The potential role of glutamate transporters in the pathogenesis of normal tension glaucoma

Abstract

Glaucoma, a progressive optic neuropathy due to retinal ganglion cell (RGC) degeneration, is one of the leading causes of irreversible blindness. Although glaucoma is often associated with elevated intraocular pressure (IOP), IOP elevation is not detected in a significant subset of glaucomas, such as normal tension glaucoma (NTG). Moreover, in some glaucoma patients, significant IOP reduction does not prevent progression of the disease. Thus, understanding IOP-independent mechanisms of RGC loss is important. Here, we show that mice deficient in the glutamate transporters GLAST or EAAC1 demonstrate spontaneous RGC and optic nerve degeneration without elevated IOP. In GLAST-deficient mice, the glutathione level in Müller glia was decreased; administration of glutamate receptor blocker prevented RGC loss. In EAAC1-deficient mice, RGCs were more vulnerable to oxidative stress. These findings suggest that glutamate transporters are necessary both to prevent excitotoxic retinal damage and to synthesize glutathione, a major cellular antioxidant and tripeptide of glutamate, cysteine, and glycine. We believe these mice are the first animal models of NTG that offer a powerful system for investigating mechanisms of neurodegeneration in NTG and developing therapies directed at IOP-independent mechanisms of RGC loss.

Figure 1. Expression of glutamate transporters in the retina.

Immunohistochemical analysis of mouse retina double-stained with GLAST and GS, a specific marker for Müller glial cells (A); EAAC1 and calretinin, a specific marker for RGCs and amacrine cells (B); and GLT-1 and calretinin antibodies (C). Scale bar: 50 μm.

Figure 2. RGC degeneration in glutamate transporter mutant mice.

(A) H&E staining of retinal sections during postnatal development. WT, GLAST^+/–, and GLAST^–/– mice are littermates. EAAC1^+/– and EAAC1^–/– mice are littermates. (B) Quantification of RGC number in glutamate transporter mutant mice. The number of neurons in the GCL was counted in the retinal section from one ora serrata through the optic nerve to the other ora serrata at 0, 1, 2, 3, 5, 8, 16, and 32 weeks of age. Each plot represents the results of 3 to 6 independent experiments. Scale bar: 50 μm (A). NBL, neuroblast layer.

Figure 3. RGC degeneration in GLAST^–/– mice.

H&E-stained sections show a decreased number of cells in the GCL in GLAST^–/– mouse (arrowhead) (A and B). Retrogradely labeled RGCs in GLAST^–/– mouse were decreased compared with those in WT mouse (C and D). E and F are magnified images of C and D, respectively. Scale bar: 100 μm (A, B, E, and F); 500 μm (C and D).

Figure 4. Normal retinal structure in GLT-1^+/– mouse.

H&E-stained sections show the absence of RGC degeneration in an 8-month-old GLT-1^+/– mouse. Scale bar: 50 μm.

Figure 5. Optic nerve degeneration and normal IOP in glutamate transporter mutant mice.

(A and B) Optic nerve atrophy in 8-month-old GLAST^–/– mice. In WT mice (A), the nasal and temporal nerve fibers (arrows) are beneath the internal limiting membrane (arrowhead) and enter a well-formed optic nerve (N). In GLAST^–/– mice (B), the nerve fiber layer has become thin and almost absent as it enters the nerve (bold arrows). Cupping extends to the posterior aspect of the inner retinal layer (arrowheads). (C and D) Thinning of optic nerve is apparent in GLAST^–/– (D) compared with WT (C) mice. (E and F) Staining of semithin sections with toluidine blue reveals the presence of abnormally dark axonal profiles (arrowheads) and decline of axons in GLAST^–/– (F) compared with WT (E) mice. (G and H) Aqueous humor drainage structures in WT (G) and GLAST^–/– (H) mice. Iridocorneal angle in GLAST^–/– mice is normal with an obvious Schlemm’s canal (arrow) and trabecular meshwork (asterisk) compared with those in WT mice. The angle recess between the cornea (C) and iris is wide open. (I) IOP of young (4 weeks old) and adult (9–11 months old) mice. Sample numbers are indicated in parentheses. Scale bar: 200 μm (A, B, G, and H); 130 μm (C and D); 9 μm (E and F).

Figure 6. Impaired multifocal electroretinogram in GLAST^–/– mice.

(A) Averaged responses of 2K from 10 mice. The visual stimulus was applied to 7 different areas in the retina. The 7 individual traces demonstrate the average responses to the visual stimulus at the corresponding stimulus area. (B) Three-dimensional plots showing the amplitude variation across the arrays in A. (C) Quantitative analysis of 2K amplitude. The response amplitudes for each stimulus element were added and the result was divided by the total area of the visual stimulus. n = 10 per group; *P < 0.005. Values in B and C are given in nV per square degree (nV/deg²).

Figure 7. Effect of glutamate neurotoxicity on RGC degeneration.

(A) Intravitreal glutamate concentration in glutamate transporter mutant mice. Sample numbers are indicated in parentheses. (B) H&E-stained P14 retinal sections from WT and GLAST^–/– mice with or without treatment with memantine (10 mg/kg, i.p.) daily from P7 to P13. (C) Quantitative analysis of RGC number following memantine administration. The number of neurons in the GCL was counted in the retinal section from one ora serrata through the optic nerve to the other ora serrata. n = 6 per group. ^#P < 0.05; *P < 0.005. Scale bar: 50 μm (B).

Figure 8. Increased oxidative stress in glutamate transporter mutant mice.

(A) Lipid hydroperoxide concentration in whole retina of WT, EAAC1^–/–, and GLAST^–/– mice. Sample numbers are indicated in parentheses. (B) Immunohistochemical analysis of mouse retina double-stained with glutathione and GS. Glutathione-like immunoreactivity was observed in Müller glial cells (arrowheads). Scale bar: 20 μm. (C) Mean glutathione concentration in whole retina and cultured Müller cells. Sample numbers are indicated in parentheses. (D) Lactate dehydrogenase (LDH) release from H₂O₂-treated RGCs cocultured with WT or GLAST^–/– Müller cells. n = 3 per group. (E) Lactate dehydrogenase release from H₂O₂-treated RGCs from WT or EAAC1^–/– mice. n = 4 per group. ^#P < 0.05; *P < 0.005.