Changes in glutamate homeostasis cause retinal degeneration in Royal College of Surgeons rats

  • Authors:
    • Kang Liu
    • Yi Wang
    • Zhengqin Yin
    • Chuanhuang Weng
    • Yuxiao Zeng
  • View Affiliations

  • Published online on: March 12, 2013     https://doi.org/10.3892/ijmm.2013.1297
  • Pages: 1075-1080
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Abstract

The aim of the present study was to investigate glutamate homeostasis in retinal degeneration-induced changes and the potential mechanisms of glutamate-mediated excitotoxicity in a rat model. The expression of vesicular glutamate transporter-1 (VGLUT-1) and protein kinase Cα (PKCα) in wild-type and Royal College of Surgeons (RCS) rat retinas, at postnatal Day 15 (P15), P30, P60 and P90, were detected using quantitative real-time polymerase chain reaction and immunohistochemistry. The levels of glutamine synthetase (GS) and L-glutamate/L-aspartate transporter (GLAST) were evaluated by western blotting. Compared with wild-type rats, outer nuclear layer thickness was significantly thinner and VGLUT-1 expression was upregulated in a time-dependent pattern in RCS rats. The ratio of VGLUT-1 to PKCα in RCS rats peaked at P60 (p<0.01) and subsequently decreased by P90 (p<0.01), while it remained constant in wild-type rats. The expression of GS increased gradually from P30 to P90 in RCS rats (p<0.01), while it remained constant in wild-type rats at various time-points. No significant difference in GLAST expression was found between RCS and wild-type rats at all stages of retinal degeneration. Our results confirm the occurrence of glutamate-mediated excitotoxicity to RCS rat retinas and provide an experimental foundation for safeguarding the remnant visual function in retinal degenerative disorders.

Introduction

Retinitis pigmentosa (RP) is one of the leading causes of vision impairment and blindness affecting millions of people worldwide (1,2). The Royal College of Surgeons (RCS) rat is a widely used animal model to study retinal degeneration diseases and the process of retinal remodeling; it inherits a null mutation in MER, a member of the Axl/Mer/Tyro3 receptor tyrosine kinase family gene (MERTK) of the retinal pigment epithelium (RPE) (3). This mutation results in RPE dysfunction and photoreceptor degeneration (1). Congenic strains of RCS rats, genetically similar to the inbred RCS strain, were produced in the 1970s and early 1980s (4,5). These strains comprise: i) pink-eyed rats that are wild-type (+/+) at the retinal dystrophy locus (rdy locus; the strain is designated as RCS-rdy+) and serve as control animals for the pink-eyed dystrophic, inbred RCS strain; ii) pigmented (black-hooded) dystrophic rats (the strain is designated as RCS-p+); and iii) pigmented, non-dystrophic rats for use as normal controls for the pigmented dystrophic rat (the strain is designated as RCS-rdy+ p+).

Mammalian retinal degeneration is involved in extensive structural changes in retinal neurons (6), loss of bipolar (7) and ganglion cells (8), heterotopic migration of neurons (9), and the presence of ectopic neurites and microneuromas (10). The action potentials of retinal ganglion cells were reduced in amplitude and frequency during retinal degeneration, although morphological changes of retina ganglion cells are not apparent (11). To date, the mechanism(s) responsible for retinal neuronal dysfunction and loss of retinal neurons at the terminal stage of degeneration remain unclear.

Glutamate is a major excitatory neurotransmitter in the vertebrate retina, responsible for the input of visual signals (12). In the normal state, photoreceptors (rods and cones) and bipolar cells release glutamate, which is toxic to retinal cells (13,14), glial L-glutamate/L-aspartate transporter (GLAST), which is expressed in the membrane of Müller cells, and glutamine synthetase (GS), which is expressed in the cytoplasm of Müller cells to maintain the requisite glutamate homeostasis (15,16). The glutamate in retinal neurons is bound with vesicular glutamate transporter-1 (VGLUT-1) reserved in the synaptic vesicle and is released during neuronal excitation. After being released by neurons, free glutamate in the extracellular space is bound with GLAST and taken up by Müller cells. Glutamate is then transformed to glutamine by GS in Müller cells. Subsequently, glutamine is released by Müller cells to the extracellular space, and the glutamatergic neurons uptake glutamine in order to synthesize glutamate. Finally, the glutamate reserves in the synaptic vesicle bind with VGLUT-1. As a result, finely tuned glutamate release, uptake, and degradation are essential for avoiding neurotoxicity and transmitting normal signals from photoreceptors to bipolar and ganglion cells.

In the present study, we investigated the expressions of key factors involved in glutamate metabolism in the degenerating RCS rat retina. Both mRNA and protein levels of VGLUT-1, protein kinase Cα (PKCα), GS, and GLAST were evaluated by quantitative real-time polymerase chain reaction (qPCR), immunohistochemistry, and western blot analyses.

Materials and methods

Animals

All experiments were conducted with the approval of the Animal Ethics Committee of the Southwest Hospital, Chongqing, China, and the ethics guidelines set forth by the Laboratory Animal Care and Use Committee of the Association for Research in Vision and Ophthalmology were followed. All efforts were made to minimize animal suffering and to use the fewest number of animals in the study.

Black-eyed dystrophic strain (RCS-p+) and black-eyed non-dystrophic control strain (RCS-rdy+ p+) rats were anesthetized by intraperitoneal injection of 10% chloral hydrate, after which both eyes were enucleated. Animals were then euthanized with an overdose of anesthetic. Retinas from both groups of rats were analyzed at four scheduled time-points: postnatal Days 15 (P15), P30, P60 and P90. At least six rat retinas were used for each time-point.

Immunohistochemistry

Eyes for immunohistochemical analysis were fixed in 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS) for 2 h at 4°C, and then cryoprotected in 30% sucrose in 0.1 M PBS for 1–2 days at 4°C. Eyecups were embedded with optimum cutting temperature (OCT) compound prior to cryostat sectioning at 10 μM, and they were stored at −80°C for subsequent use. Frozen tissue sections were initially thawed and rehydrated in 0.01 M PBS (pH 7.2). Following incubation in 5% normal goat serum (Jackson ImmunoResearch, USA) for 1 h at room temperature, sections were incubated with mouse monoclonal PKCα antibody (1:500) (Abcam, UK) overnight at 4°C. Following three rinses (5 min each) with PBS, fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG antibody (1:200) (Jackson ImmunoResearch) was added to the slides and the slides were incubated for 1 h at room temperature. Finally, 4′,6-diamidino-2-phenylindole (DAPI) staining was performed for labeling cell nuclei. After several rinses with PBS, the sections were covered with coverslips.

Quantitative real-time polymerase chain reaction

After enucleating, retinas were isolated and frozen in liquid nitrogen until use. For qPCR analysis, total RNA was isolated with TRIzol reagent, according to the manufacturer’s instructions. After contaminated DNAs were removed using the TURBO DNA-free™ kit (Applied Biosystems), cDNAs were synthesized from 2 μg of total RNA using the PrimeScript™ RT Reagent kit (Takara Bio, Inc., Japan) in 20 μl of reaction mixture. qPCR was carried out using the Bio-Rad 5-Color System (Bio-Rad, USA). All primers were designed online using SciTools software, Integrated DNA Technologies, Inc., (IDT). The sequences are listed in Table I. The expression change of a target gene in RCS rats relative to control rats was calculated as follows: fold change = 2−(ΔCT,Tg−ΔCT,control). The following PCR scheme was used: 5 min at 94°C, (30 sec at 94°C, 30 sec at 63°C, 30 sec at 72°C) for 35 cycles, 10 min at 72°C, and then 4°C thereafter.

Table I

qPCR primers used in the present study.

Table I

qPCR primers used in the present study.

Gene (rat)Forward primerReverse primer
PKCα (144 bp) GGGGGAAAGGGATGTCAGAG CTGCCCTCGTGTGAAGAACTT
VGLUT-1 (122 bp) TGCTGCTGGTGGTCGGATAC AGGGGCGATGTCCAAGTGGT
GAPDH (158 bp) GCCCATCACCATCTTCCAGGAG GAAGGGGCGGAGATGATGAC
Western blot analysis

For detection of GLAST and GS protein expression levels, RCS rat retinas from each group were homogenized in ice-cold radio-immunoprecipitation assay (RIPA) lysis buffer [50 mM of Tris-HCl buffer (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM sodium orthovanadate, 25 mM sodium fluoride, 1 mM EDTA, and 1 μg/ml leupeptin]. Homogenates were then centrifuged at 12,000 × g for 5 min at 4°C, and the clear supernatants were stored at − 80°C until use. Protein concentrations were determined using the Bicinchoninic Acid kit (Beyotime Institute of Biotechnology, China). Samples (50 μg of protein/lane) were loaded and electrophoresed on 12% SDS-polyacrylamide gels (SDS-PAGE) for 40 min at 120 V. Proteins were then transferred to nitrocellulose (NC) membranes for 70 min at 120 V. After transferring, the NC membranes were blocked with blocking solution containing Tris-buffered saline, 0.1% Tween-20 (TBST), and 5% free-fat milk for 1 h at room temperature. Blots were then washed three times (5 min each) with TBST and incubated with rabbit polyclonal GLAST antibody or GS antibody (Abcam) overnight at 4°C. After washing, all membranes were again incubated with mouse monoclonal GAPDH antibody for 1 h at room temperature. Subsequently, all membranes were incubated with the following secondary antibodies: IRDye 680-labeled donkey anti-rabbit IgG and IRDye 800-labeled donkey anti-mouse IgG (LI-COR Biosciences, Lincoln, NE, USA) in turn for 1 h at room temperature with shaking. Membranes were rinsed between incubations. Finally, NC membranes were scanned for GLAST, GS and GAPDH bands using the Odyssey infrared imaging system with Odyssey Application software V1.2.15. Densitometry ratios between GLAST and GAPDH, and GS and GAPDH were obtained to semi-quantify the relative levels of GLAST and GS.

Statistical analysis

All data are presented as the means ± standard error (SE). Data were evaluated by one-way analysis of variance (ANOVA) and the LSD-q test, with post hoc statistical analysis for the same group, and t-tests for comparing means between experimental and control groups at the same time-points. Values of p<0.05 were considered to indicate statistically significant differences.

Results

Changes in outer nuclear layer thickness during retinal degeneration

Immunofluorescence staining showed that the thickness of the outer nuclear layer (ONL) in control group retinas remained unchanged at all time-points (Fig. 1A–D). In the RCS rats, the ONL thickness at P15 was similar to that of the control group, but a thinner ONL was observed at P30. In addition, the number of photoreceptor cells in RCS rats was decreased at P60, and disappeared by P90 (Fig. 1E–H). Meanwhile, in RCS rats, a significant fibrotic glial seal layer (FGSL) between the ONL and outer plexiform layer (OPL) had gradually formed. There were no significant differences in the mean thickness of the inner nuclear layer (INL) between the two groups.

Changes in glutamate release during retinal degeneration

qPCR results showed that PKCα and VGLUT-1 mRNA were present in the retinas. No significant difference was found at P15 between RCS and control rats, although different tendencies existed in both groups. In the control group, VGLUT-1 expression reached a peak at P15 (Fig. 2A), decreased rapidly at P30, and then stabilized to a lower level. By contrast, VGLUT-1 expression in the RCS rats decreased slightly at P30, then gradually increased at P60, and increased rapidly (as much as 3-fold) at P90. Differences were also seen in the PKCα expression tendencies (Fig. 2B). In the control group, the level of VGLUT-1 relative to PKCα remained unchanged at all time-points (Fig. 2C), while in the RCS rats, it reached a peak at P60 and then decreased at P90.

Transport and conversion capacity of glutamate

Western blot analysis was used to analyze the expression levels of GLAST and GS. Odyssey infrared imaging showed that the GLAST levels did not change significantly in either group at any time-point (Fig. 3). However, analysis of GS expression relative to that of GAPDH showed a time-dependent increase in the control group (Figs. 3 and 4), while it gradually increased from P15 to its peak value at P90 (p<0.05) in the degeneration group.

Discussion

Glutamate is involved in neuronal survival during postnatal development of the retina (17,18) and plays a key role in the formation of retinal synaptic circuitry (1821). In rats, the normal death periods of bipolar cells and rods extend from birth to P4. Subsequently, there is a gradual decrease in the incidence of cell death, and the degeneration of bipolar cells continues until P48 (22). The apoptosis phase of photoreceptor cells in the normal rat retina occurs between P12 and P72, peaking at P23 (23). These occurrences lead to decreasing VGLUT-1 and PKCα levels in normal and degenerating rat retinas, consistent with our data showing that the expression of VGLUT-1 and PKCα was significantly decreased at P30 compared to P15.

It is known that retinal degeneration in RCS rats involves initial rod photoreceptor loss (24,25). A very high percentage of inner retinal neurons remained histologically intact, although loss of cells in all retinal layers was found (8). The bipolar cells in the inner plexiform layer (IPL) gradually became the predominant cells and became the main source releasing glutamate following retinal degeneration (25). The number of rod bipolar cells in the IPL is relatively stable during retinal degeneration, due to their characteristics of terminal differentiation and incapability of proliferation. Since PKCα exists mainly in bipolar cells in the retina, we indirectly detected the number of bipolar cells in the INL by detecting the levels of PKCα. VGLUT-1 was specifically localized to photoreceptor and bipolar cell terminals in the retina (26) and accounts for the ability of excitatory neurons to release glutamate by exocytosis (27). VGLUT-1 exhibits the glutamate levels released and stored in the whole retina. Therefore, we detected the ratio of VGLUT-1 to PKCα to indirectly show whether or not the glutamate levels released and stored in the whole retina had changed. Our data demonstrated that the ONL thickness decreased, while the ratio of VGLUT-1 to PKCα increased during retinal degeneration up to P60. These results indicate that the percentage of living rod bipolar cells increased, while the bipolar cells simultaneously became the main glutamate-releasing neurons in the retina during retinal degeneration. Thus, the ability of bipolar cells to release and store glutamate increased.

The relative level of VGLUT-1 to PKCα reached a peak at P60, but subsequently decreased at P90, which indicated that glutamate release increases gradually until the middle phase and decreases during the latter phases of retinal degeneration. These findings may be helpful in clarifying a contradiction regarding glutamate changes during retinal degeneration. Certain studies have reported that glutamate increases (28), whereas others have reported a decreasing trend during later periods of degeneration (29). In this study, at the late stage of retinal degeneration, the glutamate-releasing neurons in the retina were mainly bipolar cells. Therefore, the decrease of this ratio demonstrated that the release of glutamate from bipolar cells to ganglion cells decreased, which suggests the deafferentation of ganglion cells.

Glutamate toxicity has been demonstrated in both inner retinal cells and photoreceptor terminals (15,16). Since there is no apparent extracellular conversion of glutamate, retinal tissues require a powerful uptake mechanism capable of quickly removing extracellular glutamate to protect itself. Müller cells play a critical role in the inactivation of glutamate released by glutamatergic neurons, since they have a high affinity for Na+-linked transporters that take up glutamate (30). In addition, unprotected neurons in culture may be killed by even lower concentrations of glutamate in the medium (3133).

Glutamate uptake is accomplished by glutamate transporter proteins located in the plasma membranes of retinal Müller cells (34,35). GLAST has been described in retinal Müller, astrocyte, and RPE cells in rats (36,37) and the consequences induced by its absence from neuronal elements have also been reported (38). Our data show that GLAST expression levels remain unchanged throughout retinal degeneration in RCS rats, which is different from previous reports (28). We hypothesize that such a discrepancy likely arises from the different animal models and infrared imaging systems used. For instance, the probing of the target and reference proteins simultaneously in a particular sample indicates blots at ~60 kDa, without GLAST multimeric forms in our results to abate any possible measurement errors. In addition, the retinal degeneration mouse is a rapid retinal degeneration model, unlike the RCS rat, which is a slow degeneration animal model. Despite these differences, our results indicate that glutamate uptake decreases (with GLAST levels remaining unchanged) lead to inefficient extracellular glutamate transport into Müller cells and, consequently, induce the accumulation of glutamate in the extracellular space, resulting in toxicity to living neurons.

In our study, GS expression remained constant in the control group at all time-points, while differences in the RCS rats appeared from P30 onwards, with a significant increase at P90. These findings are consistent with results of a previous study, in which GS increased during the terminal stages of retinal degeneration (28). In our study, the GS expression pattern was similar to that of VGLUT-1 from P30 to P90. Increased GS expression does not result from Müller cell proliferation or from any concentration change in the RCS retinas (28). By P15 and P30, the conversion of glutamate into glutamine via GS activity in Müller cells was efficient; however, from P60 to P90, the level of GS in Müller cells was gradually increased and the glutamate balance was eventually disrupted, suggesting the existence of glutamate conversion enhanced within Müller cells. In general, expression of GS is regulated by glutamate (39). When the major glutamate-releasing population of neurons and photoreceptors degenerate, GS expression in Müller cells is reduced (4042). However, in the present study, the number of glutamatergic neurons reduced, while the level of GS increased, indicating that intracellular glutamate likely increases in Müller cells.

In conclusion, our results demonstrate that VGLUT-1 expression is high at P15 in both RCS and control rats. However, a gradual increase in VGLUT-1 levels, accompanied by photoreceptor degeneration, was observed after P30, while the relative expression of VGLUT-1 to PKCα levels increased up to P60, and decreased in P90, suggesting that glutamate release and storage gradually increases until the middle stage of retinal degeneration, but it decreases in the latter stage of retinal degeneration. Finally, GLAST expression levels remained unchanged, while GS expression increased gradually during the early stages and reached peak levels during the latter stages of retinal degeneration.

Acknowledgements

This study was supported by the National Basic Research Program of China (973 program) (Grant no. 2007CB512203). The authors thank Jianrong He for technical assistance, and Lifeng Chen and Yaochen Li for their suggestions and discussion on the manuscript.

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Liu K, Wang Y, Yin Z, Weng C and Zeng Y: Changes in glutamate homeostasis cause retinal degeneration in Royal College of Surgeons rats. Int J Mol Med 31: 1075-1080, 2013
APA
Liu, K., Wang, Y., Yin, Z., Weng, C., & Zeng, Y. (2013). Changes in glutamate homeostasis cause retinal degeneration in Royal College of Surgeons rats. International Journal of Molecular Medicine, 31, 1075-1080. https://doi.org/10.3892/ijmm.2013.1297
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Liu, K., Wang, Y., Yin, Z., Weng, C., Zeng, Y."Changes in glutamate homeostasis cause retinal degeneration in Royal College of Surgeons rats". International Journal of Molecular Medicine 31.5 (2013): 1075-1080.
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Liu, K., Wang, Y., Yin, Z., Weng, C., Zeng, Y."Changes in glutamate homeostasis cause retinal degeneration in Royal College of Surgeons rats". International Journal of Molecular Medicine 31, no. 5 (2013): 1075-1080. https://doi.org/10.3892/ijmm.2013.1297