1Department of Optometry and Vision Science; and 2Biochemistry and Cell Biology Section, School of Biological Sciences, University of Auckland, Auckland, New Zealand
Submitted 23 March 2005 ; accepted in final form 23 May 2005
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ABSTRACT |
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photoreceptor; development; glutamine synthetase; neurochemistry
The retina contains high levels of creatine. In chicken, the total retinal creatine (Cr/PCr) concentration is 3 mM and may be 15 mM within photoreceptor cells (47). PCr is also relatively high in the adult mouse (17.6 µmol/g dry wt) and in the dark-adapted (25.19 µmol/g dry wt) or light-adapted (30.48 µmol/g dry wt) frog retina (25, 26). Phosphocreatine is produced by phosphorylation of creatine by creatine kinase (CK) after ATP synthesis in mitochondria. Reversal of this reaction by cytoplasmic creatine kinase regenerates ATP at sites of high energy utilization (54). The localization of creatine kinase isoforms supports the existence of a PCr circuit in the highly polar photoreceptor cells (20). Mitochondrial CK was present in the inner segments of bovine rod and cone cells, whereas the cytoplasmic brain isoform of CK was also located in the rod outer segments. This suggested that the outer segment CK and Cr/PCr play an important role in phototransduction by providing energy for the visual cycle.
Most tissues take up creatine from the blood. Molecular cloning studies identified rabbit muscle and brain cDNAs encoding a high-affinity Na+- and Cl-dependent creatine transporter (18). The creatine transporter (CRT) exhibited significant homology to the GABA and norepinephrine transporters (17, 34) and other members of the Na+ and Cl-dependent neurotransmitter (1, 31) or solute-carrier 6 (SLC6) family of transporters (10). The Na+, Cl-dependent creatine transporter (SLC6A8) has been shown to be responsible for the absorption of creatine by intestinal epithelia (35) and the transport of creatine across the blood-brain barrier (29, 33). Mutations in the CRT gene (SLC6A8) result in the absence of creatine in the brain and a novel form of X-linked mental retardation, characterized by expressive speech-language delay, epilepsy, developmental delay, and autistic behavior (27, 38, 39). Symptoms from CRT deficiency cannot be corrected by creatine supplementation and persist despite the presence of creatine biosynthetic enzymes, L-arginine:glycine amidinotransferase (AGAT) and S-adenosyl-L-methionine:N-guanidinoacetate methyltransferase (GAMT) in the brain (7).
The blood-retinal barrier (BRB) shields the retina from the circulating blood. Nutrients must be obtained from the choroidal circulation in avascular retinas (e.g., rabbit), while vascularized retinas (e.g., human and rat) also receive a blood supply from vessels that enter the retina inner surface. Tight junctions are present between retinal pigment epithelial cells (outer BRB) and retinal capillary endothelial cells (inner BRB) (21). Cr/PCr are lost from tissues due to spontaneous conversion to creatinine (1.7%/day), which diffuses out of cells and is excreted by the kidney (60). Creatine required by the retina must be obtained by transport across the BRB. The CRT is present in both luminal and abluminal membranes of rat retinal capillary endothelial cells, where it mediates creatine transport across the inner BRB (29). However, the distribution of the CRT protein within the retina has not been studied. CRT mRNA is present in bovine and mouse retina (23, 40). The inability to detect the CRT in photoreceptor cells by in situ hybridization (23) was a surprising result, given the known importance of the Cr/PCr in these cells (20, 47). To clarify the supply of creatine to the retina, we applied an affinity-purified CRT antibody to avascular and vascular retinas and the developing mouse retina. The transporter was localized to photoreceptors, a variety of inner neuronal cells and to sites for blood-to-retina transport (blood vessels, perivascular astrocytes, and retinal pigmented epithelium), but not in Müller cells. The CRT distribution in adult mouse retina mirrors that at found at very early stages of development. These studies highlight the importance of Cr/PCr for ATP homeostasis in the retina.
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MATERIALS AND METHODS |
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Antibody against CRT. A rabbit polyclonal antibody was raised against a purified COOH-terminal fragment of the bovine CRT protein, expressed as a fusion protein with glutathione-S-transferase (GST) (13). Antibodies specific for the COOH-terminal 21 amino acids were affinity purified on a column containing immobilized fusion protein (GST-CC2) and stored in phosphate buffer at 20°C. Antibody specificity for the retina was assessed by preincubating 0.45 µg of the CRT antibody with 25 µg of GST-CC2 fusion protein at 4°C for 2 h. The preincubated antibody was diluted to 1:100 and applied to 12-µm-thick retinal sections of different species and age groups.
Analysis of the CRT in membranes from retina and choriocapillaris by Western blotting.
Retinas and the choriocapillaris (RPE and choroid) were quickly removed from bovine eyes and stored in phosphate-buffered saline during transport back to the laboratory. The tissues were homogenized separately in 5 volumes of ice-cold homogenization buffer [10 mM HEPES-NaOH, pH 7.4, and 0.25 M sucrose containing a cocktail of protease inhibitors (Roche Complete Mini)] with a 15-s burst on a Polytron homogenizer at speed setting 5. The homogenate was centrifuged at 1,000 g for 10 min at 4°C (model SS-34 rotor, Sorvall) to remove cell debris and nuclei. The supernatant was centrifuged at 10,000 g for 10 min and further centrifuged at 100,000 g (Beckman 70Ti rotor) for 1 h at 4°C to produce a crude membrane fraction. The supernatant was decanted, and the crude membranes were resuspended in homogenization buffer and aliquots were stored at 70°C. The protein content was determined using a commercial assay kit (Bio-Rad DC Protein Assay). Samples of retinal and choriocapillaris crude membranes were run on 10% SDS polyacrylamide gels and subjected to Western blot analysis as described previously (13). For comparison, membranes from a clonal human embryonic kidney (HEK)-293 cell line, stably expressing high levels of the CRT [3% of the membrane protein (50)] were run as a positive control. The CRT was detected by incubating the membrane with the affinity-purified, COOH-terminally directed antibody (0.25 µg/ml for 2 h), followed by incubation with a 1:3,000 dilution of goat anti-rabbit, horseradish peroxidase-labeled antibody (Bio-Rad) and chemiluminescent reagent (Amersham Pharmacia Biotech, ECL Plus). Control experiments were carried out by preabsorbing the CRT antibody with the GST-CC2 fusion protein. The CRT antibody (2.5 µg) was incubated with the GST-CC2 protein (50 µg) in 1 ml of 20 mM Tris·HCl-0.137 M NaCl, pH 7.6, containing 0.05% Tween (TBST) and 1% nonfat milk powder for 1 h at 4°C. The antibody was diluted 10 times with TBST/1% nonfat milk powder before incubation with the membrane blots.
Immunocytochemistry.
Immunocytochemical labeling was performed by indirect immunofluorescence against the CRT antibody. The antibodies were diluted in 3% normal goat serum, 1% bovine serum albumin, and 0.5% Triton X-100. A mixture of the CRT antibody and a range of cell markers were applied overnight to the retina. The labeling was detected using secondary antibodies conjugated to Alexa Fluor 488 (green fluorescence) or Alexa Fluor 594 (red fluorescence). A selection of antibodies were employed in the identification of retinal cell types (19); amacrine and ganglion cells were identified with calretinin (BD Transduction, 1:1,000) and parvalbumin (Sigma, 1:1,000). Calbindin (Sigma, 1:1,000) was used as a marker of horizontal cells and amacrine cells, whereas brain nitric oxide synthase (1:1,000, Sigma) and glutamic acid decarboxylase (1:500; GAD65; BD Pharmingen) were used as markers of amacrine cells. Markers for bipolar cells were anti-Go protein, -subunit (1:150; Chemicon), and
-isoform of PKC (PKC clone MC5, Sigma, 1:100). Müller cells were identified by using glutamine synthetase (BD Transduction Labs, 1:3,000); astrocytes were identified by glial fibrillary acidic protein (GFAP, Chemicon, 1:1,000); recoverin (Chemicon, 1:1,000)-detected photoreceptor cells, and by some bipolar cells. For double immunocytochemistry, recoverin was conjugated to Alexa fluor dye A594 using a protocol adapted from the Zenon rabbit IgG labeling kit (Molecular Probes). All fluorescent specimens were viewed using confocal microscopy. Images correspond to a maximum intensity projection of a 5-µm-thick stack of images unless otherwise specified. The images were optimized by adjusting the brightness and contrast using Adobe PhotoShop software (Mountain View, CA).
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RESULTS |
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Throughout mouse development, the pattern of Müller cell labeling closely matched the pattern of CRT labeling in the outer nuclear layer, especially at P8, when increased labeling of outer and inner retina was observed (Fig. 6, PR). However, high-resolution images did not show colocalization of the glutamine synthetase marker with CRT at P8 (Fig. 6O) or at P17 (Fig. 6, TV). At P8, recoverin labeled the somata of developing photoreceptor cells (Fig. 6N), and CRT was delimiting the recoverin-labeled cells. At P17, double labeling of the outer nuclear layer with recoverin and CRT showed colocalization of both signals in the photoreceptor plasma membrane of the somata and at the inner segments (Fig. 6S).
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DISCUSSION |
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The identification of the CRT in tissues has been problematic because of the specificity of available antibodies (44). The affinity-purified antibodies used in the present study are directed toward a unique COOH-terminal epitope and have been shown to recognize both transiently expressed (13) and purified recombinant CRT (50). Differences between the 65-kDa CRT detected in retina and choriocapillaris membranes and the 85- and 71-kDa forms seen in rat brain and retina (29, 33) may reflect tissue-specific glycosylation. We have estimated the CRT to represent 3% of HEK-293-CRT cell membrane protein (50). Because
100 times more retina/choriocapillaris than HEK-293-CRT membrane protein was needed to detect the CRT on Western blots (Fig. 1), we estimate the CRT to represent <0.03% of retina membrane protein. A similar low abundance in membranes from other tissues may account for difficulties in the detection of the CRT (44).
The CRT was expressed in key locations (retinal pigmented epithelium, inner retinal blood vessels, and perivascular astrocytes) for the transport of creatine from the circulation into the retina. In the chicken, strong CRT labeling was found in the pecten, which contains both endothelial cells lining blood vessels and pigmented cells (Fig. 3C). Localization of the CRT to astrocytes of the inner retinal blood vessels of mammalian retina (Fig. 2, A and E) agrees well with the recent study (29) showing CRT expression in rat retinal capillary endothelial cells and a critical role for the CRT in the uptake of creatine across the inner BRB. In addition, we have shown that only perivascular astroglia express the transporter, indicating a specialized function for the different astrocytes within the nerve fiber layer. Perivascular astroglia are intricately related to the development of inner retinal vessels (57) and have recently been linked to regulation of communication within the nerve fiber layer (48). Our identification of the CRT in the retinal pigmented epithelium indicates that creatine can also be obtained from the choroidal circulation.
Localization of the CRT in photoreceptors links predominant creatine uptake to sites of high-energy phosphate production and is consistent with the Cr/PCr shuttle being used to transfer high-energy phosphates from inner to outer segments of photoreceptors. Creatine kinase activity is found in the inner segment of bovine, mouse, and chicken cone and rod photoreceptors, in close association with the mitochondria-rich portion of the inner segment, and in the membranous disk and plasma membrane of the outer segment of the bovine photoreceptors (20, 43, 47, 49). Stable levels of ATP are encountered in the photoreceptor outer segments in the frog, salamander, and mouse even after stimulation of the phototransduction pathway (12, 26, 28, 36).
CRT labeling was not restricted to any particular type of cell. Most types of neurons in the vertebrate retina contained the CRT, suggesting that most cells in the inner retina can take up creatine. The Müller glia cells play an important role in the delivery of nutrients to neurons and supply lactate to photoreceptors (37). Without transport by Müller cells, creatine would have to enter the inner retina through the CRT in astrocytes. The absence of the CRT in Müller cells also suggests that neurons may be more metabolically independent of glia than has been suggested previously and that Müller cells do not depend on Cr/PCr to maintain ATP levels. It remains possible that Müller cells or other cell types not containing the CRT may be able to synthesize creatine. To our knowledge, there have been no studies identifying the creatine biosynthesizing enzymes, AGAT and GAMT, in the retina. In brain, however, GAMT has been shown to be highly expressed in oligodendrocytes and olfactory ensheathing glia, moderately in astrocytes, and at very low levels in most neurons, leading to the suggestion of a novel neuron-glial relationship for brain energy homeostasis (45). Additional studies are required to investigate whether Müller cells can synthesize creatine and release it by a mechanism not involving the CRT.
The metabolic requirement of the mouse retina progressively increases with development, as measured by the increasing levels of oxygen uptake, CO2 production, and lactic acid formation (25, 32). However, overall PCr and ATP levels in the developing mouse retina decrease with age (25). It appears that the CRT expression has a similar tendency: PCr and CRT expression show a small peak at P10 before decreasing gradually with age (25). At P10, rods are still differentiating in the outer part of the outer nuclear layer (56), suggesting increased retinal metabolism before the onset of mature photoreceptor metabolism (26). Similarly to the developmentally related levels of creatine kinase mRNA in the chicken retina (42, 49), the changes in CRT immunolabeling in the mouse retina suggest that the PCr-Cr cycle is in place before photoreceptor function is established. Thus early immunolabeling for the CRT is consistent with a dependence on PCr not only for photoreceptor metabolic demand but also for the early development of the outer nuclear layer. Though CRT immunolabeling is observed as early at embryonic day 15.5, an age at which the photoreceptor development has not started and retinal energy requirements are not yet related to retinal function, labeling is confined to the developing ganglion cells (5, 56). In particular, increase in the immunolabeling of the transporter in the inner retina seems to be associated with the expansion of the inner blood vessels at approximately P4 through P7 and particularly associated with the pattern of selective regression of capillaries in the second and third weeks of development (6, 11, 15). Indeed, we observed that in the adult retina, CRT-positive bipolar cells were always in close proximity to capillaries, indicating that factors present in the blood, even creatine itself, may be regulating CRT expression. However, the stratified staining of the inner plexiform layer in all vertebrate retinas, including the avascular chicken retina, suggests that creatine uptake is not restricted to those cells in contact with or in close proximity to the blood vessels.
In summary, we have identified the creatine uptake sites involving the transfer of creatine from the blood into the retina and also to neural cells within the retina. The failure of creatine supplementation to reduce the progression of eye symptoms in patients with gyrate atrophy of the choroid and retina may be due to progressive damage from the inability to maintain ATP levels from early stages of development. It is also known that oral administration of creatine brings only small increases to creatine levels in the human brain (55), so further studies of factors that regulate creatine uptake may be warranted.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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