO of FOXO4 reduced the infarct volume, improved animal survival, decreased \nneurological deficits, and enhanced functional recovery compared to WT mice. \nImmunohistochemical staining of astrocytes and microglia revealed that KO brains showed a \nreduced number of astrocytes and microglia in the peri-infarcted area two days after I/R. Western \nblot analysis of proinflammatory cytokines, IL-1β, IL-6, and TNF-α, indicated decreased levels \nof proinflammatory cytokines two days following I/R. The identified FOXO4 inhibitor, ActD, \nattenuated oxidative stress- and OGD-induced neuronal death. ActD also reduced neuronal injury \nof the brain and enhanced functional recovery in WT mice following tMCAO.  \nConclusion:  We conclude that disrupting FOXO4 is neuroprotective and the identified \ninhibitor, ActD, may be a therapeutic agent for treating ischemic stroke-induced brain injury.  \n \nKeywords: FOXO4; stroke; neuroinflammation; ischemia; reperfusion; transient middle cerebral \nartery occlusion; inhibitor; therapy; functional recovery \n \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted March 14, 2025. ; https://doi.org/10.1101/2025.03.13.643180doi: bioRxiv preprint \n\n3 \n \nIntroduction  \nStroke is a major health problem: it is the fifth leading cause of death and the leading cause of \ndisability in the United States. Ischemic stroke is the most frequent type of stroke, affecting \nmillions of people across the globe.1 Despite intensive research, there is still no effective \ntreatment for this condition, and tissue plasminogen activator remains the only Food and Drug \nAdministration-approved medication for treating ischemic stroke.2 While it is necessary to \nrestore cerebral blood flow, reperfusion itself induces the production of large amounts of \nreactive oxygen species that further damage proteins and other intracellular macromolecules to \nexacerbate brain injury.3 Moreover, ischemia/reperfusion (I/R) also disrupts the blood-brain \nbarrier, resulting in infiltration of leukocytes and inflammatory responses to aggravate brain \ninjury.4 To develop effective therapeutics, it is necessary to identify and validate additional \ntherapeutic targets.  \nTranscriptional gene regulation, governed primarily by numerous transcriptional factors, \nis one of the upstream parts potentially modulating I/R-induced brain injury. The Forkhead Box \nProteins O (FOXOs), consisting of four structurally and functionally related proteins, FOXO1 \n(also referred to as FKHR), FOXO3 (also known as FOXO3a), FOXO4 (also known as AFX1), \nand FOXO6, are one family of transcription factors, representing mammalian homologs of daf-\n16 in C. elegans and playing a crucial role in cell survival, cell proliferation, metabolism, \nresponse to oxidative stress, apoptosis, and aging.5 Under oxidative stress or the absence of the \ncellular survival drive of growth factors, FOXOs translocate to the nucleus and upregulate a \nseries of target genes, thereby promoting cell growth arrest and apoptosis.6,7  \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted March 14, 2025. ; https://doi.org/10.1101/2025.03.13.643180doi: bioRxiv preprint \n\n4 \n \nDespite the highly structural and functional similarities of FOXOs, previous data have \nsuggested that the physiological roles of FOXOs are functionally diverse in mammals.8,9  For \ninstance, FOXO1 deficiency is embryonically lethal and FOXO3 deficiency exhibits age-\ndependent infertility in females in mice; however, the loss of FOXO4 does not cause any notable \nchanges in the mouse, suggesting that FOXO4 may play a different role from other FOXO \nmembers.8,10 FOXO4 was found to promote cell death in the heart and the liver following I/R, \nwhile downregulation of FOXO4 confers protection against I/R-induced tissue injuries in these \norgans.11,12 Moreover, following cardiac ischemia, FOXO4 enhances the interaction of \nleukocytes with the endothelial cells of blood vessels to promote early tissue inflammation.13 In \ncontrast, downregulation of FOXO4 suppresses oxidative stress-induced cell death in \nproangiogenic cells and promotes neovascularization in ischemic limbs.14 Functionally, FOXO \nactivity is negatively regulated by phosphorylation via the phosphoinositide 3-kinase-Akt \npathway, a well-known cell survival pathway.6,7 Ischemic pre-conditioning upregulates Akt \nactivity, leading to FOXO inhibition and promotion of neuronal survival against a subsequent \nsevere ischemic insult.15 These prior studies suggest that FOXO4 may be a therapeutic target for \nischemic stroke, yet this has not been tested. In this study, we examined the role of FOXO4 in \nneuronal injury in vitro and in vivo following oxidative stress, oxygen-glucose deprivation \n(OGD), and I/R, using the primary neuronal cultures and FOXO4 knockout (KO) mice combined \nwith different approaches.  \nMaterials and Methods  \nMice  \nAll animal-related experiments and procedures were approved by the Institutional Animal Care \nand Use Committee of the Texas Tech University Health Science Center and were in compliance \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted March 14, 2025. ; https://doi.org/10.1101/2025.03.13.643180doi: bioRxiv preprint \n\n5 \n \nwith the National Institute of Health Guide for the Care and Use of Laboratory Animals. Animals \nwere maintained on a 14-/10-hour light/dark cycle with a maximum of four mice per cage able to \naccess water and food ad libitum. FOXO4 global knockout (KO) mice in the background of FVB \nhave been described before13 and were used for ischemic stroke-induced brain infarct studies, \nwhile the same strain of wild-type (WT) mice was used as the control. For animal behavioral \nstudies, the KO and WT male mice with a hybrid background (F1 generation) produced from the \nheterozygous KO FVB females crossed with the WT C57BL/6 males were used, because of the \nretina degeneration problem in the FVB mice. As FOXO4 is an X chromosome-linked gene, in \nthis breeding strategy, 50% of males would be FOXO4 KO males and 50% would be WT males \nthat were used as controls for behavioral studies following ischemic stroke. More than one litter \nof animals was used in each experiment. The male animals between 8–12 weeks of age with a \nbody weight of 25-30 g were used in the study. Sample size calculations and power analysis \nwere performed according to our previously described methods16 using the statistical software \nStata (StataCorp LP, College Station, TX, USA).  \nPrimary neural cell culture  \nPrimary cortical neuronal cultures were prepared from wild-type and FOXO4 KO mice at \npostnatal day 0 according to our previously described methods.17 Briefly, the cerebral cortex was \nisolated and digested with 5 ml of 0.25% trypsin/EDTA supplemented with 75 µl 0.1% DNase \n(2000 UI/mg). After 15-20 minutes of digestion at 37°C, the digested tissues were centrifuged at \n1000 × g for 3 minutes at room temperature. The supernatant was discarded, and the resulting \ntissue pellets were resuspended with fetal bovine serum (FBS) to stop the digestion. After \ncentrifugation, the tissues were resuspended with primary neuronal culture medium (Neurobasal \nmedium supplemented with 2% B27, 2 mM L-glutamine, and penicillin/streptomycin) and then \npipetted up and down several times using serological pipets to disrupt tissues. After filtering \nthrough a 70 μm nylon cell strainer, the cell suspension was adjusted and plated in poly-DL-\nlysine-coated 12-well plates. After 7 days of incubation at 37°C in a 5% CO2 incubator, the cells \nwere treated with 20 µM menadione (MD) for 24 h or oxygen-glucose deprivation (OGD) for 3 h \nand then in normal culture condition for 21 h in the absence or presence of an identified FOXO4 \ninhibitor, actinomycin D (ActD, 0.1 µM).  \nOxygen-glucose deprivation (OGD)  \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted March 14, 2025. ; https://doi.org/10.1101/2025.03.13.643180doi: bioRxiv preprint \n\n6 \n \nWT and FOXO4 KO primary neuronal cultures were subjected to OGD at 7-10 in vitro days \naccording to previous methods.17 Briefly, the neuronal culture medium was replaced with a \nglucose-free Hank’s Balance Salt Solution (HBSS) and then the culture plates were placed into a \nhypoxic chamber where the air was replaced with a 5% N2 and 95% CO2. After 3 h of OGD \ncondition, the neuronal cultures were returned to the normal culture condition, and after 21 h cell \nviability was measured via ATP and MTT assays.  \nATP assay  \nWe used an ATP detection kit (Cayman Chemical, Ann Arbor, Michigan, USA) to measure ATP \nlevels in cultured neurons according to the manufacturer’s instructions. \nMTT assay \nCell viability in cell cultures was assessed using an MTT assay kit (R&D Systems, Minneapolis, \nMN, USA) based on the company’s guide. \nTransient middle cerebral artery occlusion (tMCAO)  \nThe tMCAO procedure was performed according to previously described methods.18,19 Briefly, \nanesthesia of mice was induced with 5% isoflurane and then maintained with 2% isoflurane. The \nleft hemisphere was subjected to tMCAO using a silicon-coated monofilament (RWD, Sugar \nLand, TX, USA) and after 1 hour, the monofilament was removed. A Laser Speckle Imaging \nSystem (RWD, Sugar Land, TX, USA) was used to monitor cerebral blood flow through MCA. \nOnly the mice with successful occlusion of MCA were included in the studies, as reflected by \nreduced blood flow by over 80% and reperfusion with more than 75% recovery of blood flow in \nthe MCA.  \nDrug treatment of mice \nFor drug treatment studies, WT males were randomly separated into the vehicle group and drug \ntreatment group using an online tool (http://www.graphpad.com/quickcalcs/). Either 0.25 or 0.5 \nmg/kg of actinomycin D or vehicle was intraperitoneally injected into WT 1 h before tMCAO. \nAfter 24 h, mice were euthanized to collect the brains for TTC staining. Alternatively, the mice \nwere allowed to survive for 1-10 days to assess functional recovery. \n \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted March 14, 2025. ; https://doi.org/10.1101/2025.03.13.643180doi: bioRxiv preprint \n\n7 \n \n \nTTC staining and measurement of infarct volumes \nMice were euthanized 24 h after I/R to isolate the brains. After being sectioned into 2 mm slices, \nthe brain sections were incubated with 2% of 3,5-triphenyl tetrazolium chloride (TTC, Sigma, \nSaint Louis, MO, USA) at 37°C for 20 minutes. Subsequently, the sections were fixed in 4% \nparaformaldehyde and then imaged. The infarcted volume was measured using Image J software \nand calculated as previously described.20 \nAssessment of modified neurological deficits  \nAt 1, 3, 5, and 7 days following I/R, a modified neurological severity score (mNSS) system was \napplied to assess the motor and sensory functions in mice.21 Based on this system, a scale of 0 to \n14 was given to an animal, with 0 for normal neurological behavior and 14 for the maximal \nneurological deficit.22   \nNovel object recognition (NOR) test \nA box (50 × 50 × 30 cm) containing two objects was used for the NOR test at 7 days following \nI/R. On the first day of the test, mice were placed in the box and allowed to move freely for 10 \nminutes. On the second day, one of the objects was replaced with a new one (novel object), and \nthe other remained as a familiar object. Then, mice were placed into the box to move and explore \nthe objects. By using a camera, the total time spent exploring each object was recorded, and the \nratio of the new object exploration was calculated as the discrimination index (DI = (Tnovel -\n Tfamiliar)/(Tnovel + Tfamilar).19 \nY-maze test \nIn the training session, each mouse was placed in an arm of the Y maze (start arm) with one of \nthe arms blocked with an opaque door (novel arm) and allowed to explore the start arm and \nremaining arm (other arm) for 5 minutes. The mouse was then removed and returned to its home \ncage, and the novel arm was unblocked. After one hour, in the test trial, the mouse was replaced \non the start arm and allowed to explore all three arms freely for 2 minutes. The number of arm \nentries was recorded by the experimenter blind to the genotype of the mice.  \nImmunofluorescent staining \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted March 14, 2025. ; https://doi.org/10.1101/2025.03.13.643180doi: bioRxiv preprint \n\n8 \n \nBrains were coronally cut into 3 parts and the frontal and occipital parts were used for TTC \nstaining to define the penumbral area. The middle part was washed and fixed in 4% \nparaformaldehyde. Then, the 16 μm thickness sections were prepared on a Cryostat (Leica, \nBuffalo Grove, IL, USA), and the brain sections were processed based on the prior method.23 \nThe primary antibodies used were anti-GFAP (1:50, EMD Millipore, #MAB360), anti-Iba1 \nantibody (1:50, Cell Signaling Technology, #31659), anti-CD45 (Invitrogen #MA180090), anti-\nLy6G (Cell Signaling #87048), and  anti-CD11 (Developmental Studies Hybridoma # \nM1/70.15.11.5.2). The secondary antibodies were Texas red-conjugated anti-rabbit and FITC-\nconjugated anti-mouse antibodies. DAPI (IHC-tek#IW-1404) was used to stain the nuclei. \nThe images were captured with a fluorescence microscope (Echo Revolve, San Diego, \nUSA) and quantified with Image J software.24 The ratio of the red- or green-positively stained \ncells in each field was calculated by the total red or green cells divided by the total number of \ncells (DAPI positively stained cells).25,26 \nWestern blot  \nThe cortex of the left (i.e. the I/R side) hemisphere of each mouse brain was collected for lysate \npreparation in the RIPA buffer supplemented with a protease cocktail. The brain tissues were \nsonicated in the lysate buffer and the lysates were centrifuged at 12,000 x g for 4 min at 4°C. The \nsupernatant was collected and then the total protein concentration was measured before being \nsubjected to the SDS-PAGE and transferred to the nitrocellulose membrane according to \nprevious methods.17 The antibodies used were anti-IL-1β (1:1000, Cell Signaling, #63124), anti-\nIL-6 (1:1000, Cell Signaling, #12912), anti-TNFα (1:1000, Cell Signaling, #11948), and anti-β-\nactin (1:1000, Santa Cruz Biotechnology, #sc-1616). The secondary antibodies used were anti-\nrabbit IgG, HRP-conjugated antibody (Cell Signaling, #7074), and anti-mouse IgG, HRP-\nconjugated antibody (Cell Signaling, #7076). The western blot results were documented using an \nimaging system (C400 Azure Biosystem), and protein band intensities were measured using the \nNIH Image J software.  \nStructure-based drug screening  \nWe utilized the Autodock Vina in the PyRx 0.9.8 software (https://pyrx.sourceforge.io/) to \nperform the docking-based virtual screening of several small compound libraries against FOXO4 \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted March 14, 2025. ; https://doi.org/10.1101/2025.03.13.643180doi: bioRxiv preprint \n\n9 \n \nDNA-binding domain (DBD, Protein Data Bank,  https://www.rcsb.org/; PDB: 3L2C), with a \ngrid box of 20 x 20 x 20 Å positioned within the box center coordinates -1.0, 13.0 and -15.0 \nfocusing on the DBD of FOXO4. The FOXO4 DBD was modified by removing the DNA chains \nduring computational analyses. We screened 7,742 compounds from four different chemical \nlibraries, including the NIH Clinical Collections  \nhttps://pubchem.ncbi.nlm.nih.gov/source/NIH%20Clinical%20Collection), the FDA-approved \nand clinical drugs (https://www.targetmol.com/library-collection-2/sorted-by-clinical-phase and \nhttps://www.apexbt.com/ discoveryprobetm-clinical-fda-approved-drug-library.html), and a \nnatural product library (https://www.apexbt. com/discoveryprobetm-natural-product-library-plus-\n1.html). The lead compounds were then docked onto FOXO1 DBD (PDB: 3COA) and FOXO3 \nDBD (PDB: 2UZK) and any candidates that also showed a high affinity to FOXO1 or FOXO3 \nwere eliminated from the list.  \nCell-based reporter assay \nHEK293 cells expressing a human FOXO4 plasmid (Addgene #17549)27 and a luciferase \nreporter plasmid (Addgene, # 34571) driven by a FOXO4 target gene Arginase-1 promoter13,28, \nwere treated with triplates with 100 nM of identified compounds or vehicle (DMSO) for 8 h and \nthe treated cells were then subjected to a luciferase-based reporter assay using a luciferase \nreporter substrate kit (Abcam, Waltham, MA, USA).  Moreover, to eliminate the compounds that \nalso showed inhibitory effect on FOXO1 and FOXO3, we performed additional reporter assays \nwith HEK293 cells co-transfected with the 3XIRS luciferase (Luc) reporter that contains 3 \ncopies of the insulin-responsive sequence29 or FHRE-Luc30 together with FOXO3 plasmid \n(Addgene #8360)30.  \n \nMolecular docking \nWe used SMILES31 notation for ActD (CHEMBI: 27666) ligand against the FOXO4 (PDB: \n3L2C) to predict the protein-ligand binding sites in SwissDock 202432 and Protenix \n(https://protenix-server.com/add-prediction), keeping multiple sequence alignment and seed \n95326 as a parameter. AutoDock Vina has applied a method for SwissDock, a grid box of 20 x \n20 x 20 Å positioned within box center coordinates -1.0, 13.0, and -15.0 focusing on the DNA-\nbinding domain of FOXO4 for accurate docking predictions of actinomycin D. The PDB model \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted March 14, 2025. ; https://doi.org/10.1101/2025.03.13.643180doi: bioRxiv preprint \n\n10 \n \n(3L2C) was modified by removing the DNA chains during compuational analyses. A separate \nanalysis was executed with the matching coordinates keeping Attracting Cavities for higher \nconfidence predictions in SwissDock. The results were visualized using UCSF ChimeraX33 on \nMacintosh System.  \nStatistical analysis  \nStatistical analyses were conducted using GraphPad Prism version 9.0 statistical software. \nDifferences between the two groups were assessed using an unpaired t-test. For comparisons \nbetween different groups of animals in the RAWM, repeated measures, and two-way ANOVA \nwith Tukey’s post hoc test were utilized. All numerical data were presented as mean ± SD or \nSEM. P < 0.05 was regarded as statistically significant.  \n \nResults  \nKO of FOXO4 reduces neuronal death caused by oxidative stress and OGD in primary \ncortical neuronal cultures \nTo assess the effect of KO of FOXO4 on oxidative stress-induced cell death, we treated the \nprimary cortical neuronal cultures with an oxidative stress inducer, menadione (MD)18, and then \nmeasured the viability of the cells via ATP and MTT assays. Our results indicated that the KO \nneurons showed increased cell survival as reflected by higher ATP levels in KO cells compared \nto WT cells (Fig. 1a). Increased viability in the KO cells was also supported by the MTT assay \n(Fig. 1b). However, in those neuronal cultures without MD treatment, the cellular viability did \nnot differ between WT and KO neurons (Fig.1a, 1b). To validate these results, we further treated \nthe two types of cultured neurons with an in vitro model of ischemic stroke, OGD, and then \nexamined cell viability. In both the ATP assay (Fig. 1c) and MTT assay (Fig. 1d), KO neurons \nshowed reduced cell death compared to WT neurons. These results indicate that KO of FOXO4 \nis neuroprotective against oxidative stress- and OGD-induced neuronal death.   \nKO of FOXO4 reduces the infarct volume and enhances functional recovery after tMCAO \nTo determine whether FOXO4 influences the outcome of I/R-induced brain injury in vivo, we \nperformed tMCAO to FOXO4 KO and WT mice and after 24 h following the reperfusion, mice \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted March 14, 2025. ; https://doi.org/10.1101/2025.03.13.643180doi: bioRxiv preprint \n\n11 \n \nwere euthanized to assess brain injury by the TTC staining (Fig. 2a). Our results showed a \nsignificant decrease in infarct volume in the KO group compared to the WT group (Fig. 2b, 2c), \nsuggesting that disrupting the FOXO4 gene is neuroprotective against I/R-induced neuronal \ninjury. \nTo determine whether KO of the FOXO4  influences animal survival following I/R, \nanimal death, and survival were recorded on days 1, 2, 3, 4, 5, and 7 after tMCAO. As shown in \nFig. 2d, KO of FOXO4 increases animal survival rate. Moreover, we also assessed the \nneurological deficits and found that KO of FOXO4 facilitated animal functional recovery, as \nreflected by the reduced mNSSs compared to the WT mice following the tMCAO procedure on \ndays 1, 3,5, and 7 (Fig. 2e). Improved functional recovery in the KO mice was also supported by \nthe cognitive function test, including the memory capabilities that were evaluated 10 days after \nMCAO. We observed better learning and memory in the KO mice compared to WT mice in the \nNOR test (Fig. 2f).  Therefore, KO of FOXO4 reduces neuronal death, improves animal survival, \nand enhances functional recovery following I/R. \nKO of FOXO4 attenuates neuroinflammation in the brain following I/R \nNeuroinflammation is a hallmark following I/R in the brain. To assess whether KO of FOXO4 \nalters the inflammatory responses, brain tissues on the ipsilateral side were isolated from KO and \nWT mice 48 h after I/R.  Western blot analysis of the pro-inflammatory cytokines, including \nTNF-α, IL-1β, and IL-6, revealed a significant reduction in the KO brains compared to WT \nbrains (Fig. 3a-3d).  These data indicate that KO of FOXO4 attenuates I/R-induced \nneuroinflammation. \nKO of FOXO4 suppresses activation of astrocytes and microglia in the brain following I/R \nTo assess the reactive astrocytes and microglia, we performed immunostaining of the astrocyte \nmarker, GFAP (glial fibrillary acidic protein), and microglia marker, Iba1 (ionized calcium-\nbinding adaptor molecule 1) to the mouse brain 48 h following I/R. As shown in Fig. 4, the \nnumbers of GFAP and Iba1 positively stained cells were significantly reduced in the KO brain \ncompared to the WT brain, suggesting that the KO of FOXO4 suppresses the activation of \nastrocytes and microglia.  \nKO of FOXO4 reduces leukocyte infiltration in the brain following I/R \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted March 14, 2025. ; https://doi.org/10.1101/2025.03.13.643180doi: bioRxiv preprint \n\n12 \n \nAnother pathological feature following I/R is the infiltration of leukocytes in the brain. To \ndetermine the leukocyte infiltration, the brain sections were immunohistochemically stained \neither with the CD45 (a marker for the total hematopoietic cells), CD11b (a marker for \nmacrophages, granulocytes, and NK cells), or Ly6G (a marker for monocytes, granulocytes, and \nneutrophils). As shown in Fig. 5a and 5b, the number of the CD45 positively stained cells in the \nKO brain showed a reduction trend compared to the WT brain, despite the lack of statistical \ndifference between the two groups. In contrast, the number of positively stained CD11b was \nsignificantly decreased (Fig. 5c and 5d) in the KO brain compared to the WT brain. Similarly, \nthe number of positively stained Ly6G (leukocytes) was also significantly decreased (Fig. 5e  \nand 5f) in the KO brain compared to the WT brain. These data indicate that KO of FOXO4  \nattenuates leukocyte infiltration. \nIdentification of Actinomycin D as a FOXO4 inhibitor \nThe results demonstrated above strongly suggest that disrupting FOXO4 function is beneficial \nfor neuronal survival following oxidative stress or I/R. To translate the results into potentially \nclinical treatments, we identified compounds that could directly inhibit the interaction between \nFOXO4  and its DNA substrate. The crystal structure of the FOXO4 protein is unavailable; \nhowever, the 1.9 Å resolution 3D crystal structure of the DNA-binding domain (DBD, amino \nacid sequence 93-196) of human FOXO4 bound to a 103 bp DNA duplex has been reported (Fig. \n6a).34 Additionally, human FOXO4 DBD is identical to mouse FOXO4. We utilized this FOXO4 \nDBD and performed virtual docking of several small molecular libraries against FOXO4 DBD \n(Protein Data Bank, PDB: 3L2C). The top-scoring compounds were then docked onto FOXO1 \nDBD (PDB: 3COA) and FOXO3 DBD (PDB: 2UZK) and any candidates that also showed a \nhigh affinity to FOXO1 or FOXO3 were eliminated from the list. This led to Actinomycin D \n(ActD), an FDA-approved antibiotic, as one of the top candidates (Fig. 6b). ActD is used for \nchemotherapy to treat many different types of cancer35 and was the first natural antibiotic found \nto have anti-cancer activity36.  \nIn a cell-based reporter assay, ActD showed significant inhibitor activity against FOXO4 \n(Fig. 6c). Structurally, FOXO4 DBD is more similar to FOXO3 DBD than to FOXO1 \nDBD37. However, our reporter assay revealed that ActD did not inhibit FOXO3 activity at the \nsame concentration (0.1 µM) (Fig. 6d).  \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted March 14, 2025. ; https://doi.org/10.1101/2025.03.13.643180doi: bioRxiv preprint \n\n13 \n \nThe results of computational protein-ligand docking advocated helix H3 as the major site \nof inhibition (Fig. 6e, 6f). SwissDock-based AutoDock Vina execution exhibited active residues \nArg78, His79 and Ser82 interacting with ActD (Fig. 6g). AlphaFold3-based Protenix38  predicted \nsimilar heavy atom contacts with ligand to H2 (Leu47), H3 (Asn75, Arg78, His79, Ser82 and \nLeu83) and S3 (Lys89) domains of FOXO4 (Fig. 6h). It is important to note that the sequential \nnumbering of these receptor residues is based on the active DBD of FOXO4 and does not \ncorrespond to the entire protein from the N to C terminal. Interaction of ActD with Arg78 is \ncrucial as two hydrogen bonds are indicated (Fig. 6g) with donor-acceptor distances of 3.480 Å \n(NH2) and 3.451 Å (NE), whereas the distance between ActD to Ser82 and Lys89 is 2.735 Å and \n3.334 Å (Fig. 6g, 6h), respectively. The corresponding seven fundamental amino acid residues \nbound to the ligand are L124, N152, R155, H156, S159, L160, and K166 for FOXO4.  \nPharmacological blockage of FOXO4 attenuates oxidative stress - and OGD -induced \nneuronal death in vitro  \nTo determine the effect of ActD on primary neuronal cultures in response to oxidative stress, we \ntreated cultured neurons  with MD in the presence of 0.1 µM ActD. Both ATP assay ( Fig. 7a) \nand MTT assay results (Fig. 7b) indicated a protective effect of ActD on MD-induced cell death. \nLikewise, ActD also protected neurons from OGD-caused neuronal death, as shown by increased \nATP level ( Fig. 7c ) and  neuronal survival  rate ( Fig. 7d ), suggesting  that ActD attenuates \noxidative stress- and OGD-induced neuronal death in vitro. \nTreating WT mice with ActD reduces tMCAO -induced brain injury and promotes \nfunctional recovery  \nTo further assess the therapeutic effect of ActD on I/R-induced brain injury in mice following \ntMCAO, we treated WT mice with ActD (0.25 or 0.5 mg/kg, i.p.) or vehicle 1 h before tMCAO \n(Fig. 8a). Following 1 h of ischemia and 24 h reperfusion, mice were sacrificed for TTC staining \n(Fig. 8b). Our data showed that ActD treatment significantly reduced I/R-induced brain injury at \n0.5 mg/kg (Fig. 8b, 8c).  Moreover, ActD treatment also enhanced the animal function recovery, \nas shown by reduced mNSS scores (Fig. 8d) and increased cognitive function reflected by the Y-\nmaze test compared to the vehicle treatment. These data indicate that ActD is neuroprotective \nagainst I/R-induced brain injury. \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted March 14, 2025. ; https://doi.org/10.1101/2025.03.13.643180doi: bioRxiv preprint \n\n14 \n \n \nDiscussion  \nIn this study, we investigated the role of FOXO4 in oxidative stress-, OGD-, and cerebral I/R-\ninduced neuronal injury and validated that FOXO4 is a therapeutic target. Moreover, we \nidentified ActD as a FOXO4 inhibitor that showed a therapeutic effect on neurons following \noxidative stress, OGD, or I/R insults. While the protective role of FOXO4 in non-neuronal \ncells/tissues was previously reported in vitro and in vivo39-41, to our knowledge, this is the first \nstudy of FOXO4 in I/R-induced neuronal injury in neurons and animal brains.  \n   Unlike FOXO1 KO mice, which are embryonic lethal42, FOXO4 KO mice do not differ \nfrom their WT littermates. Our findings revealed that KO of FOXO4 yielded a better outcome \nafter cerebral I/R, significantly reducing the infarct volume 24 h after I/R, and improving \nfunctional recovery after that. KO of FOXO4 also enhanced learning and memory capability in \nmice after one week following the I/R. To understand possible mechanisms, we found that the \nFOXO4 KO mice showed a significant decrease in inflammatory cytokines in the brain cortex 48 \nh after I/R. The inflammatory cytokines come from two sources, immune cells attracted from \nsystemic circulation, and locally activated glial cells.43 Since we observed a significant decrease \nin CD11- and Ly6G-positive cells in the KO brain following I/R, leukocyte infiltration in the \nFOXO4 KO brain was reduced. Since CD11b is highly expressed by myeloid cells, the first line \nin the immune response44, the significant decrease in CD11b in the FOXO4 KO brain should \nreflect reduced leukocyte infiltration through the microvessels.  \nAdditionally, our results also support reduced activation of local microglia and astrocytes \nin the KO compared to the WT mouse brain. These findings are in good accordance with \nprevious data showing the anti-inflammatory effect of deleting FOXO4 in an ischemic condition \nin another organ in vivo45, and in cerebral endothelial cells in an in vitro model of I/R.39 After \nI/R, ROS, cell debris, mitochondrial dysfunction, excitotoxicity, and disrupted blood-brain \nbarrier (BBB) work together to trigger inflammatory responses, involving both local and \nsystemic immune factors. As a transcription factor, FOXO4 regulates many pathological \nprocesses, such as apoptosis, endothelial function, ROS production, and BBB integrity to \nfacilitate cell death, whereas KO of FOXO4 leads to neuroprotective effects and impeding the \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted March 14, 2025. ; https://doi.org/10.1101/2025.03.13.643180doi: bioRxiv preprint \n\n15 \n \nprogression of neuronal death and infarction, ultimately preserving functional performance, as \nsupported by our findings. \n Although DBDs of transcription factors were generally considered “undruggable” due to \ntheir relatively flat and similar surface areas.46 However, recent studies have challenged this \ninterdiction by developing an improved in-silico screening approach to eliminate potential non-\nselective inhibitors.47 For instance, the identified top-scoring compounds targeting the STAT3 \nDBD from an in-silico screening of a chemical library were docked onto the STAT1 DBD48,49. \nAny compounds predicted to also bind to the STAT1 DBD were eliminated due to potential lack \nof selectivity and this improved approach helped the authors identify a STAT3-selective hit \nmolecule.49 Using a similar strategy, we identified a FOXO4 inhibitor, ActD, which could be \npotentially specific for FOXO4, because our results suggest that it did not suppress FOXO3 \nactivity at the same concentration in vitro. Importantly, ActD confers neuroprotection against \noxidative stress- and OGD-induced neuronal death in the primary neuronal cultures reduces \ninfarct volume, and enhances functional recovery following I/R in the brain of mice. These data \nsuggest that ActD may be used as a therapeutic to treat the disease.  \n \nIn conclusion,  using a genetically modified mouse combined with primary neuronal \ncultures and other approaches, we validated FOXO4 as a therapeutic target for I/R-induced brain \ninjury. Our data suggest that KO of FOXO4 reduces neuronal injury, promotes functional \nrecovery, suppresses leukocyte infiltration, and attenuates neuroinflammation. Moreover, we \nalso identified ActD as a therapeutic for I/R-induced neuronal death, which has the potential to \nbe used in the clinic to treat acute ischemic stroke or other neurological disorders. \n \nAcknowledgments: This work was supported by NIH/NINDS NS124846 . 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All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted March 14, 2025. ; https://doi.org/10.1101/2025.03.13.643180doi: bioRxiv preprint \n\n19 \n \nFig. 1. Effect of KO of FOXO4 on oxidative stress- and OGD-induced neuronal death in \nprimary neuronal cultures. a. Western blot analysis of FOXO4 protein levels in primary \nneuronal cultures to validate FOXO4 KO. b. ATP  assay results of KO and WT neurons \nfollowing MD treatment. c. MTT assay results of KO and WT neurons following MD treatment. \nd. ATP assay results of KO and WT neurons following OGD treatment. e. MTT assay results of \nKO and WT neurons following OGD treatment. Data are shown as mean ± SD. N = 10-14, ***p \n< 0.001. ns, no significant difference. \n \nFig. 2. KO of FOXO4 reduces brain infarct volume, improves animal survival, and \nenhances functional recovery following I/R. a. Western blot analysis of FOXO4 protein levels \nin mouse brain to validate FOXO4 KO. b. A schematic of experimental design. c. Representative \nTTC staining of mouse brains from the sham surgery, WT, and KO animal groups after \nMCAO. d. Quantitation of TTC staining results. e. KO mice increased the animal survival rate \ncompared to WT mice after tMCAO. f. KO mice improved mNSSs compared to WT mice after \ntMCAO. g. KO mice showed better memory compared to WT mice in the NOR test after \ntMCAO. Data are shown as mean ± SD. N = 10-14 for (d), ****p < 0.0001. N = 14– 15 for (e)-\n(g), * p < 0.05, **p < 0.01, ***p < 0.001. \n \nFig. 3. KO of FOXO4 attenuates neuroinflammation following I/R. a. Representative western \nblot results of the indicated proteins. b-d. Quantified results of the indicated proteins. Data are \nshown as mean ± SEM, * p < 0.05, **p < 0.01.  \n \nFig. 4. FOXO4 KO mouse brains showed reduced astrocytes and microglia two days after \nI/R compared to WT mouse brains. a. Representative images showing GFAP staining in the \nWT and FOXO4 KO brains. Scale bar, 50 µm. b. Representative images showing Iba1 stai ning \nin the WT and FOXO4 KO brain sections following I/R. Scale bar, 50 µm.  c. Quantitation of \nGFAP positively stained cells ( green). d. Quantitation of Iba1 positively stained cells ( red). All \nnumeric data are shown as mean ± SD; n = 7 for each group. *p < 0.05, **p < 0.01. \n \nFig. 5. FOXO4 KO brain shows reduced leukocyte infiltration following I/R. a. \nRepresentative image showing CD45 staining. b. Quantitation of CD45-positively stained cells \nin the brain. c. Representative image showing CD11b staining. d. Quantitation of CD11b-\npositively stained cells. e. Representative image showing Ly6G staining. f. Quantitation of \nLy6G-positively stained cells. Scale bar, 50 µm. Data are shown as mean ± SD; n = 5-7 for each \ngroup. ns, no significant difference; *p < 0.05, ***p < 0.001. \n \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted March 14, 2025. ; https://doi.org/10.1101/2025.03.13.643180doi: bioRxiv preprint \n\n20 \n \nFig. 6. Identification of ActD as a FOXO4 inhibitor. a. Structure of the FOXO4 DBD–DNA \ncomplex. The FOXO-DBD is shown in blue ribbon (upper) and the DNA double helix is shown \nas orange and green (lower)  (https://www.rcsb.org/structure/3L2C). b. Structure of ActD. c. The \nresult of a luciferase reporter assay shows that ActD inhibited FOXO4 activity. d. The result of a \nluciferase reporter assay shows that ActD did not inhibit FOXO3 activity. e & f. The results of \ncomputational protein-ligand docking advocated helix H3 as the major site of inhibition. g. \nSwissDock-based AutoDock Vina execution exhibited active residues Arg78, His79 and Ser82 \ninteracting with ActD. h. AlphaFold3-based Protenix predicted similar heavy atom contacts with \nligand to H2 (Leu47), H3 (Asn75, Arg78, His79, Ser82 and Leu83) and S3 (Lys89) domains of \nFOXO4. All numerical data are shown as mean ± SD. N = 3, *p <0.05, ns, no significant \ndifference. \n \nFig. 7. Effect of ActD on oxidative stress- and OGD-induced neuronal death in the WT \nprimary neuronal cultures. a. ATP  assay results of WT neurons treated with 20 µM of MD in \nthe presence of 0.1 µM of ActD. b. MTT assay results of WT neurons treated with 20 µM of MD \nin the presence of 0.1 µM of ActD. d. ATP  assay results of WT neurons treated with OGD in the \npresence of 0.1 µM of ActD. e. MTT assay results of WT neurons treated with OGD in the \npresence of 0.1 µM of ActD. Data are shown as mean ± SD. N = 10-14, *p <0.05, **p < 0.01, \n***p < 0.001,  ****p < 0.0001.  \n \nFig 8. Effect of ActD on I/R-indued brain injury in the WT following tMCAO. a. A b. A \nschematic of experimental design. b. Representative TTC staining of mouse brains from the \nsham surgery, vehicle-treated, and ActD (0.25 or 0.5 mg/kg)-treated mouse brains after \nMCAO. c. Quantitation of TTC staining results. d. ActD improved mNSSs compared to vehicle-\ntreated mice after tMCAO. e. ActD-treated mice showed better memory compared to vehicle-\ntreated mice in the Y-maze test after tMCAO. Data are shown as mean ± SD. N = 12 for (c); n = \n14-15 for (d & e); *p < 0.05, **p < 0.01, ****p < 0.0001. \n \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted March 14, 2025. ; https://doi.org/10.1101/2025.03.13.643180doi: bioRxiv preprint \n\na\nd\nWT KO WT KO\n-1\n0\n1\n2\n3\n4\n  NC                       OGD.\nCell viability (A. u.)\n✱✱✱ns\nOGD-Treated Cells\n     MTT Assay\nWT KO WT KO\n0.0\n0.2\n0.4\n0.6\n0.8\nNC                       OGD.\nATP level (A. U.)\n✱✱✱ns\nOGD-Treated Cells\n     ATP Assay\nWT KO WT KO\n0.0\n0.2\n0.4\n0.6\n0.8\nVehicle                     MD....\nATP level (A. U.)\n✱✱✱ns\nMD-Treated Cells\n     ATP Assay\nb\nWT KO WT KO\n0\n1\n2\n3\nMD-Treated cell\nMTT assay\nCell viability(A. U.)\n✱✱✱ns\n   Vehicle                   MD....\nWT KO \nFOXO4\nActin \nc\ne\nFig. 1\n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted March 14, 2025. ; https://doi.org/10.1101/2025.03.13.643180doi: bioRxiv preprint \n\na b\nFig. 2\ne\ng\nSham WT KO\nf\nWT KO \nFOXO3\nFOXO4\nActin \nFOXO1\nd\nSham WT KO \n-20\n0\n20\n40\n60\n80\n100\nInfarct volume (%)\n✱✱✱✱\n0 1 2 3 4 5 6 7\n20\n40\n60\n80\n100\nSurvival\nDays after surgery\nSurvival rate (percentage) WT\nKO\n✱\n✱\n✱\nns\n✱\n✱✱✱\nns\nWT KO \n0\n20\n40\n60\n80\nNORT\nPercentage of tuching novel\nobject\n✱\nPercentage of touching novel \nobject\nNOR\n0 2 4 6 8\n0\n5\n10\n15\nmNSS\nDays after MCAO\nModified Neurological\nSeverity Score\nWT\nKO\n✱\n✱\n✱✱\n✱✱mNSS\nc\n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted March 14, 2025. ; https://doi.org/10.1101/2025.03.13.643180doi: bioRxiv preprint \n\nc\na\nFig. 3\nWT KO \n0\n5\n10\n15\nIL- 6\nIL- 6/GAPDH ratio\n✱\nWT KO\n0\n2\n4\n6\nIL1b\nIL-1b/GAPDH ratio\n✱✱\nIL-1b\nb\nd\nWT KO \n0.0\n0.5\n1.0\n1.5\nTNF-α\nTNFa/GAPDH ratio\n✱\nTNFα/GAPDH ratio\n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted March 14, 2025. ; https://doi.org/10.1101/2025.03.13.643180doi: bioRxiv preprint \n\nc d\na\nb\nWT KO\nGFAP Staining (Green)\nIba1 Staining (Red)\nWT KO\nFig. 4\nWT KO \n0\n10\n20\n30\n40\n50\nGFAP positive cells(%) ✱✱\nWT KO \n0\n10\n20\n30\nIba1 positive cells(%)\n✱\n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted March 14, 2025. ; https://doi.org/10.1101/2025.03.13.643180doi: bioRxiv preprint \n\nWT KO\nCD45 Staining (Green, Hematopoietic Cells)\nWT KO\nCD11b Staining (Green, Macrophages, \nGranulocytes, and NK cells)\nWT KO\nLy6G (Green, Leukocytes)\na b\nc d\ne\nFig. 5\nWT KO \n0\n20\n40\n60\nCD45 positive cells(%)\nns\nWT KO \n0\n20\n40\n60\n80\n100\nCD11b positive cells (%)\n✱✱✱\nf\nWT KO \n0\n10\n20\n30\nly6G positive cells(%)\n✱Ly6G positive cells (%)\n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted March 14, 2025. ; https://doi.org/10.1101/2025.03.13.643180doi: bioRxiv preprint \n\na\nb\nDMSO ActD \n0\n50\n100\n150\nFOXO3\nluciferease activity\nns\nd FOXO3\nLuciferase activity\nDMSO ActD \n0\n50\n100\n150\nFOXO4\nluciferease activity ✱✱\nc\nLuciferase activity\ne f\ng h\nFig. 6\n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted March 14, 2025. ; https://doi.org/10.1101/2025.03.13.643180doi: bioRxiv preprint \n\nVeh OGD OGD-ActD\n0.0\n0.1\n0.2\n0.3\n0.4\nOGD-treated cell\nMTT assay\nMTT production(A.U.)\n✱✱✱✱ ✱✱\nVeh MD ActD\n0\n1\n2\n3\nMD-Treated cell\nATP assay\nMTT production(A.U.)\n✱✱✱✱\nVeh MD MD-ActD\n0\n1\n2\n3\n4\nMD-Treated cell\nATP assay\nATP production(A.U.)\n✱✱\nFig. 7\na b\nc d\nCell viability (A.U.)\nATP level (A.U.)\nMD-treated cell \nMTT assay\nMD-treated cell\nATP assay\nVeh OGD OGD-ActM\n0.0\n0.5\n1.0\n1.5\n2.0\n2.5\nOGD-Treated cell\nATP assay\nATP production(A.U.)\n✱✱✱ ✱\nCell viability (A.U.)\nATP level (A.U.)\nVeh OGD OGD-ActD\n0.0\n0.1\n0.2\n0.3\n0.4\nOGD-treated cell\nMTT assay\nMTT production(A.U.)\n✱✱✱✱ ✱✱\n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted March 14, 2025. ; https://doi.org/10.1101/2025.03.13.643180doi: bioRxiv preprint \n\na c\nFig. 8\nActD TTC or behavior tests \nbetween 1-10 days\nd e\nSham Vehicle ActDb\nVehicle ActD \n0\n20\n40\n60\n80\n100\nY maze\nNumber of entry\n✱\n0 1 2 3 4 5 6 7\n0\n5\n10\n15\nmNSS\nDays after MCAO\nModified Neurological Severity\n Score\nVehicle\nActD\n✱\n✱\n✱\n✱\n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted March 14, 2025. ; https://doi.org/10.1101/2025.03.13.643180doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}