Pre-B-cell colony-enhancing factor protects against apoptotic neuronal death and mitochondrial damage in ischemia

Abstract

We previously demonstrated that Pre-B-cell colony-enhancing factor (PBEF), also known as nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in mammalian NAD(+) biosynthesis pathway, plays a brain and neuronal protective role in ischemic stroke. In this study, we further investigated the mechanism of its neuroprotective effect after ischemia in the primary cultured mouse cortical neurons. Using apoptotic cell death assay, fluorescent imaging, molecular biology, mitochondrial biogenesis measurements and Western blotting analysis, our results show that the overexpression of PBEF in neurons can significantly promote neuronal survival, reduce the translocation of apoptosis inducing factor (AIF) from mitochondria to nuclei and inhibit the activation of capase-3 after glutamate-induced excitotoxicity. We further found that the overexpression of PBEF can suppress glutamate-induced mitochondrial fragmentation, the loss of mitochondrial DNA (mtDNA) content and the reduction of PGC-1 and NRF-1 expressions. Furthermore, these beneficial effects by PBEF are dependent on its enzymatic activity of NAD(+) synthesis. In summary, our study demonstrated that PBEF ameliorates ischemia-induced neuronal death through inhibiting caspase-dependent and independent apoptotic signaling pathways and suppressing mitochondrial damage and dysfunction. Our study provides novel insights into the mechanisms underlying the neuroprotective effect of PBEF, and helps to identify potential targets for ischemic stroke therapy.

Figure 1. Overexpression of PBEF ameliorates neuronal apoptosis after glutamate excitotoxicity.

(A,B) Fluorescent images showing the triple staining of TUNEL, PBEF and DAPI in control neurons (A) and neurons treated with 30 μM glutamate together with 3 μM glycine for 24 h (B). Neuronal cultures were transfected by WT, H247A and H247E PBEF as indicated before the glutamate treatment. During imaging in (A), we adjusted exposure time and light power to avoid PBEF signal saturation in transfected neurons, so the endogenous PBEF signals in non-transfected neurons are weak but are visible. (C) Neuronal survival rates under different conditions. Notice neurons overexpressed with WT PBEF have much higher survival rate than non-transfected neurons and neurons overexpressed with H247A and H247E PBEF after glutamate stimulation. Data were analyzed from n = 3 independent experiments. **P < 0.01 versus Ctrl and WT PBEF with glutamate stimulation (Glu), ANOVA test.

Figure 2. Overexpression of PBEF prevents AIF translocation from mitochondria to the nucleus after glutamate excitotoxicity.

(A,B) Confocal fluorescent images showing the triple staining of AIF, PBEF and DAPI in neurons under normal (A) and glutamate excitotoxicity (B) conditions. Neurons co-transfected by WT PBEF or H247A PBEF with EGFP and were treated with 100 μM glutamate together with 10 μM for 3 h. Transfected neurons were identified by EGFP. Translocation of AIF from mitochondria to nuclei is illustrated by the overlap of AIF (red) and DAPI (blue) signals. (C,D) Linescan analysis of AIF and DAPI fluorescence from the neurons indicated in (A,B). The fluorescence was normalized to the background. Notice the prevention of glutamate-induced AIF translocation by WT PBEF overexpression. (E) Summary of AIF translocation after glutamate stimulation. Data was quantified by the number of cells with overlap of AIF and DAPI among the total number of cells determined by DAPI staining. n = 3 three independent experiments. **P < 0.01 versus H247A PBEF with Glu, ANOVA test.

Figure 3. Overexpression of PBEF by AAV infection reduces neuronal death and caspase-3 activation after glutamate and OGD stimulations.

(A,B) Phase contrast images of cortical neurons without (A) and with (B) AAV infection under conditions of control, 24 h glutamate treatment (30 μM glutamate together with 3 μM glycine) and 1 h OGD exposure followed by 24 h reperfusion. (C,D) Summary of neuronal viability after glutamate treatment (C) and OGD (D) by MTT assay. Data are shown as mean ± SE; n = 3–5 independent experiments. **P < 0.01 versus Glu, *P < 0.05, versus OGD ANOVA test. (E,F) Fluorescent images of double staining of PI and DAPI or PI and His-tag in neurons without (E) and with (F) AAV infection under conditions of control and glutamate treatment. (G,H) Summary of cell survival of neurons without (G) and with (H) AAV infection after glutamate stimulation based on PI staining. n = 3 independent experiments. **P < 0.01, t-test. (I,J) Western blot images of cleaved caspase-3 and β-actin (A) and summary data of cleaved capase-3 expression (B). The data were presented as the ratio of caspase-3 to β-actin and normalized to the control condition; n = 4 independent experiments. **P < 0.01 versus OGD without AAV infection (AAV-PBEF), ANOVA test.

Figure 4. PBEF prevents mitochondrial fragmentation after glutamate stimulation.

(A,B) Maximal projection confocal images of primary cortical neuron transfected with mito-mRFP alone and cotransfected with mito-mRFP and WT or H247A PBEF. Neurons were treated with or without 30 μM glutamate together with 3 μM glycine for 6 h. The upper panels are the high-resolution images of the boxed regions in the lower panels. (C) Cumulative frequency distribution curves of mitochondrial length and area under different conditions. The values of histogram intervals (bins) are 0.25 μm for mitochondrial length and 0.5 μm² for mitochondrial area. The data show that glutamate treatment caused significant increase in the number of short and small mitochondria and this effect was inhibited by the overexpression of WT PBEF, but not by H247A PBEF. (D–F) The summary of average mitochondrial length, area and density in the dendrites for different conditions. *P < 0.05, **P < 0.01, ANOVA test. Data were collected from 10–15 neurons and a total of 220–326 mitochondria for each condition. Results are shown as mean ± SE; **P < 0.01, *P < 0.05, versus Non-transf with Glu and H247A with Glu, ANOVA test.

Figure 5. Overexpression of PBEF by viral transduction reduces mtDNA loss after glutamate and OGD stimulations.

(A,B) Agarose gel images of mtDNA and nucDNA. Neurons without and with AAV infection were treated with 3 μM glutamate together with 0.3 μM glycine for 24 h (A) or were exposed to 1 h OGD followed by 24 h reperfusion (B). (C,D) Quantification of mtDNA amount after glutamate (C) and OGD (D) stimulations. The data were presented as the ratio of mtDNA/nucDNA and normalized to the control condition using nucDNA as an internal control. Notice that the overexpression of PBEF by viral infection prevented the glutamate-induced decrease of mtDNA content. Data are shown as mean ± SE; n = 3 independent experiments. (C) **P < 0.01 versus Glu, ANOVA test. (D) **P < 0.01, *P < 0.05 versus OGD, ANOVA test.

Figure 6. Overexpression of PBEF by viral transduction suppresses OGD-induced impairment of neuronal mitochondrial biogenesis.

(A) Western blot images for PGC-1, NRF-1 and β-actin in control and OGD stimulated primary neuronal cultures. The neuronal cultures without and with AAV infection were subjected to OGD for 1 h followed by 24 hreperfusion. (B,C) Summary of quantitative analysis of PGC-1 (B) and NRF-1 (C) expression levels. The data were presented as the ratio of the proteins to β-actin and normalized to the control condition. n = 4 independent experiments. **P < 0.01, *P < 0.05 versus OGD, ANOVA test.