NFATc4 knockout promotes neuroprotection and retinal ganglion cell regeneration after optic nerve injury

NFATc4 knockout promotes neuroprotection and retinal ganglion cell regeneration after optic nerve injury | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article NFATc4 knockout promotes neuroprotection and retinal ganglion cell regeneration after optic nerve injury Joanna Mackiewicz, Julia Tomczak, Malwina Lisek, Agata Sakowicz, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3813885/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 19 Apr, 2024 Read the published version in Molecular Neurobiology → Version 1 posted 9 You are reading this latest preprint version Abstract Retinal ganglion cells (RGCs), neurons transmitting visual information via the optic nerve, fail to regenerate their axons after injury. The progressive loss of RGC function underlies the pathophysiology of glaucoma and other optic neuropathies, often leading to irreversible blindness. Therefore, there is an urgent need to identify the regulators of RGC survival and the regenerative program. In this study, we investigated the role of the family of transcription factors known as nuclear factor of activated T cells (NFAT), which are expressed in the retina; however, their role in RGC survival after injury is unknown. Using the optic nerve crush (ONC) model, widely employed to study optic neuropathies and central nervous system axon injury, we found that NFATc4 is specifically but transiently up-regulated in response to mechanical injury. In the injured retina, NFATc4 immunolocalized primarily to the ganglionic cell layer. Utilizing NFATc4 −/− and NFATc3 −/− mice, we demonstrated that NFATc4, but not NFATc3, knockout increased RGC survival, improved retina function, and delayed axonal degeneration. Microarray screening data, along with decreased immunostaining of cleaved caspase-3, revealed that NFATc4 knockout was protective against ONC-induced degeneration by suppressing pro-apoptotic signaling. Finally, we used lentiviral-mediated NFATc4 delivery to the retina of NFATc4 −/− mice and reversed the pro-survival effect of NFATc4 knockout, conclusively linking the enhanced survival of injured RGCs to NFATc4-dependent mechanisms. In summary, this study is the first to demonstrate that NFATc4 knockout may confer transient RGC neuroprotection and decelerate axonal degeneration after injury, providing a potent therapeutic strategy for optic neuropathies. optic nerve injury NFATc4 transcription factor retinal ganglion cell survival intravitreal gene delivery apoptotic gene expression Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 INTRODUCTION Retinal ganglion cells (RGCs), a highly specialized type of neurons, transmit visual information from the retina to the visual processing centers of the brain. Because the unidirectional optic nerve is formed exclusively by axons projecting by RGCs, it is highly vulnerable to various injuries, leading to irreversible loss of RGC function. The progressive death of RGCs is a crucial element in the pathophysiology of glaucoma, characterized by the progressive degeneration of the optic nerve and resulting in irreversible blindness. Over the decades, numerous studies have investigated the mechanisms underlying RGC death and identified several neuronal abnormalities associated with antioxidant imbalance [ 1 ], neuroinflammation [ 2 ], mitochondrial metabolism alterations [ 3 ], ischemia/hypoxia [ 4 ], or vascular deficits [ 5 ]. Recent studies have also demonstrated a promising effect of small molecules and virus-based gene therapies in pre-clinical models of glaucoma [ 6 – 9 ]. Considerable effort is currently dedicated to unraveling molecular changes underlying disease progression and understanding signaling pathways that can be manipulated to enhance RGC survival. The most effective approaches involve the exogenous administration of neurotrophic factors or apoptotic pathway inhibitors, such as brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), neurotrophin-4, or their combination with oncomodulin or osteopontin [ 10 – 16 ]. In the majority of cases, the effects are only transitory, even with long-lasting treatments. Similarly, caspase inhibitors provide only transient neuroprotection [ 17 – 23 ]. Strategies based on the knockdown or overexpression of other prominent regulators of RGC growth and survival, such as Elk-1 [ 24 ], Sigma-1R (σ-1r) [ 25 ], MEF2 [ 26 ], or PTEN [ 27 , 28 ] have also been tested. Despite the tremendous progress in several molecular interventions targeting RGC survival following injury, most of them present only limited effectiveness in clinical interventions. This limitation likely arises from the choice of inappropriate target(s) or time window. Therefore, the elucidation of mechanisms leading to RGC death is essential to target the right signaling molecule or decipher the therapeutic time-window. Originally described as important regulators of immune function [ 29 ], NFATs are now implicated in the regulation of neuronal morphogenesis, plasticity, and the response to neurotrophin and electrical stimulation [ 30 – 32 ]. In humans, the NFAT family comprises five transcription factors named NFAT1 (NFATc2), NFAT2 (NFATc1), NFAT3 (NFATc4), NFAT4 (NFATc3), and NFAT5, which, all but the last one, are regulated by Ca 2+ - activated protein phosphatase-2B/calcineurin (CaN). The nature, source and timing of upstream signals regulating Ca 2+ /CaN activity and the flexibility of NFAT in cooperating with other transcriptional activators or repressors make an important contribution to neuronal response to external stimuli. This relationship is seen in both developing and mature neurons. For instance, profound defects in axon projections were observed in embryos with combined deletion of either NFATc3 or NFATc4 (c3/c4 mutants) or NFATc2, NFATc3, and NFATc4 (c2/c3/c4 mutants) [ 33 ].These defects were reproduced by in utero administration of cyclosporine A (CsA) – a potent inhibitor of CaN. CaN and NFATs are also essential for neurotrophin-induced neuronal outgrowth and survival [ 34 ]. The role of NFATs in RGCs is not yet fully understood, as it becomes increasingly apparent that these cells can selectively activate specific NFAT isoforms. Recently, NFATc2 and NFATc3 have been identified as the major isoforms expressed in retina [ 35 ]. NFATc4, present in low amounts, was significantly upregulated in RGCs following light-induced damage and was associated with increased neuronal apoptosis [ 36 ]. Despite the obvious role of NFAT in neuronal function, the contribution of NFAT isoforms to retinal degeneration following pro-death insults has not been widely studied. This is of paramount importance as the progressive loss of RGCs is a hallmark common to the majority of optic neuropathies, including glaucoma, often leading to permanent blindness [ 37 – 40 ]. Traumatic optic neuropathy and glaucomatous injury can be mimicked by mechanical optic nerve crush (ONC), which serves as a preclinical model of neuronal survival and regeneration, as it similarly induces RGC death and degeneration. In this model, the lesion severs all of the RGCs’ axons, ensuring high reproducibility and precise control of the injury site [ 41 – 45 ]. As optic neuropathy can be linked with other CNS diseases, this model can also be used in CNS degeneration studies to unravel degenerative mechanisms and test neuroprotective therapies. Using ONC, we provide evidence for the critical role of NFATc4 in RGC survival following injury. MATERIALS and METHODS Animals All animal procedures were carried out according to the Association for Research in Vision and Ophthalmology (ARVO) guidelines for the use of animals in ophthalmic and vision research. The experimental protocols were approved by the Institutional Animal Care and Use Committee at the Medical University of Lodz. All mice used in this project were C57BL/6. The animals were group-housed in laboratory cages and kept under a controlled temperature (23 ± 2°C) with a 12-h light/dark cycle and with food and water provided ad libitum. Nfatc4 −/− mice (B6;129S- Nfatc4 tm1Grc /J, strain #027581) and Nfatc3 −/− mice (B6;129S2- Nfatc3 tm1Glm /J, strain #010589) were obtained from the Jackson Laboratory (USA). Both males and females were used in all experimental procedures. Lentiviruses Lentiviruses were employed for in vivo delivery of NFATc4-GFP to NFATc4 −/− mouse retina, owing to the limited capacity of adeno-associated virus serotype 2. Lenti ORF particles, Nfatc4 (GFP-tagged) transcript variant 2 (reference sequence NM_001168346.1, 4036 bp), as well as Lenti ORF control particles of pLenti-C-mGFP-P2A-Puro, were generated by Origene (USA). NFAT luciferase reporter lentivirus and firefly luciferase lentivirus were sourced from BPS Bioscience (USA). Lenti-VIVIT-GFP was produced with pLV-VIVIT-GFP plasmid (Addgene#188707) using Lenti-X Packaging Single Shots technology from Takara Bio (USA). All viral titers were > 10 7 TU/ml. In vitro or in vivo transduction was caried out as specified for each experiment. Isolation of primary neurons RGCs were purified from postnatal (P8-P10) mouse pups using a procedure essentially described in [ 46 ] with some modifications. Isolated retinas were washed 3 times with Dulbecco Phosphate Buffer Saline (DPBS) and digested with papain (16.5 U/ml) for 30 min at 37 o C. After trituration, papain activity was inhibited by adding ovomucoid solution (1.5 mg/ml), and the cell suspension was centrifuged at 250 x g for 10 min. The resulting pellet was mixed with DPBS supplemented with 5 µg/ml insulin and transferred to anti-macrophage antibody-coated petri dishes for a 45 min incubation. Suspended cells were subsequently transferred to petri dishes containing anti-Thy 1.2 antibody conditioned media and further incubated for 45 min to isolate RGCs. Petri dishes were washed multiple times, and RGCs were released by trypsinization. RGCs were routinely seeded at the density of 50.000 cells/well in a 24 well plate coated with poly-D-lysine (10 µg/ml) and laminin (1 µg/ml). Cells were cultured in serum-free media supplemented with sodium pyruvate (1 mM), B27 (1:50), BDNF (50 ng/ml), CNTF (10 ng/ml), forskolin (5 M), insulin (5 µg/ml), N-acetyl cysteine (5 µg/ml), L-glutamine (1 mM) and triiodothyronine (40 ng/ml), unless otherwise specified, at 37 o C/5% CO 2 and in a humidified atmosphere. The purity of isolated RGCs was verified by staining with antibodies recognizing RNA binding protein with multiple splicing (RBPMS) and typically exceeded 95%. Primary hippocampal neurons were prepared following the methodology outlined in our previous publication [ 47 ]. In brief, hippocampal cultures were derived from Sprague-Dawley E18 embryos. Hemispheres were dissected in HBSS buffer on ice, trypsinized for 30 min at 37 0 C, centrifuged at 250 x g for 2 min, and then triturated with a fire-polished glass pipette. The dissociated neurons were seeded on nitric acid-soaked glass coverslips coated with poly-L-lysine in plating medium (10% v/v horse serum in DMEM). After 4 h, the medium was replaced with Neurobasal maintenance medium supplemented with 2% B27, 1 mM glutamine, 1 mM sodium pyruvate, and 5 µg/ml insulin. For cultures lasting beyond 4 days, half of the medium was removed on day 3 or 4 and replaced with an equal volume of fresh medium. In vitro survival and neurite outgrowth Isolated RGCs (~ 200,000 cells) were promptly electroporated with NFATc4 ON-TARGETplus siRNA oligonucleotides or ON-TARGETplus scrambled siRNA, both administered at 1 nmol per electroporation (Horizon Discovery, USA). The electroporation was conducted according to the method detailed elsewhere [ 48 ]. Subsequently, cells were cultured for up to 3 days, and stained for annexin V for 10 min in growth media to label apoptotic cells. Images of 10–12 randomly selected fields were captured in growth media at 2 and 72 h using a Leica DMi8 inverted microscope to quantify cell survival. The survival was quantified using ImageJ and was normalized to scrambled siRNA-treated RGCs. For the neurite outgrowth assay, isolated RGCs were electroporated and seeded at a low density of ~ 5000–6000 cells/well in a 48 well plate, followed by a 3-day culture period. RGCs were then fixed with 3.8% paraformaldehyde (PFA) and permeabilized with 0.2% Triton X-100 in PBS. After several washes with PBS, cells were labelled with anti-βIII tubulin (1:500, Cell Signaling technology, USA) overnight at 4 0 C. The plates were next probed with secondary antibodies conjugated to Alexa Fluor 488 (1:500) for 6 h at room temperature. Nuclei were counterstained with DAPI at a dilution of 1:5000. Images were acquired on a Leica DMi8 inverted microscope, and the longest neurite per cell (~ 20 cells on average in each experiment) was measured using the ImageJ Neurite Tracker tool. The results were normalized to the average axonal length in scrambled siRNA-treated RGCs. Optic nerve crush (ONC) and intravitreal injections For optic nerve crush, C57BL/6 male and female mice at the age 7–8 weeks were randomly assigned to the treatment group. The crush procedure followed protocols essentially described in previous studies [ 28 , 49 ] with minor modifications. Mice were anesthetized with 20 mg/kg IP xylazine and 100 mg/kg IP ketamine. A drop of 0.5% proparacaine was applied to the eye. The optic nerves were exposed from the outer canthus behind the globe, and the crush was performed ~ 2 mm behind the eyeball for 3 sec using extra-fine forceps. The contralateral control eye underwent the same procedure but was spared from the crush. Care was taken to avoid damaging blood vessels in the retina. Post-operative analgesia was provided with 0.5 mg/ml buprenorphine. In lentivirus transduction experiments, mice under isoflurane anesthesia were intravitreally injected through the sclera with 2 µl of lentivirus in PBS using a 31-gauge needle (Hamilton) connected to a 5 µL Hamilton syringe. Care was taken to avoid damage to the lens. Viral injections were performed approximately 2 weeks before ONC to allow for sufficient gene expression. Animals experiencing any postoperative complications such as excessive bleeding or swelling, retinal ischemia or cataract were excluded from the cohort at any time after procedure. Retinal flat-mount and RGC count The procedure for retinal flat-mount preparation was conducted on deeply anesthetized mice using isoflurane and intraperitoneal administration of ketamine/xylazine, followed by sacrifice through transcardial perfusion with 4% paraformaldehyde (PFA). The methodology for retinal flat mount was adapted from [ 50 ] and used in our previous study [ 47 ]. In brief, the eyes were removed, post-fixed with 4% PFA for 2 h at room temperature, and the retinas were dissected. After several washes with PBS, retinas were permeabilized with Triton X-100 and incubated overnight at 4 0 C with a primary anti-RBPMS antibody (diluted at 1:500, 1832, Aves Labs) in a blocking buffer (PBS with 10% goat serum). Following washing with PBS, retinas were incubated with secondary antibodies conjugated to Alexa Fluor 488 (1:500, Invitrogen) for 2 h at room temperature. Subsequently, retinas were flat-mounted in H-1000 mounting medium on glass slides. Scan images were acquired with Leica SP8 confocal laser scanning microscope. The counting of RBPMS-positive cells was carried out in a manner described in a previous publication [ 47 ]. This process, performed by an experienced researcher in a masked fashion, involved assessing RGC cell density per mm 2 or percentage change relative to the sham-operated contralateral eye or ONC-treated wild-type. Anterograde labelling, quantification of regeneration and axon degeneration analysis Two days prior to optic nerve harvesting, 2 µl of cholera toxin subunit B (CTB, 2 µg/µl, Invitrogen, USA) were intravitreally injected to visualize axons and nerve terminals of surviving RGCs. Animals were perfused with 4% PFA before the collection of optic nerves. The optic nerves were cryopreserved overnight in 30% sucrose at 4 0 C and then mounted in Optimal Cutting Temperature mounting medium (Thermo Fisher Scientific, USA). Longitudinal sections, 10 µm thick, were cut for optic nerves and imaged using a DMi8 fluorescence microscope (Leica, Germany). The sections were analyzed as described previously [ 41 ]. The number of CTB-positive axons passing 0.1, 0.25, 0.5, 0.75, 1.0, 1.25 mm from the crush site was manually counted. The total number of CTB-positive axons per optic nerve was calculated using methods outlined in a previous study [ 51 ]. This approach provides a quantitative assessment of axon survival and regeneration in response to experimental conditions. Axonal integrity was assessed one week after ONC through βIII-tubulin staining. This technique enables the measurement of protein abundance within axons [ 52 ] and has recently been employed to demonstrate delayed optic nerve degeneration in response to pharmacological inhibition of aldolase reductase [ 53 ]. For immunostaining, 10-µm-thick cryosections were probed with anti-βIII-tubulin antibodies (1:500, Cell Signaling, USA), followed by incubation with secondary antibodies conjugated to Alexa Fluor 594 (Thermo Fisher, USA). The density of βIII-tubulin was measured in a 500x200 µm area immediately after the crush site [ 54 ], following the protocol described in [ 53 ]. Optic nerves were imaged at the same intensity using a Leica DMi8 fluorescence microscope with a 10x objective. This approach provides insights into the preservation or alterations in βIII-tubulin expression, reflecting axonal structural integrity following the ONC procedure. Western blotting Retinas were isolated and lysed using RIPA buffer supplemented with a protease and phosphatase inhibitor cocktail. The total protein content was quantified colorimetrically with the Bio-Rad Protein Kit Assay. Subsequently, 10–30 µg of the protein samples were separated in 4–20% gradient polyacrylamide gels and transferred to a nitrocellulose membrane using a semi-dry method. The membranes were blocked with 10% goat serum in TBST-T buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.05% Tween-20) for 2 h at room temperature. Next, the membrane was incubated with primary antibodies recognizing NFATc4 (1:750, ab183117, Abcam), caspase-3 (1:1000, PA5-77887, Thermo Fisher), or GAPDH (1:3000, G8795, Merck) for 24 h at 4 o C. Following three washes in TBS-T, the membrane was probed with secondary antibodies (1:5000) conjugated to horse radish peroxidase for 2 h at room temperature. ECL western blot system was used to visualize immunoreactive bands. The membranes were scanned densitometrically, and the optical density of bands was quantified using ImageJ. The results are expressed as arbitrary units after normalization to the endogenous GAPDH level, providing a quantitative assessment of protein expression levels. Total RNA isolation, real-time PCR and microarray screening Total RNA was extracted from the retina using Trizol reagent following the manufacturer’s protocol. Single-stranded cDNA was synthesized from 1 µg of isolated RNA using M-MLV reverse transcriptase with oligo(dT) primers. Real-time PCR reactions were carried out under the following conditions: an initial denaturation at 95°C for 15 min, followed by 40 cycles at 95°C for 15 s, 60°C for 30 s, and 72°C for 30 s, using the Abi Prism 7000 sequence detection system using Eva Green Master Mix. Primers used in the reactions: Nfatc1 (NM_198429), Nfatc2 (NM_010899), Nfatc3 (NM_010901), Nfatc4 (NM_023699), Gapdh (NM_008084) were purchased from Origine (Germany). The specificity of the PCR product was assessed by running a melting curve. The relative expression of the gene was determined using the ΔCt method [ 55 ], with endogenous Gapdh expression used for data normalization. For microarray screening, cDNA amplified from 2 µg of retinal RNA was hybridized with RT² Profiler™ PCR Array Mouse Apoptosis (Qiagen, USA), and the reaction was performed using HOT FIREPol® EvaGreen® qPCR Mix Plus (Solis Biodyne, Estonia The real-time PCR conditions included an initial cycle at 95°C for 10 min, followed by cycles at 95°C for 15 s, 60°C for 1 min, and a dissociation curve at 95°C for 1 min, 55°C for 30 s, and 95°C for 30 s. The fold change was calculated by a method of Livak and Schmittgen [ 55 ]. Data were analyzed using Qiagen PCR Array Data Analysis Web Portal. The microarray analysis was run in triplicate, and the RT 2 software averaged the triplicate normalized expression for each gene (ΔCt) before calculating ΔΔCt between the control (WT after ONC) and experimental group (NFATc4 −/− after ONC). Housekeeping genes used for normalization were selected based on the recommendations of Vandesompele et al. [ 56 ]. Two Microarray Quality Control studies demonstrated that a P-value calculation based on fold change could be considered sufficient for obtaining reproducible results across microarray analyses, including RT 2 Profiler PCR Arrays [ 57 , 58 ]. Retina cryosection staining One or five days after ONC, eyes were removed, incised at the cornea for better penetration, and immersed in a 3.8% PFA solution for 48 h at 4 o C. Subsequently, the eyes were incubated in a 30% sucrose solution for an additional 4 h, embedded in OCT medium, and cryosectioned into 10-µm thickness. Retinal sections were blocked with 5% bovine serum albumin (BSA)/0.3% Triton X-100 in PBS for 10 min at room temperature. Primary antibodies against NFATc4 (1:500, SAB4501982, Merck), RBPMS (1:500, 1832, Aves Labs), or cleaved caspase-3 (1:200, 9661, Cell Signaling) were applied in BSA-containing blocking buffer for 1 h at room temperature. Following several washes with PBS, sections were stained with Alexa Fluor 488-conjugated secondary antibodies (1:500, Invitrogen) for 1 h before final washing and mounting. Images were acquired with a Leica SP8 confocal laser scanning microscope. The nuclei of retinal cells were counterstained with DAPI (1:5000). For quantitative analysis, caspase-3 positive cells were counted in RGC layer of the retina using ImageJ counting plugin 1.41 software. The density profiles were expressed as the mean number of caspase-3 positive cells per mm2, providing a quantitative assessment of apoptotic cell density in the RGC layer. In vitro luciferase reporter assay NFAT transcriptional activity was assessed following a protocol similar to [ 59 ] with some modifications. In brief, lentiviral particles were designed to carry a firefly luciferase gene under the control of the NFAT response element positioned upstream of the minimal TATA promoter. Primary hippocampal neurons were transduced with Lenti-NFAT luciferase reporter and Lenti-luciferase at DIV0, and the neurons were cultured for 3 days. NFAT transcriptional activity in control cells, NFATc4-overexpressing cells, or VIVIT-expressing cells was measured in cell lysates using the Dual-Glo Luciferase Assay System (Promega) according to the manufacturer's instructions. The expression of the NFAT luciferase reporter was normalized to the expression of firefly luciferase. The fold increase of normalized NFAT luciferase reporter was then calculated over the baseline values. Electroretinography (ERG) ERG was conducted following a protocol similar to [ 60 ] with some modifications. Mice were dark-adapted overnight and then anesthetized with intraperitoneal administration of ketamine/xylazine (100 mg/kg; 20 mg/kg). Both eyes were treated with 1% atropine sulfate, 2.5% phenylephrine hydrochloride, and 0.5% proparacaine hydrochloride for approximately 2 min. Electrodes were carefully positioned onto the corneas of both eyes using hypromellose ophthalmic solution. Single flashes of 10 ms duration with an intensity of 2.48 cd-s/m 2 were applied for stimulation under scotopic conditions. The recordings were performed using the UTAS-E2000 (Universal Testing and Analysis System Electrophysiologic 2000) equipment (LKC Technologies, USA). This method allows for the assessment of retinal function through the measurement of electrical responses to light stimuli, providing valuable information on the integrity and activity of the retina. Statistics Statistical analysis was performed using GraphPad Prism 8.0.1 version. The normality of data was checked with Shapiro-Wilk test. Statistical significance was determined using Student’s t-test, one- or two-way ANOVA with multiple comparison post hoc correction. RESULTS NFATc4 is transiently increased after optic nerve injury The expression of NFAT isoforms has been previously established in an intact mouse retina [ 35 ]. At the mRNA level, NFATc3 was identified as the predominant isoform, although the expression of NFATc2 was also readily detected. In contrast, the expression of NFATc1 and NFATc4 was relatively low [ 35 ]. To investigate the potential involvement of the NFAT transcription factor family in RGC survival and regeneration following injury, we initially assessed the changes in NFATc1-c4 expression following ONC. This model was chosen due to the predictable and consistent pattern of RGC death after optic nerve lesion, facilitating the tracking of molecular events underlying RGC loss [ 61 ]. The expression of NFATc4 increased significantly following ONC, peaking on day 1 and returning to baseline levels on day 5 when compared to contralateral control and GAPDH (Fig. 1 ). This suggests that NFATc4 may be involved in RGC response to injury. NFATc4 staining conducted on day 1 post-crush revealed a concentration of the signal within the GCL of the retina. We also observed a more muted expression of NFATc4 within the inner plexiform layer, which could contribute to the changes detected by Western blot. However, the alterations identified after optic nerve crush are likely attributed to responses from RGCs, as optic nerve injury is a well-characterized model of RGC degeneration [ 62 ]. We additionally examined the expression of NFATc1-c3, but no significant changes at the mRNA level were detected after ONC (see Supplementary Fig. 1). NFATc4 is important for RGC growth and survival in vitro In an initial exploration of NFATc4's significance in optic nerve degeneration, purified RGCs were cultured, and NFATc4 level was selectively reduced using siRNA (Fig. 2 ). Given previous findings indicating the pro-survival effects of neurotrophins and elevated cAMP level, RGCs were initially cultured in the presence of forskolin (an adenylyl cyclase activator), BDNF, and CNTF. It is noteworthy that in vitro culturing and electroporation of RGCs can induce ongoing cell death even in a rich maintenance media, partially mimicking conditions during in vivo optic nerve injury. Two days post-electroporation, NFATc4 expression was suppressed by nearly 80% relative to the control siRNA group. NFATc4 silencing slightly increased RGC viability but did not impact axonal outgrowth, suggesting a role for NFATc4 in neuronal survival. To confirm the specificity of the observed change, we subsequently investigated whether a similar effect could be achieved by silencing NFATc3, the main NFAT isoform expressed in the retina (Fig. 2 .). In contrast to NFATc4, silencing NFATc3 with an efficiency of approximately 70% did not exert a pro-survival effect. Control experiments performed 2 h following electroporation showed no differences between NFATc4, NFATc3 and control siRNA groups, indicating that the observed effects were not due to differential electroporation. Nfatc4 −/− mouse has higher baseline RGC survival after injury While the conditions of RGC culturing are termed “stressed”, it is essential to note that there are differences in the underlying mechanisms between RGC growth and survival in vitro and those governing RGC survival in vivo. Therefore, the observed significance of NFATc4 to RGC survival in vitro may not necessarily be replicated in vivo during optic nerve injury. To explore this hypothesis, we utilized a mutant mouse with genetically ablated Nfatc4 (Nfatc4 −/− mouse). For rescue experiments, we delivered GFP-tagged NFATc4 to the retina using lentiviral vectors as the size of NFATc4 exceeds the capacity of adeno-associated virus, serotype 2 (AAV2). The transgene delivered via lentiviral-mediated transfer has been demonstrated to be expressed preferentially in retinal neurons of GCL and a small population of retinal pigment epithelial cells and lasts at least, up to 3 weeks [ 63 ]. Nfatc3 −/− mouse was included in the study to determine the specificity of observed changes. First, we tested the lentiviruses in vitro using primary hippocampal neurons. We observed notable viral efficiency, evidenced by an approximately 10-fold increase in NFATc4 mRNA level in GFP-NFATc4 overexpressing cells, as compared to Lenti-GFP transduced controls (Fig. 3A). Additionally, utilizing a lentiviral NFAT reporter, we confirmed that Lenti-GFP-NFATc4 overexpression was linked to higher baseline NFAT transcriptional activity (Fig. 3B). When Lenti-GFP-NFATc4 was intravitreally injected, NFATc4 −/− retinas exhibited significant NFATc4 expression three weeks post-transduction, contrasting with control retinas transduced with Lenti-GFP (Fig. 3C). These experiments highlight the effectiveness of Lenti-GFP-NFATc4 lentiviral injections to stably express functional NFATc4 in various systems, enabling rescue experiments in NFATc4 knockout mice. To assess the impact of NFATc4 knockout on RGC survival after injury, retinal flat mounts from adult NFATc4 −/− and wild-type controls were stained with RBPMS, and the labelled RGCs were quantified. The average number of RGCs in the NFATc4 −/− group (3159 ± 66 cells/mm 2 ) did not show a significant difference from the wild-type group (3243 ± 103 cells/mm 2 ), suggesting that NFATc4 is not essential for the generation of the normal RGC count (not shown). The enhancement in the survival of RBPMS-labeled RGCs became evident on day 5 following optic nerve crush in Nfatc4 −/− mice compared to wild-type mice (Fig. 4 ). The rescue of NFATc4 expression through intravitreal injections of Lenti-GFP-NFATc4 abolished this effect and reduced RGC survival rate to the level observed in RGCs transduced with the Lenti-GFP control virus. Due to the expression of active caspase-3 by RGCs following axotomy [ 23 ], we next immunodetected caspase-3 and its cleaved form in whole retinas at 5 days after ONC. In NFATc4 −/− retinas, there was a significantly lower level of cleaved caspase-3 compared to the wild-type group. Immunocytochemical staining of retinal cryosections on day 5 following the crush revealed a visibly lower signal for cleaved caspase-3 in the ganglion cell layer of NFATc4 −/− mice compared to the wild-type. As illustrated in Fig. 4 F, the density profiles reflecting the immunoreactivity of caspase-3 were significantly decreased in the Nfact4 −/− group compared to the wild-type control. These findings suggest that the neuroprotective effect of Nfatc4 knockout is exerted, at least partly, through the modulation of apoptosis-related factors. NFATc4 regulates apoptotic signaling in the injured retina As the activation of caspase-3 significantly decreased in NFATc4 −/− mice after ONC, we hypothesized that signaling pathways associated with apoptosis might be downregulated, thereby delaying RGC death. To investigate this, we conducted a microarray screening (the full list of genes can be found in Table 1 ) and observed a trend toward an increase in the average C t values in the NFATc4 −/− group compared to the WT control, although the significance did not reach the 0.05 threshold (Fig. 5 ). However, this trend could indicate a global decrease in apoptotic gene expression in NFATc4 knockout mice subjected to ONC. Table 1 The list of screened apoptotic genes. The experiment was performed in triplicate and the data were analyzed using SABiosciences PCR Array Data Analysis Web Portal as described in Materials and Methods section. Gene bank Gene symbol Description NM_001100850 Abl1 C-abl oncogene 1, receptor tyrosine kinase NM_031356 Aifm1 Apoptosis-inducing factor, mitochondrion-associated 1 NM_033230 Akt1 V-akt murine thymoma viral oncogene homolog 1 NM_013132 Anxa5 Annexin A5 NM_023979 Apaf1 Apoptotic peptidase activating factor 1 NM_001127379 Api5 Apoptosis inhibitor 5 NM_001107757 Aven Apoptosis, caspase activation inhibitor NM_022698 Bad BCL2-associated agonist of cell death NM_001106647 Bag1 BCL2-associated athanogene NM_053812 Bak1 BCL2-antagonist/killer 1 NM_017059 Bax Bcl2-associated X protein NM_031328 Bcl10 B-cell CLL/lymphoma 10 NM_016993 Bcl2 B-cell CLL/lymphoma 2 NM_133416 Bcl2a1d B-cell leukemia/lymphoma 2 related protein A1d NM_031535 Bcl2l1 Bcl2-like 1 NM_022612 Bcl2l11 BCL2-like 11 (apoptosis facilitator) NM_021850 Bcl2l2 Bcl2-like 2 NM_022684 Bid BH3 interacting domain death agonist NM_053704 Bik BCL2-interacting killer (apoptosis-inducing) NM_021752 Birc2 Baculoviral IAP repeat-containing 2 NM_023987 Birc3 Baculoviral IAP repeat-containing 3 NM_022274 Birc5 Baculoviral IAP repeat-containing 5 NM_001106835 Bnip2 BCL2/adenovirus E1B interacting protein 2 NM_053420 Bnip3 BCL2/adenovirus E1B interacting protein 3 NM_017312 Bok BCL2-related ovarian killer NM_001130554 Card10 Caspase recruitment domain family, member 10 NM_012762 Casp1 Caspase 1 NM_130422 Casp12 Caspase 12 XM_234878 Casp14 Caspase 14 NM_022522 Casp2 Caspase 2 NM_012922 Casp3 Caspase 3 NM_053736 Casp4 Caspase 4, apoptosis-related cysteine peptidase NM_031775 Casp6 Caspase 6 NM_022260 Casp7 Caspase 7 NM_022277 Casp8 Caspase 8 NM_001107921 Casp8ap2 Caspase 8 associated protein 2 NM_031632 Casp9 Caspase 9, apoptosis-related cysteine peptidase NM_134360 Cd40 CD40 molecule, TNF receptor superfamily member 5 NM_053353 Cd40lg CD40 ligand NM_057138 Cflar CASP8 and FADD-like apoptosis regulator NM_001170467 Cidea Cell death-inducing DFFA-like effector a NM_001108869 Cideb Cell death-inducing DFFA-like effector b NM_012839 Cycs Cytochrome c, somatic NM_138910 Dad1 Defender against cell death 1 NM_001107335 Dapk1 Death associated protein kinase 1 NM_053679 Dffa DNA fragmentation factor, alpha subunit NM_053362 Dffb DNA fragmentation factor, beta polypeptide (caspase-activated DNase) NM_001008292 Diablo Diablo homolog (Drosophila) NM_152937 Fadd Fas (TNFRSF6)-associated via death domain NM_080895 Faim Fas apoptotic inhibitory molecule NM_139194 Fas Fas (TNF receptor superfamily, member 6) NM_012908 Faslg Fas ligand (TNF superfamily, member 6) NM_024127 Gadd45a Growth arrest and DNA-damage-inducible, alpha NM_057130 Hrk Harakiri, BCL2 interacting protein (contains only BH3 domain) NM_012854 Il10 Interleukin 10 NM_080769 Lta Lymphotoxin alpha (TNF superfamily, member 1) NM_053842 Mapk1 Mitogen activated protein kinase 1 NM_053777 Mapk8ip1 Mitogen-activated protein kinase 8 interacting protein 1 NM_021846 Mcl1 Myeloid cell leukemia sequence 1 XM_226742 Naip2 NLR family, apoptosis inhibitory protein 2 XM_342346 Nfkb1 Nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 NM_053516 Nol3 Nucleolar protein 3 (apoptosis repressor with CARD domain) NM_017141 Polb Polymerase (DNA directed), beta NM_017169 Prdx2 Peroxiredoxin 2 NM_012630 Prlr Prolactin receptor NM_172322 Pycard PYD and CARD domain containing XM_342810 Ripk2 Receptor-interacting serine-threonine kinase 2 NM_001012066 Sphk2 Sphingosine kinase 2 NM_012675 Tnf Tumor necrosis factor (TNF superfamily, member 2) NM_001108873 Tnfrsf10b Tumor necrosis factor receptor superfamily, member 10b NM_012870 Tnfrsf11b Tumor necrosis factor receptor superfamily, member 11b NM_013091 Tnfrsf1a Tumor necrosis factor receptor superfamily, member 1a NM_130426 Tnfrsf1b Tumor necrosis factor receptor superfamily, member 1b NM_145681 Tnfsf10 Tumor necrosis factor (ligand) superfamily, member 10 NM_001001513 Tnfsf12 Tumor necrosis factor ligand superfamily member 12 NM_030989 Tp53 Tumor protein p53 XM_223012 Tp53bp2 Tumor protein p53 binding protein, 2 NM_019221 Tp63 Tumor protein p63 NM_001108696 Tp73 Tumor protein p73 NM_001100480 Tradd TNFRSF1A-associated via death domain NM_001107815 Traf2 Tnf receptor-associated factor 2 NM_001108724 Traf3 Tnf receptor-associated factor 3 NM_022231 Xiap X-linked inhibitor of apoptosis Considering a 2-fold change as a minimum and P < 0.05, we identified 7 genes with significantly downregulated expression: Ltbr , Bok , Casp2 , Bak1 , Bid , Anxa5 , Tp53bp2 . These genes include components of the tumor necrosis factor receptor superfamily, Bcl-2 protein family members, caspase superfamily, and apoptosis-stimulating protein of p53 family. Generally, the function of proteins encoded by these genes is considered pro-apoptotic. Therefore, the downregulation of apoptosis-promoting genes in response to NFATc4 −/− knockout may restrict ONC-induced RGC death. NFATc4 knockout improves retina function following optic nerve crush To investigate whether the enhanced RGC survival in Nfatc4 −/− mice correlated with the retina’s response to flash stimuli, we utilized scotopic ERG (Fig. 6 A-E). In wild-type mice, the average baseline amplitudes of a- and b-waves were 383.7 ± 9.6 µV and 813 ± 20.3 µV, respectively. In Nfatc4 −/− mice, the average baseline amplitudes of a- and b-waves were 397.2 ± 6.5 µV and 787.5 ± 24.8 µV, respectively. In wild type mice, a- and b-waves recorded 5 days post-crush were decreased by 53.5 ± 7.2% and 58 ± 3.1%, respectively, when compared to the sham control. ERG performed in Nfatc4 −/− mice on the same post-crush day showed a reduction in a- and b-waves by 18.2 ± 2.7% and 29.6 ± 2.8%, respectively. Although the ERG waves were also reduced in Nfatc4 −/− mice when compared to the sham group, these deficits were significantly less than in wild-type mice, demonstrating the positive effect of NFATc4 knockout on the function of the injured retina. NFATc4 controls axon regeneration after optic nerve injury Next, we investigated the effect of NFATc4 knockout on short-term axon regeneration (Fig. 7 ). To address this question, RGCs were labelled by intravitreal injection of cholera toxin-B subunit (CTB) and the number of axons was quantified at day 7 after ONC. The number of regenerating axons that extended 100, 250, 500, 750 µm beyond the crush point was significantly higher in the NFATc4 −/− group than in the wild type. We also measured βIII-tubulin expression within the axons to determine axonal integrity. The density of βIII-tubulin assessed 500 µm from the crush point was significantly higher in the NFATc4 −/− group compared to the wild type, suggesting delayed axonal degeneration. Interestingly, no enhanced regeneration after ONC was seen in NFATc3 −/− mice. Taken together, our results suggest that NFATc4 may promote RGC death and repress regeneration of the injured optic nerve. DISCUSSION The research on NFAT transcription factors in RGC survival and regeneration following injury has been largely discontinued since 2014 when Xu and colleagues demonstrated an overlapping pattern of NFATc4, cleaved caspase-3, and FasL in a light-induced model of retinal degeneration [ 36 ]. Using purified RGCs, as well as Nfatc4 −/− or NFATc3 −/− knockout mice and lentiviral-mediated gene delivery, we demonstrate that NFATc4 plays a crucial role in RGC survival in a model of optic nerve crush. The knockout of NFATc4 significantly improved RGC function and enhanced axonal regeneration in the injured retina. The critical role of NFATc4 is highlighted by the fact that no similar changes were observed in Nfatc3 −/− knockout mice. This suggests that NFATc4 in the retina is downstream of divergent signaling pathways mediating survival and regeneration in the presence or absence of neurotrophic factors. NFATc4 belongs to the family of Rel homology domain (RHR) and NFAT homology domain (NHR)-containing transcription factors (NFATc1-c4), whose activity is controlled in a Ca 2+ - and CaN-dependent manner. The NHR contains two CaN-binding motifs: a Ca 2+ - independent PXIXIT motif in the N terminus and a Ca 2+ -dependent LxVP motif in the C-terminal portion of NHR [ 64 ]. Despite shared activation by CaN-dependent dephosphorylation, the activity of specific NFAT isoforms within distinct populations of neuronal cells is controlled through poorly understood mechanisms. For example, NFATc4’s activity was selectively required for the survival of adult-born neurons in response to BDNF [ 65 ] and mediated anti-apoptotic transcription in NMDA receptor-stimulated cortical neurons [ 66 ]. Depending on its transcriptional activity, NFATc4 may also participate in pro-apoptotic signaling, usually combined with an extrinsic pathway-dependent increase in Fas ligand (FasL) expression. Gomez-Sintes and Lucas demonstrated that increased nuclear NFATc4 translocation correlated with elevated FasL levels and Fas activation, an effect absent in Fas-deficient Ipr mice and following cyclosporine administration [ 67 ]. Similarly, NFATc4-mediated FasL up-regulation has been proposed to underlie methamphetamine-induced neuronal loss [ 68 ]. Furthermore, deafferentiation-induced neuronal apoptosis in the cochlear nucleus has also been suggested to be mediated by NFATc4/FasL activation [ 69 ]. Hence, the opposite functions played by NFATc4 may be attributed to the upstream stimulus controlling its phosphorylation/dephosphorylation ratio or be cell-specific, as different cells can selectively activate specific NFAT isoforms depending on environmental cues [ 70 – 75 ]. The role of NFATs in RGCs is still not fully understood. Our results demonstrate that NFATc4 is specifically and transiently up-regulated in the retina after optic nerve injury. The time-course of NFATc4 increase in our experimental model is similar to the one observed in [ 36 ], suggesting a more general phenomenon. Another research group has also demonstrated a change in NFATc4 expression in response to optic nerve injury. The microarray hybridization screen performed by Lukas and colleagues within 6 h post injury revealed early downregulation of NFATc4 in the ganglion cell layer [ 76 ]. This observation was confirmed by a more recent analysis of the retinal transcriptome performed at the same time point after ONC [ 77 ]. Both studies clearly demonstrate changes in NFATc4 expression; however, they focus either on changes occurring early after ONC or performed the injury in embryonic (E20) and postnatal animals (P1-P3). Moreover, there were significant differences between postnatal and embryonic NFATc4 expression. It is known that capacity of RGC for axonal growth and the regeneration of injured axons sharply decreases soon after birth, and this age-dependent decline is associated with a profound reorganization of retinal transcriptome [ 43 , 78 ]. A growing body of evidence indicates that molecular changes in the injured retina are progressive and many of them appear later in time [ 76 , 79 – 81 ]. Therefore, it is not unexpected that the NFATc4 expression profile changes over time as RGC death becomes prominent. Consistent with our study, none of the transcriptional profiling analyses revealed changes in other NFAT isoforms after ONC. Based on our data, wherein NFATc4 knockdown promotes RGC survival in vivo, and lentiviral-mediated NFATc4 expression in Nfatc4 −/− mouse reverses this pro-survival effect, the up-regulation of NFATc4 following injury likely represents an attempted pro-apoptotic response. This NFATc4-mediated response seems to be specifically induced by the injury, as the number of RGCs in uninjured wild type and Nfatc4 −/− groups was unchanged and similar to the results previously reported for the C57BL/6 mouse [ 82 ]. This would indicate that NFATc4 expression is dispensible for normal retina development or in uninjured RGCs. The importance of CaN/NFAT signaling in retinal degeneration has been suggested by several groups. Freeman and Grosskreutz demonstrated that the administration of the FKBP12 ligand FK506 increased the number of RGCs following optic nerve crush [ 83 ]. The FK506-FKBP12 complex is expected to inhibit CaN phosphatase activity and decrease NFAT dephosphorylation, thus preventing its nuclear import. Moreover, it has been demonstrated that CaN is activated in response to ocular hypertension in the mouse model of glaucoma [ 84 ] and is responsible for RGC degeneration [ 85 ]. In view of that, knockdown of NFATc4 in vivo may disrupt calcineurin/NFATc4 downstream signaling and, at least in part, attenuate massive apoptosis of injured RGCs. This posits NFATc4 as one of the important mediators of RGC death following optic nerve crush. While the data suggests NFATc4’s involvement in RGC death, the relevance of NFATc4 function as a potential target for axonal regeneration after retina injury has not been previously explored. Using CTB and βIII-tubulin staining, we demonstrated that NFATc4 knockout delayed axon degeneration. Labeling axons with CTB is a reliable technique based on axonal transport that is widely used for monitoring axonal regeneration [ 41 ]. However, around day 7 post-crush, axonal transport is significantly altered, leading to distal axon terminal degeneration [ 86 ]. Because, in our experiment, CTB was injected 2 days before retina collection, it is also plausible that NFATc4 knockout may affect dye transport, eventually influencing the labelling pattern one week after ONC. Nonetheless, visualization of remaining axons with βIII-tubulin, which is a marker of axonal integrity [ 52 ], seems to confirm that NFATc4 plays a role in delaying axonal disintegration one week after ONC. It is hypothesized that axonal transport breakdown is preceded by a lesion-induced signaling, triggering axon swelling and irreversible changes in neurofilaments and microtubules integrity [ 87 – 89 ]. In their elegant set of experiments, Knöferle and colleagues linked axotomy-induced intraaxonal Ca 2+ elevation to a secondary generation of autophagosomes that participate in axonal degradation [ 90 ]. The initial increase in Ca 2+ concentration activating CaN is an obligatory step for the activation of NFAT-dependent transcription. Moreover, recent reports suggest an important contribution of NFAT to autophagy in retinal pigmental epithelial cells [ 91 ] as well as in other cell types [ 92 ]. In view of this, it is tempting to speculate that Ca 2+ -dependent activation of NFATc4 and NFATc4 downstream signaling should be placed among important events restricting axonal regeneration after mechanical injury. The remaining question is how NFATc4 knockout slows down the time-dependent apoptosis of injured RGC. NFAT proteins can directly regulate the expression of apoptosis-related genes along with the induction of pro-inflammatory cytokine production [ 93 – 98 ]. Both apoptosis and neuroinflammation are frequently associated with multiple neurodegenerative diseases [ 99 – 101 ]. Although it would be interesting to explore whether the modulation of retinal inflammation underlies enhanced RGC survival in NFATc4 −/− mouse, our observation of lowered caspase-3 cleavage directed us toward studying apoptosis-related genes. The microarray analysis revealed that certain pro-apoptotic genes are downregulated in NFATc4 −/− mice, indicating that increased RGC survival observed in this group after ONC may arise from blocking the apoptotic program. This is consistent with a prior study showing reduced sensitivity of sensory hair cells to TNF-mediated apoptosis in NFATc4 −/− mice [ 102 ]. In addition, Bak1, Bok, and Bid, part of the Bcl-2 family of apoptosis regulators, were downregulated in the NFATc4 −/− retina after ONC. Selective repression of BAK1 protein attenuated neuronal apoptosis [ 103 ], similar to Bax/Bak1 double knockout cells that are resistant to multiple apoptotic inducers [ 104 , 105 ]. Like BAK1 and BAX, BOK is a pro-apoptotic protein that can induce mitochondrial apoptosis [ 106 ]. In line with this finding, Bok −/− cells were partially protected from ER stress-induced apoptosis elicited by thapsigargin or bortezomib [ 107 ]. On the contrary, other studies suggested a lack of its role in apoptosis as Bok knockout does not alter responsiveness to various apoptotic stimuli [ 108 , 109 ]. Similarly, Bid-deficient mice are resistant to Fas-induced apoptosis [ 110 ], and Tp53bp2 downregulation protected from apoptosis in certain cell types [ 111 , 112 ]. However, which NFATc4-dependent changes in gene expression reflect a pro-survival response, improving RGC survival and delaying axonal degeneration, needs further attention. It has been recently demonstrated that among 46 different RGC subtypes distinguished by high-throughput single-cell RNA-seq [ 113 ],some types exhibit selective resilience to injury while others are more susceptible to degeneration and die quickly [ 114 ]. Since NFATc4 may affect the expression profile of genes involved in apoptosis, certain types of RGCs may be more vulnerable because of their NFATc4 expression, consistent with our data that NFATc4 up-regulation peaked 1 day after ONC. NFATc4 is unique among other NFAT isoforms in its regulation by upstream signaling in neurons. Unlike NFATc3, activation of NFATc4 requires a coincident elevation in intracellular Ca 2+ and suppression of glycogen synthase kinase 3β (GSK-3β) [ 74 ]. GSK-3β and other kinases are known to phosphorylate multiple serines in the NFAT regulatory domain, leading to the termination of NFAT-dependent gene expression [ 115 – 117 ]. It has not been fully resolved whether the activity of phosphorylating/dephosphorylating enzymes is an organized mechanism. In view of this, an interesting question that remains elusive is how the activity of NFATc4 is orchestrated to direct the RGC response to injury and affect the regeneration of injured axons. Our previous study [ 47 ] demonstrated that manipulation within A-kinase anchoring protein 6 (AKAP6)-organized pro-survival signaling significantly enhanced RGC survival following ONC. AKAP6 brings together calcineurin [ 118 ], ERK5 [ 119 ] and NFAT transcription factor (unpublished data), providing a platform for the integration of pro-survival an pro-death signaling. Depending on the upstream stimuli, ERK5 activity can be effectively counterbalanced by locally anchored CaN, with the relevant outcome toward NFATc4 downstream signaling. Up to now, more than fifty AKAPs have been identified that are involved in different cellular processes. This abundance allows for efficient spatial and temporal control of intracellular signaling, but which AKAPs may potentially participate in RGC survival requires further investigation. It has been demonstrated that distinct NFAT isoforms may antagonize each other in the control of gene expression in retina degeneration [ 120 ]. For instance, siRNA-mediated NFATc3 knockdown increased the expression of TNFα-induced inflammatory response, whereas downregulation of NFATc4 has the opposite effects. Several molecular therapies based on pharmacological NFAT inhibition have been described to carry substantial potential toward retina degeneration [ 35 , 121 ]. It is highly likely that greater efficacy could be achieved by identifying the NFAT isoform’s role in RGC degeneration, which would give rise to development of isoform-specific therapies. Therefore, our intent was to investigate how NFAT isoforms contribute to the pathological events underlying injury-mediated RGC loss. To our best knowledge, no similar study with NFATc4 or NFATc3 knockout animals has been performed up to now. In summary, our data suggest that NFATc4 should be considered one of the major regulators of adult RGC survival following injury, and central to the complex interplay of multiple molecular events in axonal regeneration. Further studies on NFATc4 and, in particular, the co-regulators of its transcriptional activity are essential, as they may lead to new therapeutic interventions allowing for the preservation of RGC function. Despite accumulating studies on gene therapy enhancing RGC survival and axon regeneration, the search for novel target molecules is of paramount importance, as the functional restoration of visual pathways still remains a challenge. The synergistic effect of NFATc4 downregulation along with other known axon regeneration promoters may provide an effective combinatorial strategy to improve vision impairments in optic neuropathies. Declarations Conflict of Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Funding This work was supported by the National Science Centre (Narodowe Centrum Nauki) grant no. UMO-2019/33/B/NZ4/00587. Author Contribution JM, ML, JT, FG, AS performed experiments and analyzed the data. TB, AS and FG wrote and edited the manuscript. TB was responsible for funding acquisition. Data Availability Statement The datasets used and/or analyzed during the study are available upon requests from the corresponding authors. 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Biochim Biophys Acta Mol Basis Dis 1867(12):166238. 10.1016/j.bbadis.2021.166238 Additional Declarations No competing interests reported. Supplementary Files Supplementaryfiles.docx Cite Share Download PDF Status: Published Journal Publication published 19 Apr, 2024 Read the published version in Molecular Neurobiology → Version 1 posted Editorial decision: Revision requested 19 Feb, 2024 Reviews received at journal 18 Feb, 2024 Reviewers agreed at journal 05 Feb, 2024 Reviewers agreed at journal 05 Feb, 2024 Reviewers agreed at journal 29 Jan, 2024 Reviewers invited by journal 25 Jan, 2024 Editor assigned by journal 10 Jan, 2024 Submission checks completed at journal 10 Jan, 2024 First submitted to journal 27 Dec, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3813885","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":266511187,"identity":"1ec11fec-f98f-4f2c-9478-8e8354ace231","order_by":0,"name":"Joanna Mackiewicz","email":"","orcid":"","institution":"Medical University of Lodz","correspondingAuthor":false,"prefix":"","firstName":"Joanna","middleName":"","lastName":"Mackiewicz","suffix":""},{"id":266511188,"identity":"6971010b-d9a0-4985-89e9-3736456f5d68","order_by":1,"name":"Julia Tomczak","email":"","orcid":"","institution":"Medical University of Lodz","correspondingAuthor":false,"prefix":"","firstName":"Julia","middleName":"","lastName":"Tomczak","suffix":""},{"id":266511189,"identity":"e1e75a24-ab3c-4fde-bf7b-1e1ec7f07696","order_by":2,"name":"Malwina Lisek","email":"","orcid":"","institution":"Medical University of Lodz","correspondingAuthor":false,"prefix":"","firstName":"Malwina","middleName":"","lastName":"Lisek","suffix":""},{"id":266511190,"identity":"69d3b705-df08-4668-ac4b-ed15cfb4c826","order_by":3,"name":"Agata Sakowicz","email":"","orcid":"","institution":"Medical University of Lodz","correspondingAuthor":false,"prefix":"","firstName":"Agata","middleName":"","lastName":"Sakowicz","suffix":""},{"id":266511191,"identity":"1d1d3df0-fd05-4da6-878f-8b5b4ba3df4c","order_by":4,"name":"Feng Guo","email":"","orcid":"","institution":"China Medical University","correspondingAuthor":false,"prefix":"","firstName":"Feng","middleName":"","lastName":"Guo","suffix":""},{"id":266511192,"identity":"4c2cda69-d9e4-4dfa-88ce-8a51865c17fd","order_by":5,"name":"Tomasz Boczek","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBklEQVRIiWNgGAWjYBCDBBDB2FABZjMeYGAD0uzYlfKgajkDYUO0MBOjpbGNCC327N2JH3/8YcjjZz/+8OHMeYej+dmBWj6U1TGY47KF5+xmaR4ehmLJnoRkw43bDufO7HnAcHDGOTYGy2YcWiRyN0gzSDAkbrjBcEzyIVDLhhsJDId523gYDA7j1LL55w8DkBbG9p8P5xzO3Q/RIoFPyzYJngSQFmY2xo0NQFskwFoMcGs5c3abNc8BCaBf0pglZxxLz51x5mED0C8JPLj8wt7eu/nmjz824BD72FNjndvfnnzwATDE5MzZG7DrgQAJZA4jWCmPAT4N2AEZWkbBKBgFo2B4AgBACGDh2D9oWQAAAABJRU5ErkJggg==","orcid":"","institution":"Medical University of Lodz","correspondingAuthor":true,"prefix":"","firstName":"Tomasz","middleName":"","lastName":"Boczek","suffix":""}],"badges":[],"createdAt":"2023-12-27 21:59:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3813885/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3813885/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12035-024-04129-0","type":"published","date":"2024-04-19T23:06:31+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":49515937,"identity":"6c1b1390-2bc6-4ddd-8d71-94a6e6d8b363","added_by":"auto","created_at":"2024-01-12 08:14:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":393847,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe changes in NFATc4 in the adult retina following optic nerve crush (ONC). \u003c/strong\u003e(A)\u003cstrong\u003e \u003c/strong\u003eExperimental design scheme. Retinas were collected at different time points (either 12h, 1, 3 or 5 days) after ONC for subsequent experiments. (B) Changes in NFATc4 mRNA expression level measured by real-time PCR. Raw data were normalized to Gapdh\u003cem\u003e \u003c/em\u003eexpression, and the relative fold change was calculated using 2\u003csup\u003e-ΔCt\u003c/sup\u003e method, n=3 at different time points after ONC. (C) Representative western blot for NFATc4 and quantification of protein expression after normalization to GAPDH protein level, n=3 at different time points after ONC. Data are presented as means ± SEM with individual values indicated on graphs. * P\u0026lt;0.05, ** P\u0026lt;0.01, *** P\u0026lt;0.001. (D) Quantification of NFATc4 protein level in Sham-operated or ONC\u003csup\u003e \u003c/sup\u003eretinas following normalization to endogenous GAPDH level, n=3. AU – arbitrary units. (E) NFATc4 expression in sectioned adult retina following optic nerve injury. Representative micrographs of retina sections were evaluated for NFATc4 expression on day 1 after optic nerve crush. RBPMS was stained to visualize RGCs, and DAPI was used to locate ganglion cell layer (GCL), inner nuclear layer (INL), and outer nuclear layer (ONL). Strong immunoreactivity was present within the GCL. Scale bar: 50 μm.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-3813885/v1/6c7c5ec92c02182c66927a3c.png"},{"id":49515678,"identity":"7f03fce8-3cf4-4f72-9cf5-cce32025552c","added_by":"auto","created_at":"2024-01-12 08:06:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":198797,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe requirement for NFATc4 in RGC survival and axonal outgrowth in vitro.\u003c/strong\u003e (A) The efficiency of Nfatc4 silencing calculated using 2\u003csup\u003e-ΔΔCt\u003c/sup\u003e method relative to scrambled siRNA control. The data were normalized to the endogenous Gapdh expression level, n=4. (B) Purified RGCs were electroporated with either NFATc4 siRNA or control and stained with Annexin V to visualize apoptotic cells at different time points. Scale bar: 100 μm. (C) RGC survival normalized to scrambled siRNA-treated RGCs for indicated time points. n=3. (D) Average neurite length of the Nfatc4 siRNA group normalized to control RGCs. n=3. (E) The efficiency of Nfatc3 silencing calculated with 2\u003csup\u003e-ΔΔCt \u0026nbsp;\u003c/sup\u003emethod after normalization to Gapdh expression. The expression level in scrambled siRNA-transfected cells was taken as 1. \u0026nbsp;n=4. (F) Purified RGCs electroporated with either control or NFATc3 siRNA stained with Annexin V. Scale bar: 100 μm. (G) RGC survival following NFATc3 siRNA treatment normalized to scrambled siRNA control for respective time points. n=3. (H) Average neurite length of Nfatc3 siRNA-treated cells normalized to control RGCs. n=3. The data are presented as means ± SEM. * P\u0026lt;0.05, ** P\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-3813885/v1/bbb2ff3f842587d33b4b8731.png"},{"id":49515671,"identity":"222c1b8b-b208-4f4b-ac48-d4c6d68b65b4","added_by":"auto","created_at":"2024-01-12 08:06:26","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":193740,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNFATc4 expression and NFAT transcriptional activity following lentiviral transduction. \u003c/strong\u003e(A)\u003cstrong\u003e \u003c/strong\u003eNfatc4 mRNA expression was assessed in primary hippocampal neurons following transduction with either Lenti-GFP-NFATc4 or Lenti-GFP, using real-time PCR. Raw data were normalized to Gapdh endogenous expression and calculated using 2\u003csup\u003e-ΔΔCt\u003c/sup\u003e method. Nfatc4 expression level in non-transduced cells was set as 1. The data are presented as means ± SEM, with individual values obtained from n=4 replicate treatment. (B) Primary hippocampal neurons were co-transduced with NFAT dual-reporter lentivirus and Lenti-GFP-NFATc4 (or other viruses as indicated on the graph) on DIV0 and cultured until DIV3. NFAT transcriptional activity was determined in cell lysates by measuring luciferase activity (n=4). The results are expressed as a fold induction above baseline activity. The data are presented as means ± SEM, with individual values indicated on the graphs. * P\u0026lt;0.05, *** P\u0026lt;0.001. (C) Lenti-GFP or Lenti-GFP-NFATc4 were intravitreally injected into NFATc4\u003csup\u003e-/-\u003c/sup\u003e retinas, followed by NFATc4 staining three weeks later. The retinas were stained using the antibodies indicated in \u003cem\u003eRetina cryosection staining\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003e(primary: NFATc4, 1:250, Merck; secondary: anti-rabbit conjugated to Alexa Fluor 488, 1:500). Representative images are presented. Scale bar: 100 μm.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-3813885/v1/15d59f862215027d86b91afb.png"},{"id":49515677,"identity":"60f1757e-257c-4c26-9f00-30878c74a4e9","added_by":"auto","created_at":"2024-01-12 08:06:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":300009,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNfatc4\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e-/-\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e mice exhibit increased RGC survival after optic nerve injury. \u003c/strong\u003e(A) The time course of RGC death after optic nerve injury in WT or Nfatc4\u003csup\u003e-/- \u003c/sup\u003emice with or without intravitreal injection with Lenti-GFP-NFATc4 or Lenti-GFP control virus. All points are n=4 animals per point, normalized to naïve WT eyes. (B) The time course of RGC death after ONC showing no difference between WT and Nfatc3\u003csup\u003e-/- \u003c/sup\u003egroup, n=4 animals per time point. RGC survival was normalized to WT naïve eyes. (C) Western blot analysis of caspase-3 and cleaved caspase-3 in a whole retina isolated on day 5 after ONC. GAPDH was used as a loading control. Representative blots are shown. (D) Quantification of cleaved caspase-3 protein level in WT or Nfatc4\u003csup\u003e-/- \u003c/sup\u003eretinas following normalization to endogenous GAPDH level, n=4. AU – arbitrary units. (E) Representative micrographs showing active caspase-3 staining in retina cryosections done on day 5 following ONC. The arrows indicate puncta corresponding to cleaved caspase-3 located in the ganglion cell layer (GCL). (F) Bar charts showing the quantitative analyses of average cleaved caspase-3–positive cell counts in the retina (n = 4, two images per sample). The data on the graph are presented as means ± SEM. * P\u0026lt;0.05, ** P\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-3813885/v1/cb2ad99669f863e0a9edd431.png"},{"id":49515936,"identity":"67e169a0-2342-4628-9a9e-f597e300c3fa","added_by":"auto","created_at":"2024-01-12 08:14:26","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":322190,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNFATc4 knockout downregulates pro-apoptotic signaling pathways 5 days after ONC. \u003c/strong\u003e(A)\u003cstrong\u003e \u003c/strong\u003eHeat map adjusted to reflect variations in individual genes for each run. The map was generated using GraphPad Prism based on the microarray data. (B) Dot plot comparison of average C\u003csub\u003et\u003c/sub\u003e values of screened genes. The average is presented as a black horizontal line. (C) The volcano plot showing whole retina gene expression in WT and NFATc4\u003csup\u003e-/-\u003c/sup\u003e mice following ONC. Genes (NFATc4\u003csup\u003e-/-\u003c/sup\u003e vs. WT) with P\u0026lt;0.05 and fold change less than -2 are highlighted in red.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-3813885/v1/d9d0a81965ee76debfd28a1a.png"},{"id":49515672,"identity":"3f06c931-3a0a-422d-b77a-9ff41e04d443","added_by":"auto","created_at":"2024-01-12 08:06:26","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":124729,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of NFATc4 knockout on electroretinogram responses. \u003c/strong\u003e(A) Representative ERG traces for wild-type mice recorded before and 5 days after ONC. (B) Representative ERG traces for Nfatc4\u003csup\u003e-/-\u003c/sup\u003e mice recorded before and 5 days after ONC. Each ERG was obtained by averaging two responses to 2.48 cd-s/m\u003csup\u003e2\u003c/sup\u003e flashes with an interstimulus interval of 2 minutes. (C) Analysis of ERG a-wave amplitudes in WT and Nfatc4\u003csup\u003e-/- \u003c/sup\u003emice before and 5 days after ONC. (D) Analysis of ERG b-wave amplitudes in WT and Nfatc4\u003csup\u003e-/- \u003c/sup\u003emice before and 5 days after ONC. (E) Normalized a-wave and b-wave amplitudes 5 days after ONC. The data on the graph are presented as means ± SEM. *** P\u0026lt;0.001, n=4.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-3813885/v1/b89c55afe164bf9a8c8de40e.png"},{"id":49515676,"identity":"6811b2c7-2951-46c8-be8d-b4519f928274","added_by":"auto","created_at":"2024-01-12 08:06:26","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":586600,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of NFATc4 knockout on axon regeneration after optic nerve crush. \u003c/strong\u003e(A) Representative images showing CTB-labelled regenerating axons in NFATc4\u003csup\u003e-/-\u003c/sup\u003e mice on day 7 after optic nerve crush. (B) Quantification of regenerating axons from the injury site, n=6 per group. (C) βIII-tubulin staining showing delayed optic nerve degeneration in NFATc4\u003csup\u003e-/-\u003c/sup\u003e mice. (D) Quantification of fiber density, n=4. (E) Representative images of regenerating axons in NFATc3\u003csup\u003e-/-\u003c/sup\u003e mice on day 7 post-crush. (F) Quantification of regenerating axons, n=6 per group. Asterisks mark the crush site. The data on the graph are presented as means ± SEM. ** P\u0026lt;0.01, *** P\u0026lt;0.001. Scale bar: 250 μm.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-3813885/v1/c51b42682d37e4dac7bba1fc.png"},{"id":55691169,"identity":"9a12ffdd-db66-4011-ba89-a737b42b753d","added_by":"auto","created_at":"2024-05-01 23:06:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3268099,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3813885/v1/4f69469a-a82d-4a72-ae8c-81fa8d350080.pdf"},{"id":49515673,"identity":"3be83960-c445-400c-a72a-0f0a21b518ee","added_by":"auto","created_at":"2024-01-12 08:06:26","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":101455,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfiles.docx","url":"https://assets-eu.researchsquare.com/files/rs-3813885/v1/eb81e12e011e4d4cfe1c1009.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"NFATc4 knockout promotes neuroprotection and retinal ganglion cell regeneration after optic nerve injury","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eRetinal ganglion cells (RGCs), a highly specialized type of neurons, transmit visual information from the retina to the visual processing centers of the brain. Because the unidirectional optic nerve is formed exclusively by axons projecting by RGCs, it is highly vulnerable to various injuries, leading to irreversible loss of RGC function. The progressive death of RGCs is a crucial element in the pathophysiology of glaucoma, characterized by the progressive degeneration of the optic nerve and resulting in irreversible blindness. Over the decades, numerous studies have investigated the mechanisms underlying RGC death and identified several neuronal abnormalities associated with antioxidant imbalance [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], neuroinflammation [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], mitochondrial metabolism alterations [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], ischemia/hypoxia [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], or vascular deficits [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Recent studies have also demonstrated a promising effect of small molecules and virus-based gene therapies in pre-clinical models of glaucoma [\u003cspan additionalcitationids=\"CR7 CR8\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eConsiderable effort is currently dedicated to unraveling molecular changes underlying disease progression and understanding signaling pathways that can be manipulated to enhance RGC survival. The most effective approaches involve the exogenous administration of neurotrophic factors or apoptotic pathway inhibitors, such as brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), neurotrophin-4, or their combination with oncomodulin or osteopontin [\u003cspan additionalcitationids=\"CR11 CR12 CR13 CR14 CR15\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In the majority of cases, the effects are only transitory, even with long-lasting treatments. Similarly, caspase inhibitors provide only transient neuroprotection [\u003cspan additionalcitationids=\"CR18 CR19 CR20 CR21 CR22\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Strategies based on the knockdown or overexpression of other prominent regulators of RGC growth and survival, such as Elk-1 [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], Sigma-1R (σ-1r) [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], MEF2 [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], or PTEN [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] have also been tested. Despite the tremendous progress in several molecular interventions targeting RGC survival following injury, most of them present only limited effectiveness in clinical interventions. This limitation likely arises from the choice of inappropriate target(s) or time window. Therefore, the elucidation of mechanisms leading to RGC death is essential to target the right signaling molecule or decipher the therapeutic time-window.\u003c/p\u003e \u003cp\u003eOriginally described as important regulators of immune function [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], NFATs are now implicated in the regulation of neuronal morphogenesis, plasticity, and the response to neurotrophin and electrical stimulation [\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In humans, the NFAT family comprises five transcription factors named NFAT1 (NFATc2), NFAT2 (NFATc1), NFAT3 (NFATc4), NFAT4 (NFATc3), and NFAT5, which, all but the last one, are regulated by Ca\u003csup\u003e2+\u003c/sup\u003e - activated protein phosphatase-2B/calcineurin (CaN). The nature, source and timing of upstream signals regulating Ca\u003csup\u003e2+\u003c/sup\u003e/CaN activity and the flexibility of NFAT in cooperating with other transcriptional activators or repressors make an important contribution to neuronal response to external stimuli. This relationship is seen in both developing and mature neurons. For instance, profound defects in axon projections were observed in embryos with combined deletion of either NFATc3 or NFATc4 (c3/c4 mutants) or NFATc2, NFATc3, and NFATc4 (c2/c3/c4 mutants) [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].These defects were reproduced by in utero administration of cyclosporine A (CsA) \u0026ndash; a potent inhibitor of CaN. CaN and NFATs are also essential for neurotrophin-induced neuronal outgrowth and survival [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe role of NFATs in RGCs is not yet fully understood, as it becomes increasingly apparent that these cells can selectively activate specific NFAT isoforms. Recently, NFATc2 and NFATc3 have been identified as the major isoforms expressed in retina [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. NFATc4, present in low amounts, was significantly upregulated in RGCs following light-induced damage and was associated with increased neuronal apoptosis [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Despite the obvious role of NFAT in neuronal function, the contribution of NFAT isoforms to retinal degeneration following pro-death insults has not been widely studied. This is of paramount importance as the progressive loss of RGCs is a hallmark common to the majority of optic neuropathies, including glaucoma, often leading to permanent blindness [\u003cspan additionalcitationids=\"CR38 CR39\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Traumatic optic neuropathy and glaucomatous injury can be mimicked by mechanical optic nerve crush (ONC), which serves as a preclinical model of neuronal survival and regeneration, as it similarly induces RGC death and degeneration. In this model, the lesion severs all of the RGCs\u0026rsquo; axons, ensuring high reproducibility and precise control of the injury site [\u003cspan additionalcitationids=\"CR42 CR43 CR44\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. As optic neuropathy can be linked with other CNS diseases, this model can also be used in CNS degeneration studies to unravel degenerative mechanisms and test neuroprotective therapies. Using ONC, we provide evidence for the critical role of NFATc4 in RGC survival following injury.\u003c/p\u003e"},{"header":"MATERIALS and METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003e All animal procedures were carried out according to the Association for Research in Vision and Ophthalmology (ARVO) guidelines for the use of animals in ophthalmic and vision research. The experimental protocols were approved by the Institutional Animal Care and Use Committee at the Medical University of Lodz. All mice used in this project were C57BL/6. The animals were group-housed in laboratory cages and kept under a controlled temperature (23\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C) with a 12-h light/dark cycle and with food and water provided ad libitum. Nfatc4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice (B6;129S-\u003cem\u003eNfatc4\u003c/em\u003e\u003csup\u003etm1Grc\u003c/sup\u003e/J, strain #027581) and Nfatc3\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice (B6;129S2-\u003cem\u003eNfatc3\u003c/em\u003e\u003csup\u003etm1Glm\u003c/sup\u003e/J, strain #010589) were obtained from the Jackson Laboratory (USA). Both males and females were used in all experimental procedures.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eLentiviruses\u003c/h2\u003e \u003cp\u003eLentiviruses were employed for in vivo delivery of NFATc4-GFP to NFATc4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mouse retina, owing to the limited capacity of adeno-associated virus serotype 2. Lenti ORF particles, Nfatc4 (GFP-tagged) transcript variant 2 (reference sequence NM_001168346.1, 4036 bp), as well as Lenti ORF control particles of pLenti-C-mGFP-P2A-Puro, were generated by Origene (USA). NFAT luciferase reporter lentivirus and firefly luciferase lentivirus were sourced from BPS Bioscience (USA). Lenti-VIVIT-GFP was produced with pLV-VIVIT-GFP plasmid (Addgene#188707) using Lenti-X Packaging Single Shots technology from Takara Bio (USA). All viral titers were \u0026gt;\u0026thinsp;10\u003csup\u003e7\u003c/sup\u003e TU/ml. In vitro or in vivo transduction was caried out as specified for each experiment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eIsolation of primary neurons\u003c/h2\u003e \u003cp\u003eRGCs were purified from postnatal (P8-P10) mouse pups using a procedure essentially described in [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] with some modifications. Isolated retinas were washed 3 times with Dulbecco Phosphate Buffer Saline (DPBS) and digested with papain (16.5 U/ml) for 30 min at 37\u003csup\u003eo\u003c/sup\u003eC. After trituration, papain activity was inhibited by adding ovomucoid solution (1.5 mg/ml), and the cell suspension was centrifuged at 250 x g for 10 min. The resulting pellet was mixed with DPBS supplemented with 5 \u0026micro;g/ml insulin and transferred to anti-macrophage antibody-coated petri dishes for a 45 min incubation. Suspended cells were subsequently transferred to petri dishes containing anti-Thy 1.2 antibody conditioned media and further incubated for 45 min to isolate RGCs. Petri dishes were washed multiple times, and RGCs were released by trypsinization. RGCs were routinely seeded at the density of 50.000 cells/well in a 24 well plate coated with poly-D-lysine (10 \u0026micro;g/ml) and laminin (1 \u0026micro;g/ml). Cells were cultured in serum-free media supplemented with sodium pyruvate (1 mM), B27 (1:50), BDNF (50 ng/ml), CNTF (10 ng/ml), forskolin (5 M), insulin (5 \u0026micro;g/ml), N-acetyl cysteine (5 \u0026micro;g/ml), L-glutamine (1 mM) and triiodothyronine (40 ng/ml), unless otherwise specified, at 37\u003csup\u003eo\u003c/sup\u003eC/5% CO\u003csub\u003e2\u003c/sub\u003e and in a humidified atmosphere. The purity of isolated RGCs was verified by staining with antibodies recognizing RNA binding protein with multiple splicing (RBPMS) and typically exceeded 95%.\u003c/p\u003e \u003cp\u003ePrimary hippocampal neurons were prepared following the methodology outlined in our previous publication [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. In brief, hippocampal cultures were derived from Sprague-Dawley E18 embryos. Hemispheres were dissected in HBSS buffer on ice, trypsinized for 30 min at 37\u003csup\u003e0\u003c/sup\u003eC, centrifuged at 250 x g for 2 min, and then triturated with a fire-polished glass pipette. The dissociated neurons were seeded on nitric acid-soaked glass coverslips coated with poly-L-lysine in plating medium (10% v/v horse serum in DMEM). After 4 h, the medium was replaced with Neurobasal maintenance medium supplemented with 2% B27, 1 mM glutamine, 1 mM sodium pyruvate, and 5 \u0026micro;g/ml insulin. For cultures lasting beyond 4 days, half of the medium was removed on day 3 or 4 and replaced with an equal volume of fresh medium.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eIn vitro survival and neurite outgrowth\u003c/h2\u003e \u003cp\u003eIsolated RGCs (~\u0026thinsp;200,000 cells) were promptly electroporated with NFATc4 ON-TARGETplus siRNA oligonucleotides or ON-TARGETplus scrambled siRNA, both administered at 1 nmol per electroporation (Horizon Discovery, USA). The electroporation was conducted according to the method detailed elsewhere [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Subsequently, cells were cultured for up to 3 days, and stained for annexin V for 10 min in growth media to label apoptotic cells. Images of 10\u0026ndash;12 randomly selected fields were captured in growth media at 2 and 72 h using a Leica DMi8 inverted microscope to quantify cell survival. The survival was quantified using ImageJ and was normalized to scrambled siRNA-treated RGCs.\u003c/p\u003e \u003cp\u003eFor the neurite outgrowth assay, isolated RGCs were electroporated and seeded at a low density of ~\u0026thinsp;5000\u0026ndash;6000 cells/well in a 48 well plate, followed by a 3-day culture period. RGCs were then fixed with 3.8% paraformaldehyde (PFA) and permeabilized with 0.2% Triton X-100 in PBS. After several washes with PBS, cells were labelled with anti-βIII tubulin (1:500, Cell Signaling technology, USA) overnight at 4\u003csup\u003e0\u003c/sup\u003eC. The plates were next probed with secondary antibodies conjugated to Alexa Fluor 488 (1:500) for 6 h at room temperature. Nuclei were counterstained with DAPI at a dilution of 1:5000. Images were acquired on a Leica DMi8 inverted microscope, and the longest neurite per cell (~\u0026thinsp;20 cells on average in each experiment) was measured using the ImageJ Neurite Tracker tool. The results were normalized to the average axonal length in scrambled siRNA-treated RGCs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eOptic nerve crush (ONC) and intravitreal injections\u003c/h2\u003e \u003cp\u003eFor optic nerve crush, C57BL/6 male and female mice at the age 7\u0026ndash;8 weeks were randomly assigned to the treatment group. The crush procedure followed protocols essentially described in previous studies [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] with minor modifications. Mice were anesthetized with 20 mg/kg IP xylazine and 100 mg/kg IP ketamine. A drop of 0.5% proparacaine was applied to the eye. The optic nerves were exposed from the outer canthus behind the globe, and the crush was performed\u0026thinsp;~\u0026thinsp;2 mm behind the eyeball for 3 sec using extra-fine forceps. The contralateral control eye underwent the same procedure but was spared from the crush. Care was taken to avoid damaging blood vessels in the retina. Post-operative analgesia was provided with 0.5 mg/ml buprenorphine. In lentivirus transduction experiments, mice under isoflurane anesthesia were intravitreally injected through the sclera with 2 \u0026micro;l of lentivirus in PBS using a 31-gauge needle (Hamilton) connected to a 5 \u0026micro;L Hamilton syringe. Care was taken to avoid damage to the lens. Viral injections were performed approximately 2 weeks before ONC to allow for sufficient gene expression. Animals experiencing any postoperative complications such as excessive bleeding or swelling, retinal ischemia or cataract were excluded from the cohort at any time after procedure.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eRetinal flat-mount and RGC count\u003c/h2\u003e \u003cp\u003eThe procedure for retinal flat-mount preparation was conducted on deeply anesthetized mice using isoflurane and intraperitoneal administration of ketamine/xylazine, followed by sacrifice through transcardial perfusion with 4% paraformaldehyde (PFA). The methodology for retinal flat mount was adapted from [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e] and used in our previous study [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. In brief, the eyes were removed, post-fixed with 4% PFA for 2 h at room temperature, and the retinas were dissected. After several washes with PBS, retinas were permeabilized with Triton X-100 and incubated overnight at 4\u003csup\u003e0\u003c/sup\u003eC with a primary anti-RBPMS antibody (diluted at 1:500, 1832, Aves Labs) in a blocking buffer (PBS with 10% goat serum). Following washing with PBS, retinas were incubated with secondary antibodies conjugated to Alexa Fluor 488 (1:500, Invitrogen) for 2 h at room temperature. Subsequently, retinas were flat-mounted in H-1000 mounting medium on glass slides. Scan images were acquired with Leica SP8 confocal laser scanning microscope. The counting of RBPMS-positive cells was carried out in a manner described in a previous publication [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. This process, performed by an experienced researcher in a masked fashion, involved assessing RGC cell density per mm\u003csup\u003e2\u003c/sup\u003e or percentage change relative to the sham-operated contralateral eye or ONC-treated wild-type.\u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003eAnterograde labelling, quantification of regeneration and axon degeneration analysis\u003c/h2\u003e \u003cp\u003eTwo days prior to optic nerve harvesting, 2 \u0026micro;l of cholera toxin subunit B (CTB, 2 \u0026micro;g/\u0026micro;l, Invitrogen, USA) were intravitreally injected to visualize axons and nerve terminals of surviving RGCs. Animals were perfused with 4% PFA before the collection of optic nerves. The optic nerves were cryopreserved overnight in 30% sucrose at 4\u003csup\u003e0\u003c/sup\u003eC and then mounted in Optimal Cutting Temperature mounting medium (Thermo Fisher Scientific, USA). Longitudinal sections, 10 \u0026micro;m thick, were cut for optic nerves and imaged using a DMi8 fluorescence microscope (Leica, Germany). The sections were analyzed as described previously [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. The number of CTB-positive axons passing 0.1, 0.25, 0.5, 0.75, 1.0, 1.25 mm from the crush site was manually counted. The total number of CTB-positive axons per optic nerve was calculated using methods outlined in a previous study [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. This approach provides a quantitative assessment of axon survival and regeneration in response to experimental conditions.\u003c/p\u003e \u003cp\u003eAxonal integrity was assessed one week after ONC through βIII-tubulin staining. This technique enables the measurement of protein abundance within axons [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e] and has recently been employed to demonstrate delayed optic nerve degeneration in response to pharmacological inhibition of aldolase reductase [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. For immunostaining, 10-\u0026micro;m-thick cryosections were probed with anti-βIII-tubulin antibodies (1:500, Cell Signaling, USA), followed by incubation with secondary antibodies conjugated to Alexa Fluor 594 (Thermo Fisher, USA). The density of βIII-tubulin was measured in a 500x200 \u0026micro;m area immediately after the crush site [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e], following the protocol described in [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Optic nerves were imaged at the same intensity using a Leica DMi8 fluorescence microscope with a 10x objective. This approach provides insights into the preservation or alterations in βIII-tubulin expression, reflecting axonal structural integrity following the ONC procedure.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting\u003c/h2\u003e \u003cp\u003eRetinas were isolated and lysed using RIPA buffer supplemented with a protease and phosphatase inhibitor cocktail. The total protein content was quantified colorimetrically with the Bio-Rad Protein Kit Assay. Subsequently, 10\u0026ndash;30 \u0026micro;g of the protein samples were separated in 4\u0026ndash;20% gradient polyacrylamide gels and transferred to a nitrocellulose membrane using a semi-dry method. The membranes were blocked with 10% goat serum in TBST-T buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.05% Tween-20) for 2 h at room temperature. Next, the membrane was incubated with primary antibodies recognizing NFATc4 (1:750, ab183117, Abcam), caspase-3 (1:1000, PA5-77887, Thermo Fisher), or GAPDH (1:3000, G8795, Merck) for 24 h at 4\u003csup\u003eo\u003c/sup\u003eC. Following three washes in TBS-T, the membrane was probed with secondary antibodies (1:5000) conjugated to horse radish peroxidase for 2 h at room temperature. ECL western blot system was used to visualize immunoreactive bands. The membranes were scanned densitometrically, and the optical density of bands was quantified using ImageJ. The results are expressed as arbitrary units after normalization to the endogenous GAPDH level, providing a quantitative assessment of protein expression levels.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eTotal RNA isolation, real-time PCR and microarray screening\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from the retina using Trizol reagent following the manufacturer\u0026rsquo;s protocol. Single-stranded cDNA was synthesized from 1 \u0026micro;g of isolated RNA using M-MLV reverse transcriptase with oligo(dT) primers. Real-time PCR reactions were carried out under the following conditions: an initial denaturation at 95\u0026deg;C for 15 min, followed by 40 cycles at 95\u0026deg;C for 15 s, 60\u0026deg;C for 30 s, and 72\u0026deg;C for 30 s, using the Abi Prism 7000 sequence detection system using Eva Green Master Mix. Primers used in the reactions: Nfatc1 (NM_198429), Nfatc2 (NM_010899), Nfatc3 (NM_010901), Nfatc4 (NM_023699), Gapdh (NM_008084) were purchased from Origine (Germany). The specificity of the PCR product was assessed by running a melting curve. The relative expression of the gene was determined using the ΔCt method [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e], with endogenous Gapdh expression used for data normalization.\u003c/p\u003e \u003cp\u003eFor microarray screening, cDNA amplified from 2 \u0026micro;g of retinal RNA was hybridized with RT\u0026sup2; Profiler\u0026trade; PCR Array Mouse Apoptosis (Qiagen, USA), and the reaction was performed using HOT FIREPol\u0026reg; EvaGreen\u0026reg; qPCR Mix Plus (Solis Biodyne, Estonia The real-time PCR conditions included an initial cycle at 95\u0026deg;C for 10 min, followed by cycles at 95\u0026deg;C for 15 s, 60\u0026deg;C for 1 min, and a dissociation curve at 95\u0026deg;C for 1 min, 55\u0026deg;C for 30 s, and 95\u0026deg;C for 30 s. The fold change was calculated by a method of Livak and Schmittgen [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Data were analyzed using Qiagen PCR Array Data Analysis Web Portal. The microarray analysis was run in triplicate, and the RT\u003csup\u003e2\u003c/sup\u003e software averaged the triplicate normalized expression for each gene (ΔCt) before calculating ΔΔCt between the control (WT after ONC) and experimental group (NFATc4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e after ONC). Housekeeping genes used for normalization were selected based on the recommendations of Vandesompele et al. [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Two Microarray Quality Control studies demonstrated that a P-value calculation based on fold change could be considered sufficient for obtaining reproducible results across microarray analyses, including RT\u003csup\u003e2\u003c/sup\u003e Profiler PCR Arrays [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eRetina cryosection staining\u003c/h2\u003e \u003cp\u003eOne or five days after ONC, eyes were removed, incised at the cornea for better penetration, and immersed in a 3.8% PFA solution for 48 h at 4\u003csup\u003eo\u003c/sup\u003eC. Subsequently, the eyes were incubated in a 30% sucrose solution for an additional 4 h, embedded in OCT medium, and cryosectioned into 10-\u0026micro;m thickness. Retinal sections were blocked with 5% bovine serum albumin (BSA)/0.3% Triton X-100 in PBS for 10 min at room temperature. Primary antibodies against NFATc4 (1:500, SAB4501982, Merck), RBPMS (1:500, 1832, Aves Labs), or cleaved caspase-3 (1:200, 9661, Cell Signaling) were applied in BSA-containing blocking buffer for 1 h at room temperature. Following several washes with PBS, sections were stained with Alexa Fluor 488-conjugated secondary antibodies (1:500, Invitrogen) for 1 h before final washing and mounting. Images were acquired with a Leica SP8 confocal laser scanning microscope. The nuclei of retinal cells were counterstained with DAPI (1:5000). For quantitative analysis, caspase-3 positive cells were counted in RGC layer of the retina using ImageJ counting plugin 1.41 software. The density profiles were expressed as the mean number of caspase-3 positive cells per mm2, providing a quantitative assessment of apoptotic cell density in the RGC layer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eIn vitro luciferase reporter assay\u003c/h2\u003e \u003cp\u003eNFAT transcriptional activity was assessed following a protocol similar to [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e] with some modifications. In brief, lentiviral particles were designed to carry a firefly luciferase gene under the control of the NFAT response element positioned upstream of the minimal TATA promoter. Primary hippocampal neurons were transduced with Lenti-NFAT luciferase reporter and Lenti-luciferase at DIV0, and the neurons were cultured for 3 days. NFAT transcriptional activity in control cells, NFATc4-overexpressing cells, or VIVIT-expressing cells was measured in cell lysates using the Dual-Glo Luciferase Assay System (Promega) according to the manufacturer's instructions. The expression of the NFAT luciferase reporter was normalized to the expression of firefly luciferase. The fold increase of normalized NFAT luciferase reporter was then calculated over the baseline values.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eElectroretinography (ERG)\u003c/h2\u003e \u003cp\u003eERG was conducted following a protocol similar to [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e] with some modifications. Mice were dark-adapted overnight and then anesthetized with intraperitoneal administration of ketamine/xylazine (100 mg/kg; 20 mg/kg). Both eyes were treated with 1% atropine sulfate, 2.5% phenylephrine hydrochloride, and 0.5% proparacaine hydrochloride for approximately 2 min. Electrodes were carefully positioned onto the corneas of both eyes using hypromellose ophthalmic solution. Single flashes of 10 ms duration with an intensity of 2.48 cd-s/m\u003csup\u003e2\u003c/sup\u003e were applied for stimulation under scotopic conditions. The recordings were performed using the UTAS-E2000 (Universal Testing and Analysis System Electrophysiologic 2000) equipment (LKC Technologies, USA). This method allows for the assessment of retinal function through the measurement of electrical responses to light stimuli, providing valuable information on the integrity and activity of the retina.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStatistics\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed using GraphPad Prism 8.0.1 version. The normality of data was checked with Shapiro-Wilk test. Statistical significance was determined using Student\u0026rsquo;s t-test, one- or two-way ANOVA with multiple comparison post hoc correction.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eNFATc4 is transiently increased after optic nerve injury\u003c/h2\u003e \u003cp\u003eThe expression of NFAT isoforms has been previously established in an intact mouse retina [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. At the mRNA level, NFATc3 was identified as the predominant isoform, although the expression of NFATc2 was also readily detected. In contrast, the expression of NFATc1 and NFATc4 was relatively low [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. To investigate the potential involvement of the NFAT transcription factor family in RGC survival and regeneration following injury, we initially assessed the changes in NFATc1-c4 expression following ONC. This model was chosen due to the predictable and consistent pattern of RGC death after optic nerve lesion, facilitating the tracking of molecular events underlying RGC loss [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. The expression of NFATc4 increased significantly following ONC, peaking on day 1 and returning to baseline levels on day 5 when compared to contralateral control and GAPDH (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This suggests that NFATc4 may be involved in RGC response to injury.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNFATc4 staining conducted on day 1 post-crush revealed a concentration of the signal within the GCL of the retina. We also observed a more muted expression of NFATc4 within the inner plexiform layer, which could contribute to the changes detected by Western blot. However, the alterations identified after optic nerve crush are likely attributed to responses from RGCs, as optic nerve injury is a well-characterized model of RGC degeneration [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. We additionally examined the expression of NFATc1-c3, but no significant changes at the mRNA level were detected after ONC (see Supplementary Fig.\u0026nbsp;1).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eNFATc4 is important for RGC growth and survival in vitro\u003c/h2\u003e \u003cp\u003eIn an initial exploration of NFATc4's significance in optic nerve degeneration, purified RGCs were cultured, and NFATc4 level was selectively reduced using siRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Given previous findings indicating the pro-survival effects of neurotrophins and elevated cAMP level, RGCs were initially cultured in the presence of forskolin (an adenylyl cyclase activator), BDNF, and CNTF. It is noteworthy that in vitro culturing and electroporation of RGCs can induce ongoing cell death even in a rich maintenance media, partially mimicking conditions during in vivo optic nerve injury.\u003c/p\u003e \u003cp\u003eTwo days post-electroporation, NFATc4 expression was suppressed by nearly 80% relative to the control siRNA group. NFATc4 silencing slightly increased RGC viability but did not impact axonal outgrowth, suggesting a role for NFATc4 in neuronal survival.\u003c/p\u003e \u003cp\u003eTo confirm the specificity of the observed change, we subsequently investigated whether a similar effect could be achieved by silencing NFATc3, the main NFAT isoform expressed in the retina (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.). In contrast to NFATc4, silencing NFATc3 with an efficiency of approximately 70% did not exert a pro-survival effect. Control experiments performed 2 h following electroporation showed no differences between NFATc4, NFATc3 and control siRNA groups, indicating that the observed effects were not due to differential electroporation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eNfatc4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mouse has higher baseline RGC survival after injury\u003c/h2\u003e \u003cp\u003eWhile the conditions of RGC culturing are termed \u0026ldquo;stressed\u0026rdquo;, it is essential to note that there are differences in the underlying mechanisms between RGC growth and survival in vitro and those governing RGC survival in vivo. Therefore, the observed significance of NFATc4 to RGC survival in vitro may not necessarily be replicated in vivo during optic nerve injury. To explore this hypothesis, we utilized a mutant mouse with genetically ablated Nfatc4 (Nfatc4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mouse). For rescue experiments, we delivered GFP-tagged NFATc4 to the retina using lentiviral vectors as the size of NFATc4 exceeds the capacity of adeno-associated virus, serotype 2 (AAV2). The transgene delivered via lentiviral-mediated transfer has been demonstrated to be expressed preferentially in retinal neurons of GCL and a small population of retinal pigment epithelial cells and lasts at least, up to 3 weeks [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. Nfatc3\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mouse was included in the study to determine the specificity of observed changes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFirst, we tested the lentiviruses in vitro using primary hippocampal neurons. We observed notable viral efficiency, evidenced by an approximately 10-fold increase in NFATc4 mRNA level in GFP-NFATc4 overexpressing cells, as compared to Lenti-GFP transduced controls (Fig.\u0026nbsp;3A). Additionally, utilizing a lentiviral NFAT reporter, we confirmed that Lenti-GFP-NFATc4 overexpression was linked to higher baseline NFAT transcriptional activity (Fig.\u0026nbsp;3B). When Lenti-GFP-NFATc4 was intravitreally injected, NFATc4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e retinas exhibited significant NFATc4 expression three weeks post-transduction, contrasting with control retinas transduced with Lenti-GFP (Fig.\u0026nbsp;3C). These experiments highlight the effectiveness of Lenti-GFP-NFATc4 lentiviral injections to stably express functional NFATc4 in various systems, enabling rescue experiments in NFATc4 knockout mice.\u003c/p\u003e \u003cp\u003eTo assess the impact of NFATc4 knockout on RGC survival after injury, retinal flat mounts from adult NFATc4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e and wild-type controls were stained with RBPMS, and the labelled RGCs were quantified. The average number of RGCs in the NFATc4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e group (3159\u0026thinsp;\u0026plusmn;\u0026thinsp;66 cells/mm\u003csup\u003e2\u003c/sup\u003e) did not show a significant difference from the wild-type group (3243\u0026thinsp;\u0026plusmn;\u0026thinsp;103 cells/mm\u003csup\u003e2\u003c/sup\u003e), suggesting that NFATc4 is not essential for the generation of the normal RGC count (not shown). The enhancement in the survival of RBPMS-labeled RGCs became evident on day 5 following optic nerve crush in Nfatc4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice compared to wild-type mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The rescue of NFATc4 expression through intravitreal injections of Lenti-GFP-NFATc4 abolished this effect and reduced RGC survival rate to the level observed in RGCs transduced with the Lenti-GFP control virus.\u003c/p\u003e \u003cp\u003eDue to the expression of active caspase-3 by RGCs following axotomy [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], we next immunodetected caspase-3 and its cleaved form in whole retinas at 5 days after ONC. In NFATc4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e retinas, there was a significantly lower level of cleaved caspase-3 compared to the wild-type group. Immunocytochemical staining of retinal cryosections on day 5 following the crush revealed a visibly lower signal for cleaved caspase-3 in the ganglion cell layer of NFATc4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice compared to the wild-type. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eF, the density profiles reflecting the immunoreactivity of caspase-3 were significantly decreased in the Nfact4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e group compared to the wild-type control. These findings suggest that the neuroprotective effect of Nfatc4 knockout is exerted, at least partly, through the modulation of apoptosis-related factors.\u003c/p\u003e\u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eNFATc4 regulates apoptotic signaling in the injured retina\u003c/h2\u003e \u003cp\u003eAs the activation of caspase-3 significantly decreased in NFATc4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice after ONC, we hypothesized that signaling pathways associated with apoptosis might be downregulated, thereby delaying RGC death. To investigate this, we conducted a microarray screening (the full list of genes can be found in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and observed a trend toward an increase in the average C\u003csub\u003et\u003c/sub\u003e values in the NFATc4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e group compared to the WT control, although the significance did not reach the 0.05 threshold (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e). However, this trend could indicate a global decrease in apoptotic gene expression in NFATc4 knockout mice subjected to ONC.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe list of screened apoptotic genes. The experiment was performed in triplicate and the data were analyzed using SABiosciences PCR Array Data Analysis Web Portal as described in \u003cspan refid=\"Sec2\" class=\"InternalRef\"\u003e\u003cem\u003eMaterials and Methods\u003c/em\u003e\u003c/span\u003e section.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene bank\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGene symbol\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDescription\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_001100850\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAbl1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC-abl oncogene 1, receptor tyrosine kinase\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_031356\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAifm1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eApoptosis-inducing factor, mitochondrion-associated 1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_033230\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAkt1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eV-akt murine thymoma viral oncogene homolog 1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_013132\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAnxa5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAnnexin A5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_023979\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eApaf1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eApoptotic peptidase activating factor 1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_001127379\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eApi5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eApoptosis inhibitor 5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_001107757\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAven\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eApoptosis, caspase activation inhibitor\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_022698\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBad\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBCL2-associated agonist of cell death\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_001106647\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBag1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBCL2-associated athanogene\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_053812\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBak1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBCL2-antagonist/killer 1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_017059\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBax\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBcl2-associated X protein\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_031328\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBcl10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eB-cell CLL/lymphoma 10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_016993\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBcl2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eB-cell CLL/lymphoma 2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_133416\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBcl2a1d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eB-cell leukemia/lymphoma 2 related protein A1d\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_031535\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBcl2l1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBcl2-like 1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_022612\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBcl2l11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBCL2-like 11 (apoptosis facilitator)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_021850\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBcl2l2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBcl2-like 2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_022684\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBH3 interacting domain death agonist\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_053704\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBik\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBCL2-interacting killer (apoptosis-inducing)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_021752\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBirc2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBaculoviral IAP repeat-containing 2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_023987\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBirc3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBaculoviral IAP repeat-containing 3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_022274\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBirc5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBaculoviral IAP repeat-containing 5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_001106835\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBnip2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBCL2/adenovirus E1B interacting protein 2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_053420\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBnip3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBCL2/adenovirus E1B interacting protein 3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_017312\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBok\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBCL2-related ovarian killer\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_001130554\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCard10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCaspase recruitment domain family, member 10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_012762\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCasp1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCaspase 1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_130422\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCasp12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCaspase 12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eXM_234878\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCasp14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCaspase 14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_022522\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCasp2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCaspase 2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_012922\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCasp3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCaspase 3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_053736\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCasp4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCaspase 4, apoptosis-related cysteine peptidase\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_031775\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCasp6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCaspase 6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_022260\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCasp7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCaspase 7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_022277\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCasp8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCaspase 8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_001107921\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCasp8ap2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCaspase 8 associated protein 2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_031632\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCasp9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCaspase 9, apoptosis-related cysteine peptidase\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_134360\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCd40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCD40 molecule, TNF receptor superfamily member 5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_053353\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCd40lg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCD40 ligand\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_057138\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCflar\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCASP8 and FADD-like apoptosis regulator\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_001170467\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCidea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCell death-inducing DFFA-like effector a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_001108869\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCideb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCell death-inducing DFFA-like effector b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_012839\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCycs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCytochrome c, somatic\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_138910\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDad1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDefender against cell death 1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_001107335\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDapk1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDeath associated protein kinase 1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_053679\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDffa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDNA fragmentation factor, alpha subunit\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_053362\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDffb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDNA fragmentation factor, beta polypeptide (caspase-activated DNase)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_001008292\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDiablo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDiablo homolog (Drosophila)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_152937\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFadd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFas (TNFRSF6)-associated via death domain\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_080895\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFaim\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFas apoptotic inhibitory molecule\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_139194\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFas\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFas (TNF receptor superfamily, member 6)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_012908\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFaslg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFas ligand (TNF superfamily, member 6)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_024127\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGadd45a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGrowth arrest and DNA-damage-inducible, alpha\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_057130\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHrk\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHarakiri, BCL2 interacting protein (contains only BH3 domain)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_012854\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIl10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eInterleukin 10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_080769\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLta\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLymphotoxin alpha (TNF superfamily, member 1)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_053842\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMapk1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMitogen activated protein kinase 1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_053777\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMapk8ip1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMitogen-activated protein kinase 8 interacting protein 1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_021846\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMcl1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMyeloid cell leukemia sequence 1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eXM_226742\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNaip2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNLR family, apoptosis inhibitory protein 2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eXM_342346\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNfkb1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNuclear factor of kappa light polypeptide gene enhancer in B-cells 1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_053516\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNol3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNucleolar protein 3 (apoptosis repressor with CARD domain)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_017141\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePolb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePolymerase (DNA directed), beta\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_017169\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePrdx2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePeroxiredoxin 2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_012630\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePrlr\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eProlactin receptor\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_172322\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePycard\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePYD and CARD domain containing\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eXM_342810\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRipk2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReceptor-interacting serine-threonine kinase 2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_001012066\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSphk2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSphingosine kinase 2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_012675\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTnf\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTumor necrosis factor (TNF superfamily, member 2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_001108873\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTnfrsf10b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTumor necrosis factor receptor superfamily, member 10b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_012870\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTnfrsf11b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTumor necrosis factor receptor superfamily, member 11b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_013091\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTnfrsf1a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTumor necrosis factor receptor superfamily, member 1a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_130426\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTnfrsf1b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTumor necrosis factor receptor superfamily, member 1b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_145681\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTnfsf10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTumor necrosis factor (ligand) superfamily, member 10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_001001513\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTnfsf12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTumor necrosis factor ligand superfamily member 12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_030989\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTp53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTumor protein p53\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eXM_223012\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTp53bp2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTumor protein p53 binding protein, 2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_019221\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTp63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTumor protein p63\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_001108696\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTp73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTumor protein p73\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_001100480\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTradd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTNFRSF1A-associated via death domain\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_001107815\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTraf2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTnf receptor-associated factor 2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_001108724\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTraf3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTnf receptor-associated factor 3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNM_022231\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eXiap\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eX-linked inhibitor of apoptosis\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eConsidering a 2-fold change as a minimum and P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, we identified 7 genes with significantly downregulated expression: \u003cem\u003eLtbr\u003c/em\u003e, \u003cem\u003eBok\u003c/em\u003e, \u003cem\u003eCasp2\u003c/em\u003e, \u003cem\u003eBak1\u003c/em\u003e, \u003cem\u003eBid\u003c/em\u003e, \u003cem\u003eAnxa5\u003c/em\u003e, \u003cem\u003eTp53bp2\u003c/em\u003e. These genes include components of the tumor necrosis factor receptor superfamily, Bcl-2 protein family members, caspase superfamily, and apoptosis-stimulating protein of p53 family. Generally, the function of proteins encoded by these genes is considered pro-apoptotic. Therefore, the downregulation of apoptosis-promoting genes in response to NFATc4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e knockout may restrict ONC-induced RGC death.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eNFATc4 knockout improves retina function following optic nerve crush\u003c/h2\u003e \u003cp\u003eTo investigate whether the enhanced RGC survival in Nfatc4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice correlated with the retina\u0026rsquo;s response to flash stimuli, we utilized scotopic ERG (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-E). In wild-type mice, the average baseline amplitudes of a- and b-waves were 383.7\u0026thinsp;\u0026plusmn;\u0026thinsp;9.6 \u0026micro;V and 813\u0026thinsp;\u0026plusmn;\u0026thinsp;20.3 \u0026micro;V, respectively. In Nfatc4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice, the average baseline amplitudes of a- and b-waves were 397.2\u0026thinsp;\u0026plusmn;\u0026thinsp;6.5 \u0026micro;V and 787.5\u0026thinsp;\u0026plusmn;\u0026thinsp;24.8 \u0026micro;V, respectively. In wild type mice, a- and b-waves recorded 5 days post-crush were decreased by 53.5\u0026thinsp;\u0026plusmn;\u0026thinsp;7.2% and 58\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1%, respectively, when compared to the sham control. ERG performed in Nfatc4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice on the same post-crush day showed a reduction in a- and b-waves by 18.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7% and 29.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8%, respectively. Although the ERG waves were also reduced in Nfatc4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice when compared to the sham group, these deficits were significantly less than in wild-type mice, demonstrating the positive effect of NFATc4 knockout on the function of the injured retina.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eNFATc4 controls axon regeneration after optic nerve injury\u003c/h2\u003e \u003cp\u003eNext, we investigated the effect of NFATc4 knockout on short-term axon regeneration (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003e). To address this question, RGCs were labelled by intravitreal injection of cholera toxin-B subunit (CTB) and the number of axons was quantified at day 7 after ONC. The number of regenerating axons that extended 100, 250, 500, 750 \u0026micro;m beyond the crush point was significantly higher in the NFATc4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e group than in the wild type. We also measured βIII-tubulin expression within the axons to determine axonal integrity. The density of βIII-tubulin assessed 500 \u0026micro;m from the crush point was significantly higher in the NFATc4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e group compared to the wild type, suggesting delayed axonal degeneration. Interestingly, no enhanced regeneration after ONC was seen in NFATc3\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice. Taken together, our results suggest that NFATc4 may promote RGC death and repress regeneration of the injured optic nerve.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe research on NFAT transcription factors in RGC survival and regeneration following injury has been largely discontinued since 2014 when Xu and colleagues demonstrated an overlapping pattern of NFATc4, cleaved caspase-3, and FasL in a light-induced model of retinal degeneration [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Using purified RGCs, as well as Nfatc4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e or NFATc3\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e knockout mice and lentiviral-mediated gene delivery, we demonstrate that NFATc4 plays a crucial role in RGC survival in a model of optic nerve crush. The knockout of NFATc4 significantly improved RGC function and enhanced axonal regeneration in the injured retina. The critical role of NFATc4 is highlighted by the fact that no similar changes were observed in Nfatc3\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e knockout mice. This suggests that NFATc4 in the retina is downstream of divergent signaling pathways mediating survival and regeneration in the presence or absence of neurotrophic factors.\u003c/p\u003e \u003cp\u003eNFATc4 belongs to the family of Rel homology domain (RHR) and NFAT homology domain (NHR)-containing transcription factors (NFATc1-c4), whose activity is controlled in a Ca\u003csup\u003e2+\u003c/sup\u003e- and CaN-dependent manner. The NHR contains two CaN-binding motifs: a Ca\u003csup\u003e2+\u003c/sup\u003e - independent PXIXIT motif in the N terminus and a Ca\u003csup\u003e2+\u003c/sup\u003e -dependent LxVP motif in the C-terminal portion of NHR [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. Despite shared activation by CaN-dependent dephosphorylation, the activity of specific NFAT isoforms within distinct populations of neuronal cells is controlled through poorly understood mechanisms. For example, NFATc4\u0026rsquo;s activity was selectively required for the survival of adult-born neurons in response to BDNF [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e] and mediated anti-apoptotic transcription in NMDA receptor-stimulated cortical neurons [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. Depending on its transcriptional activity, NFATc4 may also participate in pro-apoptotic signaling, usually combined with an extrinsic pathway-dependent increase in Fas ligand (FasL) expression. Gomez-Sintes and Lucas demonstrated that increased nuclear NFATc4 translocation correlated with elevated FasL levels and Fas activation, an effect absent in Fas-deficient \u003cem\u003eIpr\u003c/em\u003e mice and following cyclosporine administration [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. Similarly, NFATc4-mediated FasL up-regulation has been proposed to underlie methamphetamine-induced neuronal loss [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. Furthermore, deafferentiation-induced neuronal apoptosis in the cochlear nucleus has also been suggested to be mediated by NFATc4/FasL activation [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. Hence, the opposite functions played by NFATc4 may be attributed to the upstream stimulus controlling its phosphorylation/dephosphorylation ratio or be cell-specific, as different cells can selectively activate specific NFAT isoforms depending on environmental cues [\u003cspan additionalcitationids=\"CR71 CR72 CR73 CR74\" citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe role of NFATs in RGCs is still not fully understood. Our results demonstrate that NFATc4 is specifically and transiently up-regulated in the retina after optic nerve injury. The time-course of NFATc4 increase in our experimental model is similar to the one observed in [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], suggesting a more general phenomenon. Another research group has also demonstrated a change in NFATc4 expression in response to optic nerve injury. The microarray hybridization screen performed by Lukas and colleagues within 6 h post injury revealed early downregulation of NFATc4 in the ganglion cell layer [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e]. This observation was confirmed by a more recent analysis of the retinal transcriptome performed at the same time point after ONC [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]. Both studies clearly demonstrate changes in NFATc4 expression; however, they focus either on changes occurring early after ONC or performed the injury in embryonic (E20) and postnatal animals (P1-P3). Moreover, there were significant differences between postnatal and embryonic NFATc4 expression. It is known that capacity of RGC for axonal growth and the regeneration of injured axons sharply decreases soon after birth, and this age-dependent decline is associated with a profound reorganization of retinal transcriptome [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e]. A growing body of evidence indicates that molecular changes in the injured retina are progressive and many of them appear later in time [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e, \u003cspan additionalcitationids=\"CR80\" citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e]. Therefore, it is not unexpected that the NFATc4 expression profile changes over time as RGC death becomes prominent. Consistent with our study, none of the transcriptional profiling analyses revealed changes in other NFAT isoforms after ONC.\u003c/p\u003e \u003cp\u003eBased on our data, wherein NFATc4 knockdown promotes RGC survival in vivo, and lentiviral-mediated NFATc4 expression in Nfatc4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mouse reverses this pro-survival effect, the up-regulation of NFATc4 following injury likely represents an attempted pro-apoptotic response. This NFATc4-mediated response seems to be specifically induced by the injury, as the number of RGCs in uninjured wild type and Nfatc4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e groups was unchanged and similar to the results previously reported for the C57BL/6 mouse [\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e]. This would indicate that NFATc4 expression is dispensible for normal retina development or in uninjured RGCs. The importance of CaN/NFAT signaling in retinal degeneration has been suggested by several groups. Freeman and Grosskreutz demonstrated that the administration of the FKBP12 ligand FK506 increased the number of RGCs following optic nerve crush [\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e]. The FK506-FKBP12 complex is expected to inhibit CaN phosphatase activity and decrease NFAT dephosphorylation, thus preventing its nuclear import. Moreover, it has been demonstrated that CaN is activated in response to ocular hypertension in the mouse model of glaucoma [\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e] and is responsible for RGC degeneration [\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e]. In view of that, knockdown of NFATc4 in vivo may disrupt calcineurin/NFATc4 downstream signaling and, at least in part, attenuate massive apoptosis of injured RGCs. This posits NFATc4 as one of the important mediators of RGC death following optic nerve crush.\u003c/p\u003e \u003cp\u003eWhile the data suggests NFATc4\u0026rsquo;s involvement in RGC death, the relevance of NFATc4 function as a potential target for axonal regeneration after retina injury has not been previously explored. Using CTB and βIII-tubulin staining, we demonstrated that NFATc4 knockout delayed axon degeneration. Labeling axons with CTB is a reliable technique based on axonal transport that is widely used for monitoring axonal regeneration [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. However, around day 7 post-crush, axonal transport is significantly altered, leading to distal axon terminal degeneration [\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e]. Because, in our experiment, CTB was injected 2 days before retina collection, it is also plausible that NFATc4 knockout may affect dye transport, eventually influencing the labelling pattern one week after ONC. Nonetheless, visualization of remaining axons with βIII-tubulin, which is a marker of axonal integrity [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], seems to confirm that NFATc4 plays a role in delaying axonal disintegration one week after ONC.\u003c/p\u003e \u003cp\u003eIt is hypothesized that axonal transport breakdown is preceded by a lesion-induced signaling, triggering axon swelling and irreversible changes in neurofilaments and microtubules integrity [\u003cspan additionalcitationids=\"CR88\" citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e]. In their elegant set of experiments, Kn\u0026ouml;ferle and colleagues linked axotomy-induced intraaxonal Ca\u003csup\u003e2+\u003c/sup\u003e elevation to a secondary generation of autophagosomes that participate in axonal degradation [\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e]. The initial increase in Ca\u003csup\u003e2+\u003c/sup\u003e concentration activating CaN is an obligatory step for the activation of NFAT-dependent transcription. Moreover, recent reports suggest an important contribution of NFAT to autophagy in retinal pigmental epithelial cells [\u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e] as well as in other cell types [\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e]. In view of this, it is tempting to speculate that Ca\u003csup\u003e2+\u003c/sup\u003e-dependent activation of NFATc4 and NFATc4 downstream signaling should be placed among important events restricting axonal regeneration after mechanical injury.\u003c/p\u003e \u003cp\u003eThe remaining question is how NFATc4 knockout slows down the time-dependent apoptosis of injured RGC. NFAT proteins can directly regulate the expression of apoptosis-related genes along with the induction of pro-inflammatory cytokine production [\u003cspan additionalcitationids=\"CR94 CR95 CR96 CR97\" citationid=\"CR93\" class=\"CitationRef\"\u003e93\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e98\u003c/span\u003e]. Both apoptosis and neuroinflammation are frequently associated with multiple neurodegenerative diseases [\u003cspan additionalcitationids=\"CR100\" citationid=\"CR99\" class=\"CitationRef\"\u003e99\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR101\" class=\"CitationRef\"\u003e101\u003c/span\u003e]. Although it would be interesting to explore whether the modulation of retinal inflammation underlies enhanced RGC survival in NFATc4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mouse, our observation of lowered caspase-3 cleavage directed us toward studying apoptosis-related genes. The microarray analysis revealed that certain pro-apoptotic genes are downregulated in NFATc4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice, indicating that increased RGC survival observed in this group after ONC may arise from blocking the apoptotic program. This is consistent with a prior study showing reduced sensitivity of sensory hair cells to TNF-mediated apoptosis in NFATc4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice [\u003cspan citationid=\"CR102\" class=\"CitationRef\"\u003e102\u003c/span\u003e]. In addition, Bak1, Bok, and Bid, part of the Bcl-2 family of apoptosis regulators, were downregulated in the NFATc4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e retina after ONC. Selective repression of BAK1 protein attenuated neuronal apoptosis [\u003cspan citationid=\"CR103\" class=\"CitationRef\"\u003e103\u003c/span\u003e], similar to Bax/Bak1 double knockout cells that are resistant to multiple apoptotic inducers [\u003cspan citationid=\"CR104\" class=\"CitationRef\"\u003e104\u003c/span\u003e, \u003cspan citationid=\"CR105\" class=\"CitationRef\"\u003e105\u003c/span\u003e]. Like BAK1 and BAX, BOK is a pro-apoptotic protein that can induce mitochondrial apoptosis [\u003cspan citationid=\"CR106\" class=\"CitationRef\"\u003e106\u003c/span\u003e]. In line with this finding, Bok\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e cells were partially protected from ER stress-induced apoptosis elicited by thapsigargin or bortezomib [\u003cspan citationid=\"CR107\" class=\"CitationRef\"\u003e107\u003c/span\u003e]. On the contrary, other studies suggested a lack of its role in apoptosis as Bok knockout does not alter responsiveness to various apoptotic stimuli [\u003cspan citationid=\"CR108\" class=\"CitationRef\"\u003e108\u003c/span\u003e, \u003cspan citationid=\"CR109\" class=\"CitationRef\"\u003e109\u003c/span\u003e]. Similarly, Bid-deficient mice are resistant to Fas-induced apoptosis [\u003cspan citationid=\"CR110\" class=\"CitationRef\"\u003e110\u003c/span\u003e], and Tp53bp2 downregulation protected from apoptosis in certain cell types [\u003cspan citationid=\"CR111\" class=\"CitationRef\"\u003e111\u003c/span\u003e, \u003cspan citationid=\"CR112\" class=\"CitationRef\"\u003e112\u003c/span\u003e]. However, which NFATc4-dependent changes in gene expression reflect a pro-survival response, improving RGC survival and delaying axonal degeneration, needs further attention. It has been recently demonstrated that among 46 different RGC subtypes distinguished by high-throughput single-cell RNA-seq [\u003cspan citationid=\"CR113\" class=\"CitationRef\"\u003e113\u003c/span\u003e],some types exhibit selective resilience to injury while others are more susceptible to degeneration and die quickly [\u003cspan citationid=\"CR114\" class=\"CitationRef\"\u003e114\u003c/span\u003e]. Since NFATc4 may affect the expression profile of genes involved in apoptosis, certain types of RGCs may be more vulnerable because of their NFATc4 expression, consistent with our data that NFATc4 up-regulation peaked 1 day after ONC.\u003c/p\u003e \u003cp\u003eNFATc4 is unique among other NFAT isoforms in its regulation by upstream signaling in neurons. Unlike NFATc3, activation of NFATc4 requires a coincident elevation in intracellular Ca\u003csup\u003e2+\u003c/sup\u003e and suppression of glycogen synthase kinase 3β (GSK-3β) [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. GSK-3β and other kinases are known to phosphorylate multiple serines in the NFAT regulatory domain, leading to the termination of NFAT-dependent gene expression [\u003cspan additionalcitationids=\"CR116\" citationid=\"CR115\" class=\"CitationRef\"\u003e115\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR117\" class=\"CitationRef\"\u003e117\u003c/span\u003e]. It has not been fully resolved whether the activity of phosphorylating/dephosphorylating enzymes is an organized mechanism. In view of this, an interesting question that remains elusive is how the activity of NFATc4 is orchestrated to direct the RGC response to injury and affect the regeneration of injured axons. Our previous study [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] demonstrated that manipulation within A-kinase anchoring protein 6 (AKAP6)-organized pro-survival signaling significantly enhanced RGC survival following ONC. AKAP6 brings together calcineurin [\u003cspan citationid=\"CR118\" class=\"CitationRef\"\u003e118\u003c/span\u003e], ERK5 [\u003cspan citationid=\"CR119\" class=\"CitationRef\"\u003e119\u003c/span\u003e] and NFAT transcription factor (unpublished data), providing a platform for the integration of pro-survival an pro-death signaling. Depending on the upstream stimuli, ERK5 activity can be effectively counterbalanced by locally anchored CaN, with the relevant outcome toward NFATc4 downstream signaling. Up to now, more than fifty AKAPs have been identified that are involved in different cellular processes. This abundance allows for efficient spatial and temporal control of intracellular signaling, but which AKAPs may potentially participate in RGC survival requires further investigation.\u003c/p\u003e \u003cp\u003eIt has been demonstrated that distinct NFAT isoforms may antagonize each other in the control of gene expression in retina degeneration [\u003cspan citationid=\"CR120\" class=\"CitationRef\"\u003e120\u003c/span\u003e]. For instance, siRNA-mediated NFATc3 knockdown increased the expression of TNFα-induced inflammatory response, whereas downregulation of NFATc4 has the opposite effects. Several molecular therapies based on pharmacological NFAT inhibition have been described to carry substantial potential toward retina degeneration [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR121\" class=\"CitationRef\"\u003e121\u003c/span\u003e]. It is highly likely that greater efficacy could be achieved by identifying the NFAT isoform\u0026rsquo;s role in RGC degeneration, which would give rise to development of isoform-specific therapies. Therefore, our intent was to investigate how NFAT isoforms contribute to the pathological events underlying injury-mediated RGC loss. To our best knowledge, no similar study with NFATc4 or NFATc3 knockout animals has been performed up to now.\u003c/p\u003e \u003cp\u003eIn summary, our data suggest that NFATc4 should be considered one of the major regulators of adult RGC survival following injury, and central to the complex interplay of multiple molecular events in axonal regeneration. Further studies on NFATc4 and, in particular, the co-regulators of its transcriptional activity are essential, as they may lead to new therapeutic interventions allowing for the preservation of RGC function. Despite accumulating studies on gene therapy enhancing RGC survival and axon regeneration, the search for novel target molecules is of paramount importance, as the functional restoration of visual pathways still remains a challenge. The synergistic effect of NFATc4 downregulation along with other known axon regeneration promoters may provide an effective combinatorial strategy to improve vision impairments in optic neuropathies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflict of Interest\u003c/h2\u003e \u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by the National Science Centre (Narodowe Centrum Nauki) grant no. UMO-2019/33/B/NZ4/00587.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJM, ML, JT, FG, AS performed experiments and analyzed the data. TB, AS and FG wrote and edited the manuscript. 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Biochim Biophys Acta Mol Basis Dis 1867(12):166238. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.bbadis.2021.166238\u003c/span\u003e\u003cspan address=\"10.1016/j.bbadis.2021.166238\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-neurobiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"moln","sideBox":"Learn more about [Molecular Neurobiology](https://www.springer.com/journal/12035)","snPcode":"12035","submissionUrl":"https://submission.nature.com/new-submission/12035/3","title":"Molecular Neurobiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"optic nerve injury, NFATc4 transcription factor, retinal ganglion cell survival, intravitreal gene delivery, apoptotic gene expression","lastPublishedDoi":"10.21203/rs.3.rs-3813885/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3813885/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRetinal ganglion cells (RGCs), neurons transmitting visual information via the optic nerve, fail to regenerate their axons after injury. The progressive loss of RGC function underlies the pathophysiology of glaucoma and other optic neuropathies, often leading to irreversible blindness. Therefore, there is an urgent need to identify the regulators of RGC survival and the regenerative program. In this study, we investigated the role of the family of transcription factors known as nuclear factor of activated T cells (NFAT), which are expressed in the retina; however, their role in RGC survival after injury is unknown. Using the optic nerve crush (ONC) model, widely employed to study optic neuropathies and central nervous system axon injury, we found that NFATc4 is specifically but transiently up-regulated in response to mechanical injury. In the injured retina, NFATc4 immunolocalized primarily to the ganglionic cell layer. Utilizing NFATc4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e and NFATc3\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice, we demonstrated that NFATc4, but not NFATc3, knockout increased RGC survival, improved retina function, and delayed axonal degeneration. Microarray screening data, along with decreased immunostaining of cleaved caspase-3, revealed that NFATc4 knockout was protective against ONC-induced degeneration by suppressing pro-apoptotic signaling. Finally, we used lentiviral-mediated NFATc4 delivery to the retina of NFATc4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice and reversed the pro-survival effect of NFATc4 knockout, conclusively linking the enhanced survival of injured RGCs to NFATc4-dependent mechanisms. In summary, this study is the first to demonstrate that NFATc4 knockout may confer transient RGC neuroprotection and decelerate axonal degeneration after injury, providing a potent therapeutic strategy for optic neuropathies.\u003c/p\u003e","manuscriptTitle":"NFATc4 knockout promotes neuroprotection and retinal ganglion cell regeneration after optic nerve injury","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-12 08:06:22","doi":"10.21203/rs.3.rs-3813885/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-02-19T16:18:48+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-02-18T22:26:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"4036d093-de94-4e40-a85d-6e9a14f85203","date":"2024-02-05T21:41:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"d42118e8-93a6-439f-ab3e-0aae6d0c6b97","date":"2024-02-05T20:55:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"29426c5f-ed21-4e0b-ae5a-8cdf2992cd84","date":"2024-01-29T21:21:50+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-01-25T23:11:48+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-01-10T15:15:24+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-01-10T15:15:23+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Neurobiology","date":"2023-12-27T21:57:12+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-neurobiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"moln","sideBox":"Learn more about [Molecular Neurobiology](https://www.springer.com/journal/12035)","snPcode":"12035","submissionUrl":"https://submission.nature.com/new-submission/12035/3","title":"Molecular Neurobiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"d3f06dbc-067a-43b4-af33-6501340062d1","owner":[],"postedDate":"January 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-05-01T23:06:31+00:00","versionOfRecord":{"articleIdentity":"rs-3813885","link":"https://doi.org/10.1007/s12035-024-04129-0","journal":{"identity":"molecular-neurobiology","isVorOnly":false,"title":"Molecular Neurobiology"},"publishedOn":"2024-04-19 23:06:31","publishedOnDateReadable":"April 19th, 2024"},"versionCreatedAt":"2024-01-12 08:06:22","video":"","vorDoi":"10.1007/s12035-024-04129-0","vorDoiUrl":"https://doi.org/10.1007/s12035-024-04129-0","workflowStages":[]},"version":"v1","identity":"rs-3813885","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3813885","identity":"rs-3813885","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}