Results: 5

1.
Fig. 5.

Fig. 5. From: Neuronal activity regulates astrocytic Nrf2 signaling.

Model for regulation of Nrf2 signaling at tripartite neuron–astrocyte synapse. At a low activity synapse (A), glutamate secreted by presynaptic terminus (red arrow) mildly activates Nrf2 signaling in adjacent astrocytes through mGluR1/5 and Ca2+-dependent pathway. At a high activity synapse (B), increased secretion of glutamate and other (currently unidentified) soluble factors secreted by a presynaptic neuron (brown arrow), postsynaptic neuron (blue arrow), or astrocyte itself (green arrow) strongly activate astrocytic Nrf2 signaling. This regulatory mechanism matches Nrf2 activity in a perisynaptic astrocyte to the firing rate of the adjacent neuronal synapse, maximizing the neuroprotection while minimizing the associated physiologic costs.

Agata Habas, et al. Proc Natl Acad Sci U S A. 2013 November 5;110(45):18291-18296.
2.
Fig. 2.

Fig. 2. From: Neuronal activity regulates astrocytic Nrf2 signaling.

Nrf2 pathway activation by high K+ or Gab/4-AP requires neurons and astrocytes. Direct neuron–astrocyte cocultures were obtained by plating astrocytes into DIV10 neuronal cultures, with experiments performed 6 d after astrocyte plating. (A) Mixed cultures, neuronal cultures, and direct neuron–astrocyte cocultures were immunostained for Map2 and GFAP as in Fig. 1; DAPI nuclear staining (blue) was used to visualize all cells. A representative of three experiments is shown. (Scale bar, 50 µm.) (B and C) Culture treatments were performed as described in Fig. 1. Nuclear Nrf2 protein level was increased by high K+ and Gab/4-AP treatments to a similar extent in mixed cultures and direct neuron–astrocyte cocultures; a representative of three or more similar experiments is shown.

Agata Habas, et al. Proc Natl Acad Sci U S A. 2013 November 5;110(45):18291-18296.
3.
Fig. 3.

Fig. 3. From: Neuronal activity regulates astrocytic Nrf2 signaling.

Neuronal activity induces Nrf2 signaling in astrocytes through a diffusible messenger. (A–C) Following Gab/4-AP treatment, hPAP+ cells (arrow in C) showed astrocytic morphology (A and B, frontal cortex; C, hippocampus); neurons were not hPAP+ (arrowheads in C mark the two blades of the dentate gyrus, which contain granule neurons). (Scale bars: A and B, 200 µM; C, 40 µM.) (D) The number of hPAP+ astrocytes was significantly higher in Gab/4-AP–treated than control and Gab/4-AP+TTX–treated slices, but similar to SLF-treated slices (**p < 0.01; n = 17, Kruskal–Wallis ANOVA on ranks). The data were not normally distributed and are represented as statistical box charts (horizontal lines: 25th, 50th, and 75th percentiles; error bars: 5th and 95th percentiles). (E) Cultures were treated as described in Fig. 1C. Following high K+ treatment, nuclear Nrf2 protein was elevated in astrocytes that were cocultured with neuronal inserts, but not in neurons that were cocultured with astrocyte inserts; a representative of at least three similar experiments is shown. (F) Astrocytes were treated with either regular or neuronally preconditioned (NC) ACSF solutions for 4 h. Irrespective of the length of the conditioning step, there was no increase in nuclear Nrf2 in response to NC-high K+; 16-h treatment with SLF was used as a positive control. A representative of at least three similar experiments is shown. (G) Experiment was performed as in F, except that neurons were cocultured with astrocyte inserts and astrocytes with neuron inserts for 3 d before insert removal, ACSF preconditioning, and astrocyte treatment. Again, there was no increase in nuclear Nrf2 in astrocytes treated for 4 h with NC-high K+. A representative of at least three similar experiments is shown.

Agata Habas, et al. Proc Natl Acad Sci U S A. 2013 November 5;110(45):18291-18296.
4.
Fig. 4.

Fig. 4. From: Neuronal activity regulates astrocytic Nrf2 signaling.

Nrf2 activation depends on glutamatergic signaling and requires Ca2+. (A) Nrf2 activation by synaptic activity was attenuated following neuronal silencing, block of metabotropic and ionotropic glutamate receptors, and chelation of extracellular Ca2+. Mixed cultures were treated for 24 h with Gab/4-AP alone or with 200 µM APV, 10 µM MK801, 1 µM TTX, 50 µM MCPG, 10 µM NBQX, 10 µM CNQX, 50 µM MCPG + 200 µM APV, or 1 mM EGTA; 24-h treatment with 0.2% DMSO (Veh) was used as a negative and 16-h treatment with 2.5 µM SLF as a positive control. Representatives of four or more similar experiments are shown; for another example, see Fig. S7 A and B. (B) Nrf2 activation by global neuronal depolarization was attenuated following block of metabotropic (but not ionotropic) glutamate receptors and chelation of Ca2+. Mixed cultures were treated for 4 h with high K+ ACSF alone or in combination with 1 µM TTX, 10 µM nimodipine (NIM), 10 µM MK801, 200 µM APV, 1 mM EGTA, 250 µM MCPG, 1 µM LY341495 (a selective mGluR group II/III antagonist), 10 µM NBQX, or 10 µM CNQX; 4-h treatment with control ACSF was used as a negative and 16-h treatment with 2.5 µM SLF as a positive control. In BAPTA-AM experiments, cultures were pretreated with 50 µM BAPTA-AM for 30 min, washed, and then treated with high K+ ACSF. Representatives of three or more similar experiments are shown; for another example, see Fig. S7C. (C) Astrocytes were treated for 16 h with 0.1% DMSO (Veh), 2.5 µM SLF, 300 µM glutamate (Glu), 300 µM NMDA, 10 µM DHPG (a selective mGluR group I agonist), 300 µM NMDA + 10 µM DHPG, or 300 µM Glu + 1 mM EGTA (all in culture medium); Glu or NMDA treatments were combined with 30 µM glycine, which is NMDAR coagonist. There was no increase in nuclear Nrf2 level in response to glutamate or its analogs; this outcome was seen in four of six similar experiments. Weak activation by Glu and DHPG, but not NMDA, was seen in two of six experiments (Fig. S7D). (D) Astrocytes were treated for 4 h with control or high K+ ACSF either alone or in combination with 300 µM Glu and 30 µM glycine; 16-h treatment with 2.5 µM SLF was used as a positive control. Nuclear Nrf2 level was lower in astrocytes treated with glutamate-ACSF than in astrocytes treated with ACSF alone, but there was no difference between astrocytes treated with glutamate in control or high K+ ACSF. A representative of four similar experiments is shown.

Agata Habas, et al. Proc Natl Acad Sci U S A. 2013 November 5;110(45):18291-18296.
5.
Fig. 1.

Fig. 1. From: Neuronal activity regulates astrocytic Nrf2 signaling.

High K+ and Gab/4-AP treatments activate Nrf2 signaling in mixed hippocampal cultures. (A) Neuronal, mixed, and astrocytic cultures were immunostained for Map2 (neuronal marker, green) and GFAP (astrocytic marker, red); DAPI nuclear staining (blue) was used to visualize all cells. At DIV14, mixed cultures were composed of 49 ± 9% neurons, 17 ± 2% astrocytes, and 33 ± 11% other cells (presumably oligodendroglia and microglia); AraC-treated, neuron-enriched cultures consisted of 95 ± 2% neurons, 4 ± 2% astrocytes, and 1 ± 1% other cell types (mean ± SD, n = 3). In astrocytic cultures, no neurons were present and nearly all nuclei were associated with GFAP+ cell bodies. Note that astrocytes cultured with neurons extend many long processes, but astrocytes cultured alone exhibit a flat, epithelioid morphology. (Scale bar, 50 µm.) (B and C) Neuronal, mixed, and astrocytic cultures were treated with control or 50 mM K+ (↑K+) ACSF for 4 h (B), 0.1% DMSO (Veh), or 20 µM Gab + 2.5 mM 4-AP (Gab/4-AP) for 24 h (C), or 2.5 µM SLF for 16 h (B and C). Nuclear Nrf2 protein level (apparent molecular weight: 84 kDa) was increased by high K+ and Gab/4-AP treatments only in mixed cultures. Cytosolic Nrf2 protein level was generally below the detection threshold; however, an increase in cytosolic Nrf2 was occasionally seen in SLF-treated astrocytic cultures (C). Representative of five or more similar experiments is shown. (D and E) Mixed cultures were treated with control or high K+ ACSF for 4 h (D) or with 0.1% DMSO (Veh) or Gab/4-AP for 24 h (E). A statistically significant increase in Nqo1 and Gclc mRNA was seen with both treatments, but only high K+ treatment led to increase of Ephx1 mRNA; the level of Gclm mRNA was not significantly changed after either treatment. Note that both baseline and induced levels for all mRNAs (except Gclm) were lower in ACSF than in the culture medium, which was used for Gab/4-AP treatment. *p < 0.05; **p < 0.01 (mean ± SEM, two-tailed t test, n = 5). ns, not significant.

Agata Habas, et al. Proc Natl Acad Sci U S A. 2013 November 5;110(45):18291-18296.

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