Role of NFAT4-Trim17 axis mediates the neuronal apoptosis after subarachnoid hemorrhage

doi:10.21203/rs.3.rs-2099630/v1

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Role of NFAT4-Trim17 axis mediates the neuronal apoptosis after subarachnoid hemorrhage

https://doi.org/10.21203/rs.3.rs-2099630/v1

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Background: Although accumulating evidence indicates that Trim17 is implicated in the pathophysiology of neuronal apoptosis, the regulation mechanism on pro-apoptotic role of Trim17 after subarachnoid hemorrhage (SAH) has not been well identified so far.

Methods: Trim17 siRNA was administered to explore the detrimental role of Trim17 in mediating neuronal apoptosis in vivo and in vitro after experimental SAH. The nuclear factor of activated T cells (NFAT) signaling, involving in the underlying regulation mechanism on Trim17, was further explored.

Results: Trim17 mainly located in the neurons and presented an obvious elevated expression in vivo and in vitro after SAH. Trim17 siRNA administration could significantly ameliorate neuronal apoptosis after SAH. Moreover, inhibition the nuclear translocation of NFAT4 could effectively abolishthe pro-apoptotic activity of Trim17/Bax pathway, accompanied by ameliorated neuronal apoptosis and improved neurological function.

Conclusions: The elevation of Trim17 is sufficient for triggering the intrinsic apoptotic effect after SAH, and this pro-apoptotic activity could be mediated by the initiated of the nuclear translocation of NFAT4. Pharmacological targeting of NFAT4/Trim17 pathway can be explored for SAH therapy.

Subarachnoid hemorrhage

Neuron

Apoptosis

Trim17

NFAT4

The neuronal death following subarachnoid hemorrhage (SAH) could be triggered by the dissolving of red blood cells in subarachnoid space(1, 2). Neuronal apoptosis, one of the main neuronal death patterns, is evoked via extrinsic and intrinsic mechanisms in the early phase of SAH(3). Therefore, the therapeutic strategies targeting on neuronal apoptosis would efficiently improve neurologic outcomes after SAH. In neurons, apoptosis is predominantly driven through the intrinsic pathway, which includes mitochondrial cytochrome c release and apoptosome formation. Significantly, the intrinsic pathway appears to be entirely Bax dependent, and in deed deficiency or inhibition of Bax effectively alleviates aberrant neuronal apoptosis(2).

Trim17, a member of the Trim family, can be identified as an E3 ubiquitin-ligase. This role confers several pivotal abilities to Trim17 in mediating cellular responses, such as apoptosis, autophagy and cell division(4–6). It has been well reported that Trim17 protein specifically presents in apoptotic neurons, and its expression is necessary and sufficient to trigger neuronal apoptosis through the intrinsic pathway(7). This pro-apoptotic effect from Trim17 can be efficiently abolished in the Bax deficient neurons(8). All these might be speculated that the pro-apoptotic role of Trim17 is tightly coupled with Bax in neurons.

Although it has been reported that cytochrome c from mitochondria can trigger the activation of downstream caspases after SAH(9), the dynamic activation of neuronal Trim17 and the underlying Trim17/Bax signaling pathway have not been investigated in SAH. In addition, the previous study has suggested that the nuclear factor of activated T cell (NFAT) transcription factor 4 can effectively control the fate of neurons though driving the transcription of the pro-apoptotic gene Trim17(7). Accordingly, we hypothesized here that inhibition of the increased level of Trim17 could remarkably attenuate neuronal apoptosis induced by SAH, and that the inhibition of NFAT4 could significantly abolish Trim17/Bax pro-apoptotic pathway, accompanied by ameliorated neuronal apoptosis and improved neurological outcomes after SAH.

Animal preparation

Adult male C57BL/6 mice (8–10 weeks old) were housed in a room with constant temperature (25 ℃), humidity control and free access to food and water.

SAH model in vivo

The SAH models in vivo were performed by prechiasmatic injection of nonheparinized arterial blood. After deep anesthesia, the mouse head was fixed in a stereotactic device, followed by a hole was drilled in the skull (1 mm diameter, 4.5 mm in front of the bregma). The nonheparinized arterial blood (60 ul) was slowly drew from the left ventricle of the donor mouse, and then injected into the subarachnoid space along the previous bore hole. After injection, the hole was closed with bone wax. Each mouse in sham group was injected with saline (60 ul), instead of arterial blood(10, 11).

Tissue Preparation

After deep anesthesia, mice hearts were perfused with 0.01M PBS (4℃). The inferior basal temporal lobes adjacent to the clotted blood were harvested for extraction of protein and RNA. For immunohistochemistry analysis, the mice brains were perfused with 0.01M PBS (4℃) and 4% paraformaldehyde. After transferring to sucrose (4℃), the mice brains were embedded for cryostat sectioning.

Primary neurons culture

Primary neurons were cultured from the mice at E16-18 day. Briefly, cortical tissues were dissected in ice-cold media, chopped and digested with 0.125% trypsin for 5 min at 37℃. The trypsin was inactivated using 10% FBS, the suspension was filtered through a 40-um nylon mesh and centrifuged at 1500 rpm for 5 min. The cell pellet was resuspended in DMEM with 10% FBS and 1% penicillin-streptomycin, counted and seeded at a density of 1×10⁵ cells/cm² into poly-D-lysine-coated plates. After 2 h, the culture medium was replaced with Neurobasal Medium containing GlutaMax-I, B27 supplement, and penicillin-streptomycin. The medium was replaced with fresh medium every 2 days. Neurons were cultured for 8–10 days before being used in vitro studies(11).

SAH model in vitro

After 8–10 days culture, the primary neurons were induced the SAH models in vitro. OxyHb (MilliporeSigma, USA) was added into the medium with a final concentration of 10 mM. After SAH models induced in vitro, the primary neurons were detected at different time points according the experimental design(12).

Lentivirus transduction in vivo and in vitro

To knockdown the Trim17 and NFAT4 expression at gene level, the lentivirus vector (LV) expressing Trim17 shRNA, NFAT4 shRNA and non-targeting control (NC, Hanbio, Shanghai, China) were transfected in vivo and in vitro before SAH models induced.

In brief, the LV (1×10⁹ Tu/ml) was slowly microinjected into the mouse left ventricle (3 ul; 0.5 ul/min) by Hamilton syringe. SAH models were induced at the second day after LVs transfection in vivo. After 8–10 days culture, the primary neurons were infected with LVs (1×10⁹ Tu/ml) at a transfection coefficient of 20 according to our preliminary experiment. After 48 h incubation with LVs, the neurons were used following as the experimental design. The NFAT4 and Trim17 RNA interference target sequences were shown: 5’-UGUUUCUGGUGGAAGAAGA-3’ and 5'-CCCTTTGAGTGCCCAAGTA-3'(8, 13). The knockdown levels of NFAT4 and Trim17 were measured by PCR analysis.

Study Design and Drug Administration

In experiment 1, detected endogenous changes of Trim17 and NFAT4 at different time points after SAH. 172 mice were randomized into six groups: Sham group (n = 18) and eight SAH groups (6 h, 12 h, 1 d, 3 d, 5 d, 7 d, 10 d and 14 d, n = 18/group). And, the primary neurons were randomized into five groups: control group and four SAH groups (6 h, 12 h, and 1 d).

In experiment 2, evaluated the detrimental role of Trim17 in mediating neuronal apoptosis in vivo and in vitro after SAH. 48 mice were randomized into four groups: Sham group, SAH group, SAH + LV-NC group and SAH + LV-shTrim17 group (n = 12/group).

In experiment 3, investigated the underlying regulating mechanisms of NFAT4/Trim17 pathway on neuronal apoptosis in vivo after SAH. 120 mice were randomized into four groups: Sham group, SAH group, SAH + LV-NC group and SAH + LV-shNFAT4 group.

Western Blot Analysis

The primary neurons and temporal lobe tissues were respectively harvested in lysis buffer containing protease (Beyotime, Jiangsu, China). The cytosolic and nuclear protein extracted following the manufacturer’s protocol. After protein concentration of each sample was measured by BCA assay kit (Beyotime), equal amounts of protein samples were separated on SDS-PAGE gels and transferred to PVDF membranes. After blocked with skim milk, the membranes were incubated overnight at 4°C with the following primary antibodies: Trim17(Abnova, H00051127-M01, 1:1000), NFAT4 (Santa cruz, sc-8405, 1:100), H3 (CST, 4499, 1:2000), Bax (CST, 2772, 1:2000), cleaved-caspase3 (Abcam, ab32042, 1:2000), β-actin (Bioworld, AP0060, 1:5000). The membranes were then hatched with appropriate secondary antibodies at room temperature for 2 h. Then, the specific bands were visualized by an ECL reagent. Band densities were quantified by the ImageJ software.

Real-time PCR analysis

In brief, the RNA samples from primary neurons and temporal lobe tissues were respectively lysed by TRIzol reagent (Invitrogen, USA) according to the manufacturer’s protocol. After reverse transcription, quantitative real-time PCR was performed using primers specific for the genes encoding Trim17. Primer sequences were shown as: Trim17 5’- AGGGAGTATAAGCTCAAGTTGGA-3’ and 5’-CCTGCCACTCAGTTAAGGTCT-3’, β-actin 5’-GTCCCTCACCCTCCCAAAAG-3’ and 5’-GCTGCCTCAACACCTCAACCC-3’. The relative ratio of the control group was set to 100%, and the mRNA values in other groups were converted into multiples compared with the control group(14).

Immunofluorescence (IF) and TUNEL staining

Briefly, the samples from cultured cells or tissue sections were permeabilized with 0.1% Triton X-100 for 5 min at room temperature. After blocking overnight with 5% albumin, the samples were incubated overnight with primary antibodies against Trim17(Abnova, H00051127-M01, 1:200), NFAT4 (Santa cruz, sc-8405, 1:100), GFAP (Thermo Fisher Scientific, 53-9892-82, 1:200), neuronal nuclei (NeuN, Millipore, MAB377X, 1:200), MAP2 (CST, 8707T, 1:300) at 4°C, followed by PBS containing 0.5% Tween-20 washes and incubation with proper secondary antibodies (Alexa Fluor 488 or Alexa Fluor 594, 1:200; Jackson ImmunoResearch,West Grove, PA, USA) for 1 h at room temperature. Following washing again, coverslips or sections were counterstained by 4 ,6 -diamino-2-phenylindole (DAPI, 1:4000; Sigma) for 4 min at room temperature. No reactivity was observed when the primary antibody was omitted.

The TUNEL staining was accordingly performed following the manufacturer’s instructions (Roche Inc., Indianapolis, USA). In brief, neurons on the coverlips were incubated with primary antibody against NeuN (1:200, MAB377X; Millipore) at 4°C overnight. Following washing with PBS containing 0.5% Tween-20, the coverlips were incubated with TUNEL reaction mixture for 45 min at 37°C. After washing again, the coverlips were stained with DAPI (1:4000; Sigma). Pictures were examined under a fluorescence microscope (Scope A1; Carl Zeiss GmbH, Oberkochen, Germany). The positive cells in IF and TUNEL staining were identified, calculated, and analyzed by two investigators blinded to the experimental design.

Brain water content

Brain water content was evaluated in different groups at 24 h after SAH. The mice brains were weighed immediately after removal (wet weight) and again after being dried at 100℃ for 48 h (dry weight). The percentage of brain water content was calculated as [(wet weight-dry weight)/wet weight] ×100%(15).

Neurological function evaluation

Neurological function was evaluated at 1 d post-SAH by the Modified Garcia Score system(15, 16). The system included 6 tests about sensorimotor assessment (maximum scores = 18), with scores of 0–3 for each test.

Statistical analysis

Data were expressed as mean ± SME. Differences in multiple groups were compared by one-way ANOVA analysis followed by the Tukey post hoc test. P value < 0.05 was defined as statistically significant.

Temporal patterns and cellular expression of endogenous Trim17 after SAH

The results of western blot and quantitative real-time PCR analyses indicated that the protein and mRNA expressions of Trim17 started increasing at 6 h and peaked at 24 h following SAH in vivo (Fig. 1A-C). Double immunofluorescence staining of the Trim17 with the neuron’s marker NeuN showed that Trim17 located in the cytoplasm of neurons within cortices of sham and SAH mice at 24 h after injury. In contrast to the resting morphology in sham group, the neurons in SAH group presented the more enlarged cell bodies, with a stronger fluorescence intensity of Trim17 (Fig. 1D).

In vitro, the endogenous Trim17 protein and mRNA levels significantly increased over 2 d after SAH which peaked at 12 h and decreased gradually at 24 h (Fig. 1E-G). Double immuniofluorescene staining in vitro showed that Trim17 located in the cytoplasm of neurons with a weak fluorescence intensity in the control group. SAH caused a stronger fluorescence intensity of Trim17, but the more serious neuronal axon damage in comparison with the control level (Fig. 1H). These results provided in vitro and in vivo evidence that Trim17 was involved in the pathophysiological mechanisms of SAH.

Trim17 silencing ameliorated the neuronal apoptosis in vivo and in vitro after SAH

In order to investigate the detrimental role of Trim17 in mediating neuronal apoptosis after SAH, the LV-shRNA was transfected in vivo and in vitro to knockdown the expression of Trim17. Based on the endogenous expressions in experiment 1, the samples here were collected at 24 h in vivo and 12 h in vitro after SAH. By detecting the green fluorescent protein (GFP), it presented a strong transfection ability of LV in the cortex tissue, which confirmed the transduction efficiency of Trim17 shRNA in this study (Fig. 2A). Quantitatively, Trim17 mRNA and protein showed obviously reduced levels in SAH+LV-shTrim17 group than that of SAH+LV-NC group. Consistently, transfection of LV-shTrim17 significantly decreased the protein levels of Bax and cleaved caspase-3 in comparison with that of LV-NC administration after SAH (Fig. 2B-F).

To evaluate whether the key pro-apoptotic role of Trim17 expression described above in vivo was also evident in vitro, the LV-shRNA was transfected in the primary neurons. Significantly, compared with that of LV-NC administration, transfection of Trim17 shRNA obviously decreased the mRNA and protein expressions of Trim17 in primary neurons after SAH. Consistent with that, western blot detection similarly revealed a manifestly lower levels of Bax and cleaved caspase-3 in LV-shTrim17 group than that of LV-NC group after SAH (Fig. 2G-K). These further supported the possibility that neuronal Bax and its pro-apoptotic pathway regulation might have a Trim17 sensitivity profile in vivo and in vitro after SAH.

Temporal patterns and cellular expression of endogenous brain NFAT4 after SAH

Of note, it has been well demonstrated that the nuclear NFAT4 can be re-phosphorylated by diverse kinases, which leads to NFAT4 inactivation and relocation to the cytoplasm(17). After the cytosolic/nuclear protein extraction, the western blot results found that the protein level of nuclear NFAT4 was markedly enhanced, while the cytoplasmic NFAT4 was dramatically reduced after SAH (Fig. 3A-D). In contrast to the levels in the sham group, mRNA level of NFAT4 was manifestly increased and peaked at 1 d after SAH (Fig. 3 E). As revealed by the localization of immunofluorescence staining for NFAT4 and the neuronal marker NeuN and astrocytic marker GFAP, it was found that NFAT4 was mainly expressed in neurons but not in astrocytes in vivo. Meanwhile, consistent with the results of western blot, the nuclear NFAT4 staining was obviously enhanced after SAH (Fig. 3 F-G). Taken together, these data indicated that NFAT4 dephosphorylation was essential for the nuclear transfer after SAH, which might confer NFAT4 more opportunities to bind to the promoter of Trim17 specifically initiating the transcription of Trim17.

Neuronal Trim17 regulation had a NFAT4 sensitivity profile in vivo after SAH

In order to confirm the regulation of NFAT4 on Trim17 after SAH, the LV-shRNA was administrated to knockdown the expression of NFAT4 in the mice cortex. In light of the endogenous expressions in experiment 1, the samples here were collected at 24 h in vivo after SAH. Dramatically, it showed lower expressions of nuclear and the cytoplasmic NFAT4 with the LV-shNFAT4 treatment than that of LV-NC administration after SAH (Fig. 4 A-D). Accompanied by the knockdown of NFAT4, the expressions of Trim17, Bax and cleaved caspase-3 were efficiently decreased of the SAH+LV-shNFAT4 group in comparison with that in the SAH+LV-NC group (Fig. 4 E-H).

Consistent with that, NFAT4 shRNA injection further exacerbated the neuronal apoptosis displayed by the TUNEL staining in comparison with that by LV-NC administration after SAH (Fig. 5A-B). Genetic knockdown of NFAT4 induced a further decrease in brain water content, which was more ameliorated than that in the negative control group (Fig. 5C). Furthermore, SAH markedly aggravated neurological function, and mice with the NFAT4 shRNA injection showed a better neurological dysfunction score (Fig. 5D). These results indicated that NFAT4 knockdown had a protective function against neuronal apoptosis though inhibiting the activation of pro-apoptotic pathway Trim17/Bax during SAH.

In the present study, we explored the pro-apoptotic activity of Trim17 and the potential underlying regulation mechanisms after experimentally after SAH. Our results demonstrated that (1) Trim17 and NFAT4, located in the neurons, were obviously increased and peaked at 24 h after SAH. (2) inhibition the nuclear translocation of NFAT4 could effectively reduce the pro-apoptotic activity of Trim17/Bax pathway, which might describe a novel mechanism regulating neuronal apoptosis after SAH. (3) Inhibition NFAT4/Trim17/Bax pro-apoptotic pathway could improve neurobehavioral functions and ameliorate brain edema, making it an attractive new clinical target for the treatment of SAH (Fig. 5E).

The Trim17 Pathway In The Pathogenesis Of Neuronal Apoptosis After Sah

The principal pathway of apoptosis comprises the intrinsic pathway and the extrinsic pathway(2). The pro-apoptotic members Bax and Bak, contain Bcl-2 homology, are thought to be almost entirely essential for the execution of apoptosis though the intrinsic pathway(18). However, it has been reported that Bak is translationally repressed and does not participate in neuronal apoptosis(2). Thus, the initiation of intrinsic apoptosis in neurons presents Bax-dependent, and indeed deletion and inhibition of Bax effectively alleviates neuronal apoptosis. It has been well demonstrated an obviously increased expression of Bax after SAH, which fully confirms the driving of the intrinsic apoptosis though Bax-dependent pathway following SAH insult(9). Consistent with that, we similarly observed an increased protein level of Bax after SAH, accompanying by an exaggerated neuronal apoptosis.

Although the alleviation of intrinsic apoptosis in neurons appears to center on control of Bax activation, the underlying regulation mechanisms of Bax in SAH neuronal apoptosis remains elusive. Significantly, it has been shown that Trim17, as a critical E3 ubiquitin ligase, is sufficient for triggering the intrinsic pathway of neuronal apoptosis. Furthermore, the pro-apoptotic activity of Trim17 is tightly coupled with the intrinsic apoptosis though Bax-dependent pathway(7). Consistent with that, we significantly observed that the endogenous Trim17 was dramatically increased following SAH, and that Trim17 knockdown by RNA interference displayed remarkably reduced activity of neuronal apoptosis as shown by TUNEL immunofluorescence, Bax and cleaved caspase3 expressions in vivo and in vitro. These data in our study confirmed the pro-apoptotic activity of Trim17 on an endogenous level, and clearly supported that Trim17/Bax signaling likely modulated pro-apoptotic activity originating from SAH via intracellular the intrinsic pathway.

Possible neuroprotection by NFAT4 inhibition through regulating the Trim17 pathway in the pathogenesis of neuronal apoptosis

Of note, Lassot et al. reasoned that Trim17 may play a decisive role in the initiation of neuronal apoptosis upstream of Bax and that its expression is controlled by the PI3K/AKT/GSK3 pathway(8). It might predominantly attribute to the depolarization-induced calcium entry, which could trigger the PI3K/AKT/GSK-3 pathway playing a crucial role in neuronal survival. In addition, it has also been shown that GSK-3 inhibition increases translocation of NFATc3/4 transcription factors to the nucleus, leading to an aggravated neuronal apoptosis(19). On the one hand, NFAT dephosphorylation triggered by activated calcium was essential for the nuclear transfer after SAH insult(12). On the other hand, compared with that in control level, SAH induced a low expression of GSK-3 at 1d time point(20, 21). Therefore, this might support the possibility that the nuclear transfer NFAT tightly associate with a low expression of GSK-3 after SAH, which ultimately activate the Trim17/Bax pro-apoptotic pathway.

NFATc3, also named as NFAT4, is mainly located in the cytoplasm of neurons, and can be efficiently stimulated to nuclear translocation by the calcium signaling pathways(22). After nuclear translocation, NFAT4 can bind to the Trim17 promoter specifically(23). Similarly, we recently characterized the expression of NFAT4 in vivo and found that the nuclear NFAT4 obviously increased after SAH. One possibility in our study was that GSK-3 inhibition led to more nuclear translocation of NFAT4 after SAH, which gave NFAT4 more opportunity to initiate the transcription of Trim17 resulting in neuronal apoptosis after SAH. In an effort to investigate the molecular mechanism by which NFAT4 modulated Trim17-mediated pro-apoptotic signaling, we impeded the nuclear translocation of NFAT4 by genetic knockdown. Significantly, NFAT4 inhibition mice displayed markedly reduced pro-apoptotic activity of Trim17/Bax pathway, accompanied by ameliorated brain water content and improved neurological function. In parallel, we also observed a manifestly reduced accumulation of nuclear translocation of NFAT4 by genetic knockdown in comparison with that in negative control group after SAH. The fact that accumulation of nuclear translocation of NFAT4 obviously drove the pro-apoptotic activity of Trim17/Bax pathway after SAH, supporting the recent observation that PI3K/Akt/GSK3 pathway mediate pro-apoptotic activity of Trim17/Bax pathway. With these data, it was plausible to deduce that the strong activation of Trim17/Bax pro-apoptotic pathway was associated with the nuclear translocation of NFAT4 after SAH, and that inhibition the nuclear translocation of NFAT4 would be expected to repair early brain injury, making NFAT4 an attractive new clinical target for the treatment of SAH.

Remarkably, there was also a limitation in the present study. A previous study reported that NFAT4 and Trim17 had a reciprocal regulation on controlling of neuronal apoptosis(24). Therefore, there may be other involved signaling pathways underlying the regulatory mechanism between NFAT4 and Trim17 after SAH. Future studies are required to fully elucidate the potential mechanisms associated with neuronal apoptosis in the setting of experimental SAH.

Conflict of interests

The authors declare that they have no conflict of interests.

Author Contributions

All authors contributed to the study conception and design. Shibin Song: Conceptualization, Methodology, Software; Xiaoming Zhou: Conceptualization and Writing- Original draft preparation; Wenjin Qiu and Zhixiong Xu: Visualization, Investigation; Yimin Chen: Supervision; Minghao Dong: Formal analysis; Hua Yang: Writing- Reviewing and Editing. All authors read and approved the final manuscript.

Funding: This work was supported by the project funded of the Affliated Hospital of Guizhou Medical University (NO. I-2019-09), and the project funded of Jinling Hospital (NO. YYBJ2021041).

Ethical approval: All animal experiments were approved by the Institutional Animal Care Committee of Guizhou Medical University and were performed in accordance with the guidelines of the National Institutes of Health.

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Role of NFAT4-Trim17 axis mediates the neuronal apoptosis after subarachnoid hemorrhage

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Version 1

Abstract

Figures

Introduction

Materials And Methods

Animal preparation

SAH model in vivo

Tissue Preparation

Primary neurons culture

SAH model in vitro

Lentivirus transduction in vivo and in vitro

Study Design and Drug Administration

Western Blot Analysis

Real-time PCR analysis

Immunofluorescence (IF) and TUNEL staining

Brain water content

Neurological function evaluation

Statistical analysis

Results

Temporal patterns and cellular expression of endogenous Trim17 after SAH

Trim17 silencing ameliorated the neuronal apoptosis in vivo and in vitro after SAH

Temporal patterns and cellular expression of endogenous brain NFAT4 after SAH

Neuronal Trim17 regulation had a NFAT4 sensitivity profile in vivo after SAH

Discussion

The Trim17 Pathway In The Pathogenesis Of Neuronal Apoptosis After Sah

Declarations

References

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