Apurinic/apyrimidinic endonuclease APE1 is required for PACAP-induced neuroprotection against global cerebral ischemia

Abstract

Inducible DNA repair via the base-excision repair pathway is an important prosurvival mechanism activated in response to oxidative DNA damage. Elevated levels of the essential base-excision repair enzyme apurinic/apyrimidinic endonuclease 1 (APE1)/redox effector factor-1 correlate closely with neuronal survival against ischemic insults, depending on the CNS region, protective treatments, and degree of insult. However, the precise mechanisms by which this multifunctional protein affords protection and is activated by upstream signaling pathways in postischemic neurons are not well delineated. Here we show that intracerebral administration of pituitary adenylate cyclase-activating polypeptide (PACAP), an endogenously occurring small neuropeptide, induces expression of APE1 in hippocampal neurons. Induction of APE1 expression requires PKAand p38-dependent phosphorylation of cAMP response-element binding and activating transcription factor 2, which leads to transactivation of the APE1 promoter. We further show that PACAP markedly reduces oxidative DNA stress and hippocampal CA1 neuronal death following transient global ischemia. These effects occurred,atleastinpart,viaenhancedAPE1expression.Furthermore, the DNA repair function of APE1 was required for PACAP-mediated neuroprotection. Thus, induction of DNA repair enzymes may be a uniquestrategy forneuroprotectionagainsthippocampalinjury.activating transcription factor 2 | cAMP response-element binding | delayed neurodegeneration | DNA repair | oxidative stress O xidative stress is a hallmark of neurological disorders, including cerebral ischemia. Ischemic injury rapidly elicits oxidative DNA damage either directly via reactive oxygen species attack or indirectly via oxidization of lipids or proteins (1-3). These actions induce DNA lesions, involving base modifications and damage to the sugar moiety in the DNA, formation of abasic (apurinic/apyrimidinic, AP) sites, single strand breaks, and DNA intra-or interstrand crosslinks. Of these lesions, AP sites and single strand breaks are highly prevalent types of DNA damage in insulted neurons (4). Whereas the accumulation of unrepaired DNA ultimately induces cell death, active repair of DNA promotes cell survival (4-6).In neurons, base-excision repair (BER) is the predominant mechanism for repair of oxidative DNA lesions (7). BER is a three-step process. First, 5′-acting AP endonuclease 1 (APE1, also known as redox-effector factor 1) cleaves the phosphodiester backbone, then β-polymerase or alternative repair proteins, such as FEN1 or PCNA, fill in the gap with newly synthesized nucleotides, and finally a DNA ligase seals the nick. In several models of ischemia, oxidative DNA damage is reversible in regions that survive (4,6,8), raising the possibility that repair of DNA damage via BER is required for cell survival following ischemia.APE1 is an attractive target for neuroprotection, as it is a critical component of the BER process (9) and as also has redox and transcription-regulation activities. Following cerebral ischemi...

Human Apurinic/Apyrimidinic Endonuclease 1

“…Jiang et al found that inhibition of AP site repair using the inhibitor, methoxyamine, and not inhibition of the redox activity of APE1, induced hypersensitivity of differentiated (post-mitotic) SH-SY5Y neural cells to a range of oxidizing agents (92). APE1 protein levels were also shown to be correlated with the degree of neuroprotection against the deleterious effects of both transient focal and global cerebral ischemia in rodent models (104,197), observations which are consistent with the emerging evidence that BER plays an important role in the defense against stroke [reviewed in Sykora et al (201)]. In total, the experiments support the hypothesis that APE1, apparently in a DNA repair-dependent mechanism, and presumably other proteins which process oxidative DNA lesions, contributes to the maintenance of brain cell function, particularly when challenged with conditions of oxidative stress.…”

Apurinic/Apyrimidinic Endonuclease 1 Upregulation Reduces Oxidative DNA Damage and Protects Hippocampal Neurons from Ischemic Injury

“…Similarly, adenovirus-mediated APE1 upregulation reduces AP sites, 8-OHdG formation, DNA fragmentation, and infarct volume after ischemia/ reperfusion injury (31). A previous study of ours demonstrated that pituitary adenylate cyclase activating polypeptide (PACAP) protects neurons against ischemia in an APE1-dependent manner (57). We discovered that APE1 was raised by PACAP through protein kinase A-and p38-dependent phosphorylation of cyclic adenosine monophosphate response element binding protein and activating transcription factor 2.…”

“…AP sites were measured with the biotin-labeled aldehyde reactive probe in a colorimetric assay (Dojindo Molecular Technologies), as described previously (57). TUNEL staining was performed with a kit according to the manufacturer's instructions (Roche Applied Science).…”

“…The scrambled sequence was used as the control. Lentiviral vectors were infused into CA1 of the dorsal hippocampus using a convection-enhanced microinfusion system (12,57). Transgenic APE1 overexpressors were generated in collaboration with the Transgenic Service Core at the University of Michigan.…”

“…shRNA vectors and transgenic animals shRNA sequences against rat APE1 and OGG1 were inserted into lentiviral transfer vectors FSW under control of the U6 promoter (12,57). The scrambled sequence was used as the control.…”