Concurrent Downregulation of a Glutamate Transporter and Receptor in Glaucoma

Abstract

purpose. Elevated levels of extracellular glutamate have been implicated in the pathophysiology of neuronal loss in both central nervous system and ophthalmic disorders, including glaucoma. This increase in glutamate may result from a failure of glutamate transporters, which are molecules that ordinarily regulate extracellular glutamate. Elevated glutamate levels can also lead to a perturbation in glutamate receptors. The hypothesis for the current study was that glutamate transporters and/or receptors are altered in human glaucoma.

methods. Immunohistochemical analyses of human eyes with glaucoma and control eyes were performed to evaluate glutamate receptors and transporters. These molecules were also assayed in rat eyes injected with glial-derived neurotrophic factor (GDNF).

results. Glaucomatous eyes had decreased levels of both the glutamate transporter, excitatory amino acid transporter (EAAT)-1, and the glutamate receptor subunit N-methyl-d-aspartate (NMDA)-R1. Eyes treated with GDNF had elevated levels of both EAAT1 and NMDAR1.

conclusions. The loss of EAAT1 in glaucoma may account for the elevated level of glutamate found in glaucomatous vitreous and lead to a compensatory downregulation of NMDAR1. Inasmuch as GDNF can increase levels of both EAAT1 and NMDAR1, it may be a useful therapeutic approach to restore homeostatic levels of these in glaucoma.

Glutamate is the principal excitatory neurotransmitter in the mammalian central nervous system. ¹ Extracellular glutamate is normally tightly regulated through glutamate transporters located in the plasma membrane of neurons and glia. Excessive levels of glutamate have been implicated in the pathogenesis of many neurologic and ophthalmic diseases, including stroke, trauma, epilepsy, dementia, and glaucoma. ^{1 2 3 4} Glutamate can be toxic to neurons through an excitotoxic pathway, mediated primarily through the N-methyl-d-aspartate (NMDA) subtype of glutamate receptor. ¹ Increased extracellular glutamate is generally assumed to result from the death of neurons with the subsequent release of intracellular contents. Under normal conditions, however, glutamate transporters rapidly transport glutamate into the intracellular space and maintain physiologic glutamate concentrations. ⁵ Glutamate can reach potentially toxic concentrations when released synaptically. Furthermore, in the developing mammalian retina, up to 50% of the retinal ganglion cells die by programmed cell death. However, in both cases, this transient release of glutamate is not associated with a significant elevation in extracellular glutamate, inasmuch as normally functioning transporters can rapidly restore homeostatic levels. ⁶ Consequently, if elevated extracellular glutamate is involved in neuronal loss, the possibility of a transporter abnormality must be considered. 

Glutamate transporter malfunction plays a role in excess glutamate levels (and corresponding neuronal loss) in amyotrophic lateral sclerosis, dementia, and stroke. ^{7 8 9 10} Elevated concentrations of glutamate have been found in the vitreous of glaucomatous eyes. ^{2 4 11} Transporter malfunction may therefore account for the elevated glutamate found in glaucomatous vitreous. 

To date, five excitatory amino acid transporters (EAAT1–5)have been identified that may be significant in the clearance of glutamate in the nervous system. ^{12 13} In the retina, EAAT1 (also referred to as GLAST) is found in Müller cells and astrocytes ¹³ ; EAAT2 (GLT-1) is localized to cones and two types of bipolar cells ¹⁴ ; EAAT3 (EAAC-1) is found on horizontal, amacrine, and ganglion cells and, rarely, on bipolar cells ¹³ ; and EAAT5 is localized to photoreceptors and bipolar cells. ¹⁵ EAAT4 has not been found in retinal tissue. 

An elevation in extracellular glutamate can perturb other aspects of neuronal glutamatergic systems. NMDA receptor subunits are altered in excitotoxic disease states. ^{16 17 18 19 20} For example, there is a significant loss of the NMDAR2A subunit in amyotrophic lateral sclerosis. ²¹ Optic nerve crush alters splicing of the NMDAR1 subunit in the retina. ²² Ischemia increases expression of NMDAR2C. ²³ The interrelationship of NMDA receptor subunits and splice variants is not fully understood, nor is it known whether any change constitutes a compensatory effort on the part of the cell or is part of the pathologic process. It has been hypothesized that, in the face of elevated extracellular glutamate, neurons may downregulate or alter the NMDA receptor to decrease sensitivity to excess glutamate. ^{22 24}  

We have suggested that toxic levels of glutamate may contribute to retinal ganglion cell death in glaucoma. ² We therefore analyzed two glutamate transporters and the NMDAR1 glutamate receptor subunit in human glaucoma. 

Methods

Human tissue was provided by the Glaucoma Research Foundation and the Scheie Eye Pathology Laboratory. For glaucomatous eyes, the diagnosis was confirmed in all cases by histopathologic analysis (of ganglion cell loss and optic nerve excavation) in addition to a review of all available medical records. Details of demographics of the patient are provided in Table 1 . 

Eyes were immersion fixed in formalin and then embedded in paraffin, after which sections were cut, mounted on slides, and deparaffinated in xylene before immunohistochemistry was performed. Conventional immunohistochemistry provides little evidence for the localization of ionotropic receptors, suggesting that their epitopes are not readily accessible in situ. Therefore, we used the antigen retrieval procedure based on microwave irradiation, as described by Fritschy et al. ²⁵ to enhance the immunohistochemical staining of the NMDAR1 subunit in the retina. ²⁵ Immunohistochemistry was performed according to the manufacturer’s protocol (Dako, Hamburg, Germany). In brief, after preincubation with 1% bovine serum albumin in Tris-buffered saline (TBS), sections were incubated with a rabbit antibody directed against EAAT1 (Alpha Diagnostics, San Antonio, TX), diluted at 1:40, or with a monoclonal mouse antibody directed against the NMDAR1 subunit (Chemicon, Temecula, CA). The latter was diluted at 1:200 in TBS. Incubation with the primary antibody was performed overnight at 4°C, followed the next day by incubation with either biotinylated goat anti-rabbit or goat anti-mouse IgG (1:300; Vector, Burlingame, CA) and the ABComplex (Dako) for 30 minutes at room temperature. The final step involved incubation for 15 minutes with alkaline phosphatase substrate solution, using New Fuchsin (Sigma, St. Louis, MO) as the chromogen. After the sections were rinsed in distilled water, they were mounted (Aquatex; Dako). For control sections, the primary antibody was omitted. Only minimal background staining was observed in any control section. Antibodies were selected based on availability and on our observation that specific binding was detectable in control human retina. In all cases, glaucoma and control sections were incubated simultaneously with a single set of reagents. 

All animal experiments were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Intraocular injections were performed with a heat-pulled glass capillary connected to a microsyringe (Drummond Microdispenser, Broomall, PA). The total volume injected was 2 μl. Injections were made over a period of approximately 30 seconds and were directed toward the posterior pole of the eye to avoid damage to the lens. 

For GDNF experiments, 1 μg was injected into the vitreous of a rat eye on days 1, 3, and 5; the animal was killed on day 7. Eyes were fixed overnight in 10% buffered formalin, and then sectioned in a fashion identical with human tissue. 

To quantify immunohistochemical staining, the following protocol was derived from previously published techniques. ^{26 27 28} Images were obtained through a digital image system (Image Pro Plus; Media Cybernetics, Silver Spring, MD) connected to a microscope equipped with appropriate illumination, coded, and analyzed in a masked fashion. All images were recorded under identical illumination conditions. With the use of image analysis software (Photoshop, ver. 5.0.2; Adobe, San Jose, CA), all images were pasted into a single image. A region of unambiguous staining was identified and selected using the “magic wand” tool (tolerance set to 25). The“ similar” command was used to highlight all stained regions in the composite figure simultaneously. These regions were then cut and pasted into a new image. The “invert” command was applied to the entire, flattened new composite (so that stained regions would be brighter than the background), and the intensity of each original retinal image was quantified with the “histogram” command. Three retinal sections from each of three eyes were analyzed for human tissue; four eyes were analyzed for rat experiments. Values were compared by Student’s t-test. 

Results

In normal human retina, EAAT1 was present from the inner limiting membrane to the outer plexiform layer (Fig. 1A ). EAAT1-positive structures bordering the inner limiting membrane appeared to represent the end feet of Müller cells. A dense network of EAAT1-positive fine processes throughout the inner and outer plexiform layers indicated labeling of the Müller cells. 

In contrast, EAAT1 was dramatically reduced in glaucomatous retina (Figs. 1B 2) . The maximal level of EAAT1 seen in any glaucomatous eye evaluated was less than the lowest level found in any control retina. A second glutamate transporter, EAAT2, was found in amacrine and bipolar cells at similar levels in both glaucomatous and control retinas (Fig. 2) . The similar levels of EAAT2 between glaucomatous and control eyes indicates the appropriateness of our selection of controls. 

As noted, glutamate receptors can also be altered in excitotoxic states, perhaps to compensate for elevated extracellular glutamate. We therefore explored expression of the NMDAR1 glutamate receptor subunit. In normal retina, most neurons in the inner nuclear and ganglion cell layers expressed NMDAR1 (Fig. 1C) . The photoreceptor outer segments were also strongly positive for NMDAR1. However, glaucomatous retina had little or no evidence of NMDAR1 immunoreactivity (Figs. 1D 4) . As we had seen with EAAT1, the maximal level of NMDAR1 in any glaucomatous eye was less than the lowest level found in any control retina. EAAT2 levels did not differ between glaucomatous and control eyes. 

As shown in Figures 3A and 4 , GDNF increases expression of EAAT1 in the retina. If intensities are normalized to a control value of 1.0 ± 0.3 (SD), the GDNF treated eyes had a staining intensity of 2.0 ± 0.4. Most intriguingly, GDNF also increased levels of NMDAR1 in the retina (Fig. 3B 4) . If NMDAR1 levels in control eyes are normalized to 1.0 ± 0.3, the GDNF-treated eyes had a staining intensity of 2.6 ± 0.2. These data suggest that GDNF may compensate for the observed changes noted in glaucomatous tissue in the present study. 

Discussion

The glutamate transporter, EAAT1 is diminished in glaucomatous eyes. Because its absence decreases the ability of the cell to regulate extracellular glutamate levels, this may account wholly or in part for the elevated levels of glutamate found in eyes with glaucoma. Cebers et al., ²⁹ in studies on cultured cerebellar granule cells, have demonstrated that pharmacologic blockade of glutamate transporters leads to decreased levels of NMDAR1. Downregulation of the glutamate transporter EAAT1 and the subsequent loss of glutamate reuptake could therefore precede the loss of NMDAR1 in glaucomatous eyes. It is possible that a neuron, when faced with elevated levels of extracellular glutamate, may attempt to compensate by lowering levels of glutamate receptors. Excessive stimulation of the glutamate receptor could lead to its internalization and subsequent desensitization. We will be able to expand our understanding of alterations in glutamatergic biology in glaucoma as other antibodies become available. 

Our findings of diminished NMDAR1 and EAAT1 levels spanned the entire retina and were not limited to the ganglion cell layer. Although the primary retinal cell loss in glaucoma is of the ganglion cell layer, that does not preclude perturbations elsewhere in the retina (for example, increased levels of GFAP in Müller cells noted by Hiscott et al., in glaucomatous eyes ³⁰ ). Other investigators have reported a loss of NMDAR1 reactivity in regions of Alzheimer’s disease–affected brains without corresponding neuronal loss. ³¹ It should be noted that our findings are at variance with results reported by Hof et al., ³² in experimental glaucoma in the macaque monkey. They found little or no loss of NMDAR1 in monkey retina. This discrepancy may reflect a difference between the human and monkey response to a glaucomatous insult or a difference in experimental technique. Future investigations may provide an explanation. 

Several neuroprotective growth factors have been shown to increase glutamate transporter expression in culture. ^{33 34} Glial-derived neurotrophic factor (GDNF) is a well-characterized neuroprotective agent that can increase neuronal survival in the face of several insults, including excitotoxicity. ^{35 36 37 38 39} We suggest that part of GDNF’s neuroprotective ability is a consequence of its ability to upregulate EAAT1. Our findings further suggest that increasing levels of EAAT1, through administration of GDNF, may be a valid therapeutic approach in glaucoma and related conditions. 

Glutamate receptor–mediated excitotoxicity has been implicated in many neurologic conditions. The results in the present study indicate a loss of EAAT1 in glaucomatous retina, which may explain the elevated extracellular glutamate seen in this disease. The loss of EAAT1 in glaucoma may also account for the downregulation of NMDAR1 in glaucoma. Furthermore, GDNF increased levels of both EAAT1 and NMDAR1, suggesting that this growth factor may be a useful therapeutic approach in the management of glaucoma and other diseases mediated by chronic excitotoxicity. 

¹  Present address: Department of Ophthalmology, Otto-von-Guericke University, D-39120 Magdeburg, Germany.

 Supported by National Institutes of Health Grant R01 EY10009; a Merit Grant from the Veteran’s Administration; and grants from Research to Prevent Blindness; Potts Foundations; Allergan, Irvine, California; and the Jody Lynn Sack Memorial Fund. EBD is the recipient of a Research to Prevent Blindness Lew R. Wasserman Merit Award. CKV was supported by a Theodor Leber Stipend from the Basotherm Förderkreis, Germany and from the Ernst and Berta Grimmke Stiftung, Germany.

 Submitted for publication September 1, 1999; revised December 20, 1999; January 11, 2000.

 Commercial relationships policy: N.

 

Table 1.

 

Demographics of Patients

	Age at Death	Years Diagnosed with Glaucoma	Diagnosis	Other Ophthalmic Diagnosis	Ophthalmic Procedures	Time from Death to Fixation
Glaucoma	81	16	Chronic angle closure	Cataract	Cataract extraction	12 hours
	82	7	Open angle	Cataract	Cataract extraction, trabeculectomy	6 hours
	90	4	Open angle	Cataract	Cataract extraction	4 hours
Control	71			Cataract		12 hours
	71			Cataract	Cataract extraction	12 hours
	90			Cataract	Cataract extraction	4 hours

Figure 1.

 

Alterations in glutamate transporters and the NMDAR1 subunit of the NMDA glutamate receptor in human glaucoma. Sections of normal (A) and glaucomatous (B) human retina were stained immunohistochemically for EAAT1. Glaucomatous retina had significantly lower levels of EAAT1 than normal retina. Similar findings were seen with retinas stained for NMDAR1 (C, D). Glaucomatous retina (D) had significantly lower levels of the glutamate receptor subunit, NMDAR1 than normal retina (C). The maximal level of either EAAT1 of NMDAR1 seen in any glaucomatous eye evaluated was less than the lowest level found in any control retina. gcl, ganglion cell layer; ipl, inner plexiform layer; onl, outer nuclear layer; prl, photoreceptor layer.

Figure 2.

 

Quantification of EAAT1, EAAT2, and NMDAR1 in human eyes with glaucoma. Immunohistochemical staining for the glutamate transporters EAAT1 and EAAT2 and the glutamate receptor subunit NMDAR1 were quantified. Both EAAT1 and NMDAR1 were diminished in glaucomatous eyes; EAAT2 did not differ between glaucomatous and control eyes. Values are normalized to a control intensity of 1 and are means ± SD. Significant difference from control at *P < 0.01;** P < 0.001.

Figure 3.

 

GDNF increases levels of both EAAT1 and NMDAR1. Eyes were injected with GDNF, and retinal sections were then stained for EAAT1 and NMDAR1. As shown in (A; control) and (B; GDNF), GDNF treatment led to upregulation of the glutamate transporter EAAT1. Similarly, GDNF increased levels of NMDAR1 (D) over those in vehicle-injected eyes (C).

Figure 4.

 

Quantification of the effect of GDNF on EAAT1 and NMDAR1 levels. Immunohistochemical staining for the glutamate transporter EAAT1 and the glutamate receptor subunit NMDAR1 were quantified in rat eyes treated with GDNF. GDNF increased levels of both proteins. Values are normalized to control intensity of 1 and are means ± SD. Significant difference from control at *P < 0.01;** P < 0.001.

The authors thank Jeffrey Rothstein, Johns Hopkins University, Baltimore, MD, for helpful discussions and Mark Bove for technical assistance.