Identification of a novel neurotrophic factor from primary retinal Müller cells using stable isotope labeling by amino acids in cell culture (SILAC)

Abstract

Retinal Müller glial cells (RMGs) have a primary role in maintaining the homeostasis of the retina. In pathological situations, RMGs execute protective and regenerative effects, but they can also contribute to neurodegeneration. It has recently been recognized that cultured primary RMGs secrete pro-survival factors for retinal neurons for up to 2 weeks in culture, but this ability is lost when RMGs are cultivated for longer durations. In our study, we investigated RMG supernatants for novel neuroprotective factors using a quantitative proteomic approach. Stable isotope labeling by amino acids in cell culture (SILAC) was used on primary porcine RMGs. Supernatants of RMGs cultivated for 2 weeks were compared with supernatants from cells that had already lost their protective capacity. Using this approach, we detected established neurotrophic factors such as transferrin, osteopontin, and leukemia inhibitory factor and identified C-X-C motif chemokine 10 (CXCL10) as a novel candidate neuroprotective factor. All factors prolonged photoreceptor survival in vitro. Ex vivo treatment of retinal explants with leukemia inhibitory factor or CXCL10 demonstrated a neuroprotective effect on photoreceptors. Western blots on CXCL10- and leukemia inhibitory factor-stimulated explanted retina and photoreceptor lysates indicated activation of pro-survival signal transducer and activator of transcription signaling and B-cell lymphoma pathways. These findings suggest that CXCL10 contributes to the supportive potential of RMGs toward retinal neurons.

Fig. 1.

Identification of neurotrophic candidate proteins using SILAC quantification. A total of 376 protein groups were reliably identified in the RMG supernatants. A, protein abundances are displayed as log transformed heavy-to-light ratios between cells cultured for 21 days and cells cultured for 14 days, independent of their intensity. Negative ratio values represent a greater abundance of proteins on day 14 than on day 21 (black dots). B, for the identification of neurotrophic candidates, stringent filtering criteria were applied to proteins with differential abundances with respect to quality of quantification, secretion, and fold change.

Fig. 2.

Transferrin, LIF, and CXCL10 directly promote PR survival in vitro. Isolated and cultured porcine PRs were used to assess the neurotrophic capacities of either RMG-conditioned medium or the individual candidate neurotrophic factors identified via the quantitative SILAC approach. PRs were treated with supernatant from day 14 or day 21 cultured RMGs or medium containing neurotrophic candidates transferrin (1000 ng/ml transferrin), LIF (1 ng/ml), or CXCL10 (100 ng/ml). After 6 days in vitro, the day 14 supernatants and all three candidates were found to significantly enhance PR survival relative to controls and day 21 supernatant (t test p < 0.05). FC, fold change.

Fig. 3.

CXCL10 expressed in porcine retina in vivo. Fluorescent labeling of CXCL10 was diffusely present throughout the retina (A) and was particularly intense in the cells of the ganglion cell layer and some amacrine cells in the inner nuclear layer. In comparison, only background fluorescence was present in the photoreceptors in the negative control (B). Scale bar: 50 μm. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer.

Fig. 4.

LIF and CXCL10 showed protective effects in ex vivo retina explants. Retinas from mice with the Pde6b^rd1 mutation (rd1-positive) were isolated 5 days after birth and cultivated for 14 days. All micrographs are of the central retina. A, rd1-positive mice lost 90% of photoreceptors, as reflected by the small size of the ONL, which was reduced to a single cell layer. Rd1-positive retinas treated for 12 days with (B) 50 ng/ml of CXCL10 or (C) 100 ng/ml LIF demonstrated rescue of ONL size. D, a combination treatment of 50 ng/ml CXCL10 and 100 ng/ml LIF resulted in slightly increased ONL size relative to the singly treated explants. E, a retinal section of a 20-day-old C57/Bl6 mouse showing the normal size of the ONL in a healthy, non-explanted retina. F, mean ONL thickness for treated and untreated explants. All treatments resulted in significantly increased ONL thicknesses relative to untreated samples (untreated, 9 μm ± 3.9; CXCL10, 21 μm ± 5.5, p < 0.05; LIF, 35 μm ± 7.3, p < 0.05), as well as to the combined treatment (CXCL10/LIF, 38 μm ± 7.9, p < 0.05). None of the treatments restored ONL thickness to the level of C57/Bl6 (wild-type) mice (62 μm ± 11.7). All data presented as mean ± S.D. Scale bar: 50 μm. ONL, outer nuclear layer; INL, inner nuclear layer; wt, wild type.

Fig. 5.

CXCL10 and LIF stimulated anti-apoptotic signaling in the retina. Western blot analyses from porcine explant lysates or primary PR lysates showing levels of pSTAT3 and BCL-2 with α-tubulin as the loading control. pSTAT3 and BCL-2 abundance increased after stimulation with CXCL10 and LIF in the retinal explants (A) and in the isolated porcine PRs (pooled from 20 eyes to ensure adequate material) (B). Signal intensities were determined using ImageJ, normalized against α-tubulin intensities, and are depicted as graphs below the respective Western blot results.