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.

Melatonin improves stroke through MDM2-mediated ubiquitination of ACSL4

“…Protein ubiquitination is a process where intracellular proteins are classified and modified specifically through a series of enzymes including ubiquitin-activating enzymes, conjugating enzymes, ligases and degrading enzymes. This process plays an important role in protein localization, metabolism, function, regulation and degradation as well as almost all life activities such as cell cycle progression, proliferation, apoptosis, differentiation, metastasis and gene expression [21][22][23][24]. Furthermore, it is involved in transcriptional regulation signal transduction damage repair inflammation and immunity.…”

Gene therapy of adeno‐associated virus (AAV) vectors in preclinical models of ischemic stroke

“…with PCNA, thereby enhancing the survival and proliferation of endogenous NSCs. 70 The application of gene therapy to induce the transforming astrocytes to neurons in the damaged brain after ischemic stroke in adult animals has been studied as a potential strategy for promoting brain recovery. Research has demonstrated that targeted expression of achaete-scute complex homolog-like 1 (Ascl1) through the use of GFAP-AAV vectors can convert astrocytes in the dorsal midbrain of mice into fully functional induced neurons in vivo.…”

“…50,52,71,79 AAV2 is currently considered the most extensively studied serotype, but AAV9 appears to be used most frequently in ischemic stroke. 59,62,70,73,80,81 When delivered intravenously, AAV9 can traverse the BBB to target newborn neurons and adult glial cells, 29 and drive axonal transport. 104 A newly engineered capsid, AAV-PHP.B, has also been used in stroke research.…”

“…In the rat MCAO model, high expression of p‐KAP1 gene through AAV2/9 mediation was achieved by injecting it into the lateral ventricle 2 weeks prior to onset of stroke. Further studies demonstrated that p‐KAP1 could bind with proliferating cell nuclear antigen (PCNA) and inhibit the binding of E3 ubiquitin ligase CUL4A with PCNA, thereby enhancing the survival and proliferation of endogenous NSCs 70 …”

“…After injection, most transgenes are expressed around the injection site, with not only main neuron tropism but also transduction of astrocytes or microglia 50,52,71,79 . AAV2 is currently considered the most extensively studied serotype, but AAV9 appears to be used most frequently in ischemic stroke 59,62,70,73,80,81 . When delivered intravenously, AAV9 can traverse the BBB to target newborn neurons and adult glial cells, 29 and drive axonal transport 104 .…”