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(Investigative Ophthalmology and Visual Science. 2008;49:1660-1664.)
© 2008 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.07-1094
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Carnitine Palmitoyltransferase I and Acyl-CoA Dehydrogenase 9 in Retina: Insights of Retinopathy in Mitochondrial Trifunctional Protein Defects

Eva Roomets,1,2 Tero Kivelä,3 and Tiina Tyni1

1From the Department of Pediatric Neurology, Hospital for Children and Adolescents, and the 3Department of Ophthalmology, Helsinki University Central Hospital, Helsinki, Finland; and the 2University of Helsinki, Helsinki Biomedical Graduate School, Helsinki, Finland.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. Progressive pigment chorioretinopathy is a major long-term complication of mitochondrial trifunctional protein (MTP) defects, disorders of mitochondrial fatty acid β-oxidation. To better understand the pathogenesis of the retinopathy component, the authors studied expression of the main regulatory protein of the β-oxidation pathway, carnitine palmitoyltransferase (CPT) 1, and acyl-CoA dehydrogenase (ACAD) 9 in retinal sections and cultured cells.

METHODS. Immunoblotting was performed with polyclonal antibodies to ACAD9 and the three isoforms of CPT1. In quantitative real-time PCR (QRT-PCR), predesigned gene-specific probes and primer sets for human CPT1 isoforms were used. In situ hybridization (ISH) and immunohistochemistry was performed on formalin-fixed, paraffin-embedded sections of the rat and human eye.

RESULTS. The predominant CPT1 mRNA types detected by QRT-PCR in cultured human retinal pigment epithelial cells were of the liver (CPT1A) and brain (CPT1C) isotypes. CPT1A and ACAD9 protein expression was found in cultured human and rat RPE and rat neural retinal precursor cells. ISH of rat retinal sections showed CPT1A and CPT1C expression in the retinal pigment epithelium (RPE), the inner nuclear layer, and the ganglion cell layer. CPT1A expression was also detected in the Müller cell microvilli, and CPT1C expression was detected in the photoreceptor inner segments. ACAD9 immunolabeling was detected in rat and human RPE, human photoreceptor inner segments, and ganglion cell layer.

CONCLUSIONS. These findings imply that the mitochondrial fatty acid β-oxidation pathway probably is active in metabolism of the RPE and certain neuroretinal cell types. Accumulation of 3-hydroxylated intermediates of long-chain fatty acids may contribute to the pathogenesis of retinopathy in MTP deficiencies.

Human mitochondrial trifunctional protein (MTP) is a multienzyme complex with three enzyme activities bound to the inner mitochondrial membrane: long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD), long-chain 3-enoyl-CoA hydratase, and long-chain 3-ketoacyl-CoA thiolase. Defects in the MTP complex cause autosomal recessive inherited disorders of the mitochondrial fatty acid β-oxidation (FAO) cycle, a major energy-producing pathway in the skeletal muscle and the heart. Biochemically, two subgroups of MTP deficiency are distinguished, isolated LCHAD and complete MTP.1 In the latter, all three enzyme activities are reduced, whereas in the former, thiolase and hydratase activities are normal or slightly reduced. Although energy production of the brain and the retina rely mostly on glucose oxidation, during prolonged fasting the brain also uses ketone bodies, which are converted by the liver from acetyl-CoA produced by FAO.

The natural history of LCHAD deficiency is severe; hypoglycemic coma, cardiomyopathy, hepatopathy, and, frequently, death occur during the first years of life. Unlike other β-oxidation defects, isolated LCHAD and complete MTP deficiency are both associated with pregnancy complications, peripheral neuropathy, and progressive pigment chorioretinopathy. The chorioretinopathy emerges in early childhood as granular pigmentation of the central fundus with or without pigment clumping, and it may advance to chorioretinal atrophy, high myopia, posterior staphyloma, and low vision.2 Current treatment, including a low-fat, high-carbohydrate diet and the avoidance of fasting, dramatically improves the overall prognosis of MTP deficiencies and allows long-term survival.3 The efficacy of the diet in preventing the progression of chorioretinopathy remains inconclusive.4

Pathogenesis mechanisms proposed to explain the chorioretinopathy of LCHAD and MTP deficiencies include secondary deficiency of fatty acid docosahexaenoic acid (DHA), energy deprivation, and toxicity of accumulating intermediates.4 Resolving the pathogenesis of the chorioretinopathy might provide new therapeutic opportunities and would be important because these disorders are now screened in neonates among other "treatable" inherited metabolic defects. Several β-oxidation proteins, including MTP, are expressed in human ocular tissues, particularly in the retinal pigment epithelium (RPE).5 6

We studied ocular expression of carnitine palmitoyltransferase (CPT) 1 and acyl-CoA dehydrogenase 9 (ACAD9) to understand better the retinopathy component in mitochondrial trifunctional protein defects. The former is the major regulatory enzyme of FAO, which controls access into the mitochondria of acyl-CoAs—activated fatty acids for β-oxidation. It has three tissue-specific isoforms: CPT1A in the liver, CPT1B in the muscle, and CPT1C in the brain. Genes for CPT1 isoforms are located in different chromosomes: CPT1A in 11q13.1-q13.2, CPT1B in 22q13.3-ter, and CPT1C in 19q13.33. CPT1A has two and CPT1B four transcriptional variants. CPT1A and CPT1B catalyze the transfer of acyl groups from fatty acyl-CoAs to carnitine, whereas CPT1C appears to be involved in the regulation of energy homeostasis by an unknown mechanism.7 ACAD9 is a recently identified long-chain acyl-CoA dehydrogenase involved in the first step of mitochondrial FAO. Of the three known long-chain ACADs, ACAD9 is particularly expressed in the brain.8 Only a few patients with ACAD9 deficiency have been reported, and none of them is known to have retinopathy.9


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture
Human (ARPE19) and rat (RPE-J) retinal pigment epithelial cell lines were purchased from American Type Culture Collection (Manassas, VA). The rat neural retina precursor cell line (R28) was a kind gift of Gail M. Seigel (University of Rochester School of Medicine and Dentistry, Rochester, NY).10 Human skin fibroblasts and myoblasts were obtained from a healthy volunteer control subject. All cell culture media and supplements were bought from Invitrogen (Paisley, UK). ARPE19 cells were grown in DMEM/F12 medium, RPE-J cells in high glucose (4.5 g/L) DMEM, and R28 cells and human fibroblasts in DMEM. Media were supplemented with 2 mM L-glutamine, 0.1 mg/mL streptomycin, and 100 U/mL penicillin. RPE-J medium contained 4% and other media contained 10% fetal bovine serum (FBS). The R28 medium was also supplemented with 0.1 mM MEM nonessential amino acids and 1x MEM vitamins. Media were changed every 3 to 4 days. RPE-J cells were cultured at a permissive temperature of 33°C, allowing expression of the transformed phenotype. The other cells were grown at 37°C.

Immunoblotting
Mitochondrial protein was isolated from the livers and brains of Wistar rats (Harland, The Netherlands), as described earlier.11 Shortly after rinsing in ice-cold isolation medium containing 250 mM mannitol, 5 mM Tris-HCl, and 0.5 mM EGTA (final pH 7.4), the tissues were homogenized and centrifuged at 600g (10 minutes, 4°C). The first supernatant was further centrifuged at 3600g (10 minutes, 4°C), and the resultant pellet was resuspended in the isolation medium and recentrifuged at 2700g (10 minutes, 4°C). The final pellet was taken up in the isolation medium and kept on ice.

Protein from cultured human fibroblasts, ARPE19 cells, and rat RPE-J and R28 cells was extracted by a method described earlier.12 Then the detached cells were homogenized in a medium containing 150 mM NaCl, 5 mM EDTA, 10 mM Tris/HCl (pH 7.5), 1% Triton X-100, and a protease inhibitor (Complete; Roche, Penzberg, Germany). The homogenate was centrifuged at 20,800g (10 minutes, 4°C), and supernatants were used for SDS-PAGE and Western blotting. Polyclonal rabbit antibodies raised to rat CPT1A were a kind gift of Carina Prip-Buus (Institut Cochin, Meudon, France), polyclonal sheep antibodies raised to mouse CPT1B and CPT1C were a generous gift of Victor A. Zammit (Hannah Research Institute, Scotland, UK), and polyclonal rabbit antibody raised to human ACAD9 was a gift from Jerry Vockley (Children’s Hospital of Pittsburgh, Pittsburgh, PA). After blotting, the immune complex was stripped from nitrocellulose by incubation in 62.5 mM Tris, pH 6.7, 100 mM 2-mercaptoethanol, and 2% SDS for 30 minutes at 50°C, and the blot was reprobed with polyclonal rabbit antibodies raised to rat MTP, a generous gift of Bruce Middleton (University of Nottingham Medical School, Nottingham, UK)

Quantitative Real-Time PCR
Fresh human skeletal muscle was obtained from tissue surrounding a surgically removed tumor of the quadriceps femoris. Total cellular RNA was isolated from cultured human fibroblasts, myoblasts, ARPE19 cells, and fresh muscle using an RNA isolation kit (RNeasy Mini; Qiagen Sciences, Valencia, CA). The concentration of the extracted RNA was determined spectrophotometrically, and 500 ng total RNA was used for each RT reaction. cDNA templates for PCR were prepared with random primers and M-MLV RT (Promega, Madison, WI). The resultant cDNA (diluted 1:5) was used for PCR reaction carried out for 2 minutes at 50°C and for 10 minutes at 95°C, followed by 44 cycles for 15 seconds at 95°C and for 1 minute at 60°C. Quantitative real-time PCR (QRT-PCR) was performed on a detection system (ABI PRISM 7000 Sequence Detection System; Applied Biosystems, Foster City, CA) and predesigned, gene-specific probe and primer sets for human CPT1A, CPT1B, and CPT1C (TaqMan; Assays-on-Demand; Applied Biosystems). Primer sets included the following primers: 5'-AAACGGCCAACTGCATGTCCAGCCA-3' (Hs00157079_m1) for CPT1A, covering both transcriptional variants, 5'-TGTCATGGACCTTGTGCTCATCAAG-3' (Hs00189258_m1) for CPT1B, covering all four transcriptional variants, and 5'-GTCCAATTATGTCAGTGACTGGTGG-3' (Hs00380581_m1) for CPT1C (Hs00380581_m1).

Expression of mRNA was assessed by evaluating threshold cycle (CT) values in quadruplicate reactions. Values were normalized against the expression level of a house-keeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Gene expression levels of each CPT1 isoform were evaluated by comparing their expression with that in fibroblasts using the comparative 2–{Delta}{Delta}CT method of quantification.13

In Situ Hybridization and Immunohistochemistry
In situ hybridization (ISH) was performed with digoxigenin (DIG)-labeled RNA probes on formalin-fixed, paraffin-embedded, 5-µm-thick sections of the albino Wistar rat eye. The protocol for the RNA probe synthesis was based on the method described by Wilkinson.14 Approximately 1 kb cDNA was reverse transcribed from CPT1A and CPT1C RNA, as described earlier, with the following primers: 5'-AAGAACATTGTGAGCGGCGTC-3' and 5'-ATTTGGCGTAGCTGTCGATGG-3' for CPT1A; 5'-GGCATTGGTCAGAATCTTTTCG-3' and 5'-ACTCCCATAAATGTCCCGCGA-3' for CPT1C. The cDNA was cloned to TOPO blunt vector (pCR4Blunt-TOPO; Invitrogen, Carlsbad, CA). The plasmid DNA was amplified in Escherichia coli using antibiotic selection according to the guidelines of the manufacturer. Antisense and sense RNA probes, serving as controls, were synthesized from 1 µg linearized plasmid DNA by T7 or T3 RNA polymerases in the presence of transcription mix, RNase inhibitor, and nucleotide mix (all from Roche) containing DIG-11-UTP. Synthesis of adequate amounts of the probe (10–15 µg) was confirmed by running 1-µL aliquot on 1% agarose gel. The remaining DNA was destroyed by RNase free-DNase I (Qiagen, Hilden, Germany). The resultant RNA probe was precipitated with LiCl and ethanol, and the pellet was redissolved in 50% formamide and stored at –20°C.

Nonradioactive in situ hybridization on sections was performed with automate (Discovery; Ventana Medical Systems, Tucson, AZ). In brief, samples were deparaffinized with heat treatment, followed by postfixation and pretreatment (RiboClear; Ventana). After enzymatic predigestion (proteinase K, 350 ng/µL; Roche) for 4 minutes, the sections were hybridized with antisense and sense probes for 6 hours at 55°C. Before detection, the sections were washed for three times with 0.1x SSC at 65°C or 70°C. The detection step included incubation with biotinylated anti-DIG antibody (Jackson ImmunoResearch Laboratory, West Grove, PA; diluted 1:2000) for 20 minutes followed by 1- to 1.5-hour incubation with BCIP/NBT alkaline phosphatase substrate for the color reaction. After hybridization, the sections were washed, dehydrated, and mounted (Mountex; HistoLab, Gothenburg, Sweden). Analysis and documentation of the results was performed with microscope (Axioplan 2; Carl Zeiss Microscopy, Jena, Germany) equipped with a camera (Axio Cam; Carl Zeiss Vision, Hallbergmoos, Germany). All reagents for the automate were provided by Ventana Medical Systems except for proteinase K (Roche).

ACAD9 immunohistochemistry of rat and human eyes was performed exactly as described earlier.6 Ocular immunohistochemistry with CPT1A antibodies was not performed because the antibodies showed nonspecific labeling in control tissues.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Human skeletal muscle and all human cell types cultured expressed all three isoforms of CPT1. The relative amounts of CPT1A-, CPT1B-, and CPT1C-specific mRNA differed from one cell line or tissue to another. Total expression of CPT1 and expression of each isoform was lower than the expression in fibroblasts, except for CPT1B in the skeletal muscle (Fig. 1A) . Calculating the percentage of each CPT1 isoform (total expression, 100%) revealed that CPT1A was the predominant isoform in fibroblasts, myoblasts, and ARPE19 cells. The major CPT1 isoform in skeletal muscle was CPT1B (Fig. 1B) .


Figure 1
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  		FIGURE 1. CPT1 isoform mRNA expression detected by QRT-PCR in cultured (c) or fresh (f) fibroblasts (FBL), myoblasts (MBL), skeletal muscle (SKM), and retinal pigment epithelial (RPE) cells. (A) Using the comparative 2–{Delta}{Delta}CT method of quantification, the RT-PCR results are presented as the fold change in CPT1A, CPT1B, and CPT1C gene expression, normalized to the endogenous reference gene GAPDH, relative to their expression in fibroblasts for which the expression of each isoform was set to 1. (B) Percentages of normalized CPT1A, CPT1B, and CPT1C expression of the total CPT1 expression in the corresponding samples. (C) Difference in threshold cycle ({Delta}ct) of the target gene versus the housekeeping gene GAPDH (set to 0); all {Delta}ct values were multiplied by –1 to show a positive shift in the {Delta}ct value with an increase in the amount of RNA.

 
A band corresponding to CPT1A was found by immunoblotting in rat RPE-J and R28 cells (results not shown), human fibroblasts, and ARPE19 cells (Fig. 2) . A double band was seen in ARPE19 cell and fibroblast samples, which probably resulted from different conformational states of CPT1A15 after different protocols used in the extraction of protein from tissue samples compared with cultured cells. Another explanation could be the different nutritional state of the donor animals.16 No band corresponding to CPT1B or CPT1C was detected in any of the cell lines studied (results not shown). A band corresponding to ACAD9 was found in ARPE19, RPE-J (not shown), and R28 cells (Fig. 3) .


Figure 2
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  		FIGURE 2. Immunoblot analysis of CPT1A and MTP protein expression with polyclonal rabbit antibodies raised to rat CPT1A and MTP. Five micrograms of protein extracted from human retinal pigment epithelial cells (ARPE), human fibroblasts (fibro), rat brain, and 1 µg rat liver sample was applied per lane. CPT1A and MTP {alpha}- and β-subunit expression were detected in all samples.

 

Figure 3
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  		FIGURE 3. Immunoblot analysis of ACAD9 and MTP protein expression with polyclonal rabbit antibodies raised to human ACAD9 and MTP. Three micrograms of protein extracted from fibro and 5 µg from ARPE and neural retina cells (R28) were applied per lane. ACAD9 and MTP {alpha}- and β-subunit expression were detected in all the samples.

 
By ISH of the rat eye, the most robust CPT1A expression was revealed in the retinal pigment epithelial cells and in the inner nuclear and ganglion cell layers of the neuroretina (Fig. 4) . The labeling pattern observed in the photoreceptor cell layer suggested expression of CPT1A in the microvilli of Müller cells rather than in the inner and outer segments of photoreceptors, whereas CPT1C expression was found in the inner segments of photoreceptors and in the outer nuclear layer of the retina (Fig. 4) . In the anterior segment of the eye, CPT1A expression was detected predominantly in the epithelium of the iris and the ciliary body (not shown). CPT1C was also detected in the ciliary epithelia. Despite several trials with various CPT1B probes, we could not achieve an acceptable quality of labeling.


Figure 4
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  		FIGURE 4. Nonradioactive in situ hybridization with digoxigenin-labeled RNA probes of rat retina: nfl, nerve fiber; gcl, ganglion cell; ipl and opl, inner and outer plexiform; inl and onl, inner and outer nuclear layers; is and os, inner and outer segments of photoreceptors; rpe, retinal pigment epithelium; ch, choroid. (A) CPT1A hybridization signal is found in rpe, inl, and gcl. Labeling at the level of the inner portion of the photoreceptor inner segments is consistent with labeling of Müller cell microvilli. (B) The CPT1C hybridization signal is found in the rpe, is, inl, and gcl. Signal is detected throughout the photoreceptor inner segments. Control sections are labeled with the respective sense probes. Labeling of onl in (A) and (B) is equivocal compared with control. Scale bar, 30 µm.

 
By immunohistochemistry, antibodies to ACAD9 labeled the inner limiting membrane, which corresponds to the basal lamina of Müller cells, ganglion cells, RPE, and inner segments of photoreceptor cells in the human retina (Fig. 5) , whereas only the RPE was labeled in the rat retina. In addition, the external muscles and ciliary nerves of the eye showed prominent labeling.


Figure 5
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  		FIGURE 5. Immunohistochemistry of the human retinal specimen stained with polyclonal rabbit antibody raised to human ACAD9 and control section with no primary antibody. ACAD9 antibody labeled inner limiting membrane; ilm, gcl, is, and rpe. Scale bar, 30 µm.

 

   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Retinal cells that are morphologically and functionally affected in the retinopathy associated with LCHAD deficiency are photoreceptor cells and retinal pigment epithelial cells.17 We detected RNA expression specific for CPT1A and CPT1C in the rat RPE, as well as in retinal ganglion cells and the inner nuclear layer, which are morphologically unaffected in LCHAD retinopathy. CPT1C and CPT1A expression was also found in the inner segments of photoreceptors and in the Müller cell microvilli located between them, respectively. Antibodies to ACAD9 labeled the rat and human RPE, the inner segments of human photoreceptor cells, and the human retinal ganglion cells. This labeling pattern largely paralleled the binding of MTP antibodies to the RPE, photoreceptor cells, inner nuclear layer, ganglion cells, and nerve fiber layer of the human retina.6 The outer retina, especially the photoreceptor cells, depend on oxidative phosphorylation,18 and CPT1 isoform expression and ACAD9 immunolabeling are in accordance with the location of mitochondria in the retina.19

Immunoblotting showed CPT1A and ACAD9 in cultured rat neural retina, and in rat and human retinal pigment epithelial cells. ACAD9 expression was more prominent than MTP labeling in R28 cells.

The difference detected in CPT1 isoform expression in QRT-PCR between cultured human myoblasts and fresh skeletal muscle implies that cell culture conditions and dedifferentiation of the cells in culture modify CPT1A RNA and, potentially, protein expression. In cell culture, a clear difference could be detected in protein expression between human fibroblasts and retinal pigment epithelial cells. CPT1 expression in the RPE is consistent with earlier findings of CPT1A in RNA extracted from the mouse (GEO GSE1133), rat (GSE2868), and human (GSE740) retina. CPT1B expression has been detected in the rat retina (GSE2868) and CPT1C expression in the mouse retina (GSE3554).

Detection of CPT1 and ACAD9 expression in cultured human RPE and neural retinal cells raises another question of their potential role in these cells. Three long-chain acyl-CoA dehydrogenases involved in the first step of mitochondrial FAO, namely ACAD9, very long-chain (VLCAD), and long-chain acyl-CoA (LCAD), have complementary expression patterns in different tissues.9 In vitro studies showed ACAD9 RNA particularly in the brain, and ISH demonstrated ACAD9 in human embryonic brain and retina.8 Interpretation of the RPE labeling was complicated by the strong pigmentation of the eye. In addition to the saturated fatty acids, ACAD9 is active against DHA,20 a polyunsaturated long-chain fatty acid highly abundant in membranes and especially enriched in photoreceptor outer segments. Our finding of CPT1 and ACAD9 in the human neuroretina suggests that these FAO enzymes may participate in its metabolism but not necessarily in energy production. RPE is known to perform numerous metabolic functions essential for the photoreceptors, including outer segment renewal. The newly detected role of CPT1C in the regulation of energy homeostasis in the central nervous system by an unknown mechanism21 shows that enzymes of β-oxidation have unexpected functions that do not involve energy production.

Even if mitochondrial fatty acid β-oxidation has a role in metabolism of the retina, it does not explain why the pigment retinopathy complicates only LCHAD and complete MTP deficiencies. Therefore, long-chain 3-hydroxy-intermediates accumulating in the blood and tissues may have a deleterious effect on retinal function. Thus far, there has been no experimental evidence to prove this hypothesis. Palmitate and its derivatives, enoyl-CoA palmitate and hydroxypalmitate compounds, inactivate adenine nucleotide transport, resulting in an oxidative phosphorylation defect in mitochondria.22 Because palmitate accumulates in VLCAD deficiency and retinopathy is not a feature of this disorder, a simple oxidative phosphorylation defect cannot be the sole pathogenic mechanism of the retinopathy in LCHAD and complete MTP deficiencies.

In conclusion, our study showed evidence of CPT1A and CPT1C expression at the RNA level by both ISH and QRT-PCR in the human and rat retina, particularly in the RPE, which is affected early in the retinopathy of LCHAD and complete MTP deficiencies. CPT1A expression could be verified at the protein level in cultured human retinal pigment epithelial cells, as could ACAD9 expression in retinal sections. We propose that mitochondrial β-oxidation has a role in the metabolism of the RPE and that the toxicity of 3-hydroxylated intermediates of long-chain fatty acids that accumulate in these deficiencies contribute to the pathogenesis of the associated retinopathy.


   Footnotes
 
Supported by grants from Helsinki Biomedical Graduate School, Helsinki, Finland; Oskar Öflund Foundation, Espoo Finland; Helsinki University Central Hospital Research Fund (TYH4115), Helsinki, Finland; the Arvo and Lea Ylppö Foundation, Espoo, Finland; Evald and Hilda Nissi Foundation, Espoo, Finland; and the Finnish Medical Foundation, Helsinki, Finland.

Submitted for publication August 21, 2007; revised November 16, 2007; accepted February 27, 2008.

Disclosure: E. Roomets, None; T. Kivelä, None; T. Tyni, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Eva Roomets, Department of Pediatric Neurology, Hospital for Children and Adolescents, Helsinki University Central Hospital, FI-00029 HUS, Helsinki, Finland; eva.roomets{at}helsinki.fi.


   References
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 References
 

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