ProNGF induces TNFalpha-dependent death of retinal ganglion cells through a p75NTR non-cell-autonomous signaling pathway

Abstract

Neurotrophin binding to the p75 neurotrophin receptor (p75(NTR)) activates neuronal apoptosis following adult central nervous system injury, but the underlying cellular mechanisms remain poorly defined. In this study, we show that the proform of nerve growth factor (proNGF) induces death of retinal ganglion cells in adult rodents via a p75(NTR)-dependent signaling mechanism. Expression of p75(NTR) in the adult retina is confined to Müller glial cells; therefore we tested the hypothesis that proNGF activates a non-cell-autonomous signaling pathway to induce retinal ganglion cell (RGC) death. Consistent with this, we show that proNGF induced robust expression of tumor necrosis factor alpha (TNFalpha) in Müller cells and that genetic or biochemical ablation of TNFalpha blocked proNGF-induced death of retinal neurons. Mice rendered null for p75(NTR), its coreceptor sortilin, or the adaptor protein NRAGE were defective in proNGF-induced glial TNFalpha production and did not undergo proNGF-induced retinal ganglion cell death. We conclude that proNGF activates a non-cell-autonomous signaling pathway that causes TNFalpha-dependent death of retinal neurons in vivo.

Fig. 1.

Exogenous proNGF leads to marked RGC loss in the adult rodent eye. Quantitative analysis is shown of RGC survival in rat (A) and mouse (B) retinas at 1 week after intraocular injection of proNGF (solid bars), vehicle (PBS, shaded bars), or NGF (bars with horizontal lines). The density of RGCs in intact, noninjected retinas is shown as reference (open bars). Data are expressed as RGC densities (RGCs/mm²; mean ± SEM; ANOVA, *P < 0.001). The number of animals used in each experiment is shown above the corresponding graph bar.

Fig. 2.

p75^NTR, sortilin, and NRAGE are required for proNGF-induced death of RGCs. (A) Treatment with REX, a p75^NTR function-blocking antibody, promoted RGC survival in the presence of proNGF whereas coadministration of a nonspecific, control Ig did not exert a neuroprotective effect (ANOVA, *P < 0.001). RGCs from mice eyes deficient in p75^NTR (B), sortilin (C), or NRAGE (D) were resistant to proNGF-induced death (Student’s t test, P > 0.05). Values are expressed as RGC densities (RGCs/mm²; mean ± SEM).

Fig. 3.

p75^NTR and p75^NTR-induced TNFα are expressed by Müller cells in the adult mouse retina. (A) Confocal microscopy images show absence of p75^NTR within Fluorogold-positive RGCs, demonstrating that adult RGCs are devoid of p75^NTR. (B) Double immunolabeling with antibodies against p75^NTR (REX) and the Müller cell marker cellular retinaldehyde-binding protein (CRALBP) shows strong expression of p75^NTR in Müller cell processes surrounding RGCs. (C) Fluorescent microscopy images show that NRAGE protein colocalizes with Müller cell soma and processes, visualized with CRALBP. (D) Robust TNFα protein induction was observed in retinas exposed to proNGF compared to low, basal levels in eyes injected with vehicle or recombinant, mature NGF. (E) Confocal microscopy images show that TNFα colocalizes with CRALBP-positive Müller cells. GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer. (Scale bars: A and B, 10 μm; C–E, 50 μm.)

Fig. 4.

Sortilin and NRAGE cooperate with p75^NTR to transduce proNGF-induced signaling events required for TNFα production. (A) Coadministration of proNGF with the TNFα inhibitor Etanercept leads to striking RGC neuroprotection, but RGCs did not survive in Fc-treated control retinas (ANOVA, *P < 0.001). (B) RGCs from TNFα null mice were resistant to proNGF-induced death (Student’s t test, P > 0.05). (C) Western blot analyses of retinal extracts show that whereas TNFα protein levels are below the detection limit in wild-type retinas, exposure to proNGF elicits robust TNFα protein up-regulation within 48 h of proNGF injection. ProNGF-induced TNFα production is completely lost in p75^NTR null, sortilin null, and NRAGE null animals (n = 5 mice/group). Lower blots were probed with antibodies that recognize p75^NTR, sortilin, or NRAGE, respectively, and actin was used as a control for equal protein loading.