KAP1 Phosphorylation Promotes the Survival of Neural Stem Cells after Ischemia/Reperfusion by Maintaining the Stability of PCNA

doi:10.21203/rs.3.rs-1142716/v1

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KAP1 Phosphorylation Promotes the Survival of Neural Stem Cells after Ischemia/Reperfusion by Maintaining the Stability of PCNA

https://doi.org/10.21203/rs.3.rs-1142716/v1

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Aims: To explore the function of phosphorylation of KAP1 (p-KAP1) at serine-824 site (S824) in the proliferation and apoptosis of endogenous neural stem cells (NSCs) after cerebral ischemic/reperfusion (I/R).

Methods: The apoptosis and proliferation of C17.2 cells transfected with the p-KAP1-expression plasmids and the expression of proliferation cell nuclear antigen (PCNA) and p-KAP1 were detected by immunofluorescence and Western blotting after the Oxygen Glucose deprivation/reperfusion model (OGD/R). The interaction of p-KAP1 and CUL4A with PCNA were analyzed by immunoprecipitation. In the rats MCAO model, we performed the lentiviral-mediated p-KAP1 mutants to verify the proliferation of endogenous NSCs and the colocalization of PCNA and CUL4A by immunofluorescence.

Results: The level of p-KAP1 was significantly down-regulated in the stroke model in vivo and in vitro. Simulated p-KAP1(S824) significantly increased the proliferation of C17.2 cells and the expression of PCNA after OGD/R. Simulated p-KAP1(S824) enhanced the binding of p-KAP1 and PCNA and decreased the interaction between PCNA and CUL4A in C17.2 cells subjected to OGD/R. With the lentiviral AAV2/9-mediated p-KAP1(S824), there was increased endogenous NSCs proliferation, PCNA expression, p-KAP1 binding to PCNA, and improved neurological function in the rat MCAO model.

Conclusions: Our findings confirmed that simulated p-KAP1(S824) improved the survival and proliferation of endogenous NSCs. The underlying mechanism involved in that highly expressed p-KAP1(S824) promoted to bind to PCNA, inhibiting the binding of CUL4A to PCNA. This reduced CUL4A-mediated ubiquitination degradation to increase the stability of PCNA and promote survival and proliferation of NSCs.

Stem Cell & Developmental Cell Biology

Cerebral ischemia/reperfusion (I/R)

Neural Stem Cells (NSCs)

Proliferation

KRAB domain protein 1(KAP1)

PCNA

Stroke is a major cause of mortality and disability worldwide ^[1]. Ischemic stroke accounts for more than 87% of stroke events and is an acute cerebral arterial obstruction characterized by abrupt onset and focal neurological deficits ^[2]. Cerebral ischemia-reperfusion (I/R) injury after vascular recanalization further amplifies the death of nerve cells ^[3]. Therefore, strategies to promote recovery from stroke require enhanced neurogenesis to compensate for damaged nerve cells. Numerous studies have explored the use of neural stem cells (NSCs) for the treatment of stroke, as NSCs can proliferate and differentiate into neurons and glial cells to reconstruct destroyed neuronal circuits ^[4–8]. With the continued development of stem cell technology, the transplantation of exogenous NSCs has been used to treat various diseases of the central nervous system ^[9–11]. However, there remain limitations of exogenous NSCs therapy, such as transplant rejection, poor stability, difficulty in getting materials, and ethical controversies ^{[12, 13]}. Given these factors, it is important to investigate how to enhance the proliferation of endogenous NSCs to improve survival and recovery from neurological deficits after cerebral I/R.

The Krueppel-associated box (KRAB) is a domain of around 75 amino acids that is found in the N-terminal part of about one third of eukaryotic Krueppel-type C2H2 zinc finger proteins (ZFPs)^[14]. KRAB domain protein 1(KAP1), also known as TRIM28 (tripartite motif-containing protein 28) or TIF1β (transcriptional intermediary factor 1β), is a transcriptional cofactor that was originally isolated, cloned, and purified by affinity chromatography in 1996 ^[15]. KAP1 is involved in regulation of the embryonic stem cell cycle and control of proliferation and apoptosis ^{[16, 17]}. Post-translational modifications of KAP1, including phosphorylation and ubiquitination, are critical for its function in stem gene expression, embryonic development, immune regulation, tumor occurrence and development, and DNA damage response ^{[18, 19]}. The phosphorylation of KAP1 (p-KAP1) acts as an interpreter of cell signaling and modulates the ability of MyoD to drive myogenesis ^[20]. The p-KAP1 occurred at Tyr-449, Tyr-458, and Tyr-517 residues inhibit the association of KAP1 and heterochromatin protein 1α with heterochromatin ^[21]. Additionally, the p-KAP1(S824) has been reported to participate in embryonic stem cell proliferation and differentiation, maintaining stemness gene expression ^[22]. However, whether KAP1 and its phosphorylation state can regulate the functions of NSCs has not been reported.

Proliferating cell nuclear antigen (PCNA), a marker of proliferation of NSCs, is a key factor in DNA replication and cell cycle regulation ^[23]. Increased expression of PCNA indicates increased proliferation ability of NSCs, required for successful clinical treatment of central nervous system diseases^{[24, 25]}. Jang et al. reported that p-KAP1(S473) can interact with PCNA to restore heterochromatin after DNA replication during S phase ^[26]. Additionally, p-KAP1 recruits DNA repair-related factors after damage to facilitate DNA repair ^[27]. Therefore, we hypothesized that there might be a relationship between the phosphorylation level of KAP1 and the proliferation of NSCs after cerebral I/R injury, so that strategies targeting p-KAP1 could be used to prevent or treat ischemic stroke.

In this study, we found that p-KAP1 at serine 824 (S824) could promote the proliferation of NSCs and reduce apoptosis and neurobehavioral deficits, thereby promoting the recovery of cerebral I/R-induced neurological dysfunction. The underlying mechanism is that p-KAP1(S824) act to bind to PCNA and inhibit binding of PCNA to E3 ubiquitin ligase CUL4A, reducing CUL4A's degradation of PCNA and thereby promoting the survival and proliferation of NSCs during I/R. Analysis of the KAP1/PCNA signaling pathway suggested that p-KAP1 may be a target for endogenous neurogenesis in cerebral I/R treatment.

Culture of the C17.2 cells and establishment model of OGD/R

The C17.2-NSCs was purchased from the American Type Culture Collection (Rockville, MD, USA) and were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco, Carlsbad, USA) with 10% (v/v) fetal bovine serum (FBS; Gibco, Carlsbad, USA) in a humidified incubator with 5% CO2 and 95% air at 37°C. In the Oxygen Glucose deprivation/reperfusion model (OGD/R), cells were washed three times with sterilized PBS and the medium was replaced with sugar-free medium culturing in O2/CO2 (1% / 5%) three-gas incubator. After 6 h, the cells medium was replaced with DMEM containing 10% FBS for another 24 h in a humidified incubator with 5% CO2 and 95% air at 37°C.

Lentiviral-mediated KAP1 mutations and plasmid transfection

Lentiviral-mediated KAP1 (AAV2/9-KAP1-GFP) and its mutations (AAV2/9-KAP1-S824A-GFP, AAV2/9-KAP1-S824D-GFP) were purchased from Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. Viral-mediated KAP1 and its mutations (injected at a titer of 109 VG/µl) were administered to the rats intracerebroventricularly (i.c.v) (bregma, 1.5 mm lateral, 0.8 mm posterior, and 3.8 mm deep) 14 days before ischemia.KAP1 plasmids (WT, S824D, S824A) were obtained from Shanghai Sangon, and were transiently transfected into cells with Lipo3000 liposome transfection reagent following the manufacture’s protocols. Briefly, 60% - 80% confluent cells were prepared for transfection. Abandon the medium and wash with PBS for 3 times. Then, add Opti-MEM into every well. Next, prepare lipofectamine 3000 reagent and plasmid mixture. The corresponding plasmid DNA (including P3000) and liposome were diluted with Opti-MEM medium respectively according to the size of the perforated plate, and then the two were mixed after standing at room temperature for 5 min. After that, incubate at room temperature for 15 min. Finally, the plasmid DNA-liposome complex was added into the cells in each well and cultured in the cell incubator for 24 - 48 h. The transfection efficiency was confirmed by Western blot analysis.

Western Blotting

Cells were lysed in RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) containing 1% protease inhibitor cocktail (VICMED, VPI012), and the cell lysates were then centrifuged at 14000 × g at 4°C for 15 min. Protein concentrations were quantified using the Bicinchoninic Acid protein assay kit (BCA; Beyotime, P0010). Total proteins (50 µg) were separated by SDS-PAGE and electro-transferred to polyvinylidenedifluoride membranes (PVDF; Millipore, Billerica, MA, USA). Then the membranes were blocked with TBST buffer (NaCl 150 mmol/L, Tris 10 mmol/L, Tween-20 0.05% (v/v) pH = 7.6) containing 5% non-fat milk powder at room temperature for 2 h. The membranes were then probed overnight at 4°C with primary antibodies including phospho-KAP1(1:1000, Abcam, ab70369), KAP1(1:1000, Cell Signaling Technology, #4123), PCNA (1:2000, Servicebio, GB11010), Bcl-2 (1:1000, ABclonal, A0208), Bax(1:1000, ABclonal, A0207) followed by incubation with corresponding HRP-conjugated secondary antibodies (1:5000, Proteintech, SA00001-2) at room temperature for 2 h. Proteins on the membranes were visualized by adding the enhanced chemiluminescence reagent (Millipore, USA). Band intensities were analyzed using Image J 1.25 software (National Institutes of Health, Bethesda, USA) and normalized to that of loading controls.

Immunoprecipitation

Immunoprecipitation experiments were carried out with protein lysates using Beyotime Western and IP cell lysis buffer containing 1% protease inhibitor cocktail (VICMED, VPI012), and the cell lysates were then centrifuged at 14000 × g at 4°C for 15 min. Protein concentrations were quantified using the Bicinchoninic Acid protein assay kit (BCA; Beyotime, P0010). Add a certain amount of the antibody into the protein lysates and shake at 4 ℃ overnight. The next day, wash the protein A/G magnetic beads (MCE) with 0.5% PBST (1 × PBS + 0.5% Triton X-100) for three times, and then add it into the protein antigen-antibody mixture. Then, continue to shake at 4 ℃ for 2.5 h. After that, wash the protein A/G magnetic beads-protein antigen-antibody complex with 0.5% PBST for five times. Finally, add 20-30 µl 2 × protein loading buffer into the complex, and then heat it in 100 ℃ water for 5 min. Samples were centrifuged at 12000 g for 2 min, and the supernatant was collected. Primary antibodies used in this stage including Flag (1:100, Proteintech, 20543-1-AP), KAP1 (1:100, CST, #4123), PCNA (1:100, Abcam, ab29), PCNA (1:2000, GB11010, Servicebio), Flag (1:10000, D110005, Sangon Biotech), CUL4A (Proteintech, 66038-1-Ig), α-Tubulin (1:1000, AF0001, Beyotime Biotechnology).

CCK-8 cell proliferation assay

The cells transfected with mutant plasmids were grown in 96-well plate. After attachment, cells were subjected to OGD/R or not. CCK-8 reagent and DMEM medium were mixed in 1:10. 100 µl of the mixture was added to each well, and then incubated in 5% CO2 incubator at 37 ℃ for 0.5 - 1 h. Then, the absorbance was determined at 450 nm with a microplate analyzer.

Bromodeoxyuridine (Brdu) staining

The cells transfected with mutant plasmids were grown in 24-well plate. After attachment, cells were subjected to OGD/R or not and were treated with or without Brdu for 8 h before staining. Brdu staining steps were as follows. Firstly, cells were washed with 0.3% TBS for 5 min × 3 times. Then, fix with 4% paraformaldehyde for about 20 min at room temperature. Next, cells were washed with TBS for 5 min × 3 times. Subsequently, 2 N hydrochloric acid was added to cells denaturing at room temperature for 1 h. After that, wash with TBS for 5 min × 3 times, and seal 1 h at room temperature (sealing fluid is 10% goat serum + 0.3% TritonX-100 PBS). Finally, incubate cells with the anti-Brdu antibody (1:500, Abcam, ab152095) at 4 ℃ overnight (diluent with 1% goat serum + PBS + 0.3% TritonX-100). The next day, wash with TBS for 5 min × 3 times and incubate with secondary antibody (1:1000, Invitrogen, A-21207) at room temperature for 2 h. Then, wash with TBS for 5 min × 3 times and stain with DAPI dye for 10 min. Finally, wash with TBS for 5 min × 3 times and photograph under Olympus IX73 inverted fluorescence microscope.

Annexin V-APC/7-AAD apoptosis assay

The cells transfected with mutant plasmids were grown in middle-plates, followed by OGD/R treatment or not after adherence. Digest and collect with trypsin free of EDTA, and wash with PBS twice to collect 1 - 5 × 105 cells. Cells were suspended with 500 µl binding buffer. 5 µl Annexin V-APC and 5 µl 7-Aminoactinomycin D (7-AAD) dye were added into cells. Incubate at room temperature in dark for 5 - 15 min. Then, samples were detected with Flow cytometry within 1 h.

Establishment of rat middle cerebral artery occlusion (MCAO) model

Male Sprague-Dawley rats (Beijing Vital River Laboratory Animal Technology Co., Ltd.) aged between 7 and 8 weeks old (180 g - 200 g) were used in this study. Rats were housed in the vivarium managed by the Division of Animal Lab Center of Xuzhou Medical University with a 12 h light/dark cycle and ad libitum access to food and water.

The MCAO model refers to the established method (Gong et al. 2015) in the laboratory, and the specific steps are as follows. After isoflurane inhalation anesthesia, the rat’s neck was vertically cut with scissors. Separate the surrounding tissue and expose rats left common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA). Then, fasten CCA and ECA near the heart with sutures, clip ICA with arteriole clip. Subsequently, a small incision was made with ophthalmic scissors at an oblique 45° between the CCA ligation point and the three fork-lets at the upper end. The standard cord plug corresponding to body weight was carefully inserted at the incision point. The micro-arteriole was loosened, and the direction of the inlet line was adjusted to avoid the pterygopalatine artery, and the ICA was entered until the beginning of the middle cerebral artery (MCA). After ischemia for 1.5 h, the rats were under reperfusion for 24 h by pulling out the plug. The insertion depth of the thread plug in the sham operation group was about 5 mm, and the rest of the treatment was the same as that in the model group.

Balance beam test

The balance beam test was performed to assess the ability of rats to maintain balance and motor function while walking along an elevated and narrow strip of wood (122 × 2.5 × 42 cm). The performance was scored from 0 to 6 (no attempt to stay on the beam as 0 point); attempted to stay on the beam but no movement as 1 point; attempted to cross the beam but failed as 2 points; crossed the beam with contralateral hindlimb slips > 50% of the time as 3 points; crossed the beam with contralateral hindlimb slips < 50% of the time as 5 points; crossed the beam without slips as 6 points (Splenectomy fails to provide long-term protection against ischemic stroke. Aging Dis. 2018).

Open Field Test

A square cage (1 m × 1 m, with walls 45 cm high) containing an opaque acrylic container and a video camera were applied in the test. A rat was placed in the center of the square cage. The locomotive activities, including travel distance, speed, and time in corner were recorded (Breviscapine Alleviates Cognitive Impairments Induced by Transient Cerebral Ischemia/Reperfusion through Its Anti-Inflammatory and Anti-Oxidant Properties in a Rat Model). The whole arena was wiped clean with 70% ethyl alcohol between each test.

Frozen section

Frozen sections of brain tissue were performed using a Leica freeze slicer. Firstly, the brain was removed from the sucrose solution, and the bottom of brain was cut flat with a knife. Then, the back of the brain was placed facing the notch on the freezing platform and the abdomen facing operator vertically. Wrap the head from top to bottom with the embedding agent and freeze it in the machine for at least 2 h. After that, put the head on the freezer and cut into 20 µm thick slices. The brain slices were then placed in phosphate buffer saline (PBS; NaCl 137 mmol/L, KCl 2.7 mmol/L, Na₂HPO₄·12H₂O 8 mmol/L, NaH₂PO₄·2H₂O 1.5 mmol/L). If stored for a long time, the slices needed to be placed in PBS solution containing 60% glycerin and stored in a refrigerator at – 80 ℃.

Immunofluorescence staining of frozen sections

The rats were sacrificed 7 days after the MCAO model and the brain tissue was taken for follow-up study. The rat brain slices attached to viscous slide were washed with TBS (0.3% Triton X-100 PBS) for 5 min × 2 times, and sealed with blocking solution (0.3% Triton X-100 + 10% PBS of goat serum) at room temperature for 1 h. The rat brain frozen sections were then added with primary antibody working solution (Beyotime, P0023A) and incubated at 4 ℃ in a wet box overnight. The following day, wash the brain slides with TBS for 5 min × 5 times and incubated with secondary antibody solution (Beyotime, P0023D) in a wet box at room temperature for 1 - 2 h. Afterwards, wash the brain slides with TBS for 5 min × 5 times, and then DAPI staining was added to the brain slides incubating for 10 min. Finally, wash the brain slides with TBS for 5 min × 5 times, seal the slide with a sealing tablet and photograph under Olympus IX73 inverted fluorescence microscope.

Statistical Analysis

The statistical analysis of data was conducted using GraphPad Prism 7. Experimental data were expressed as Means ± SEM. Students’ T test and One-way ANOVA was used for statistical analysis. Dunnet T test was used for comparison between multiple experimental groups and a control group. The data were checked for normally distributed by Kolmogorov-Smirnov test (with Dallal-Wikinson-Lillie for P value), before using parametric tests. P < 0.05 indicated that the difference was statistically significant.

The total expression of KAP1 remained unchanged but the level of p-KAP1 decreased after cerebral I/R.

To explore the role of KAP1 in cerebral I/R injury, the rat MCAO model was established for immunofluorescence staining. Compared with the sham group, there was no obvious difference in overall expression of KAP1 in the MCAO group, but the expression level of p-KAP1 was significantly reduced (Fig. 1B, C). Next, the OGD/R model of C17.2 cells was used to simulate cerebral I/R injury in vitro and we again detected the expression of KAP1 and p-KAP1. As shown in Fig. 1D and E, there was no difference in KAP1 expression in C17.2 cells between the normoxia group and the OGD/R group. However, compared with the normoxia group, the expression of p-KAP1 was significantly decreased in the OGD/R group, and this result was confirmed by immunofluorescence staining (Fig. 1F, G). Collectively, these results showed that the total expression of KAP1 remained unchanged but p-KAP1 declined significantly during cerebral I/R.

Simulated p-KAP1 improved the proliferation of endogenous NSCs after cerebral I/R.

We next explored the function of p-KAP1 in endogenous NSCs. We simulated phosphorylation of KAP1 (p-KAP1 is phosphorylated at serine 824, and S824D and S824A respectively simulated the state of phosphorylation and non-phosphorylation) in vitro and in vivo. Using BrdU staining and CCK-8 analysis, we found that the proliferation ability of C17.2 cells transfected with wild-type (WT) KAP1 and KAP1 (S824A) in the OGD/R group was significantly lower than that of the normoxia group. Transfected with p-KAP1 (S824D) mimic, the decrease in C17.2 cells proliferation was reversed (Figure 2A, B and C). We also detected the expression of PCNA by Western blotting. OGD/R treatment led to down-regulation of PCNA expression in C17.2 cells, but the use of the p-KAP1 (S824D) mimic abrogated this down-regulation (Figure 2D, E). We further used immunofluorescence staining to analyze the proliferation of endogenous NSCs in rats with p-KAP1 mutation were transfected with the lentiviral AAV2/9. As shown in Fig. 2F, compared with other groups subject to cerebral I/R, colocalization of Ki67 and Nestin in the Subependymal Ventricular Zone (SVZ) region was significantly increased in the viral-mediated KAP1 mutation (S824D) group. These results suggested that simulated p-KAP1 (S824) promoted the proliferation and regeneration of endogenous NSCs after cerebral I/R.

Simulated p-KAP1 alleviated OGD/R-induced C17.2 cells apoptosis.

We then determined the relationship between p-KAP1 and apoptosis of C17.2 cells after OGD/R by Western blotting and flow cytometry. Flow cytometry results indicated that the apoptosis rates of C17.2 cells transfected with KAP1 WT or KAP1 S824A in OGD/R groups were higher than that of the normoxia group, while the KAP1 S824D group could reverse the apoptosis of C17.2 cells induced by OGD/R (Figure 3A, B). We explored the possible pathways involved in the apoptosis of NSCs treated with p-KAP1 (S824) mimic. Western blotting showed the Bcl-2/Bax ratio of C17.2 cells were significantly decreased after exposure to OGD/R, while simulating p-KAP1 could increase the ratio (Fig. 3C and D). These results indicated that the p-KAP1 mimic might decrease OGD/R-induced apoptosis of C17.2 cells through the Bcl-2/Bax signaling pathway.

Simulated p-KAP1 enhanced binding to PCNA and decreased the interaction of CUL4A under cerebral I/R.

KAP-1 can act as a ubiquitin-like ligase of PCNA and mediate its ubiquitination degradation ^[28]. So, we assayed PCNA ubiquitination in endogenous NSCs after cerebral I/R. To do this, RT-PCR was used to detect the PCNA mRNA level. We found that both the normoxia group and the OGD/R group did not exhibit altered mRNA level (Fig. 4A). C17.2 cells were treated with or without proteasome inhibitor MG132 (20 µM) for 6 h, and then expression of PCNA was detected. The down-regulation of PCNA in C17.2 cells induced by OGD/R was improved when pre-incubated with MG132 (Fig. 4B, C). We also performed immuno-precipitation and found that after OGD/R, the level of PCNA poly-ubiquitination at K48 and K63 sites increased in C17.2 cells, while the p-KAP1 mimic could alleviate this poly-ubiquitination (K48) (Fig. 4D). However, simulated p-KAP1(S824D) had no effect on poly-ubiquitination (K63). This result indicates that the effects of p-KAP1 might depend on regulating the stability of PCNA by post-transcriptional modification, reducing its ubiquitination degradation by some ubiquitin enzymes. The expression of PCNA in the SVZ region of cerebral I/R rats was detected by immunofluorescence (Fig. 4E). As shown in Fig. 4E, the KAP1 mutation (S824D) mimic promoted its colocalization with PCNA and decreased the colocalization of PCNA with CUL4A in the SVZ region. The results in vivo were consistent with cellular experiments. Thus, we concluded that the p-KAP1 mimic may help regulate the proliferation of endogenous NSCs by influencing the stability of PCNA after cerebral I/R.

Simulated p-KAP1 promoted the stability of PCNA and ultimately competitively inhibited the degradation of CLU4A to PCNA.

To determine the regulatory relationship between p-KAP1 and PCNA in cerebral I/R, we studied their interaction by molecular docking and observed colocalization Fig. 5A, B). Confocal microscopy showed that KAP1 co-localized with PCNA in cellular nucleus (Fig. 5C). We also confirmed the interaction of KAP1 with PCNA in C17.2 cells by immuno-precipitation, and found that this interaction decreased after OGD/R (Fig. 5D, E). For this condition, we found that simulated p-KAP1 could increase its association with PCNA (Fig. 5F). Binding of PCNA to its E3 ubiquitin ligase CUL4A increased in C17.2 cells exposed to OGD/R, while the p-KAP1 mimic suppressed binding of CULA4 (Fig. 5G). In detail, the increased binding of PCNA to p-KAP1 resulted in decreased interaction of CUL4A, which weakened the ability of CUL4A to degrade PCNA, thus further stabilizing PCNA. This suggested that simulated p-KAP1 could stabilize PCNA expression by competitively inhibiting CUL4A ubiquitin degradation.

Simulated p-KAP1 (S824) improved long-term neurological deficits after cerebral I/R.

We explored whether p-KAP1 (S824) could achieve a long-term therapeutic effect on rats against cerebral I/R-induced behavioral deficits. Behavioral tests were performed 6 days after cerebral I/R. A schematic of the study design is depicted in Fig. 6A. In the open-field test, the locomotor activity levels were severely impaired in the KAP1-NC group after I/R, but treatment with KAP1-WT and its S824D mutation increased the recovery of neurological function as assessed by the total distance and speed (Fig. 6B–D). The KAP1-S824D mutant was better, but there was no difference in time in center of open-field test and balance beam test (Fig. 6E and F). Collectively, these data indicated that mimicked p-KAP1 achieved a long-term protective effect against cerebral I/R injury.

Simulated p-KAP1(S824) promoted the survival and proliferation of endogenous NSCs through increasing and stabilizing PCNA expression against cerebral I/R injury. We found no change in total KAP1 expression in endogenous NSCs in cerebral I/R, but its p-KAP1 level was decreased. The simulated p-KAP1 could promote the proliferation of NSCs and reduce its apoptosis and improve the behavioral deficits of rats after cerebral I/R. In the proposed mechanism, p-KAP1 increased the binding of PCNA and competitively inhibits ubiquitination of PCNA by CUL4A in NSCs (Fig. 7). Overall, the results show that p-KAP1 is promising for use as a new strategy to promote NSCs proliferation by the KAP1/PCNA signaling pathway.

Enhancing the proliferation of endogenous NSCs is an important component of stroke treatment. KAP1 is a transcription cofactor ^[29], and its structural domain, mainly localized in the cellular nucleus, can bind to members of the zinc finger protein family (ZFPs) ^{[15, 30–32]}. The function of KAP1 depends mainly on its phosphorylation state; p-KAP1 participates in human cytomegalovirus activation, and inhibition of p-KAP1 can depress viral replication ^[33]. Early infection with human adenoviruses (HAdV5) increases levels of p-KAP1 in cells and creates favorable conditions for viruses to replicate and enhance viral gene transcription ^[34]. Similarly, p-KAP1 is involved in Epstein Barr virus replication ^[35]. Phosphorylation of KAP1 at S473 and S824 is involved in the regulation of DNA damage repair ^[36]. In addition, p-KAP1 (S824) regulates the biological behavior of malignant tumors. Compared with non-invasive breast cancer, both KAP1 and p-KAP1 (S824) are increased in highly invasive breast cancer tissues ^[37]. Melanoma antigen encoding genes (melanoma-associated antigen encoding gene-C2, MAGE-C2) can bind KAP1, increasing p-KAP1 (S824) levels, promoting tumor cells proliferation and restoration of the genes that inhibit apoptosis, accelerating overall malignant tumor progression ^[38]. Although there have been many studies of p-KAP1 on virus and cancers, little is known of KAP1 function in the neuronal system. KAP1 is highly expressed in CNS ^[39]. Herein, in a model of rat MCAO, we found that p-KAP1 (S824) decreased significantly despite unchanged total expression of KAP1. Subsequently, using an in vitro model of OGD/R, we also found significantly decreased level of p-KAP1 (S824) in C17.2 cells. We speculated whether simulated p-KAP1 could restore the deficit of neural function induced by cerebral I/R. Transient transfection of p-KAP1 (S824) mimic plasmid significantly reversed OGD/R-depressed cell proliferation. These results were further validated by the in vivo Ki-67 and Nestin colocalization assay. Thus, simulated p-KAP1 could enhance the proliferation of endogenous NSCs after cerebral I/R.

PCNA is a core component of the eukaryotic replication complex with a special ring tertiary structure. PCNA can bind to different replication-related proteins to coordinate the DNA replication process ^{[26, 40–42]}. PCNA also acts as a functional conversion factor, interacts with a variety of cytokines through different regulatory modes, and participates in many important cellular events including DNA damage repairment and refinement, cell cycle regulation, and apoptosis^{[40, 43–45]}. As an indicator of cell proliferation, PCNA correlates with the occurrence and development of diseases linked to cell proliferation ^[45–47]. Therefore, we inferred that the effects of p-KAP1 on NSC proliferation might be related to regulation of PCNA. Western blotting revealed that the p-KAP1 mimic could improve OGD/R-induced suppression of PCNA, but it was unclear how this regulation occurs. Overall, there is significant interest in the potential relationship of p-KAP1 and expression of PCNA in NSCs subjected to cerebral I/R. Suk Min Jang et al. reported binding of KAP1 to PCNA and p-KAP1(S473) enhanced this binding ^[26]. Here, we confirm the interaction of KAP1 and PCNA. The association of KAP1 with PCNA significantly decreased in NSCs after cerebral I/R, while p-KAP1(S824) enhanced this interaction. KAP1 can also act as an E3 ubiquitin ligase in some cellular pathways ^[48–51], so we asked if it was a E3 ubiquitin ligase of PCNA. Unexpectedly, increased association between KAP1 and PCNA did not lead to a decrease in the expression of PCNA. Therefore, we deduced that regulation of PCNA may be indirect, where p-KAP1 influences other ubiquitin enzymes involved in mediating the stability of PCNA. Residue Tyr-322 of p-PCNA by the EGF receptor (EGFR) protected PCNA from poly-ubiquitylation and degradation mediated by E3 ubiquitin ligase CUL4, and promoted cell proliferation ^[52]. Molecular docking revealed that KAP1 could bind to PCNA at Arg-210, which is just adjacent to Tyr-211. The interaction of KAP1 with PCNA may disrupt the interaction between CUL4A and PCNA, thereby influencing the proteolysis of PCNA mediated by CUL4A. To test this idea, an immunoprecipitation experiment was employed. As shown in Figure 3F, the binding of CUL4A to PCNA increased in C17.2 cells exposed to OGD/R, while the use of the p-KAP1 mimic suppressed the interaction of CUL4A with PCNA. In the rat MCAO model, we also observed that virus-mediated KAP1 mutant (S824D) promoted the expression of PCNA; this was accompanied by a reduction in co-localization of PCNA and CUL4A. This result indicated that simulated p-KAP1 could competitively inhibit the interaction of PCNA with its E3 ubiquitin ligase CUL4A.

PCNA poly-ubiquitination has been shown to inhibit cell proliferation and regulate cell differentiation, apoptosis, and other processes ^{[44, 53]}. The decreased expression of PCNA induced by cerebral I/R could be reversed by the p-KAP1mimic, possibly due to the altered stability of PCNA. To test this, we detected the mRNA level of PCNA in C17.2 cells after OGD/R. No significant difference was found between the normoxia and OGD/R groups. The expression of PCNA decreased in C17.2 cells after OGD/R, and pre-treatment of MG132 (a proteasome inhibitor) reversed OGD/R-induction of the decrease of PCNA. Thus, we preliminarily concluded that OGD/R treatment caused the ubiquitination of PCNA. Mono-ubiquitination and poly-ubiquitination could happen at the K164 site of PCNA. Mono-ubiquitination of PCNA by E3-ubiquitin ligase Rad18 can promote translesion synthesis, and K63-linked poly-ubiquitination of PCNA by Rad6 can promote template switching ^[54]. Compared with the poly-ubiquitination formed via the K48 junction, mono-ubiquitination and poly-ubiquitination via K63 junctions generally do not cause protein degradation, but instead alter the function of the substrate or mediates the interaction between the proteins ^[55–58]. We next measured the ubiquitination of PCNA in NSCs subjected to cerebral I/R. The results showed that ubiquitination of PCNA (K48 and K63) in C17.2 cells increased significantly and the use of the p-KAP1 mimic could reverse this poly-ubiquitination (K48). This was consistent with what we have shown above, that simulated p-KAP1 promoted binding to PCNA, thereby competitively inhibiting the association of PCNA with CUL4A, involved in mediating the proteolysis of PCNA. The results show that PCNA ubiquitination related to p-KAP1 is mostly through the pathway of K48-poly-ubiquitination that mediates degradation by the proteasome system. However, the K63-polyubiquitination of PCNA also increased in C17.2 cells after OGD/R. PCNA plays a vital role in DNA damage repair and its K63-polyubiquitination is generally increased in the DNA damage response by transducing the damage signal for downstream factors^[59], so it may modulate some signal pathway. Given this, the effects of p-KAP1 on the apoptosis of C17.2 cells exposed to OGD/R were also detected. The results showed that p-KAP1 mimic may reverse apoptosis of OGD/R-induced C17.2 cells through the pathways involved in Bcl-2/Bax.

An improvement in neurological deficits will result in a better prognosis. We next detected the long-term effects of simulated p-KAP1 (S824) after cerebral I/R. We used a strategy of AAV2/9-mediated p-KAP-1 14 days before model and verified the success of lateral ventricular injection. After transfection of rats with S824A mutant viral vector, the S824A mutant rats dies due to intestinal obstruction or vascular atrophy. Here, we found that the rats treated with S824D virus vector showed good functional recovery after cerebral I/R. In terms of behavioral indicators after cerebral I/R, rats in the S824D group showed higher locomotor activity, but there was no effect on time in center and no effect on the results of the balance beam test. These data suggested the improvement of long-term neurological deficits by simulating p-KAP1.

In summary, we revealed the unique role of KAP1 in regulating the neurogenesis of endogenous NSCs after cerebral I/R though the KAP1/PCNA signaling pathway. Simulated p-KAP1 could reverse cerebral I/R-induced neural deficit and enhance proliferation of endogenous NSCs by improving the stable expression of PCNA and reducing the rate of apoptosis. Strategies targeting p-KAP1 should be useful for the prevention or treatment of ischemic stroke. Future studies are required to determine the details of the specific regulatory mechanism of the apoptosis of NSCs by p-KAP1.

KAP1

KRAB domain protein 1

p-KAP1

phosphorylation of KAP1

S824

serine 824 site

PCNA

proliferation cell nuclear antigen

NSCs

neural stem cells

I/R

ischemic/reperfusion

KRAB

Krueppel-associated box

ZFPs

zinc finger proteins

TRIM28

tripartite motif-containing protein 28

TIF1β

transcriptional intermediary factor 1β

OGD/R

Oxygen Glucose deprivation/reperfusion

GFP

green fluorescent protein

MCAO

middle cerebral artery occlusion

CCA

common carotid artery

ECA

external carotid artery

ICA

internal carotid artery

CCK-8

Cell Counting Kit-8

SVZ

Subependymal Ventricular Zone

Acknowledgements

We thank Xuewen Wei (Department of Laboratory Medicine, Xuzhou Municipal Hospital, Jiangsu, China) for his aid with some animal techniques.

Author contributions

W W, TQ Y and XJ G performed experiments, data analysis, interpretation, and manuscript writing. H C, Ch L, LY H and YL W performed data analysis. P M and SH Q designed and supervised the study.

Funding

This study was supported by Grants of the National Natural Science Foundation of China (No. 81671164), Natural Science Foundation of Jiangsu Province (No. BK20211348), and the Xuzhou Science and Technology Planning Project (No. KC18052), the Specialized Research Fund for Senior Personnel Program of Xuzhou Medical University (No. D2018015), the Natural Science Foundation of the Jiangsu Higher Education Institutions of China (No. 20KJB310018).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethical Approval

The experimental procedures were approved by the Animal Ethics Committee of Xuzhou Medical University (approval ID, SCXK (SU) 2010-0003). All animal experiments were conducted in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Author details

¹School of Medical Technology, Xuzhou Key Laboratory of Laboratory Diagnostics, Xuzhou Medical University, Xuzhou, 221004, Jiangsu, P.R China.²Pharmacology college, Xuzhou Medical University, Xuzhou, 221004, P.R. China.³School of Basic Medical Science, Xuzhou Medical University, Xuzhou, 221004, Jiangsu Province, China.⁴Department of Laboratory Medicine, Affiliated Hospital of Xuzhou Medical University, Xuzhou, 221002, P.R. China.

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KAP1 Phosphorylation Promotes the Survival of Neural Stem Cells after Ischemia/Reperfusion by Maintaining the Stability of PCNA

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Abstract

Figures

Introduction

Materials And Methods

Culture of the C17.2 cells and establishment model of OGD/R

Lentiviral-mediated KAP1 mutations and plasmid transfection

Western Blotting

Immunoprecipitation

CCK-8 cell proliferation assay

Bromodeoxyuridine (Brdu) staining

Annexin V-APC/7-AAD apoptosis assay

Establishment of rat middle cerebral artery occlusion (MCAO) model

Balance beam test

Open Field Test

Frozen section

Immunofluorescence staining of frozen sections

Statistical Analysis

Results

Discussion

Conclusion

Abbreviations

Declarations

References

Status:

Version 1