Immunohistochemical localization of phospholipase D1 in the retina of pigs

Neuroscience Letters 397 (2006) 44–47

Immunohistochemical localization of phospholipase D1 in the retina of pigs Jeeyoung Lee a , Heechul Kim a , Meejung Ahn a , Do Sik Min b , Taekyun Shin a,∗ a

b

Department of Veterinary Medicine, Cheju National University, Aradong 1, Jeju 690-756, South Korea Department of Molecular Biology, College of Natural Science, Pusan National University, Busan 609-735, South Korea Received 2 November 2005; received in revised form 19 November 2005; accepted 28 November 2005

Abstract The expression of phospholipase D1 (PLD1) was examined in the retinas of pigs. Western blot analysis detected the expression of PLD1 in the retinas of 1-day-old piglets and showed that it was enhanced in the retinas of 2 years old adult pigs. Immunohistochemically, PLD1 was mainly immunostained in ganglion cell bodies in the ganglion cell layer, in some radial processes of Muller cells in the retinal layer and in the inner and outer segments of the rod and cone layer in newborn and adult pigs, but not in astrocytic bundles in nerve fiber layers. The immunoreactivity of PLD1 in the radial processes of Muller cells across the retinal layers was enhanced in adult pig retinas compared to those of newborn piglets. This was the first demonstration to show that PLD1 is constitutively expressed in the retina of pigs, implying that retinal PLD1 expression is enhanced in radial fibers of Muller cells with age. This finding suggests that PLD1 plays an important role in signal transduction of glial cells and neuronal cells in the retina. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Development; Phospholipase D1; Pig; Retina

Phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine (PC) to phosphatidic acid (PA) and choline [11,13]. Recently, PLD1 and PLD2, two mammalian isoforms of PLD, were cloned and characterized at the molecular level [11]. These isoforms perform distinct roles, such as apoptosis, membrane trafficking and secretory events [11–13]. PLD is known to be activated via a G-protein coupled receptor and tyrosine kinase receptors [3,7,18]. It is thus proposed that PLD plays an important role in a novel receptor-mediated signal transduction pathway in the nervous system. In neuronal tissue, the activity of PLD1 is regulated in a cell-specific manner by neuronal activity, neurotransmitters, hormones, growth factors, and cytokines [3,7,8]. In astrocytes, PLD1 activity is required for proper control of cell proliferation [9]. The porcine eye has been suggested as a novel model for human eye disease because the pig eye/retina shares many similarities with the human [2,15,16]. Although pig eyes have been used to study eye disease [15], little is known about the expression of PLD1 in the retina of pigs [2,16]. PLD1, an important

∗

Corresponding author. Tel.: +82 64 754 3363; fax: +82 64 756 3354. E-mail address: [email protected] (T. Shin).

0304-3940/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2005.11.051

enzyme both in normal development and under pathological conditions, has been found only in the Muller cells, but not in ganglion cells, of the adult rat retina [9]. The goal of the present study was to examine the expression and cellular localization of PLD1 in the retinas of pigs. The retinas of newborn (postnatal, 1 day old; n = 3) and adult pigs (2 years old; n = 3) were obtained from a local farm and slaughterhouse. The anterior segments of the eyeballs were removed, and the retinas were carefully dissected. For Western blot analysis, retinal tissues were kept in a deep freezer. After removing the anterior segments of the eyes, the eyecups were fixed by immersion in 10% neutral buffered formalin in 0.1 M phosphate buffered saline (PBS; pH 7.4) for 24 h and processed for routine paraffin embedding. Antisera were raised against the C-terminal peptide of PLD1 corresponding to amino acid residues 1063–1074 of the sequence of PLD1 (TKEAIVPMEVWT). For affinity purification of the antibodies, the peptide was coupled to Affi-Gel 15 (BioRad, Hercules, CA, USA) following the manufacturer’s instructions with slight modifications. Antiserum (6 ml) was incubated with peptide-conjugated Affi-Gel 15 (5 mg of peptide/ml of Affi-Gel 15) overnight at 4 ◦ C. The column was then washed with 20 ml of buffer (20 mM HEPES/NaOH [pH 7.0],

J. Lee et al. / Neuroscience Letters 397 (2006) 44–47

200 mM NaCl, and 0.1% Triton X-100), and the antibodies were eluted with 0.1 M glycine/HCl (pH 2.5) into tubes containing 1 M Tris–HCl (pH 8.0) for neutralization. This antiserum has been used in previous retina studies [9,10]. Retinal tissues were thawed in a lysis buffer (20 mM HEPES [pH 7.2], 1% Triton X-100, 1% deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 150 mM NaCl, 10 ␮g/ml leupeptin, 10 ␮g/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride). After incubation in an ice-cold bath, the homogenates were centrifuged, and the lysate supernatant was obtained. Equal amounts of the lysates (40 ␮g) were then dissolved in the SDS sample buffer and boiled. The homogenized tissue samples were electrophoresed under denaturing conditions in 8% SDS–polyacrylamide gel. The proteins were then electrotransferred in transfer buffer to a PROTRANR nitrocellulose transfer membrane (Schleicher and Schuell; Keene, NH, USA) for 2 h at 100 V. The residual binding sites on the membrane were blocked by incubation with 5% nonfat milk in Tris-buffered saline (TBS; 10 mM Tris–HCl [pH 7.4] and 150 mM NaCl) for 1 h and then incubated with rabbit anti-PLD1 antibody for 2 h. The blots were then washed three times in TBS containing 1% Tween-20 and probed with horseradish peroxidase-conjugated anti-rabbit IgG (Vector; Burlingame, CA, USA) for 1 h. The membrane blots were developed using an enhanced chemiluminescence reagent (ECL; Amersham Pharmacia Biotech, Uppsala, Sweden) kit according to the manufacturer’s instructions. Paraffin sections (5 ␮m) of retinas were deparaffinized and pretreated with citrate buffer (0.01 M, pH 6.0) in the microwave for 3 min. After hydrating, the sections were treated with 0.3% hydrogen peroxide in distilled water for 20 min to block endogenous peroxidase activity. After three washes in PBS, the sections were exposed to 10% normal goat serum and then incubated for 1 h at room temperature (RT) with polyclonal rabbit antiPLD1 antibody. After three washes, the sections were incubated with the appropriate biotinylated second antibody, followed by formation of the avidin–biotin peroxidase complexes using the Elite kit (Vector). The peroxidase reaction was developed with a diaminobenzidine substrate kit (Vector). Before mounting, the sections were counterstained with hematoxylin. As a control, the primary antisera were omitted for a few test sections in each experiment, and specific labeling of cell bodies or fibers was not detected in these sections (data not shown). For double immunofluorescence, the sections were incubated in the following order: 10% normal goat serum for 1 h at RT, rabbit anti-PLD1 antibody overnight at 4 ◦ C, and then fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit IgG (Sigma, St. Louis, MO, USA) for 1 h at RT. They were then washed and blocked with 10% normal goat serum for 1 h at RT, incubated with mouse monoclonal anti-glial fibrillary acidic protein (GFAP) overnight at 4 ◦ C, and washed and incubated with tetramethyl rhodamine isothiocyanate (TRITC)labeled goat anti-mouse IgG (Sigma). To reduce or eliminate lipofuscin autofluorescence, the sections were washed in PBS (three times for 1 h) at RT and then dipped briefly in distilled H2 O, treated with 10 mM CuSO4 in ammonium acetate buffer (50 mM CH3 COONH4 , pH 5.0) for 20 min, dipped briefly again in distilled H2 O, and returned to PBS. The double

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Fig. 1. Representative Western blots for phospholipase D1 (PLD1) in the retinas of newborn piglets (1 day old) and adult pigs (2 years old). The position of the PLD1 protein (120 kDa) is indicated by the arrow. The molecular sizes were determined from standard proteins.

immunofluorescence-stained specimens were examined under an FV500 laser confocal microscope (Olympus, Tokyo, Japan). Western blot analysis showed that PLD1 was detected in the whole homogenates of retinas of newborn piglets and adult pigs (Fig. 1). The approximate molecular weight of PLD1 was 120 kDa. The expression of PLD1 in adult pigs was more intense compared to that of newborn pigs. Histologically, the retinas of newborn piglets and adult pigs (2 years old) showed typical retinal layers, including an inner limiting membrane, ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL), outer nuclear layer (ONL), and rod and cone layer (RCL). The nerve fiber layer was clearly visualized in the adult retina. In the retina of newborn piglets, PLD1 was intensely immunostained in ganglion cell bodies in the GCL (Fig. 2A , arrowheads), some somata in the INL, and in the inner and outer segments of the RCL (Fig. 2A). In the adult porcine retina, PLD1 was clearly immunostained in ganglion cell bodies in the GCL (Fig. 2B, arrowheads). PLD1immunoreactivity was intense in the radial fibers originating from somata in the INL in the adult retina, suggesting that radial fibers expressing PLD1 increased in number in the adult retina. PLD1 was immunostained in the inner and outer segments of the RCL as well (Fig. 2B, arrows). A double-labeling experiment was performed to observe the cell phenotype of PLD1 expression in porcine retina. In newborn piglets (Fig. 3A–C ), PLD1 immunofluorescence (Fig. 3A) was detected in some ganglion cells (Fig. 3A–C, arrowhead) and in a few fibers in which few were co-localized with GFAP (Fig. 3B, C). PLD1 was weakly detected in other retinal layers in postnatal piglet retina (Fig. 3A). In the adult pig retina, PLD1 immunoreactive radial fibers were found to originate from the somata located in the middle of the INL (Fig. 3D). PLD1-positive radial fibers (Fig. 3D) crossing through the whole retinal layer were co-localized with GFAP (Fig. 3E), suggesting that both PLD1-positive and GFAPpositive elements comprise Muller cells (Fig. 3F). There were two types of GFAP-positive elements in the retina. One type was GFAP-positive and PLD1-negative, which may reflect astrocytic processes in the nerve fiber layer (Fig. 3E, asterisk). The other type was composed of both GFAP- and

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J. Lee et al. / Neuroscience Letters 397 (2006) 44–47

Fig. 2. Immunohistochemical analysis of PLD1 expression in the retinas of newborn (A) and adult pigs (B). (A) PLD1 was intensely immunostained in ganglion cell bodies (arrowheads) and in some cells in the INL in the retina of a newborn piglet. (B) PLD1 was immunostained in ganglion cell bodies (arrowheads), some radial fibers with increased intensity and inner and outer segments in the RCL (arrows) in adult pigs. Each retinal layer was marked on the right side: ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL), outer nuclear layer (ONL), and rod and cone layer (RCL). Counterstained with hematoxylin. Scale bars represent 30 ␮m.

PLD1-positive fibers in the IPL (Fig. 3F, arrows), which originated from the radial processes of Muller cells in the INL. This finding suggests that GFAP-positive, PLD1-negative bundles in retinal nerve fiber layer are distinct from both GFAP-positive and PLD1-positive fibers, which may originate from the Muller cells in the INL. This is the first study to demonstrate that PLD1 is expressed in the retina of a domestic animal. In the present study, we examined the expression of PLD1 in newborn and adult pigs and found that its expression was enhanced in adult animals. This finding suggests that PLD1 is gradually activated with age in pigs.

The localization of PLD1 in porcine retina is partly similar to those of rats and mice [9,10]. In the present study, we found that PLD1 was clearly immunostained in ganglion cell bodies in the GCL and Muller cells in the INL in the porcine retina, while PLD1 was detected in Muller cells in the INL, but not in ganglion cell bodies, in the retina of rat and mice [9]. As well, we found that increased PLD1 expression in adult pigs was associated with the retinal development after birth. In the adult retina, GFAP-positive radial fibers, particularly along the inner limiting membrane, were more prominent than those of newborn piglets. Considering that Muller cells are major supportive glial cells in

Fig. 3. Immunofluorescent co-localization of PLD1 (A and D) and GFAP (B and E) in the newborn (A–C) and adult porcine retina (D–F). C and F are merged images of newborn and adult pigs, respectively. In the newborn piglet (A–C), PLD1 (A, green) was intensely immunostained in ganglion cells (arrowhead) and a few radial fibers. GFAP was immunostained in some bundles in nerve fiber layer fibers and some fibers along the inner limiting membrane (B, red). Few PLD1- and GFAP-positive fibers were found in the retinal layers (C, merge). In the adult retina (D–F), PLD1 immunofluorescence (D, green) was evident in some ganglion cells (arrowhead) and radial fibers (arrows) that were also positive for GFAP (E, red, arrows; F, merge, arrows). These PLD1-positive and GFAP-positive processes and somata were regarded as Muller cells in the INL. GFAP-immunoreactive bundles in the nerve fiber layer (E, asterisks) were negative for PLD1 in (D). Scale bars represent 50 ␮m. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

J. Lee et al. / Neuroscience Letters 397 (2006) 44–47

the retina, PLD1 plays an important role in retinal function. In fact, Muller cells are involved in the clearance of extracellular glutamate via a glutamate transporter and in the synthesis of glutamine from glutamate via glutamine synthetase [14]. An interesting finding in the present study is that PLD1 was immunostained in the inner and outer segments of RCL, as previously reported in the outer segment of RCL in the rat retina [10]. In view of the presence of PLD1 in the RCL in bovine retina [17] as well as in rat [10] and porcine retina in the present study, we postulate that PLD1 plays an important role in the signal transduction of photoreceptor cells in retina. Recent studies have suggested that PLD1 is involved in the activation of astrocytes and macrophages in the retina with experimental autoimmune uveoretinitis [5] and in central nervous system disease models, including cryogenic head injury [6], spinal cord injury [4], and experimental autoimmune encephalomyelitis [1]. Considering the pathophysiological role of PLD1 in diseased CNS tissues described above, we postulate that PLD1 in the retina has a potential to stimulate Muller cells when the retina is damaged. The exact role of PLD1 in Muller cells needs elucidation. This study showed that PLD1 is constitutively expressed in retinal cells, including ganglion cells and Muller cells, and that its expression in Muller cells is enhanced with age. Our observations imply that with aging, PLD1 plays a role in the signal transduction pathway whether it is beneficial or detrimental in retinal cells. Acknowledgement This research was supported by the Program for the Training of Graduate Students in Regional Innovation which was conducted by the Ministry of Commerce Industry and Energy of the Korean Government. References [1] M. Ahn, D.S. Min, J. Kang, K. Jung, T. Shin, Increased expression of phospholipase D1 in the spinal cords of rats with experimental autoimmune encephalomyelitis, Neurosci. Lett. 316 (2001) 95–98. [2] M. Garcia, J. Ruiz-Ederra, H. Hernandez-Barbachano, E. Vecino, Topography of pig retinal ganglion cells, J. Comp. Neurol. 486 (2005) 361–372.

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