Activation of GLP-1 Receptor Enhances Neuronal Base Excision Repair via PI3K-AKT-Induced Expression of Apurinic/Apyrimidinic Endonuclease 1

Abstract

Glucagon-like peptide-1 (GLP-1) is an intestinal-secreted incretin that increases cellular glucose up-take to decrease blood sugar. Recent studies, however, suggest that the function of GLP-1 is not only to decrease blood sugar, but also acts as a neurotrophic factor that plays a role in neuronal survival, neurite outgrowth, and protects synaptic plasticity and memory formation from effects of β-amyloid. Oxidative DNA damage occurs during normal neuron-activity and in many neurological diseases. Our study describes how GLP-1 affected the ability of neurons to ameliorate oxidative DNA damage. We show that activation of GLP-1 receptor (GLP-1R) protect cortical neurons from menadione induced oxidative DNA damage via a signaling pathway involving enhanced DNA repair. GLP-1 stimulates DNA repair by activating the cyclic AMP response element binding protein (CREB) which, consequently, induces the expression of apurinic/apyrimidinic endonuclease 1 (APE1), a key enzyme in the base excision DNA repair (BER) pathway. In this study, APE1 expression was down-regulated as a consequence phosphatidylinositol-3 kinase (PI3K) suppression by the inhibitor LY294002, but not by the suppression of MEK activity. Ischemic stroke is typically caused by overwhelming oxidative-stress in brain cells. Administration of exentin-4, an analogue of GLP-1, efficiently enhanced DNA repair in brain cells of ischemic stroke rats. Our study suggests that a new function of GLP-1 is to elevate DNA repair by inducing the expression of the DNA repair protein APE1.

Keywords: Apurinic/apyrimidinic endonuclease 1 (APE1); Base excision repair (BER); Exendin-4; Glucagon-like peptide-1(GLP-1); Oxidative DNA damage.

Figure 1

Activation of cortical neuron expressed GLP-1 receptor (GLP-1R) protected cortical neurons from oxidative insults. (A) mRNA and (B) protein expressions of GLP-1R were detected in the 8-day in vitro primary cortical neurons. (C) Immunofluorescence staining of GLP-1R (green), TUJ-1 (red), and DAPI (blue) in the 8-day in vitro primary cortical neurons. Merged bright field (100X) and immunostained (100X) images indicated that GLP-1R is mostly distributed in neuronal bodies but also exist in both axons and dendrites. (D) GLP-1or (E) EX-4 pretreated cortical neurons were continually treated with 100 nM GLP-1 or EX-4 after an oxidative insult and neuronal viability was measured at 0, 6, 12, 24 hr time points. Neuronal viability of ligand administrated neurons (GLP-1:~82%, EX-4:~67%) was approximately 20% higher than the menadione-treated group (GLP-1~65%, EX-4:~48%) 24-hour after menadione-induced oxidative insults. (M±SE; * p<0.05, ** p< 0.01, *** p< 0.001; compared to the 24-hr value of the menadione alone group, n=6).

Figure 2

APE1 expression was up-regulated by GLP-1 and it's analogue Exendin-4 (EX-4) in neurons. Rat primary cortical neurons were treated with 100 nM GLP-1 or EX-4 and were subsequently harvested at 30 min, 1 hr, 3 hr, 6 hr, 12 hr, and 24 hr time points. The APE1 protein levels were induced starting at 30 min continuing to 24 hr. These results clearly demonstrated that (A) GLP-1 or (C) EX-4 increased APE1 protein levels in cortical neurons. The immunoblots showed that phosphorylation of AKT and CREB were immediately increased 30 min after (B) GLP-1 or (D) EX-4 administration. The phosphorylation level of AKT returned to normal levels at 24 hr, and phophorylated CREB started decreasing after reaching a peak at 30 min. (M±SE; * p<0.05; ** p<0.01; ***p<0.001; compared to the value of control group, n=6).

Figure 3

Protein expressions of OGG1, NEIL1, Polβ, FEN1, UDG, and ligase III were not significantly influenced by GLP-1. Cortical neurons were treated with 100 nM GLP-1 and harvested in order to measure BER proteins at 30 min, 1 hr, 3 hr, 6 hr, 12 hr, and 24 hr time points. Nuclear fractions were collected for Western blotting; the blotting results suggested that protein levels of (A) OGG1, (B) NEIL1,and (C) Polβ were not significantly altered by GLP-1 administration. Whole cell lysates were used for FEN1, UDG, and ligase III Western blotting. Protein levels of (D) FEN1, (E) Ligase III, and (F) UDG were not affected by GLP-1. However, we observed that Polβ was slightly increased by GLP-1 treatment but not statistically significant. (M±SD; * p<0.05; ** p<0.01; ***p<0.001; compared to the value of control group, n=6).

Figure 4

Administration of GLP-1 enhanced the enzymatic activity of APE1, but not UDG, OGG1 or Polβ. Cortical neurons were treated with GLP-1 (100 nM) and harvested to measure protein activity at 1, 3, 6, 12, and 24 hr time points. The incision activities of (A) APE1, (B) UDG, and (C) OGG1 were examined by various Cy5-labeled defect-oligonucleotides which could be recognized by APE1, UDG, and OGG1 respectively. (D) The gap-filling activity of Polβ was assayed by Cy5-labeled duplex-oligonucleotides lacking deoxycytidine. The DNA incision activity of APE1 (panel A) was increased significantly by GLP-1 treatment, but not the activities of UDG (panel B), OGG1 (panel C), or Polβ (panel D). However, the incision activity of OGG1 and the gap-filling activity of Polβ were slightly increased by GLP-1 treatment but were not statistically significant. (M±SE; *p< 0.05; ** p< 0.01; ***p<0.001; compared to the value of control group, n=6).

Figure 5

The downstream PI3K-AKT signaling axis of GLP-1R is the major pathway that regulates expression of APE1. (A) Western blot analyses of pAKT, pERK1/2, pCREB, APE1, and actin levels in neuronal cultures treated with 100 nM of GLP-1(100nM), GLP-1 plus MEK inhibitors U0126, or GLP-1 plus PI3K inhibitor LY294002. (B) LY294002 specifically inhibited GLP-1-induced AKT phosphorylation. (C) U0126 specifically inhibited GLP-1-induced pERK1/2 phosphorylation. (D) pCREB levels and (E) APE1 were specifically inhibited by LY294002 but not U0126. The results suggested that PI3K-AKT-CREB is the predominant downstream signaling pathway of GLP-1R elevating APE1 expression. (M±SE; *p< 0.05; ** p< 0.01; ***p<0.001; compared to the value of control group, n=4).

Figure 6

Administration of EX-4 significantly reduced ischemia-induced nuclear DNA damage. (A) Cerebral ischemic strokes in rat brains were induced by MCAO procedures and injuries developed in the frontal cortex region. (B-C) APE1 expression was robustly elevated in EX-4 administrated animals comparing to vehicle treated animals. (D) In this study, TUNEL assay and measurement of γH2AX (Phosphorylation of histone 2AX) were applied to assess DNA damage and repair after an ischemic stroke in rats. Five regions of the brains, two in the infarct (filled-line circles) and three in the penumbra (dot-line circles), were analyzed for injured brain cells. (E-F) The results of the TUNEL assay demonstrated that daily administration of EX-4 rapidly reduced nuclear DNA fragmentation compared with the vehicle group in ischemic brains. (G-H) γH2AX is a common biomarker of nuclear DNA damage, which is wildly used to determine DNA damage. Immunohistological results of γH2AX also showed that EX-4 administrated animals had less DNA damage than vehicle treated animals. These results suggested that EX-4 treated animals had less nuclear DNA damage and better DNA repair to protect brain cells against ischemic injury. (M±SE; *p< 0.05; ** p< 0.01; ***p<0.001; compared to the number of MCAO without EX-4, n=4).

Figure 7

This schematic diagram illustrates the mechanisms by which activated GLP-1R elevate repair of oxidative DNA damage and increase neuronal survival. The expression GLP-1 and its receptors occurs in the central nervous system and is involved in neuronal protection. Two major signaling pathways are triggered, including PKA-Erk (dotted-line arrow) and PI3K-AKT (filled-line arrow), when GLP-1R is activated. Results of our study suggested that PI3K-AKT is the key downstream signaling pathway regulating APE1 expression, elevation DNA repair efficiency, and increasing neuronal survival after oxidative insults. (AC: adenylyl cyclase; cAMP: cyclic adenosine monophosphate; PKA: protein kinase A; MEK: mitogen-activated protein kinase kinase; ERK: mitogen-activated protein kinase; PI3K: phosphatidylinositide 3-kinase; AKT: protein kinase B; CREB: cAMP response element binding protein).