Creatine Transporter Immunolocalization in Aged Human and Detached Retinas

Abstract

Purpose.: To identify the distribution of creatine transporter (CRT) in the aged human retina and how this expression pattern is modified after retinal detachment.

Methods.: An affinity-purified antibody raised against the CRT was used in the immunohistochemical investigation. The anti-CRT antibody was colocalized with neuronal markers (calbindin, parvalbumin, Islet-1, calretinin, GAD₆₇, Go-alpha), glia markers (glutamine synthetase, glial fibrillary acid protein), and a blood vessel basal membrane marker (laminin). Confocal microscopy was used to visualize the labeling patterns in retinal sections. The level of CRT expression was determined in the retina using a semiquantification method.

Results.: Immunohistochemical assessment of CRT expression in the normal aged retina revealed strong labeling in photoreceptor synaptic terminals and in inner and outer segments. Labeling was also observed on subpopulations of amacrine cells and ganglion cells as well as in the outer and inner plexiform layers. CRT labeling was observed in blood vessels, although was absent in glial cells. In retinal detachment, the CRT labeling pattern was maintained, although there was an apparent decrease in labeling in inner retina and an increase in CRT expression in photoreceptors.

Conclusions.: CRT is expressed in areas of intense metabolic activity, such as photoreceptors, selected cells in the inner retina, and sites of creatine transport into the retina (inner retinal blood vessels and retinal pigment epithelium). The absence of CRT to Müller cells harmonizes with the concept that glial cells are a biosynthetic source of creatine but not a source of creatine to other retinal neurons. The increased immunolabeling of CRT localized to the outer retina in retinal detachment suggests a regional metabolic remodeling occurring in photoreceptor cells.

Introduction

Impaired energy metabolism plays an important role in the pathogenesis and progression of any neurological insult. Creatine and its high-energy phosphorylated analogue, phosphocreatine, are essential molecules that maintain ATP levels in high-energy demanding tissues such as the retina and the brain. ^1,2 Creatine level is maintained through uptake from diet and endogenous biosynthesis. ^3,4 A specific plasma membrane creatine transporter (CRT) is required for the cellular uptake of creatine from the blood. ^5,6 CRT is critical in normal brain function, as mutations in the CRT gene (SLC6A8) lead to X-linked mental retardation, epilepsy, developmental delays in speech and language, seizures, and autistic behavior. ^7–10 As an energy buffer, creatine is an efficient neuroprotective agent in animal models of neurodegeneration and brain injury. ^1,3,11–15 Creatine supplementation is currently under trial in human subjects with neurological pathologies such as Parkinson's and Alzheimer's diseases that have known bioenergetic deficits. ^16–25 Maintenance of ATP supply is of fundamental importance for the function of the retina. Hence, it is expected that the CRT transporter should be localized to areas of high energy demand in this tissue. 

Retinal detachment is associated with altered energy metabolism and oxygen tension ^26,27 because of the hypoxic environment for photoreceptor cells as they are separated from the pigmented epithelium. The hypoxic insult is followed by photoreceptor cell death, triggering cellular remodeling of the glia and inner retinal neurons. ^28–31 Although metabolic regulation is expected in remaining cells to compensate for the anaerobic environment, the role of high-energy phosphate transporters, such as CRT, in the hypoxic environment remains to be determined. 

Immunocytochemical studies have shown CRT localization to both luminal and abluminal faces of the rat retinal blood vessel endothelial cells and retinal pigmented epithelium. ³² It has also been detected in retinal neurons of different vertebrates ³³ ; however, CRT expression in human tissues has not been characterized. This study refers to the detection of CRT expression in the normal and detached human retina as a way of evaluating creatine regulation in retinal degeneration conditions. 

Methods

Collection of Human Normal and Detached Retina Samples

Retinal tissue samples were obtained after informed consent from patients. The procedures were in accordance with the tenets of the Declaration of Helsinki and approved by the Institutional Review Committee of the University of Auckland and Auckland District Health Board. Specimens used as normal aged retinas were obtained from three Caucasian patients (2 women aged 70 years old and a male individual aged 87 years old) subjected to enucleation for uveal melanoma with no other previous treatment. Human retina obtained from enucleated eyes owing to uveal melanoma has been used in other studies. ^34–37 The retinal samples were collected after enucleation from the opposite side of the tumor. A detailed description of these samples can be found in de Souza et al. ³⁸ Three rhegmatogenous retinal detachment (RRD) specimens were from patients aged 54 to 62 years. The samples were characterized in a separate publication and used here to demonstrate CRT distribution in a diseased retina. ⁶⁵ The patient demographic information is shown in Table 1. We used samples from mid-peripheral retina (area within 7 mm from the ora serrata) to illustrate the patterns of CRT expression in aged normal retina and comparison with retinal detachment sections. 

Table 1

 

Donor Subject Demographics

Donor	Age	Sex	Ethnicity
1. Control	70	Female	Caucasian
2. Control	70	Female	Caucasian
3. Control	87	Male	Caucasian
4. Retinal detachment (2 wk)	62	Male	Caucasian
5. Retinal detachment (1 mo)	62	Male	Caucasian
6. Retinal detachment (2 mo)	54	Male	Caucasian

Retinal specimens were fixed in a mixture of 4% paraformaldehyde and 0.01% glutaraldehyde in phosphate buffer for 30 minutes. Tissues were washed in PBS, pH 7.4, before cryoprotection by immersion in graded series of sucrose up to 30% wt/vol in PBS. Then, the tissues were embedded in optimum cutting temperature medium (OCT; PELCO International, Redding, CA) and sectioned at 16 μm with a Leica CM3050 S cryostat (Heidelberg, Germany). 

Immunohistochemistry

Immunohistochemical procedures for detection of creatine transporter were conducted as previously reported. ^33,39,40 Briefly, retinal vertical sections were incubated for 1 hour in a blocking solution containing 6% goat serum (Sigma, St. Louis, MO), 1% BSA (Sigma), 0.5% Triton-X-100 (Fisher Scientific Inc., Fairlawn, NJ). Retinal sections were double labeled by overnight incubation at room temperature with primary antibodies. The 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). ⁴¹ Antibody specificity for retinal tissue has been previously reported by our group. ^33,42 Details of primary antibodies used in this study are described in Table 2. Primary and secondary antibodies were diluted in a buffer containing 3% goat serum, 1% bovine serum, 0.5% Triton X-100, and 0.05% thimerasol in PBS (phosphate buffer solution, pH 7.4). Secondary antibodies were applied to the tissues for 3 hours and consisted of goat anti-mouse conjugated with Alexa TM 594 (Molecular Probes, Eugene, OR), goat anti-rat conjugated with Alexa TM 594 (Molecular Probes), goat anti-rabbit antibody conjugated to Alexa 488 (Molecular Probes), and goat anti-rat antibody conjugated to Alexa 488. An exception was the use of one primary antibody (GFAP) conjugated to Cy3 (Sigma). All secondary antibodies were used at a 1:500 concentration. Slides were washed and cover slipped using antifading reagent (Prolong Gold; Invitrogen, Eugene, OR) containing 4′, 6-diamidino-2-phenylindole (DAPI) and sealed with nail polish. Negative controls were performed by omission of the primary or secondary antibodies otherwise maintaining the protocol steps. 

Table 2.

 

Primary Antibodies Used in This Study

Antibody	Host	Source	Working Concentration
Creatine transporter	Rabbit	Assoc. Prof. David Christie (University of Auckland, NZ)	1:100
Calbindin D28K	Mouse	Sigma-Aldrich, St. Louis, MO (C9848)	1:1000
Calretinin	Mouse	BD Biosciences, San Jose, CA (610,908)	1:1000
Parvalbumin	Mouse	Sigma (P3088)	1:1000
Islet-1	Mouse	Developmental Studies Hybridoma Bank at the University of Iowa, Iowa City, IA	1:500
Go-protein (α subunit) Go-alpha	Mouse	Chemicon International Inc., Billerica, MA (MAB327)	1:500
Glutamate decarboxylase (GAD67)	Mouse	Chemicon (AB5992)	1:500
Glutamine synthetase (GS)	Mouse	BD Biosciences (610,518)	1:1000
GFAP-Cy3	Mouse	Sigma (C9205)	1:3000
Laminin	Rat	Abcam, Cambridge, UK (ab79057)	1:200
MAP-2	Mouse	Novus Biologicals (NB300-143)	1:200
Tyrosine hydroxylase (TH)	Rabbit	Chemicon (AB152)	1:1000

Confocal Microscopy

Single- and double-labeled retinal sections were visualized and images captured using an Olympus confocal scanning laser microscope (FV1000; Olympus, Tokyo, Japan). High-resolution scanning was performed using a ×40 or ×60 oil objective lens (plan apochromat). Scans were collected with z-axis sequence with a step size between 0.5 and 1.0 μm per frame and visualized in a monitor with 1024 × 1024 pixel resolution. The final images corresponded to a maximal intensity projection of 12 μm stack of scans from a selected area of the retinal specimen. The brightness and contrast of final figures were equally adjusted by using Adobe Photoshop (Adobe Systems, Mountain View, CA). 

Semiquantitative Analysis of CRT Immunolabeling

We used a semiquantification approach to determine the level of CRT immunolabeling (expressed in pixel value), within detached retina compared with mid-peripheral nondetached retina. Suitable retinal images randomly selected from two control retina and two RRD retinas, processed under identical conditions and with confocal images of identical characteristics, were converted to gray scale using the Image J program (National Institutes of Health, Bethesda, MD). Color images were converted into 8-bit grayscale by using the split-channel function. The level of immunoreactivity is therefore expressed over a 256-pixel value range. Three different retinal areas were assessed: CRT immunoreactivity over the whole retina, outer nuclear layer, and within cone photoreceptors. The overall aim was to determine the difference in pixel value for CRT immunoreactivity in normal and detached areas. To overcome the error introduced by background immunolabeling, the following method was used when comparing the images from control and RRD retina. By thresholding the immunoreactivity, we determined the area that was immunoreactive and used this value to normalize the pixel value for CRT immunoreactivity. In this way, the average pixel value reflected the average CRT immunoreactivity in labeled retinal areas alone from images acquired under identical settings. The difference in pixel value was calculated by subtracting the RRD pixel value from the control pixel value (control – RRD). Seven images from the three control and three retinal detachment samples were used in the assessment. The average area analyzed was approximately 600,000 pixels per image. The pixel value for cone photoreceptors can be directly assessed within photoreceptor somata. We made use of the unique morphological appearance of cone photoreceptors to determine the level of CRT immunolabeling in a 100-pixel area within 5 to 10 cone photoreceptors in four different regions from aged normal and the 2-weeks retinal detachment samples. 

Results

Immunolocalization of CRT in Human Retina

To determine the identity of the CRT-labeled cells, we colocalized CRT immunoreactivity with a range of cell markers. The calcium-binding protein calbindin is a known marker for cone photoreceptors, horizontal cells, and subpopulations of bipolar cells (DB3 OFF-bipolar cells), amacrine cells, and ganglion cells in the human retina (Fig. 1C). ^43–45 Strong CRT labeling was found colocalizing with calbindin-positive photoreceptor outer and inner segments and synaptic terminals (Figs. 1A–D, arrowhead). We observed CRT on the calbindin-labeled photoreceptor cell axon (Figs. 1A–D, thick arrow). A type of amacrine cell with mitral shape and lobular appendage, suggestive to be AII amacrine cells, colocalized with CRT labeling (Figs. 1B–D). CRT labeling was also observed in the inner plexiform layer (IPL) and nerve fiber layer (NFL), as well as on calbindin-negative ganglion cells (Figs. 1B–D, small arrow). Moreover, there was a uniform expression of CRT in the retinal pigmented epithelium (RPE) cells (Fig. 1F, arrows), that could not be clearly discriminated from the background fluorescence in the RPE when secondary antibody alone was added to the tissue (Fig.1E). 

Figure 1.

 

Immunofluorescence labeling in human aged normal retina. Images were captured using confocal microscopy. DAPI was used as counter-staining (A). Creatine transporter (B) colocalizes with calbindin (C). Strong immunoreactivity coexpressed in cones (arrows), photoreceptor synaptic terminals (arrowheads), AII amacrine cells, and outer and inner plexiform layers. (E) DAPI labeling of a negative control for the immunocytochemical procedure where secondary antibody was applied in the absence of a primary antibody. (F) The presence of CRT immunoreactivity at the RPE. (Scale bar: 20 μm.) HC, horizontal cell; AC, amacrine cell; OPL, outer plexiform layer; IPL, inner plexiform layer; GCL, ganglion cell layer.

To determine if CRT was expressed in retinal glial cells, we colocalized CRT with glutamine synthetase (GS), a marker of Müller cells (Figs. 2A–F). GS expression was noted throughout Müller cell from their end-feet to the outer limiting membrane. The absence of CRT expression in Müller cells was evident in the GS/CRT double-labeled retina (Figs. 2D–F). CRT negative processes of Müller cells were found traversing the neurosensory retina (Figs. 2C, 2D, yellow arrow), whereas GS labeling was seen around inner retinal cells or in projections that run in parallel to CRT labeling in the outer plexiform and outer nuclear layers (Figs. 2D–F, white arrow). In Figure 2E and Figure 2F we show that there is no overlap between GS and CRT in both plexiform layers. 

Figure 2.

 

Confocal microscopy images of human aged normal retina immunolabeled with CRT and GS. DAPI used as counter-staining (A). CRT labeling (B) and the Müller cell marker GS (C) revealed lack of colocalization of both signals. (E) and (F) inserts correspond to a magnification of outlined areas in (D). (Scale bars: A–D, 20 μm; E and F, 15 μm.) OPL, outer plexiform layer; IPL, inner plexiform layer; GCL, ganglion cell layer.

CRT Immunoreactive Cells in Inner Retina

We used a range of cellular markers for identification of the inner retina cell types colocalizing with CRT. CRT-positive cells represent several cell populations. In Figure 1, we show the expression of CRT in AII amacrine cells, although there was no colocalization with horizontal cells, and DB3 OFF bipolar cells labeled with calbindin (Figs. 1B–D). Another calcium-binding protein, parvalbumin, was used as a marker for horizontal cells; yet, there was no expression of CRT in these cells (Figs. 3A–C, arrow). Nonetheless, parvalbumin also labels ganglion cells, which colocalized with CRT (Figs. 3A–C, arrowheads). Moreover, we used the antibodies Islet-1 and Go-alpha to label ON rod and cone bipolar cells. ⁴⁴ Among Go-alpha–positive bipolar and amacrine cells, there was absence of colocalization with CRT (Figs. 3D–F, arrows); however, we found some sparse colocalization with Ilet-1–positive cells located in the outer sector of the inner nuclear layer (INL), corresponding to the ON bipolar cells (Figs. 3G–I, arrowhead). 

Figure 3.

 

Immunolabeling of CRT and cellular markers of inner retinal neurons. (A–C) Colocalization of parvalbumin (PV) and CRT shows CRT in ganglion cells (arrows), but not in horizontal cells (yellow arrows). (D–F) ON bipolar cells were identified by Go-alpha where there was no colocalization. (G–I) Few islet-1 cells expressed CRT (arrowhead). (J–L) In GAD₆₇ amacrine cells there was some CRT colocalization. (M–O) Calretinin (CR) showed complete colocalization with CRT in the mid-peripheral human retina. (P–R) Some MAP-2 positive cells expressed CRT. (Scale bar: 20 μm.) OPL, outer plexiform layer; IPL, inner plexiform layer; GCL, ganglion cell layer.

There were other CRT-positive amacrine cells not labeled with calbindin. To determine the neurochemical signature of these CRT immunoreactive amacrine cells, we used GAD₆₇, calretinin and MAP-2 antibodies, which are markers of GABAergic, AII, and tyrosine hydroxylase/dopaminergic amacrine cells, respectively. ^46–48 We found partial colocalization between GAD₆₇ and CRT-positive amacrine cells (Figs. 3J–L), mostly at large amacrine cells whose morphology suggest them to be the tyrosine hydroxylase (TH)-immunopositive amacrine cells. In fact, this notion is corroborated by the similar partial colocalization between CRT and MAP-2 (Figs. 3P–R). Accordingly, we confirmed the immunolabeling of TH amacrine cells with MAP-2 by double labeling with these two markers (Figs. 4A–C). In contrast, extensive colocalization was evident in calretinin and CRT-labeled amacrine cells (Figs. 3M–O, arrowheads). In primate peripheral retinas, essentially all calretinin-positive cells in the INL are AII amacrine cells, ⁴⁷ therefore human AII amacrine cells express CRT. Calbindin and calretinin also labeled the stratification of these amacrine cells in the inner plexiform layer (Figs. 1B–D and 3M–O) where colocalization with CRT was observed. 

Figure 4.

 

Double immunofluorescence of aged normal retinas with markers of amacrine cell types MAP-2 (A), TH (B), and a blood vessel basal membrane marker, laminin (D) and CRT (E). MAP-2 labeled big neurons corresponding to amacrine cells (arrow) and a specific labeling pattern in the IPL (arrowhead), whereas CRT specifically labeled the soma of these cells (B–C). Cell nuclei (blue) were labeled with DAPI. Multiple and punctiform expression pattern of CRT detected in a blood vessel (bv) at the INL (F). (Scale bars: 10 μm.) IPL, inner plexiform layer; GCL, ganglion cell layer.

Previous work demonstrated CRT expression at the inner blood retinal barrier, where CRT is regulating creatine entry. ^4,6,32 CRT immunolabeling was also evident in the retina blood vessels. Laminin, a blood vessel basal membrane marker, was used to outline the vascular structures of the inner retina. CRT was observed with variable expression in blood vessels located in the INL and ganglion cell layer (Figs. 4 D–F). 

CRT Expression in Retinal Detachment

To determine whether in retinal detachment there are changes in creatine transport involving the glial cells, we assessed the CRT expression pattern and used glial fibrillary acid protein (GFAP) as a marker of astrocytes or activated Müller cells. In normal aged retina (Figs. 5A–C), astrocytes are mainly located in the NFL (Fig. 5A, double arrows). Despite the strong CRT labeling observed in the NFL, the GFAP-positive astrocyte processes did not show colocalization with CRT in normal retina (Figs. 5A–F, double arrows), suggesting that CRT expression in the NFL layers corresponded to a bundle of ganglion cell axons. To confirm this observation, we individualized the series of confocal microscopy images (0.45-μm thick each) from the final superimposed images stack. There was no colocalization between CRT and GFAP (Figs. 5D–F). 

Figure 5.

 

Confocal images of normal aged (A–F) and detached retinas (G–O). Immunolabeling of GFAP in the normal aged retinas is shown in the astrocyte processes in the NFL (double arrows, A–C) not colocalizing with CRT (merged images in C). (D–F) Single confocal image sections (0.45μm) showing absence of colocalization between CRT and GFAP. (G–I) GFAP is upregulated and found in the hypertrophic trunks of Müller cells (double arrows) in retinal detachment without colocalization with CRT. (J–L) In retinal detachment specimens, CRT is expressed in calretinin cells in inner retina. (M–O) The labeling pattern of CRT and GAD₆₇ is maintained in detached retina. (Scale bars: 20 μm.) OPL, outer plexiform layer; IPL, inner plexiform layer; GCL, ganglion cell layer.

Three retinal detachment specimens were analyzed for the pattern of CRT expression. All three samples presented identical pattern of CRT distribution despite differences in the age of patients and duration of detachment. Figure 5 is representative of the CRT expression pattern found in all three RRD samples. In the detachment samples, there was expression of CRT in the outer retina, mainly in the outer nuclear layer and photoreceptor inner segments (Figs. 5H, 5K, and 5N). 

Photoreceptor outer segment loss is a widespread phenomenon after retinal detachment; however, in areas where they were present, the difference in pixel value between RRD and normal outer nuclear layer (ONL) (ie, rods) reflected increased CRT immunoreactivity (Table 3). We found that although the overall retinal CRT immunoreactivity was reduced, there was an overall increase within the ONL. Also, when specific comparisons were made, we corroborated that a specific CRT pixel value increase was observed within morphologically recognized cone photoreceptors. Table 3 shows five examples where CRT immunoreactivity was compared between samples from control and RRD retinas. A positive pixel value indicates that the level of immunoreactivity was overall higher in the control retina, whereas a negative pixel value means the RRD retina displayed a higher level of CRT immunoreactivity in the ONL (Table 3). In fact, when specific comparisons were made within cone photoreceptors, the RRD retina displayed a relatively higher level of CRT immunoreactivity (Table 3). 

Table 3.

 

Difference in Immunoreactivity Found between Control and RRD Samples

	Difference in IMR (pixel value) [Control-RRD]
	Whole Retina	Outer Nuclear Layer	Cone Photoreceptors
Example 1	24	−69	−39
Example 2	26	−50	−99
Example 3	88	−64	−149
Example 4	93	−46	−71
Example 5	78	−77	−134

An important manifestation of anatomical remodeling of the retina after detachment is the hypertrophy and proliferation of Müller cell processes, which is evident by upregulation of GFAP. Figures 5G–I show GFAP-positive hypertrophic Müller cells spanning the entire retina but no colocalization with CRT is observed (double arrows). Conversely, CRT colocalization pattern in the inner retina with cell markers of inner retinal neurons was preserved. Figures 5J–L illustrate the colocalization pattern found in retinal detachment between CRT and calretinin amacrine cells. The immunolabeling pattern of CRT and GAD₆₇ was also maintained in the inner retina (Figs. 5M–O). Therefore, the hypoxic conditions in the outer retina did not alter the immunocytochemical expression of CRT and the different macromolecular markers in inner retinal cells. 

Discussion

We have shown the presence of CRT with a specific labeling pattern in photoreceptors and selected neurons, but absent in glial cells in the human retina. In detached retina, increased immunolabeling of CRT in the ONL suggests a regional metabolic remodeling occurring in photoreceptor cells. 

CRT Distribution Pattern in the Retina

The distribution of CRT expression in the human retina shares similarities to the pattern of labeling described in the retina of other vertebrate species. ³³ CRT is expressed by photoreceptors, inner retinal neurons, blood vessels, and retinal pigment epithelium (RPE). The interspecies presence of CRT in all three retinal neuronal orders and in the retinal capillaries suggests that the CRT transports creatine into photoreceptors and neurons at the blood retinal barriers (BRBs). Accordingly, Nakashima et al. ³² documented CRT expression in a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB) using RT-PCR and Western blot. They also demonstrated that creatine transporter at the inner BRB (iBRB) works by continuously providing creatine at a near-maximum transport rate. The RPE cells represent the outer BRB in the holangiotic human retina. Our observation of partial CRT increase in human RPE, corroborated by its finding in other vertebrate retinas, suggests facilitated creatine transport into photoreceptors. This transport model is comparable to the transport systems of lactate and glucose into the retina with monocarboxylate transporter 1 and glucose transporter 1 via both iBRB and RPE. ^49,50  

Preferential CRT immunolabeling to specific neurons in the inner retina (dopaminergic, AII amacrine cells and some ganglion cells) represents a conundrum. The vicinity of some neurons to blood vessels could explain the random yet specific CRT labeling to amacrine cells; however, the fact that all AII amacrine cells express CRT and that immunolabeling is observed also in the plexiform layers suggests that the hypothesis is incorrect. We propose an alternative explanation in that the CRT immunoreactivity identifies neurons that are resistant to disturbances of the energy metabolism facilitated by the provision of creatine. Evidence shows that amacrine cells are not equally susceptible to retinal ischemia or excitotoxicity. ^51–54 Variation in number and types of glutamate receptors as well as the expression of receptors that may provide neuroprotection (e.g., purinergic receptors) ^55,56 helps to explain the variable tolerance of neurons to insult. Furthermore, structural changes in amacrine cells subsequent to deafferentation and input losses may also corroborate for variable vulnerability of amacrine cells to excitotoxic or ischemic insults. In addition, AII amacrine cells have been shown not to possess glycogen stores, thus suggesting that these cells may rely on creatine for energy. ⁵⁷  

CRT Distribution in Müller Cells

We have shown that in the human retina, similar to other vertebrates, Müller cells do not express the CRT. Glial cells have an established role in delivering nutrients to retinal neurons and providing lactate to photoreceptors. ^57,58 Therefore, our observation that CRT is absent from human glia suggests that either (1) within Müller cells there is no creatine/phosphocreatine system to preserve their ATP levels or (2) that they have a local source of creatine biosynthesis. The latter hypothesis was strongly reinforced by the demonstration with RT-PCR, Western blot, and HPLC of the creatine precursor enzyme S-adenosyl_- l _-methionine:N-guanidinoacetate methyltransferase (GAMT) in retinal Müller cells. ⁵ Immunocytochemical colocalization of GAMT with glutamine synthetase further confirmed Müller cells as a local source of creatine biosynthesis. ⁵ Nevertheless, the combined evidence for creatine biosynthesis by Müller cells and the absence of CRT in these retinal glial cells raises the question about if and how the trafficking of creatine between Müller cells and neurons occurs. One possibility is that the production of creatine by Müller cells solely serves their own metabolic needs without provision to other retinal cells. 

We also verified the absence of CRT expression in Müller cells after retinal detachment. The detachment-induced hypoxic insult causes hypertrophy and proliferation of Müller cell processes. In this hypoxic environment, there was increased CRT expression specific to photoreceptors. The overall reduction in CRT immunoreactivity complies with other studies; however, we are showing within the ONL an increase in the RRD tissue, likely because of the protein moving into the somata of photoreceptors. These suggest an increased demand for ATP synthesis from anaerobic metabolism in photoreceptors; however, it is also likely that the increased expression of CRT in the ONL and decrease in the INL represent a redistribution of CRT protein owing to remodeling of the outer segments and disruption of its compartmentalization. This redistribution could be similar to what occurs to the rod photoreceptor protein rhodopsin, which is aberrantly expressed in the ONL after retinal detachment ³⁰ ; however, because phosphocreatine/creatine regenerate ATP significantly faster than glycolysis and oxidative phosphorylation, ⁵⁹ the increased CRT expression in the sector correspondent to hypoxic insult in detached samples reinforces the concept that the creatine/phosphocreatine shuttle system plays an important role in the retinal energy homeostasis in damaging conditions. 

Metabolic Diseases

The relevance of creatine metabolism and transport is demonstrated by the cumulative evidence of its neuroprotective properties and use in major clinical trials for neurodegenerative pathologies, such as Parkinson's and Huntington's disease and amyotrophic lateral sclerosis. ^{11,12,15,16,25,60,61} The increased expression and function of CRT mRNA in the blood brain barrier under hyperammonemic conditions ⁶² suggest that CRT plays an important role in neuroprotective mechanisms in neurological disorders. For example, the deprivation of creatine because of the loss of photoreceptor contact with the RPE combined with disruption in creatine biosynthesis by a reactive and proliferative Müller cell, further support the need for creatine uptake in the detached retina. 

Moxon-Lester et al. ⁶³ showed a significant reduction in the CRT immunolabeling of rat retinas in an acute retinal ischemia experiment. Acute ischemia is a much more severe degeneration than retinal detachment. Yet, disrupted biosynthesis of creatine by hyperornithinemia affects the choroid and retina in patients with gyrate atrophy who have a reduced creatine supply from the blood and a compromised supply from the Müller cells ⁶⁴ ; however, retinal detachment represents a localized and less severe hypoxic and hypoglycemic insult where the inner retinal circulation is intact and the outer retina may still obtain nutrients in the subretinal fluid by diffusion from the choroid depending on the height and duration of the detachment. 

Footnotes

 Disclosure: C.F. de Souza, None; M. Kalloniatis, None; D.L. Christie, None; P.J. Polkinghorne, None; C.N.J. McGhee, None; M.L. Acosta, None

Footnotes

 Supported in part by the New Zealand Optometric Vision Research Foundation (NZOVRF) reference number 3625663.