NR4A orphan nuclear receptors as mediators of CREB-dependent neuroprotection

Abstract

Induced expression of neuroprotective genes is essential for maintaining neuronal integrity after stressful insults to the brain. Here we show that NR4A nuclear orphan receptors are induced after excitotoxic and oxidative stress in neurons, up-regulate neuroprotective genes, and increase neuronal survival. Moreover, we show that NR4A proteins are induced by cAMP response element binding protein (CREB) in neurons exposed to stressful insults and that they function as mediators of CREB-induced neuronal survival. Animals with null mutations in three of six NR4A alleles show increased oxidative damage, blunted induction of neuroprotective genes, and increased vulnerability in the hippocampus after treatment with kainic acid. We also demonstrate that NR4A and the transcriptional coactivator PGC-1alpha independently regulate distinct CREB-dependent neuroprotective gene programs. These data identify NR4A nuclear orphan receptors as essential mediators of neuroprotection after exposure to neuropathological stress.

Fig. 1.

NR4A-mediated neuroprotection. (A and B) MACS-sorted neurons were transfected with the indicated oligos for 5 h, received fresh medium for 19 h, and were then treated with rolipram or DMSO for 1 h and subsequently stressed with the indicated concentrations of ionomycin, H₂O₂, or glutamate for 18 h. Cell survival was assayed by adding cell titer reagent and measuring the absorbance at 490 nm. Error bars indicate SD, n = 9; *significant at P < 0.05; **significant at P < 0.01. (C) MACS-sorted neurons infected with eGFP or NR4A2 lentivirus were treated for 18 h with increasing concentrations of ionomycin, glutamate, or H₂O₂. Cell survival was assayed by adding cell titer reagent and measuring the absorbance at 490 nm. Error bars indicate SD, n = 5. (D) MACS-sorted neurons infected with eGFP or the indicated NR4A lentiviruses were treated for 18 h with the indicated concentrations of ionomycin, glutamate, or H₂O₂. Cell survival was assayed by adding cell titer reagent and measuring the absorbance at 490 nm. The values obtained with the different NR4A lentiviruses were normalized to the eGFP lentivirus values. Error bars indicate SD, n = 5.

Fig. 2.

NR4A2 activates neuroprotective gene expression. (A) Neuroprotective genes up-regulated in MACS-sorted neurons infected with L-NR4A2, n = 3. (B and C) MACS-sorted neurons were pretransfected with CRE or mutCRE oligo (B) or NBRE or mutNBRE oligo (C) and treated with 8CPT-cAMP or DMSO. Total RNA was extracted after 6 h and the expression levels of the indicated genes were determined by real-time PCR. Error bars indicate SD, n = 4; *P < 0.05; **P < 0.01. All genes as a group were significantly down-regulated by both CRE (**P < 0.01) and NBRE (*P < 0.05) decoys.

Fig. 3.

CREB-induced PGC-1α-mediated neuroprotection. (A) MACS-sorted neurons were treated with the indicated chemicals. Total RNA was extracted after 2 h and the expression levels of PGC-1α were determined by real-time PCR. Error bars indicate SD, n = 4. (B) MACS-sorted neurons were pretransfected with CRE or mutCRE oligo and treated for 2 h with 8CPT-cAMP or DMSO. Nuclear extracts were prepared, resolved on SDS/PAGE, and probed with the indicated antibodies. Expression of Histone 1 was used as a loading control. (C) MACS-sorted neurons were infected with control or PGC-1α shRNA lentivirus for 24 h, received fresh medium for another 24 h, and were then treated with rolipram or DMSO for 1 h and subsequently stressed with the indicated concentrations of ionomycin, H₂O₂, or glutamate for 18 h. Cell survival was assayed by adding cell titer reagent and measuring the absorbance at 490 nm. Error bars indicate SD, n = 9; *significant at P < 0.05. (D) MACS-sorted neurons infected with the indicated lentiviruses were treated for 18 h with the indicated concentrations of ionomycin, glutamate, or H₂O₂. Cell survival was assayed by adding cell titer reagent and measuring the absorbance at 490 nm. Error bars indicate SD, n = 5.

Fig. 4.

NR4A2 and PGC-1α have distinct neuroprotective targets. (A and B) MACS-sorted neurons were infected with the indicated lentiviruses. Total RNA was extracted after 18 h and the expression levels of the indicated genes were determined by real-time PCR. Error bars indicate SD, n = 4; **P < 0.01.

Fig. 5.

In vivo evaluation of the neuroprotective role of NR4A receptors. (A) Cresyl violet and DAPI staining of hippocampal sections from WT C57/BL and NR4Amut mice injected with saline or 25 mg/kg kainic acid and killed 72 h postinjection. (B) DAPI, 5-nitro-tyrosine, and fluoro-jade staining of the CA3 hippocampal region of WT and NR4Amut mice injected with saline or 25 mg/kg kainic acid and killed 72 h postinjection. (C) Total RNA was extracted at the indicated time points from the hippocampus of WT and NR4Amut mice injected with saline or 25 mg/kg kainic acid. The expression levels of the indicated genes were determined by real-time PCR. Error bars indicate SD, n = 3.