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. 2007 Mar;27(5):1696-705.
doi: 10.1128/MCB.01760-06. Epub 2006 Dec 18.

Neuropilin-1 is a direct target of the transcription factor E2F1 during cerebral ischemia-induced neuronal death in vivo

Susan X Jiang  1 , Melissa Sheldrick, Angele Desbois, Jacqueline Slinn, Sheng T Hou
Affiliations

Neuropilin-1 is a direct target of the transcription factor E2F1 during cerebral ischemia-induced neuronal death in vivo

Susan X Jiang et al. Mol Cell Biol. 2007 Mar.

Abstract

The nuclear transcription factor E2F1 plays an important role in modulating neuronal death in response to excitotoxicity and cerebral ischemia. Here, by comparing gene expression in brain cortices from E2F1(+/+) and E2F1(-/-) mice using a custom high-density DNA microarray, we identified a group of putative E2F1 target genes that might be responsible for ischemia-induced E2F1-dependent neuronal death. Neuropilin 1 (NRP-1), a receptor for semaphorin 3A-mediated axon growth cone collapse and retraction, was confirmed to be a direct target of E2F1 based on (i) the fact that the NRP-1 promoter sequence contains an E2F1 binding site, (ii) reactivation of NRP-1 expression in E2F1(-/-) neurons when the E2F1 gene was replaced, (iii) activation of the NRP-1 promoter by E2F1 in a luciferase reporter assay, (iv) electrophoretic mobility gel shift analysis confirmation of the presence of an E2F binding sequence in the NRP-1 promoter, and (v) the fact that a chromatin immunoprecipitation assay showed that E2F1 binds directly to the endogenous NRP-1 promoter. Interestingly, the temporal induction in cerebral ischemia-induced E2F1 binding to the NRP-1 promoter correlated with the temporal-induction profile of NRP-1 mRNA, confirming that E2F1 positively regulates NRP-1 during cerebral ischemia. Functional analysis also showed that NRP-1 receptor expression was extremely low in E2F1(-/-) neurons, which led to the diminished response to semaphorin 3A-induced axonal shortening and neuronal death. An NRP-1 selective peptide inhibitor provided neuroprotection against oxygen-glucose deprivation. Taken together, these findings support a model in which E2F1 targets NRP-1 to modulate axonal damage and neuronal death in response to cerebral ischemia.

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Figures

FIG. 1.
FIG. 1.
Identification of E2F1 target genes using DNA microarrays. (A) High-density DNA microarrays containing 3,456 brain genes were screened with 33P-labeled cDNA probes prepared from E2F1+/+ and E2F1−/− mouse brains either with or without focal cerebral ischemia. We identified 375 differentially expressed genes in the E2F1−/− mouse brains, which represented putative E2F1 target genes. By comparing them with genes having altered expression levels following cerebral ischemia, we identified 206 putative E2F1 target genes that appeared to be responsive to ischemia. (B) Plot showing no differential gene expression between the sham-operated and ischemic animals (R2 = 0.98). (C) Plot showing a drastic alteration in the level of gene expression following ischemia (R2 = 0.67). The arrows in panels B and C indicate the altered genes. (D) A few examples of differentially expressed putative E2F1 target genes, confirmed by RT-PCR.
FIG. 2.
FIG. 2.
E2F1−/− CGNs have significantly reduced NRP-1 expression. (A) mRNAs and proteins were extracted from E2F1+/+ and E2F1−/− mouse brains. Equal amounts of total RNA were used for the first-strand cDNA synthesis. PCRs were performed using the cDNAs to detect NRP-1 and β-actin levels; β-actin was used as an internal control for equal mRNA loading. (B) The intensities of the bands were quantified using densitometry, and the ratio of NRP-1 to actin was calculated and plotted. (C and D) Western blotting was performed to detect NRP-1 protein expression (C), and GAPDH was used as a control for equal protein loading. No detectable NRP-1 protein was found in E2F1−/− brain lysate (C and D). Statistical analysis was performed; **, P < 0.001 by ANOVA. The error bars indicate standard deviations. (E) Replication-defective adenoviral constructs expressing GFP only (Ad-GFP), a mutant E2F1 (Ad-Emut), and the full-length E2F1 (Ad-E2F1) were made and used to infect cultured primary glial cells derived from the brains of E2F1+/+ and E2F1−/− mice. Noninfected cells were used as the baseline control for NRP-1 expression (CTL). After 3 days of infection, cells were collected for Western blotting to detect NRP-1 expression. GAPDH Western blotting was performed to show equal protein loading.
FIG. 3.
FIG. 3.
E2F1−/− CGNs are less susceptible to semaphorin 3A-mediated inhibition of axonal outgrowth. Semaphorin 3A at the indicated concentrations was added to CGNs at the time of plating. After 3 days in culture, cells were fixed in 4% paraformaldehyde and immunostained with β-III-tubulin to detect axons. As shown in panels A and C, untreated E2F1+/+ and E2F1−/− neurons had developed long axons and neurites and started to form networks of connections at 3 DIV. However, semaphorin 3A treatment caused a significant inhibition of axonal outgrowth in E2F1+/+ CGNs (B and E), but not in E2F1−/− CGNs (D and E). Digital images of axons were taken, and axon lengths were measured using Image J software. **, P < 0.01 by one-way ANOVA. The error bars indicate standard deviations.
FIG. 4.
FIG. 4.
Identification of an E2F1 binding site in the NRP-1 promoter. (A) ChIP assays were performed on CGNs. Chromatin was isolated and immunoprecipitated with either an antibody specific to E2F1 or a nonspecific mouse IgG (Neg IgG) or no antibody (Ab) as negative controls. PCR for the NRP-1 promoter sequence was carried out to detect NRP-1 promoter sequence. Input chromatin represents the total chromatin serving as a positive control. (B) The density of each PCR band was quantified and normalized against the input band and plotted. The error bars indicate standard deviations. (C) A mouse E2F1 consensus binding sequence was used to identify homologous sequences in the NRP-1 promoter region by pairwise alignment using a BLAST search. (D) The sequence identified on the NRP-1 promoter was chemically synthesized to make a dsDNA, which was then labeled with radioactive [α-32P]dCTP for EMSA (NRP1 hot probe). Nuclear fractions were isolated from E2F1+/+ and E2F1−/− brain extracts and mixed with radioactive dsDNA NRP1 hot probes. Several controls were used in the study, including CTL hot probe (a conserved mouse E2F1 binding promoter DNA sequence as a positive control), CTL and NRP1 cold probes (100 times the amount of excess CTL and NRP1 cold probes, respectively), and E2F1 antibody supershift. Specific E2F1 binding to the NRP-1 promoter sequence was identified, as indicated by an arrow in panel D. The supershift pattern is characteristic of E2F1, which when mixed with the antibody and in the absence of poly(dI-dC), completely shifted the binding of E2F1 to the dsDNA probe. (E) Activation of NRP-1 promoter by E2F1 was demonstrated by the use of a luciferase reporter assay. The E2F1 binding site sequence from the NRP-1 promoter was subcloned into the PGL3 promoterless expression vector. Several controls were also used, including GLO buffer alone, nontransfected cells, and cells transfected with empty vector. Primary glial cells derived from E2F1+/+ and E2F1−/− mouse brains were transfected with the constructs for 3 days. After that, cells were collected, and their lysates were quantified. Equal amounts of proteins were subjected to luciferase assay using a Promega kit following the manufacturer's instructions. A 10-fold increase in luciferase activity was detected in the NRP-1 promoter construct-transfected E2F1+/+ cells.
FIG. 5.
FIG. 5.
E2F1 positively regulates NRP-1 expression during cerebral ischemia. (A) Nuclear fractions were isolated from both the ischemic (L) and contralateral (R) sides of MCAO mouse brains. EMSA was performed using these nuclear extracts. Sham-operated brains were used as a control to determine the baseline level of E2F1 binding to the promoter. (B) The E2F1 band was quantified by densitometry measurement and plotted. Temporal changes in E2F1 binding on the ischemic side of the brain were normalized against those on the contralateral side of the brain. (C) E2F1 immunoprecipitation (IP) was performed against nuclear extracts obtained from ischemic mouse brains. (D) Western blotting was then performed on equal amounts of the IP products to show increased E2F1 levels following MCAO by densitometry measurement and normalization against the E2F1 level in the sham-operated brain. (E) RT-PCR was performed using RNA from the ischemic side of the MCAO mouse brains to detect changes in NRP-1. (F) β-Actin was used as an internal control for quantification of NRP-1 levels. formula image and formula imageformula image indicate statistical significance at P < 0.05 and P < 0.01, respectively, by one-way ANOVA with a post hoc Tukey's test for significant groups. The error bars indicate standard deviations.
FIG. 6.
FIG. 6.
Colocalization of E2F1 and NRP-1 expression in the ischemic mouse brain. (A to H) E2F1+/+ and E2F1−/− mice were subjected to MCAO and reperfusion as described in Materials and Methods. Formaldehyde-fixed paraffin sections were cut and double immunostained with antibodies to E2F1 (red) and NRP-1 (green). (C and D) Increases in nuclear E2F1 (red) and cytosolic and membrane NRP-1 (green) occurred the E2F1+/+ ischemic brains. The arrows (C and D) indicate positive cells. The arrowheads (B and E to H) indicate E2F1- or NRP-1-negative cells/staining. Scale bar = 100 nm.
FIG. 7.
FIG. 7.
Semaphorin 3A causes axonal damage and neuronal death. Mature postmitotic CGNs at 3 DIV were treated with semaphorin 3A for 18 h. The CGNs were either fixed with 4% paraformaldehyde to immunostain for β-III-tubulin (A) or supplemented with 10 μg/ml PI for cell viability assays (B). (A) Axon lengths were measured on β-III-tubulin-stained slides, and the data were plotted. E2F1−/− CGN axons were not affected by semaphorin 3A treatment, while E2F1+/+ neurons had significantly shortened axons following both 0.1-μg/ml and 5-μg/ml semaphorin 3A treatment. The error bars indicate standard deviations. (B) A cell death assay was performed using PI staining, which detected significant neuronal death after 18 and 24 h of semaphorin 3A treatment. formula imageformula image, P < 0.01 by ANOVA and post hoc Tukey's test. (C) CGNs were plated in 10-cm dishes and subjected to OGD treatment for 90 min. Individual plates were reperfused for either 2, 6, or 10 h with regular culture media. At these time points, the cells were scraped from the plate and pelleted, and total protein extracts were collected for RNA or protein extraction to detect changes in NRP-1 expression. (D) For NRP-1 inhibitor studies, CGNs were preincubated for 30 min with or without 0.5 μg/ml of synthetic peptide inhibitor. The cells were subsequently subjected to 90 min of OGD and reperfused for 24 h, at which point the cells were fixed with 4% fresh paraformaldehyde and double immunostained for DAPI and MAP2. Cell death was quantified by manual counting of condensed DAPI-stained nuclei, and the induction in neuronal death (n-fold) was determined.
FIG. 8.
FIG. 8.
Schematic diagram depicting pathways of axonal damage and neuronal death mediated by E2F1 activation of NRP-1 during cerebral ischemia. Ref, references.
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