Cell-Autonomous and Non-Cell-Autonomous Neuroprotective Functions of RORα in Neurons and Astrocytes during Hypoxia

RORα in astrocytes is neuroprotective in a non-cell-autonomous manner after hypoxia. A–E, The loss of RORα in astrocytes modifies the development of the dendritic arbor of neurons in normoxia. A, Schematic diagram illustrating the experimental protocol: Rora^+/+ (wt) neurons (black cells) were plated directly onto a monolayer of Rora^+/+ (wt) or Rora^sg/sg (sg) astrocytes (as; orange and red circles). B, Survival rates of neurons in cocultures with Rora^+/+ or Rora^sg/sg astrocytes were estimated in normoxia. C, Neurons grown on Rora^+/+ or Rora^sg/sg astrocytes were transfected with GFP. D, The number of dendrites per neuron was determined. E, The total dendritic length per neuron was determined in each set of culture conditions. Dendrites from the individual neurons (numbers indicated in the bars) in four independent experiments were imaged and analyzed with NeuronJ software. F, The loss of RORα in astrocytes increases the susceptibility of neurons to hypoxia-induced neuronal death. Schematic diagram illustrating the experimental protocol: Rora^+/+ or Rora^sg/sg neurons (black cells) were plated directly onto a monolayer of Rora^+/+ or Rora^sg/sg astrocytes (orange and red circles) and were subjected to hypoxia (6, 8, or 10 h) followed by 24 h of normoxia. Neuronal death rates were estimated by quantifying LDH release (left, orange). Neuronal survival rates were estimated by counting living nuclei (manual counting) (left, purple). After 8 h of hypoxia, neurons were labeled with anti-MAP2 antibody (green) and nuclei were labeled with Hoechst 33258 (blue, right). The values shown are the means ± SEM of 4 independent cultures. (t test, *p < 0.05, ns, not significant). G, Losses of RORα function in astrocytes and neurons have additive effects on hypoxia-induced neuronal death. Cocultures were subjected to 8 h of hypoxia followed by 24 h of normoxia. Neuronal death rates were estimated by quantifying LDH release. Values are the means ± SEM of 4 independent cultures (ANOVA, de Scheffe, *p < 0.05).

Antioxidant gene expression is similar between Rora^+/+ and Rora^sg/sg neurons/astrocytes after hypoxia. Rora^+/+ (wt) or Rora^sg/sg (sg) neurons (A) and astrocytes (B) were subjected to hypoxia without reoxygenation. mRNA abundance for antioxidant genes was evaluated by real-time RT-PCR. Cat, Catalase; gpx1, glutathione peroxidase 1; sod1, superoxide dismutase 1; Prdx6, peroxiredoxin 6; NH, non hypoxia, H, hypoxia. Values are the means ± SEM of 4 independent cultures.

In cocultures, RORα inhibits HIF-1α production after hypoxia. A, Schematic diagram illustrating the experimental protocol: Rora^+/+ (wt) neurons (black cells) were plated directly on a monolayer of Rora^+/+ (wt) or Rora^sg/sg (sg) astrocytes (as; orange or red circles) and were subjected to 8 h of hypoxia. Analyses were performed immediately upon removal from hypoxia. B, Hif1a gene transcription was analyzed by real-time RT-PCR. Transcription of the Hif1a gene was found to be induced similarly in Rora^+/+ and Rora^sg/sg cocultures after hypoxia. C, Western blot analysis (top) of the HIF-1α protein in enriched nuclear extracts and relative quantification (bottom). D, Vegf, Nip3, and Hk2 gene transcription was analyzed by real-time RT-PCR. H, Hypoxia; NH, non hypoxia. Values are the means ± SEM of 4–6 independent cultures. (Mann–Whitney, *p < 0.05).

In neurons, RORα is upregulated by hypoxia and does not prevent HIF-1α expression after hypoxia. A, Schematic diagram of the experimental protocol: Rora^+/+ (wt) or Rora^sg/sg (sg) neurons were subjected to 7 h of hypoxia followed by 0–24 h of normoxia. B, Rora gene transcription was analyzed by real-time RT-PCR (t test, *p < 0.05). C, Western blot analysis (top) of RORα protein in enriched nuclear extracts from cortical neurons and relative quantification (bottom) (t test, *p < 0.05). D, Rorb gene transcription was analyzed by real-time RT-PCR. E, Hif1a gene transcription was analyzed by real-time RT-PCR. F, Western blot analysis (top) of HIF-1α protein in enriched nuclear extracts and relative quantification (bottom). G, Vegf, Nip3, and Hk2 gene transcription was analyzed by real-time RT-PCR. (Mann–Whitney, ns, not significant). Values are the means ± SEM of 4 independent cultures. H + 0, Hypoxia without reoxygenation, NH, non hypoxia.

In astrocytes, RORα is upregulated by hypoxia and inhibits HIF-1α expression after hypoxia A, Schematic diagram of the experimental protocol: Rora^+/+ (wt) or Rora^sg/sg (sg) astrocytes were subjected to 24 h of hypoxia followed by 0–24 h of normoxia. B, Rora gene transcription was analyzed by real-time RT-PCR. (t test, *p < 0.05). C, Western blot analysis (top) of RORα protein in enriched nuclear extracts from cortical astrocytes and their relative quantification (bottom) (t test, *p < 0.05). D, Rorb gene transcription was analyzed by real-time RT-PCR. E, Hif1a gene transcription was analyzed by real-time RT-PCR in Rora^+/+ (wt) and Rora^sg/sg (sg) astrocytes. F, Western blot analysis (top) of HIF-1α protein in enriched nuclear extracts and relative quantification (bottom). G, Vegf, Nip3, and Hk2 gene transcription was analyzed by real-time RT-PCR in Rora^+/+ and Rora^sg/sg cells (Mann–Whitney, *p < 0.05, ns, not significant). The values shown are the means ± SEM of 4–6 independent cultures. H + 0, Hypoxia without reoxygenation; H + 24, hypoxia and 24 h of reoxygenation; NH, non hypoxia.