GDF15 is elevated in mice following retinal ganglion cell death and in glaucoma patients

Abstract

Glaucoma is the second leading cause of blindness worldwide. Physicians often use surrogate endpoints to monitor the progression of glaucomatous neurodegeneration. These approaches are limited in their ability to quantify disease severity and progression due to inherent subjectivity, unreliability, and limitations of normative databases. Therefore, there is a critical need to identify specific molecular markers that predict or measure glaucomatous neurodegeneration. Here, we demonstrate that growth differentiation factor 15 (GDF15) is associated with retinal ganglion cell death. Gdf15 expression in the retina is specifically increased after acute injury to retinal ganglion cell axons and in a murine chronic glaucoma model. We also demonstrate that the ganglion cell layer may be one of the sources of secreted GDF15 and that GDF15 diffuses to and can be detected in aqueous humor (AH). In validating these findings in human patients with glaucoma, we find not only that GDF15 is increased in AH of patients with primary open angle glaucoma (POAG), but also that elevated GDF15 levels are significantly associated with worse functional outcomes in glaucoma patients, as measured by visual field testing. Thus, GDF15 maybe a reliable metric of glaucomatous neurodegeneration, although further prospective validation studies will be necessary to determine if GDF15 can be used in clinical practice.

Keywords: Ophthalmology.

Figure 1. Gdf15 expression is selectively increased in the retina following retinal ganglion cell (RGC) axonal injury.

(A) Venn diagram of upregulated genes in retinal cytokine/growth factor–focused PCR array (n = 3 for optic nerve crush [ONC] and its control, n = 4 for endotoxin-induced uveitis [EIU] and its control, n = 4 for light-induced retinal degeneration [RD] and its control). (B) Venn diagram of downregulated genes in retinal cytokine/growth factor–focused PCR array. (C) Retinal gene expression of Gdf15 from PCR array in ONC, EIU, and RD models 24 hours after each intervention. (D) Retinal gene expression of Tgfb2 from PCR array in ONC, EIU, and RD models 24 hours after each intervention. (E) Retinal gene expressions of growth differentiation factor family members 24 hours after ONC (n = 4–5 per group). (F) GDF15 protein level in aqueous humor (AH) 24 hours after ONC (n = 3 per group). Values are mean ± SD. **P < 0.01 and ***P < 0.001 by 2-tailed unpaired t test.

Figure 2. GDF15 in aqueous humor (AH) is not affected by aging.

(A) Retinal gene expression of Gdf15 in young (6 weeks old) and old (18 months old) mice (n = 4 per group). (B) Retinal gene expression of Tgfb2 in young and old mice (n = 4 per group). (C) GDF15 protein level in AH in young and old mice (n = 5 per group). Values are mean ± SD.

Figure 3. Ganglion cell layer (GCL) showed increased Gdf15 expression following axonal injury to the optic nerve.

(A) In situ hybridization of Gdf15 in the mid peripheral retina (n = 3 per group, representative pictures are shown). ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar: 50 μm. (B and C) Gene expression of Gdf15 in anterior segment (AS), lens, and retina (B) 6 hours (n = 3 per group) and (C) 24 hours after optic nerve crush (ONC) (n = 6 per group). (D) In situ hybridization of Gdf15 in the mid-peripheral retina 24 hours after ONC (n = 4 per group, representative pictures are shown). Scale bar: 50 μm. (E–G) High-magnification images from D. Scale bar: 50 μm. (E) In situ hybridization of Gdf15 24 hours after ONC in GCL. (F) In situ hybridization of Gdf15 24 hours after ONC in INL. (G) In situ hybridization of Gdf15 24 hours after ONC in ONL. (H) Gdf15 gene expression in GCL, INL, and ONL of the retina following the isolation by laser microdissection 24 hours after ONC (n = 4 per group). (I and J) F4/80 gene expression in the retina (I) 6 hours (n = 3 per group) and (J) 24 hours after ONC (n = 5 per group). Values are mean ± SD. ***P < 0.001 by 2-tailed unpaired t test.

Figure 4. Elevated GDF15 level in aqueous humor (AH) of rats following optic nerve crush (ONC).

(A) Gdf15 expression in the retina of rats 24 hours after ONC (n = 5 per group). (B) Tgfb2 expression in the retina of rats 24 hours after ONC (n = 5 per group). (C) GDF15 protein level in AH of rats 24 hours after ONC (n = 5 per group). Values are mean ± SD. ***P < 0.001 by 2-tailed unpaired t test.

Figure 5. Elevated GDF15 level in aqueous humor (AH) of chronic glaucoma mouse model.

(A) Intraocular pressure (IOP) of control (3-month-old DBA/2J mice) and glaucoma (1-year-old DBA/2J mice) (n = 14 per group). (B) Gdf15 expression in the retina of control and glaucoma mice (n = 10 per group). (C) Tgfb2 expression in the retina of control and glaucoma mice (n = 10 per group). (D) GDF15 protein level in AH of control and glaucoma mice (n = 12 per group). Values are mean ± SD. **P < 0.01 and ***P < 0.001 by 2-tailed unpaired t test.

Figure 6. Elevated GDF15 level in aqueous humor (AH) of primary open angle glaucoma (POAG) patients.

(A) GDF15 protein level in AH of control patients (n = 23) and POAG patients (n = 57). ***P < 0.001 by Mann Whitney U test. (B) GDF15 protein level in control patients (n = 23) and POAG stage 1 (n = 23), stage 2 (n = 15), and stage 3 (n = 19) patients. P < 0.001 by Kruskal-Wallis test. *P < 0.05 and ***P < 0.001 by Dunn’s multiple comparison. Arrows in the graph indicate an individual case for each stage of POAG with the highest GDF15 level in AH for which the visual fields are illustrated. (C–E) Representative visual field images of POAG patient for (C) stage 1: 76 y/o, male, OD, MD: –1.63, GDF15: 126.8 pg/ml; (D) stage 2: 67 y/o, male, OD, MD: –10.82, GDF15: 466.1 pg/ml; and (E) stage 3: 88 y/o, female, OS, MD: –18.01, GDF15: 703.1 pg/ml. OD, oculus dexter (right eye); OS, oculus sinister (left eye); MD, mean deviation. (F–H) GDF15 protein level in AH of POAG patients did no differ by (F) sex: male (n = 18) vs. female (n = 39), (G) diabetes status: patients without DM (no DM: n = 41) vs. with DM (DM: n = 16), or (H) race: European descent (ED: n = 42) vs. African American (AA: n = 15). (I) Correlation between age and GDF15 protein level in AH of POAG patients (n = 57, Pearson Correlation Coefficient = 0.052, 95% CI: –3.08 to 4.82, P = 0.662). (J) Correlation between average IOP and GDF15 protein level in AH of POAG patients (n = 57, Pearson Correlation Coefficient = –0.037, 95% CI: –8.72 to 6.60, P = 0.78). Values are median with interquartile range.

Figure 7. TGFB2 level in aqueous humor (AH) of primary open angle glaucoma (POAG) patients.

(A) TGFB2 protein level in AH of control patients (n = 23) and POAG patients (n = 57). *P < 0.05 by Mann Whitney U test. (B) TGFB2 protein level in control patients (n = 23) and POAG stage 1 (n = 23), stage 2 (n = 15), and stage 3 (n = 19) patients. P < 0.05 by Kruskal-Wallis test. *P < 0.05 by Dunn’s multiple comparison. Values are median with interquartile range.

Figure 8. GDF15 level in aqueous humor (AH) of primary open angle glaucoma (POAG) patients, but not TGFB2, is a significant predictor of worse mean deviation measured by visual field (VF) testing.

The results of multivariable linear regression model showed log-transformed GDF15 levels (β = –2.0, 95% CI: –3.7 to –0.2, t = –2.3, P = 0.027) but not log-transformed TGFB2 levels (β = –0.3, 95% CI: –8.0 to 7.5, t = –0.1, P = 0.945) predict worse mean deviations on VF testing after controlling for age, race, intraocular pressure (IOP), and diabetic status.