Astrocyte-derived lipoxins A4 and B4 promote neuroprotection from acute and chronic injury

Abstract

Astrocytes perform critical non-cell autonomous roles following CNS injury that involve either neurotoxic or neuroprotective effects. Yet the nature of potential prosurvival cues has remained unclear. In the current study, we utilized the close interaction between astrocytes and retinal ganglion cells (RGCs) in the eye to characterize a secreted neuroprotective signal present in retinal astrocyte conditioned medium (ACM). Rather than a conventional peptide neurotrophic factor, we identified a prominent lipid component of the neuroprotective signal through metabolomics screening. The lipoxins LXA4 and LXB4 are small lipid mediators that act locally to dampen inflammation, but they have not been linked directly to neuronal actions. Here, we determined that LXA4 and LXB4 are synthesized in the inner retina, but their levels are reduced following injury. Injection of either lipoxin was sufficient for neuroprotection following acute injury, while inhibition of key lipoxin pathway components exacerbated injury-induced damage. Although LXA4 signaling has been extensively investigated, LXB4, the less studied lipoxin, emerged to be more potent in protection. Moreover, LXB4 neuroprotection was different from that of established LXA4 signaling, and therapeutic LXB4 treatment was efficacious in a chronic model of the common neurodegenerative disease glaucoma. Together, these results identify a potential paracrine mechanism that coordinates neuronal homeostasis and inflammation in the CNS.

Keywords: Cell Biology; Cell stress; Eye; Neurodegeneration; Neurological disorders; Neuroscience; Ophthalmology.

Figure 1. Transplanted RAs protect inner retinal neurons in vivo.

(A–C) Transplantation of RAs increased survival of GCL and INL neurons following KA-induced injury, compared with PBS-injected controls (Islet1: red, arrows). INL, inner nuclear layer; ONL, outer nuclear layer. (D–F) Adjacent sections were TUNEL stained (green, arrows), and show a complementary pattern, with reduced apoptosis GCL and INL in eyes with transplanted RAs. (G–H) Rescue of Islet1-positive neurons was reduced or absent when RAs were prestressed by treatment with 300 μM PQ (RA+PQ) or heat killed (RA+HK). (I and J) Transplants of A7 or 661W cells were also not protective. (K–N) Corresponding TUNEL staining showed higher GCL or INL apoptosis in the controls compared with RA transplants. (O) Quantification of Islet1 results for each group, showing significant protection of GCL neurons by RAs that is absent in each of the controls (n = 10). (P) Quantification of TUNEL results for each group, showing a significant reduction of apoptosis by RAs that is lower or absent in the controls (n = 10). Scale bars: 50 μm. *P < 0.05; **P < 0.01; ***P < 0.005 compared with PBS. Bars represent SEM. Statistical analyses were performed by 1-way ANOVA with TUKEY post-hoc test.

Figure 2. Astrocyte neuroprotection is mediated by a small secreted activity that is enriched in lipoxins.

ACM or cell-free control media was injected intravitreally 1 day prior to KA challenge. (A) ACM treatment reduced KA-induced RGC loss compared with cell-free control media (RBPMS: green, arrows). (B) Quantification of RBPMS results showing significant RGC survival with ACM injection (n = 5). (C) Complimentary results showing ACM-mediated reduction in TUNEL-labeled cells (green, arrows) compared with control media. (D) Quantification of TUNEL results showing significant decrease in GCL apoptosis from ACM media (n = 5). (E) ACM protection was reproduced in vitro with HT22 neuronal cells challenged with 5 mM glutamate (n = 3). (F) Substantial protective activity in ACM is contained in a 3-kDa filtrate (n = 3). (G) High concentrations of LXA₄ and LXB₄ were detected in ACM compared with control media by LC-MS/MS (n = 3). Scale bar: 50 μm. *P < 0.05; **P < 0.01; ***P < 0.005. Bars represent SEM. Statistical analyses were performed by 1-way ANOVA with TUKEY post-hoc test.

Figure 3. Lipoxins are regulated in the inner retina in response to acute injury.

(A) qPCR of mouse retinal cDNAs shows significantly reduced expression of Alox5 and Fpr2, but not Alox15, 2 hours after KA insult compared with PBS controls (n = 3). (B) LXB₄ concentrations in total mouse retina are reduced at 6 hours following injury, while LXA₄ concentrations are reduced in the ON compared with PBS-injected controls (n = 10 retinas/aggregate group). (C) Confocal microscopy shows 5-LOX immunostaining in primary RAs. (D) Confocal imaging of 5-LOX immunostaining (green) shows accumulation in the GCL and NFL, with partial colocalization (yellow, arrows) in astrocytes (GFAP: red). (E) Signal for 5-LOX in the inner retina is reduced at 3 and 6 hours after injury (arrows). (F) FPR2 immunostaining (green) is prominent in cultured primary RGCs stained with β3-tubulin (red). (G) ALX/FPR2 immunostaining (green) is specific to the GCL and colocalizes (yellow, arrows) with RGCs (Brn3a: red). Scale bars: 20 μm (C and F); 10 μm (D and G). ***P < 0.005. Bars represent SEM. Statistical analyses were performed by 1-way ANOVA with TUKEY post-hoc test.

Figure 4. LXA₄ and LXB₄ promote RGC survival following acute injury.

(A) Intravitreal injection of 10 μM LXA₄ or LXB₄ prior to KA-induced insult resulted in increased RGC survival compared with control (PBS) (RBPMS: green, arrows). (B) Corresponding quantification shows significant increases in RGC survival with LXA₄ or LXB₄ treatment compared with vehicle control. Values are presented as fold change from noninjured controls (n = 8). (C and D) In contrast, intravitreal treatment with 10 μM of the 5-LOX inhibitor zileuton significantly compromised RGC survival following acute stress compared with vehicle (n = 5). (E and F) Similarly, 15 μM of the ALX/FPR2 inhibitor WRW4 significantly reduced RGC survival following acute stress compared with vehicle (n = 5). Scale bar: 50 μm. *P < 0.05. Bars represent SEM. Statistical analyses were performed by 1-way ANOVA with TUKEY post-hoc test.

Figure 5. LXA₄ and LXB₄ have direct neuroprotective activities.

(A) Treatment of HT22 neuronal cells with LXA₄ or LXB₄ significantly protected them from metabolic insult in a dose-dependent manner up to 500 nM (n = 3). (B) No protective activity was observed by treatment with up to 1 μM of the related molecules 15-HETE or RvD2 (n = 3). (C) LXB₄ protective activity at 500 nM was not blocked by treatment with increasing μM concentrations of WRW4 or the GPR18 antagonist O-1918 (n = 3). (D) MitoTracker red signal indicates protection from increased membrane potential with LXB₄ treatment (n = 3) (E) Primary RGCs labeled with β3-tubulin extend an extensive network of neurites that disintegrate dramatically after 24 hours of 30 μM PQ. Intact neurites are significantly maintained by 1 μM LXB₄, but not LXA₄ or RvD2. (F) RGC survival following oxidative stress demonstrates significant rescue with treatment by LXA₄ or LXB₄ (n = 3). (G) RGC neurite degeneration following PQ challenge was significantly rescued by LXB₄, but not LXA₄ or RvD2 (n = 3). (H) Primary cortical neurons demonstrate a similar protective effect for LXB₄ (n = 5). Scale bar: 20 μm. *P < 0.05; **P < 0.01. Bars represent SEM. Statistical analyses were performed by 1-way ANOVA with TUKEY post-hoc test.

Figure 6. Therapeutic LXB₄ protects RGC function and survival following glaucomatous injury.

(A) Experimental schematic showing ERG and OCT readings every 4 weeks following suture-induced IOP elevation. LXB₄ administration started at week 8, and retinal flatmounting and RGC counting were performed at week 15. q.a.d., every other day. (B) Average waveforms with an intensity of –4.60 log cd.s.m^–2 for RGC (pSTR) responses at week 15 for LXB₄ and vehicle groups and (C) relative change in RGC function across 15 weeks. Starting at week 12, there was significant and increasing rescue of RGC response in LXB₄-treated eyes compared with vehicle (n = 8 per group). (D) RNFL thickness was monitored by OCT across 15 weeks, comparing sutured to control eyes in LXB₄- and vehicle-treated groups. (E) RNFL loss is significantly reduced in the LXB₄ group compared with vehicle at week 15 (n = 8). (F) Representative retinal flatmounts stained for BRN3a after 15 weeks of elevated IOP compared with contralateral control eyes from LXB₄- or vehicle-treated animals. Original magnification: ×20. (G) Quantification of RGC density shows significant rescue of RGC loss by LXB₄ treatment in central and peripheral retinas compared with vehicle (n = 8). *P < 0.05; **P < 0.01; ***P < 0.005. Bars represent SEM. Shaded area indicates the treatment period (A, C, and E). Statistical values were analyzed by 2-way ANOVA with Bonferroni post-hoc test (C–E) and by t test (G).