TRIM59 suppresses the brain ischaemia/reperfusion injury and pyroptosis of microglial through mediating the ubiquitination of NLRP3

Cerebral ischaemia/reperfusion (I/R) injury induces irreversible brain injury and causes functional impairment. Ubiquitination plays a crucial role in protein degradation, but its role in cerebral I/R injury remains unclear. Differentially expressed genes in stroke were identified by analysing the microarray dataset GSE119121. Cerebral I/R was simulated in vitro by treating human microglial HMC3 cells with oxygen–glucose deprivation/reperfusion (OGD/R). Cell viability was tested by Cell Counting Kit 8 (CCK-8) assays, and pyroptosis was examined by flow cytometry. Lactate dehydrogenase (LDH) and inflammatory cytokine secretion were measured by LDH cytotoxicity assays and enzyme-linked immunosorbent assay (ELISA), respectively. The cerebral I/R animal model was established by middle cerebral artery occlusion (MCAO) surgery in rats. Bioinformatic analysis indicated that tripartite motif-containing protein 59 (TRIM59) is downregulated in stroke, which was verified in cerebral I/R models. The upregulation of TRIM59 promoted viability and inhibited pyroptosis in OGD/R-treated microglia and alleviated cerebral I/R injury in vivo. TRIM59 attenuated NOD-like receptor family pyrin domain containing 3 (NLRP3) protein expression through ubiquitination, thus degrading NLRP3 and alleviating OGD/R-induced injury. TRIM59 relieves cerebral I/R injury in vivo and in vivo. Mechanistically, TRIM59 directly interacts with NLRP3 and inhibits NLRP3 through ubiquitination. Targeting the TRIM59/NLRP3 signalling axis may be an effective therapeutic strategy for cerebral I/R.


Animals and induction of MCAO
The rat experiments were carried out in compliance with the revised Animals (Scientific Procedures) Act 1986 and the ARRIVE guidelines (http:// www.nc3rs.org.uk/ page.asp? id= 1357).In addition, the rat experiments were approved by the Animal Ethical and Welfare Committee (approval no.MDKN-2021-058).Twenty-four SPF healthy SD rats weighing 260 ± 10 g were purchased from Shanghai Slack Laboratory Animal Co., Ltd.(Shanghai, China).The production licence number was SCXK (Shanghai) 2014-0002, and the qualification licence number was 2015000518015.All animal experiments complied with ethical requirements.The rats were randomly divided into four groups, with 6 rats in each group: sham operation group (Sham), model group (MCAO), TRIM59 overexpression model group (MCAO + LV-TRIM59) and TRIM59 overexpression negative control model group (MCAO + LV-NC).
To establish MCAO in rats, the rats were anaesthetized with 2% sodium pentobarbital (2 mL/kg) after 12 h of fasting.The common carotid artery, external carotid artery and internal carotid artery were separated, a small incision was made in the common carotid artery, a thread plug was inserted, and the internal carotid artery was ligated.After 2 h of obstruction, the 4-0 (0.26 mm diameter) wire plug was removed to achieve reperfusion.The sham group underwent the same operations as the MCAO group except that the wire plug was not inserted.After the rats were awake, neurological impairment was scored according to a previous report 35 .

Stereotaxic injection of the lentivirus
LV-TRIM59 and LV-NC were injected into the left lateral ventricle one month before MCAO surgery to overexpress TRIM59 in rats.After anaesthesia by intraperitoneal injection of 1% pentobarbital sodium (40 mg/kg), the rats were fixed with a brain stereoscopic locator, and the left cerebral cortex was positioned using the following coordinates relative to the bregma: 1.0 mm posterior, 2.0 mm lateral, 3.0 mm below the horizontal plane of the

Measurement of brain infarct volume
After the evaluation of neurological impairment, the rats were euthanized with 400 mg/kg pentobarbital sodium.The euthanasia and anaesthesia methods were carried out in accordance with the guidelines of the American Veterinary Medical Association.Then, the brains were immediately collected and cut into six coronal slices with a thickness of 2 mm.Then, the slices were dyed using 2% 2,3,5-triphenyltetrazolium chloride (TTC; BIOPIKE, Guangzhou, China) at 37 °C for 10 min.Next, brain infarct size was calculated by the following formula: (volume of total brain tissue-volume of total ipsilateral hemispheric damage)/volume of total brain tissue × 100.

Nissl staining
The paraffin sections were dewaxed and stained with 10% methylene blue solution for 20 min.After being washed, the sections were treated with 95% alcohol, dehydrated, made transparent, sealed, and placed under an ordinary light microscope (Leica Microsystems, Wetzlar, Germany) for observation and photometry, and the number of intact neurons was counted.The total neuronal cells were divided into 10 areas and calculated, and then the degree of ischaemia-damaged neurons was calculated according to the percentage of damaged neurons in the total neurons 35 .

Immunofluorescence analysis
Brain sections were fixed with 4% paraformaldehyde and then incubated in PBST containing 5% BSA (Santa Cruz, CA, USA) for 0.5 h.Next, the sections were treated with primary antibodies, including anti-IBA-1 (highly expressed in microglia) and anti-NLRP3, at 4 °C overnight, followed by incubation with the corresponding secondary antibody at 37 °C for 1 h.The nuclei were stained with DAPI (Beyotime, Shanghai, China).Finally, the sections were observed with a scanning fluorescence microscope (Leica Microsystems, Wetzlar, Germany) and analysed using ImageJ software (ver.1.45r, National Institutes of Health, Bethesda, MD, USA).

Real-time quantitative polymerase chain reaction (RT-qPCR)
The mRNA expression of TRIM59 and NLRP3 in brain tissues and HMC3 cells was assessed by RT-qPCR.We extracted total RNA using TRIzol ® reagent (Thermo Fisher, Waltham, MA, USA) and then reverse transcribed the RNA into cDNA (Hi-Fi cDNA Synthesis Kit, Abcam, Cambridge, MA, USA).RT-qPCR was performed using the SYBR Green Mix kit (Beyotime) in an Eppendorf Thermol Cycler system (Eppendorf AG, Shanghai, China).The target amplification process was performed as follows: predenaturation at 95 °C for 5 min, denaturation at 95 °C for 10 s, annealing at 60 °C for 20 s, and then extension at 72 °C for 30 s, all for 40 cycles.Quantitative analysis was then performed using the 2 -ΔΔCt method.The primer sequences are listed in Table 1.

Cell viability assay
Cell counting kit 8 (CCK-8, Beyotime) was used to evaluate the viability of HMC3 cells.In brief, the cells were seeded into 96-well plates, and 10 μL/well CCK-8 reagent was added.After 4 h of culture, cell viability was detected at an absorbance of 450 nm with a microplate reader (Bio-Rad).

Lactate dehydrogenase (LDH) cytotoxicity evaluation
To evaluate cell damage and measure cell death, LDH release by HMC3 cells was determined by an LDH Cytotoxicity Assay Kit (Beyotime).In brief, HMC3 cells were seeded into a 96-well plate and incubated with the LDH reagent for 0.5 h to measure LDH release.The absorbance values were obtained at 490 nm with a microplate reader (Bio-Rad, Hercules, CA, USA).

Measurement of pyroptosis
Flow cytometry was performed to examine pyroptosis.HMC3 cells were digested with trypsin and washed with PBS.Pyroptosis was assessed with FLICA 660-YVADFMK in accordance with the manufacturer's protocol.The

Western blot analysis
HMC3 cells were collected to extract proteins (RIPA, Beyotime), and the protein concentration was determined using the BCA method.Then, the proteins were separated by 10% SDS-PAGE and transferred to PVDF membranes.After the membrane was transferred, the proteins were blocked in 5% skim milk at room temperature for 2 h.Diluted primary antibodies (Table 2) were added and incubated overnight at 4 °C.HRP-labelled secondary antibodies were then added and incubated.Finally, the protein bands were visualized using an ECL system (Thermo Fisher).

Protein stability assessment
A cycloheximide (CHX) blocking assay was performed to determine the half-life of NLRP3 as previously reported 36 .HMC3 cells were incubated with CHX (100 μg/mL, Sigma-Aldrich, St. Louis, MO,USA), and the protein level of NLRP3 was detected at different time points (0, 2, 4, 8 h) after CHX treatment.

Coimmunoprecipitation (Co-IP)
HMC3 cells were lysed in RIPA lysis buffer (Beyotime).A 1/10 volume of supernatant was collected as the input, and half of the remaining supernatant was incubated with 20 µL/mL protein A/G Sepharose beads (Beyotime) at 4 °C for 1 h to remove nonspecific hybrid proteins.Afterwards, the lysates were incubated with 2 µg anti-NLRP3 antibodies (ab263899, Abcam)/anti-TRIM59 antibodies (ab69639, Abcam) or negative control IgG (Beyotime) at 4 °C overnight and then rotated at 4 °C with a mixture of protein A/G Sepharose beads (20 µL/mL) for 4 h.The bound proteins were analysed by western blotting.

Ubiquitination assay
The cells were transfected with vectors expressing Flag-NLRP3, HA-UB, TRIM59 and the control.Whole-cell extracts were immunoprecipitated with anti-HA and analysed by immunoblotting with anti-Flag antibodies.

Statistical analysis
Each experiment was repeated three times, and all data are expressed as the mean ± SD and were calculated by GraphPad Prism 8.3.Student's t test was used to analyse the differences between two groups, and one-way analysis of variance (ANOVA) was performed for comparisons between multiple groups.All results were considered significantly different when p < 0.05.

Ethics approval
The rat experiments were approved by the Animal Ethical and Welfare Committee (approval no.MDKN-2021-058).All animal experiments complied with ethical requirements.

TRIM59 is expressed at low levels in cerebral I/R and OGD/R models
Differentially expressed genes in the blood samples of 8 spontaneously hypertensive rats at 0 and 2 h post stroke induction by MCAO were identified (Fig. 1A).As indicated in Fig. 1B, 344 aberrantly elevated genes and 267 downregulated genes were identified.Ten ubiquitination-related genes that were significantly downregulated in MCAO rats were screened (Fig. 1C-L), among which TRIM59 was selected as the most downregulated gene (Fig. 1I).Then, the low protein expression of TRIM59 was verified in the brain tissues of MCAO rats, which were established to mimic cerebral I/R in vivo (Fig. 1M, N).The OGD/R model in HMC3 cells was established, and TRIM59 mRNA and protein expression was shown to be dramatically downregulated (Fig. 1O, Q). www.nature.com/scientificreports/

TRIM59 attenuates NLRP3 protein expression through ubiquitination
As an E3 ubiquitin ligase, TRIM59 has ubiquitin protein transferase activity.Therefore, TRIM59 was thought to modulate NLRP3 stability through the ubiquitin-proteasome pathway.The co-IP results revealed the interaction between TRIM59 and NLRP3 (Fig. 3A).Then, a ubiquitination assay was performed on HMC3 cells after transfection.As shown in Fig. 3B, OGD/R knocked down the protein expression of TRIM59 and decreased the ubiquitination of NLRP3.However, the upregulation of TRIM59 enhanced the ubiquitination of NLRP3 and inhibited NLRP3 protein levels (Fig. 3B), suggesting that TRIM59 might affect the stability of NLRP3.To substantiate this conclusion, we treated HMC3 cells with OGD/R and the protein synthesis inhibitor CHX.The results suggested that OGD/R extended the half-life of the NLRP3 protein, while overexpression of TRIM59 shortened the half-life of the NLRP3 protein in HMC3 cells after CHX treatment (Fig. 3C, D).

TRIM59 alleviates cerebral I/R injury in vivo
The neurological score and infarct volume were evaluated 7 days after MCAO.The infarct volume was prominently elevated in the MCAO group, but there was almost no infarct volume in the sham-operated group.The increase in TRIM59 notably decreased the infarct volume in MCAO rats (Fig. 5A, B).Moreover, the neurological score was prominently enhanced in MCAO-treated rats, while these effects were suppressed after TRIM59 upregulation (Fig. 5C).The Nissl staining results confirmed that MCAO dramatically aggravated neuronal injury in rats, and shrunken nuclei and disorderly arrays were observed in model rats (Fig. 5D, E).Additionally, MCAO prominently promoted the levels of IL-1β and IL-18 in rat serum, while TRIM59 dramatically inhibited this increase (Fig. 5F, G).Furthermore, the immunofluorescence results demonstrated that NLRP3 expression was elevated in the MCAO group, and TRIM59 overexpression significantly downregulated NLRP3 levels (Fig. 5H).

Discussion
TRIM59 was downregulated in the brain tissues of MCAO rats and OGD/R-treated HMC3 cells.Our data suggested that the upregulation of TRIM59 promoted cell viability and inhibited pyroptosis in microglia after OGD/R treatment.Moreover, TRIM59 directly interacted with NLRP3 and inhibited NLRP3 through ubiquitination.These findings indicated that TRIM59 could alleviate cerebral I/R injury by inhibiting NLRP3 expression.Cerebral I/R injury refers to injury caused by insufficient blood supply to the brain tissue and the restoration of blood perfusion.This process triggers an innate immune response and leads to an inflammatory cascade 19 .Inhibiting NLRP3 inflammasome activation has been used as one of the therapeutic methods for cerebral I/ R 21,37 .For example, ibrutinib, a Bruton's tyrosine kinase inhibitor, can inhibit NLRP3 inflammasome, caspase-1 and IL-1β activation, thereby alleviating cerebral I/R injury 38 .
Ubiquitination can affect the biological functions of cells by regulating the stability and expression level of intracellular proteins, including cell proliferation, cell cycle, apoptosis, tumour development, metastasis, and invasion 39 .Changes in protein stability and abnormal expression are related to the development of diseases, and so the study of protein ubiquitination mechanisms can better explain the molecular mechanisms of diseases 40,41 .Ubiquitination was shown to modulate the stability of the NLRP3 inflammasome 42,43 .At present, only E3 ubiquitin ligases have been shown to negatively regulate the inflammasome by targeting NLRP3 44 .One of the main mechanisms involves controlling NLRP3 levels through the proteasome degradation pathway.Recently, TRIM59 has been identified as a novel molecular biomarker in various tumours 45 .TRIM59 promotes lung cancer progression by regulating autophagy or the NLRP3 inflammasome signalling pathway 28,46 .TRIM59 participates in tumour disease progression by regulating the ubiquitination of genes [47][48][49] .The presence of the unique N-terminal RING domain enables most TRIM family members to have extensive ubiquitination enzymatic activities and induce target proteins to enter the proteasome-dependent ubiquitination degradation process by binding them to ubiquitin.TRIM59 has the conserved RBCC domain of typical TRIM family proteins 28,46 .In addition to the three typical RBCC domains (RING finger domain, B-box domain and coiled-coil domain), the C segment of TRIM59 also has a TM domain that may be involved in the transmembrane localization of TRIM59, which is involved in regulating the intracellular localization of TRIM59.In the current work, bioinformatic analysis was carried out to screen genes in stroke related to ubiquitination, and TRIM59 was shown to be expressed at low levels in cerebral I/R animal and cell models.An increase in TRIM59 dramatically protected HMC3 cells from OGD/R injury.Afterwards, TRIM59 was found to regulate the ubiquitination of NLRP3.
Microglia, the resident immune cells tiling the whole CNS, exhibit an amazing capacity for regeneration 50,51 .Even when depleted in large quantities in the uninflamed central nervous system, microglia are able to quickly refill and return to normal density within a few days through the proliferation of residual cells.After neurodegeneration, they proliferate and accumulate in the diseased central nervous system 52 .Therefore, if the microglia are protected well, it may be possible to convert them into neurons to repair the damaged central nervous system where neurons are lost.In this study, TRIM59 promotes microglial survival and reduces infarction size and neuronal damage.Whether the protective mechanism benifits neurons should be investigated in the future.Meanwhile, which amino acid residue of NLRP3 is ubiquitination modified has not been elucidated.
Therefore, it is suggested that TRIM59 may act as a ubiquitin ligase to induce the ubiquitination-mediated degradation of NLRP3.Our data revealed that TRIM59 binds endogenously to NLRP3 and attenuates NLRP3 protein expression through ubiquitination.Furthermore, the half-life of the NLRP3 protein was decreased in TRIM59-overexpressing cells.These results suggest that the overexpression of TRIM59 contributes to NLRP3 protein degradation in microglia.

Figure 1 .
Figure 1.TRIM59 is expressed at low levels in a cerebral I/R animal model and cell model.(A) Heatmap analysis of differentially expressed genes in stroke from the microarray expression profiling dataset GSE119121.(B) A total of 344 aberrantly upregulated genes and 267 downregulated genes were identified.(C-L) The mRNA levels of ten ubiquitination-related genes in MCAO rats were detected by RT-qPCR, n = 6.(M, N) The protein expression of TRIM59 in the brain tissues of rats was detected by western blotting, n = 6.The mRNA (O) and protein expression (P, Q) of TRIM59 in HMC3 cells were evaluated by RT-qPCR and western blot analysis, respectively.n = 3. **p < 0.01, ***p < 0.001.

Figure 3 .
Figure 3. (A) TRIM59 attenuates NLRP3 protein expression through ubiquitination.The interaction between TRIM59 and NLRP3 was detected by Co-IP.(B) After transfection, IP using anti-Flag was performed.The ubiquitination and expression of HA in the pull-down product was detected by western blotting.(C, D) Western blot analysis of the half-life of NLRP3 protein in HMC3 cells under OGD/R conditions and with TRIM59 overexpression.n = 3, ***p < 0.001, versus control group.##p < 0.01, versus OGD/R + Vector group.

Figure 5 .
Figure 5. TRIM59 alleviates cerebral I/R injury in vivo.Images of TTC-stained complete coronal sections in the MCAO and sham groups.(B) The infarct volume was determined.(C) Neurologic scores in rats of the MCAO and Sham groups were evaluated.(D) Quantitative evaluation of the neuronal damage rate of the hippocampal CA3 region and (E) cerebral cortex was performed.(F, G) The expression of IL-18 and IL-1β in the serum of rats was detected by ELISA.(H) Immunofluorescence analysis was performed to evaluate the localization and expression of IBA1 and NLRP3.n = 6, ***p < 0.001, versus Sham group.##p < 0.01, ###p < 0.001, versus MCAO + LV-NC group.