KAP1 phosphorylation promotes the survival of neural stem cells after ischemia/reperfusion by maintaining the stability of PCNA

Abstract

Aims: To explore the function of phosphorylation of KAP1 (p-KAP1) at the 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 was analyzed by immunoprecipitation. In the rats MCAO model, we performed the adeno-associated virus (AAV) 2/9 gene delivery of 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. The AAV2/9-mediated p-KAP1(S824) 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 is that highly expressed p-KAP1(S824) promotes binding to PCNA, and inhibits the binding of CUL4A to PCNA. This reduced CUL4A-mediated ubiquitination degradation to increase the stability of PCNA and promote the survival and proliferation of NSCs.

Keywords: Cerebral ischemia/reperfusion (I/R); KRAB domain protein 1(KAP1); Neural stem cells (NSCs); PCNA; Proliferation.

Fig. 1

The expression of KAP1 and p-KAP1 in NSCs after cerebral I/R. A The expression and location of KAP1 in the subgranular zone (SGZ) and Subventricular zone (SVZ) were examined by immunofluorescence staining. KAP1 was dyed red, Nestin was green and the nuclear was stained blue with DAPI. 100× , scale bar = 200 μm; 200 × , scale bar = 50 μm. B The phosphorylation and protein expression of KAP1 in SVZ of rat subjected to I/R were analyzed by immunofluorescence staining. 200 × , scale bar = 200 μm. C Western blot analysis was also utilized to detect the phosphorylation and protein expression levels of KAP1 in C17.2-NSCs after OGD/R. D Bands were scanned, and the intensities were represented as folds of normoxia group, ^***P < 0.001 versus normoxia group (N means normoxia, O/R means OGD/R). Data were expressed as mean ± SEM (n = 5). E Immunofluorescence staining was also performed to detect phosphorylation and protein expression. 200 × , scale bar = 100 μm

Fig. 2

Effects of p-KAP1 on the proliferation of NSCs subjected to cerebral I/R. A BrdU immunofluorescence staining was performed to evaluate the C17.2-NSCs proliferation. 200 × , scale bar = 100 μm. B CCK-8 assay was also used to detect the proliferation of C17.2-NSCs. Data were presented as mean ± SEM (n = 3). ^**P < 0.01 versus WT and O/R group; ^&P < 0.05 versus SD and O/R group. C, D Western blot analysis was used to examine the expression of PCNA. Band intensities were represented as fold-values relative to the levels in normoxia treatment. Data were presented as mean ± SEM (n = 4), ^*P < 0.05, ^**P < 0.01 versus WT and O/R group; ^&P < 0.05 versus SD and O/R group. E Ki-67 and Nestin colocalization immunofluorescence staining was performed to evaluate the neurogenesis of endogenous NSCs. 200× , scale bar = 100 μm

Fig. 3

Effects of p-KAP1 on the apoptosis of C17.2-NSCs after OGD/R. A, B Annexin V-APC/7-AAD apoptosis detection kit was utilized to detect C17.2-NSCs apoptosis and the data were quantified as mean ± SEM (n = 3), ^*P < 0.05, ^****P < 0.0001 versus WT and O/R group; ^&&P < 0.01 versus SD and O/R group. C, D Western blot analysis was used to examine the expression of Bcl-2/Bax and band intensities are represented as fold-values relative to the levels in normoxia treatment. Results were presented as mean ± SEM (n = 4), ^*P < 0.05 versus WT and O/R group; ^&P < 0.05 versus SD and O/R group

Fig. 4

Effects of p-KAP1 on the stability of PCNA in NSCs undergoing cerebral I/R. A Real-time PCR analysis of PCNA mRNA levels in C17.2-NSCs after OGD/R. Data are the mean values ± SEM of three replicate experiments, NS indicates no significance. B Western blot analysis of PCNA protein levels in C17.2-NSCs after OGD/R. C17.2-NSCs were pre-incubated with 20 μM MG132 for 6 h before harvesting cells for protein lysis. C Band intensities are represented as fold-values relative to the levels in the normoxia group. Data were expressed as mean ± SEM (n = 4). ^##P < 0.01 versus DMSO and O/R group. D C17.2-NSCs were treated with 20 μM MG132 for 6 h before harvesting for protein lysis and PCNA was precipitated with the anti-PCNA antibody. The protein levels and ubiquitination levels of PCNA in the immune complex were determined by Western blot. The protein levels of PCNA and α-Tubulin in cell lysates (input) were also analyzed by Western blot, and three independent experiments were performed. “a” indicates the heavy chain of IgG, “b” indicates the light chain of IgG. E PCNA and CUL4A colocalization immunofluorescence staining was performed to evaluate the neurogenesis of endogenous NSCs. 200× , scale bar = 100 μm

Fig. 5

Effects of p-KAP1 on the binding of KAP1 to PCNA, PCNA to CUL4A in C17.2-NSCs after OGD/R. A Protein docking of KAP1(Green) and PCNA(Red). B Potential binding sites between KAP1 and PCNA. C Confocal immunofluorescence staining revealed the coexistence of KAP1 and PCNA in the cellular nucleus. Scale bar = 2 μm. D, E Immunoprecipitation and immunoblotting (IP–IB) were performed to confirm the association of KAP1 with PCNA in C17.2-NSCs subjected to 6 h of oxygen and glucose deprivation followed by 24 h of reoxygenation. F Site-mutant plasmids were transiently transinfected into C17.2-NSCs subjected to OGD/R, or no treatment, and the binding of KAP1 to PCNA was examined by IP–IB. G The binding of CUL4A to PCNA was investigated by IP–IB. The relative bands were selected from three independent experiments

Fig. 6

Simulated p-KAP1 (S824) improved locomotor activity of rats subjected to cerebral I/R. A Experimental design was illustrated schematically. A total of 50 rats were stochastic fall into 5 groups: the (1) NC (Scramble AAV 2/9), (2) NC + I/R (Scramble AAV 2/9 + I/R), (3) WT + I/R (AAV2/9-KAP1 + I/R), (4) S824D(AAV2/9-KAP1-S824D + I/R), (5) S824A(AAV2/9-KAP1-S824A + I/R) groups. Adeno-associated virus-mediated KAP1 and its mutation overexpression were administered 14 days before. After ischemia for 1.5 h, the rats were under reperfusion by pulling out the plug. B–E Effects of KAP1 and its mutations on locomotor activity of rats subjected to I/R. The total distance, speed, and time in the center were investigated by an open field test. Data were expressed as mean ± SEM (n = 3–6), ^*P < 0.05, ^**P < 0.01 versus NC + I/R group; ^&P < 0.05 versus WT + I/R group. F A balance beam test was performed on the sixth day after cerebral ischemia. Data were expressed as mean ± SEM (n = 3–4), ^***P < 0.001 versus NC + I/R group

Fig. 7

Schematic diagram of KAP1 phosphorylation during cerebral I/R involved in endogenous NSCs proliferation and apoptosis is summarized. Simulated p-KAP1 promoted the proliferation of endogenous neural stem cells (NSCs) and the expression of PCNA in cerebral I/R. Simulated p-KAP1 bound to PCNA, inhibiting CUL4A-mediated ubiquitination degradation to PCNA. This finding suggests that targeting the KAP-1/PCNA signaling pathway could enhance the neurogenesis of endogenous NSCs in the management of brain diseases