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OPA1 expression in the human retina and optic nerve
Experimental Eye Research 83 (2006) 1171e1178 www.elsevier.com/locate/yexer
OPA1 expression in the human retina and optic nerve An-Guor Wang a,b,c, Ming-Ji Fann c,d,e, Hsin-Yi Yu a,c, May-Yung Yen a,b,* a
Department of Ophthalmology, Taipei Veterans General Hospital, Taipei, Taiwan b Faculty of Medicine, National Yang-Ming University, Taipei, Taiwan c Institute of Neuroscience, National Yang-Ming University, Taipei, Taiwan d Faculty of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei, Taiwan e Brain Research Center, University System of Taiwan, Taipei, Taiwan Received 22 March 2006; accepted in revised form 9 June 2006 Available online 18 July 2006
Abstract Mutations in the optic atrophy type 1 (OPA1) gene give rise to human autosomal dominant optic atrophy. The purpose of this study is to investigate OPA1 protein expression in the human retina and optic nerve. A rabbit polyclonal antiserum was generated using a fusion protein covering amino acids 647 to 808 of the human OPA1 protein as the immunogenic antigen. Western blot and immunofluorescence staining were performed to examine OPA1 expression in the human retina and optic nerve. In human retina, we found that OPA1 expression was clearly present in retinal ganglion cells and photoreceptors. OPA1 immunoreactivity was also present in the nerve fiber layer, inner plexiform layer and outer plexiform layer. However, OPA1 protein was not detected in the choline acetyltransferase-positive, calretinin-positive, and calbindinpositive amacrine cells, nor in the calbindin-positive horizontal cells. In the human optic nerve, expression of OPA1 was present in the axonal tract that was labeled with neurofilament specific antibody. In conclusion, expression of OPA1 gene is present in the mitochondria-rich regions of the retina and optic nerve. This suggests that OPA1 protein might be involved in the functioning of the mitochondria that are present in both inner and outer retinal neurons. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: dominant optic atrophy; mitochondria; OPA1
1. Introduction Autosomal dominant optic atrophy (DOA) is the most common form of hereditary optic neuropathy and has a prevalence of 1:12,000 to 1:50,000 (Delettre et al., 2002); the disease has an insidious onset in early childhood. It is characterized by progressive loss of visual acuity, bilateral atrophy of the optic nerve, central visual field defects and color vision deficits (Johnston et al., 1999; Kline and Glaser, 1979; Votruba et al., 1998). This disease shows a highly variable manifestation, with the patient’s visual acuity usually between 6/9 and 6/60; but the acuity may be as good as 6/6 or as poor as light * Corresponding author. Department of Ophthalmology, Taipei Veterans General Hospital, No. 201, Section 2, Shih-Pai Road, Taipei 11217, Taiwan. Tel.: þ886 2 2875 7325; fax: þ886 2 2876 1351. E-mail address: [email protected] (M.-Y. Yen). 0014-4835/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.exer.2006.06.004
perception. There is considerable variation in the visual function among patients, even within the same family (Johnston et al., 1999; Votruba et al., 1998). Evidence from electrophysiological (Elenius, 1991; Smith, 1972) and histopathological studies (Johnston et al., 1979; Kjer et al., 1983) shows that the fundamental underlying defect is degeneration of the retinal ganglion cells (RGC) and this is followed by ascending optic atrophy. The gene responsible for DOA has been mapped to human chromosome 3q28-qter, and named Optic Atrophy Type 1 (OPA1) (Bonneau et al., 1995; Eiberg et al., 1994; Jonasdottir et al., 1997; Lunkes et al., 1995; Votruba et al., 1997). The OPA1 gene spans more than 70 kb, is composed of 31 exons, and encodes a dynamin-related guanosine triphosphatase (GTPase) with 960 amino acids (Alexander et al., 2000; Delettre et al., 2000). Ninety-eight different mutations of the OPA1 gene have been identified in DOA patients (Alexander
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et al., 2000; Baris et al., 2003; Delettre et al., 2000, 2001; Pesch et al., 2001; Puomila et al., 2005; Toomes et al., 2001). Although precise function of the OPA1 protein is not completely clear, the fact that its protein sequence is similar to those of the yeast Msp1 and Mgm1p proteins and evidence gathered from perturbation experiments suggest that it may act as a factor maintaining the integrity and function of mitochondria within cells (for review see Frank, 2006). It has been demonstrated that cells overexpressing wild-type or mutant forms of the OPA1 protein exhibit fragmented mitochondria clustering near the nucleus (Griparic et al., 2004). Loss of OPA1 expression in cultured cells by small interfering RNA leads to swelling and stretch of mitochondria at the first stage and dispersal of fragmented mitochondria throughout the cytosol at the later stage, which is followed by cytochrome c release and apoptotic events (Griparic et al., 2004; Lee et al., 2004; Olichon et al., 2003). It is likely that OPA1 regulates the processes of mitochondrial fusion and fission. OPA1 is ubiquitously expressed in all human tissue when they are examined by Northern blot (Alexander et al., 2000; Delettre et al., 2000). The highest transcript level is found in retina, followed by the brain, testis, heart and skeletal muscle (Alexander et al., 2000). In the mammalian retina, OPA1 protein has been reported to be expressed in the RGC, amacrine cells, horizontal cells, the inner plexiform layer (IPL), and the outer plexiform layer (OPL) (Aijaz et al., 2004; Ju et al., 2005; Pesch et al., 2004). It was also found that OPA1 is expressed in the axonal mitochondria of the optic nerve (Ju et al., 2005). Nevertheless, inconsistent OPA1 expression was reported by different research groups. Aijaz et al. (2004) observed OPA1 in the RGC layer and inner nuclear layer (INL) of mouse and human retina. Ju et al. (2005) reported that OPA1 immunoreactivity is present in RGC and horizontal cells in adult rat retina. Pesch et al. (2004) described staining of OPA1 protein not only in RGC, but also in starburst amacrine cells and horizontal cells of the adult mouse and rat retina. Thus, whether OPA1 is expressed in the amacrine cells or horizontal cell of mammalian retina remains controversial. In the optic nerve, Ju et al. (2005) found that OPA1 protein is expressed in the entire optic nerve of adult rat. However, there was no detectable immunoreactivity in the optic nerve of the adult rat in the study by Pesch et al. (2004). In human tissue, Aijaz et al. (2004) observed that OPA1 is expressed exclusively in the myelinated region beyond the lamina cribrosa in the optic nerve. Thus, it is not clear whether OPA1 is expressed in the prelaminar portion of mammalian optic nerve. Furthermore, investigation of OPA1 expression in human ocular tissues has been rarely carried out (Aijaz et al., 2004). To understand the exact cellular expression pattern of OPA1 in normal human retina and to explore whether OPA1 expresses in the unmyelinated prelaminar zone of human optic nerve, we generated a polyclonal antiserum against OPA1 protein and examined the cellular expression pattern of OPA1 in the retina and optic nerve of adult human eyes in conjunction with various other immunological markers.
2. Materials and methods All experiments were performed according to institutional protocol guidelines. Experiments involving human tissues conformed to the guidelines set forth in the Declaration of Helsinki for the use of human tissue in research. The Institutional Review Board of the Taipei Veterans General Hospital approved the use of the donated eyeballs for this research. Three donated eyeballs used in immunofluorescent staining were obtained from three donors with no history of ocular disease (41-year-old male, 69-year-old male and 78-year-old male). Human retinal protein used in Western blot was obtained from another eye of the 41-year-old donor. 2.1. Reverse transcription Human brain RNA was purchased from Clontech (Palo Alto, CA, USA). Three micrograms of RNA in 10 ml was denatured at 75 C for 5 min. Reverse transcription was performed in a total volume of 20 ml containing 50 mM Trise HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 0.5 mM dNTP, 0.5 mg oligo(dT), 20 U of RNasin (Promega, Madison, WI, USA), and 100 U of Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA) for 1.5 h at 50 C. Samples were then stored at 20 C until use. 2.2. Preparation of fusion protein The cDNA (495 bp) encoding amino acid 647 to amino acid 808 of human OPA1 was amplified by PCR. Amplification of cDNA was conducted in a thermal cycler (Perkin Elmer 9700) with a 25 ml mixture containing 1 ml of adult human retina cDNA, 1 PCR buffer, 0.5 U high-fidelity Taq DNA polymerase (Roche, Mannheim, Germany), 0.25 mM dNTP, and one set of primers (100 nM). The forward primer was 50 GGAACTTTTAACACCACAGTG-30 and the reverse primer was 50 -ATAAGCTGGGTGCTCCTCAT-30 . The following conditions were used for the PCR: 35 cycles of denaturation at 94 C for 15 s; annealing at 55 C for 30 s, extension at 68 C for 30 s and finally, termination extension at 68 C for 5 min. The PCR products were then digested with BamHI and XhoI restriction enzymes and cloned into pET29a vector, which had been digested with the same enzymes. Clones that contained the correct insert were selected and sequenced to confirm the insert. Fusion protein was induced by 1 mM IPTG and the protein purified by passing through a metal chelating column following manufacturer’s protocol (Novagen, Darmstadt, Germany). 2.3. Antibodies To generate OPA1 polyclonal antiserum, the fusion proteins were injected into an adult New Zealand rabbit. Booster injections were given subsequently. Sera were collected every week from the fourth week after first injection and their titers were analyzed by dot blot assay. The resulting antiserum against
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OPA1 was used at a 1:200 dilution for immunofluorescence staining and at a 1:6000 dilution for Western blotting. Other primary antibodies were anti-cytochrome c monoclonal antibody (1:500 dilution, BD PharMingen, San Jose, CA, USA), mouse anti-neurofilament 200 kDa monoclonal antibody (1:500 dilution), goat anti-ChAT polyclonal antibodies (1:100 dilution), mouse anti-calretinin monoclonal antibody (1:100 dilution), mouse anti-rhodopsin monoclonal antibody (1:300 dilution) (Chemicon International, Temecula, CA, USA), anti-glial fibrillary acidic protein monoclonal antibody (1:200 dilution, SigmaeAldrich, Saint Louis, MO, USA), and mouse anti-calbindin monoclonal antibody (1:450 dilution, Swant, Bellinzona, Switzerland). A hybridoma clone that secretes an IgM monoclonal antibody against Thy-1 was purchased from American Type Culture Collection (Manassas, VA, USA), and twofold-diluted conditioned medium was used for immunofluorescence staining. The secondary antibodies used were Alexa Fluor 488 goat anti-rabbit antibodies (at 1:600 dilution, Molecular Probes, Eugene, OR, USA), Rhodamine (TRITC)-conjugated goat anti-mouse IgG antiserum and Cy3-conjugated donkey antigoat IgG antiserum (the latter two both at 1:600 dilution, Jackson ImmunoResearch, West Grove, PA, USA), FITC-conjugated goat anti-mouse IgM antiserum (at 1:600 dilution, Bethyl, Montgomery, TX, USA), and rhodamine-conjugated goat anti-rabbit antibodies (at 1:600 dilution, Chemicon International). 2.4. Subcellular localization of OPA1 HeLa cells were cultured at 37 C with Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum in a 5% CO2 incubator. Immunolabeling was performed overnight at 4 C in phosphate-buffered saline (PBS) containing 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4, and 5% bovine serum albumin (BSA) using anti-OPA1 antiserum and anti-cytochrome c monoclonal antibody as the primary antibodies. Cells were next incubated with the matched secondary antibodies for 2 h. Each step was preceded by washing in PBS. Specimens were analyzed using a Nikon Diaphot inverted fluorescence microscope (E300, Nikon, Tokyo, Japan) equipped with a MicroMax cool CCD (Princeton Instrument, Trenton, NJ, USA). Image acquisition was carried out by MetaMorph software (Universal Image Corp., Downingtown, PA, USA) with individual filter sets for each channel and photomicrographs were assembled using Adobe PhotoShop (Adobe Systems, San Jose, CA, USA). 2.5. Immunofluorescence staining of human retina and optic nerve For the human tissue sections, the posterior segments of three normal donated eyeballs were processed after removal of the corneal buttons for cornea transplantation. These posterior segments were fixed in 4% paraformaldehyde/PBS solution at 4 C overnight. The eyeballs were then transferred to
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30% sucrose/PBS overnight. The eyeballs were then divided into small pieces, submerged in OCT compound, and frozen using dry ice. Sections were cut 12 mm thick and collected on SuperFrost slides (Fisher Scientific, Pittsburgh, PA, USA). Sections were stored at 20 C until use. Sections were fixed with 4% paraformaldehyde for 20 min, then pretreated with 0.1% trypsin for 10 min at room temperature. They were then incubated with 0.2% Triton X-100 in PBS for 10 min, and then with 5% BSA in PBS for 60 min at room temperature. Sections were incubated in the primary antibodies, diluted in 5% BSA/PBS, overnight at 4 C. Finally, they were incubated with the matched secondary antibodies for 2 h at room temperature. Each step was preceded by washes in PBS. To stain the nucleus, the sections were incubated with 0.16 mg/ml 40 ,6-diamidino-2-phenylindole (DAPI) for 15 min at room temperature. The sections were examined by fluorescence microscopy. 2.6. Western blot of OPA1 protein For the human tissue, retina was dissected out from the posterior segments of a normal donated eyeball under balanced salt solution after removal of the corneal buttons for cornea transplantation. Mouse tissues were obtained from adult Balb/C mice. Tissues were homogenized in 10 volumes of lysis buffer. Samples (50 mg of protein) of tissue or recombinant proteins were loaded with an equal volume of 2 loading buffer (125 mM TriseHCl, pH 6.8; 4% SDS; 20% glycerol and 10% b-mercaptoethanol), boiled for 5 min and resolved by SDSepolyacrylamide gel (5% for tissue protein, 10% for recombinant protein) electrophoresis. Gels were electroblotted onto Immobilon-P membranes (Millipore, Bedford, MA, USA) in 25 mM Tris HCl, pH 8.3 containing 192 mM glycine, 20% methanol. Membranes were blocked in PBS/0.1% Tween (PBST) containing 10% fat-free dry milk (Carnation) for 1 h. Primary antisera were added overnight in PBST with 1% dry milk. After washing, HRP-conjugated goat anti-rabbit antiserum (Bethyl, Montgomery, TX, USA, 1:4,000) was incubated for 1 h. Immunoreactive bands were detected by enhanced chemiluminescence (ECL, Amersham Pharmacia, Buckinghamshire, UK) followed by exposure to Hyperfilm MP (Amersham Pharmacia). 3. Results 3.1. Characterization of the OPA1 protein A rabbit polyclonal antiserum was raised against a peptide corresponding to amino acids 647 to 808 (peptide OPA1647e808) of the predicted human OPA1 protein. The specificity of this antiserum (anti-OPA1) was first verified by Western blotting. The antiserum recognized a 24-kDa band but no other bacterial proteins when total proteins from the Escherichia coli strain over-expressing the peptide OPA1647e808 were analyzed (Fig. 1A). Pre-incubation of the antiserum with purified OPA1647e808 peptide completely abolishes the 24-kDa band (right panel, Fig. 1A), suggesting that the
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Fig. 1. (A) A Western blot of OPA1 protein. Anti-OPA1 antiserum recognized a 24-kDa band during the total proteins from E. coli over-expressing the OPA1647e808 peptide. Preincubation of the antiserum with purified peptide OPA1647e808 completely abolishes the 24-kDa band. (B) In various tissues, the molecular weight of the recognized protein was about 97 kDa. This 97-kDa band was present abundantly in the human retina, mouse retina, and mouse brain. There was also another band with an approximate molecular weight of 111 kDa present in the mouse tissues that was recognized by the anti-OPA1 antiserum. The expression of the OPA1 bands was blocked by preincubation of the anti-OPA1 antiserum with purified peptide OPA1647e808, which indicates the specificity of the anti-OPA1 antiserum. (C) Immunocytochemistry of OPA1 protein. HeLa cells were stained with anti-OPA1 antiserum (green), anti-cytochrome c antibody (red), and DAPI (blue). The expression of OPA1 in HeLa cell was co-localized with the expression of cytochrome c (C1e4), and this signal could be blocked by preincubation of anti-OPA1 antiserum with the peptide OPA1647e808 (C5e8). Scale bar in C is 10 mm.
antiserum recognizes OPA1647e808 specifically. We then used this antiserum to examine expression of OPA1 in human retina, mouse retina and mouse brain by Western blotting. A major band of approximately 97 kDa was detected in all the tissues analyzed. Another band with molecular weight of 111 kDa was also detected in the mouse tissues (Fig. 1B). The 111-kDa band may represent the un-processed immature OPA1 protein as the predicted size of OPA1 protein is 111 kDa. The 97-kDa band is likely the mature form of the OPA1 protein, as suggested by previous studies (Aijaz et al., 2004; Herlan et al., 2003). No protein was detected in the Western blot when the anti-OPA1 antiserum was pre-incubated with peptide OPA1647e808 (Fig. 1B, right panel) or without addition of the primary antiserum (data not shown), indicating that the reactivity against these two bands by the anti-OPA1 antiserum was specific. Previous studies have shown that expression of OPA1 occurs in the mitochondria. Thus, we examined whether the anti-OPA1 antiserum that we had generated could also detect OPA1 proteins in the mitochondria. HeLa cells, which express
OPA1 endogenously, were incubated with anti-OPA1 antiserum. A typical tubular interconnected mitochondrial network stained by anti-cytochrome c antibody was revealed by the anti-OPA1 antiserum (Fig. 1C). In a control experiment, no staining signal was observed when cells were incubated with anti-OPA1 antiserum in the presence of the peptide OPA1647e808 (Fig. 1C). A negative control, without addition of the anti-OPA1 antiserum, did not detect any immunoreactivity in the HeLa cells (data not shown). Taken together, these results indicate that the anti-OPA1 antiserum used in this study recognizes OPA1 proteins specifically. 3.2. OPA1 in human retina To determine the precise cellular location of OPA1 expression in the adult human retina and optic nerve, sections were incubated with the anti-OPA1 antiserum together with various retinal cell-specific markers. When the primary antibody was omitted, as a negative control for the immunofluorescence staining, there was no signal detected by the secondary
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antibodies in the human retina and optic nerve (data not shown). The OPA1 staining signal in the sections was competed out when the anti-OPA1 antiserum was pre-incubated with peptide OPA1647e808 (data not shown). Sections from peripapillary retina and optic nerve were examined from the prepared blocks such that a thick layer of optic nerve fibers overlying the RGC could be observed. We found that the expression of OPA1 was abundantly detected in the nerve fiber layer (NFL) and RGC (Fig. 2A). OPA1 immunoreactivity was also observed in the inner plexiform layer (IPL), the outer
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plexiform layer (OPL) and inner segment of photoreceptor (Fig. 2A). Under high power, the expression of OPA1 was co-localized with 200-kDa neurofilament protein in the nerve fiber layer (Fig. 2B). When the sections were double stained with GFAP, a glial cell marker, immunoreactivity of OPA1 was present in the whole thickness of the nerve fiber layer, while GFAP was stained as thin strands within the axonal tracts and did not co-localize with OPA1 (Fig. 2B). In the RGC layer, we used Thy-1 protein as the marker for RGC and the results showed that Thy-1-positive cells expressed
Fig. 2. OPA1 expression in human retina. Normal retina of an adult human was stained with anti-OPA1 antiserum (green), DAPI (blue), and various antibodies (red). (A) The expression of OPA1 was distributed in the nerve fiber layer (NFL) and RGC layer (GCL), inner plexiform layer (IPL), outer plexiform layer (OPL), and inner segment (IS) of photoreceptor of retina. (B) In the NFL, the expression of OPA1 protein was co-localized with the expression of neurofilament protein (NF) in the axonal tracts (B1e3), while OPA1 expression was not co-localized with GFAP, which is expressed as thin fibers intermingled among the axonal tracts (B4e6). (C) In the RGC layer, OPA1 immunoreactivity was detected in the RGCs (GC), which was labeled by anti-Thy-1 antibody (C1e3). Displaced amacrine cells (AC) in the RGC layer were labeled with the antibody against choline acetyltransferase (ChAT), but not the OPA1 antiserum (C4e6). (D) Amacrine cells in the inner nuclear layer (INL) were labeled with anti-calretinin antibody and anti-calbindin antibody, but were negative for OPA1 expression (D1e6). Horizontal cells (HC) labeled by calbindin were also negative for OPA1 expression (D4e6). (E) There was OPA1 immunoreactivity in the outer nuclear layer (E2e3, E5e6). The inner segment (IS) of photoreceptor is also labeled by anti-OPA1 antiserum (E2e3, E5e6). The signal is aligned with the rod outer segment labeled by antirhodopsin antibody (E4e6), but not well co-localized with cone cells marked by calbindin (E1e3). Scale bar in A is 100 mm, and in B is 25 mm.
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OPA1 protein (arrows in Fig. 2C). To examine the possibility of OPA1 expression in the amacrine cells, we used anti-OPA1 antiserum along with antibodies against choline acetyltransferase (ChAT), calbindin, and calretinin. In the RGC layer, displaced amacrine cells (marked by ChAT) did not express OPA1 protein (Fig. 2C). Amacrine cells in the superficial inner nuclear layer expressed calretinin and/or calbindin, but did not express OPA1 protein (Fig. 2D). Horizontal cells, marked by calbindin in the inner nuclear layer, did not express OPA1 protein (Fig. 2D). In the outer retina, OPA1 expression was present in the outer nuclear layer (Fig. 2E). The inner segment of rod photoreceptors was also labeled by the anti-OPA1 antiserum. The signal was aligned with, but not co-localized to the outer segment marked by anti-rhodopsin antibody, which labels rod cells (Fig. 2E). Moreover, the expression of OPA1 protein did not co-localized with calbindin, a cone cell marker (Fig. 2E). A similar pattern of OPA1 expression was observed in retinas of all three of the donated normal human eyeballs studied here. 3.3. OPA1 in human optic nerve head We next determined whether OPA1 is expressed in the human optic nerve. Longitudinal sections of adult human optic nerve stained with the anti-OPA1 antiserum and showed intense OPA1 immunoreactivity. To examine the precise expression pattern of OPA1, sections were double stained with antibodies against GFAP (an astrocyte marker) and against 200-kDa neurofilament protein (an axonal marker). In a lowpower field, OPA1 protein expressed in the prelaminar and
lamina cribrosa regions of the optic nerve head and in the optic nerve beyond the lamina cribrosa (Fig. 3A). At higher magnification of the prelaminar portion (Fig. 3B), the staining of OPA1 protein showed a filamentous pattern and OPA1 was co-localized with 200-kDa neurofilament protein (Fig. 3B). However, OPA1 did not co-localized with GFAP protein, which was manifested as multiple parallel thin fibers along the axons (Fig. 3B). In the lamina cribrosa (LC), expression of OPA1 was in the axonal fibers, which were marked by 200-kDa neurofilament protein (Fig. 3C). Again, the expression of OPA1 was not co-localized with GFAP (Fig. 3C). 4. Discussion OPA1 mutations are responsible for DOA, the most common form of hereditary optic neuropathy (Alexander et al., 2000; Delettre et al., 2000, 2002; Eiberg et al., 1994; Jonasdottir et al., 1997; Votruba et al., 1997). However, the expression pattern of OPA1 protein in the human retina and optic nerve is not well characterized. To assess the expression of OPA1 in the human retina and optic nerve, we first prepared an antiOPA1 antiserum and used this antiserum to visualize the distribution of OPA1 protein. In comparison to previous studies that utilized antisera against synthesized short peptides of OPA1 to detect its expression in mammalian retinas, an antiserum against a larger fusion protein covering amino acid 647 to amino acid 808 of human OPA1 was used in this study. This was in expectation that antibodies thus generated would show greater binding and higher specificity against OPA1. Two bands with apparent molecular weights of 111 kDa and
Fig. 3. OPA1 expression in human optic nerve. Normal optic nerve of adult human was stained with anti-OPA1 antiserum (green), anti-GFAP antibody and/or antineurofilament antibody (red), and DAPI (blue). (A) OPA1 protein was expressed in a filamentous pattern from the prelaminar region (PL), through lamina cribrosa (LC), to the myelinated optic nerve (ON) beyond the lamina cribrosa. There were small blood vessels (BV) in the prelaminar portion of the optic nerve head. (B) In the high-power fields of the prelaminar region, the expression of OPA1 protein was co-localized with neurofilament protein (B1e3), but not with GFAP (B4e6). (C) In the lamina cribrosa, OPA1 expresses in the axonal tracts between the glial trabeculae of the cribriform plate and co-localized with neurofilament protein (C1e3). The expression of GFAP does not co-localize with the expression of OPA1 (C4e6). Scale bar in A is 100 mm, and in B is 50 mm.
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97 kDa were detected in mouse tissue suggesting that the antiserum recognizes unprocessed OPA1 (predicted size of 111 kDa) and mature OPA1 (the signal-sequence cleaved form with a predicted size of 99 kDa). In the previous studies, two bands have also been detected by antisera against short peptides (McQuibban et al., 2003; Misaka et al., 2002). Several studies have reported OPA1 expression in the mammalian retina. We noted that discrepancies concerning OPA1 expression in retinal interneuron exist among these previous studies (Aijaz et al., 2004; Ju et al., 2005; Pesch et al., 2004). As our antiserum also recognizes mouse OPA1 (Fig. 1B), mouse retina was examined with this antiserum. We found that OPA1 expression is in mouse calretinin-positive amacrine cell, starburst amacrine cell, and horizontal cell, in addition to RGC and photoreceptors (data not shown). In human retina, we observed OPA1 immunoreactivity in RGC and photoreceptors. The detection of OPA1 immunoreactivity in the human photoreceptors has not been reported previously. Nevertheless, we did not observe OPA1 immunoreactivity in the calretinin-positive or calbindin-positive amacrine cells, nor in the ChAT-positive starburst amacrine cells of the human retina. Furthermore, we did not observe OPA1 expression in calbindin-positive horizontal cells. Thus, amacrine cells and horizontal cells, both involved in the lateral signal processing, do not express OPA1 protein in human retina based on our study. Since we used the same antiserum in two species, the discrepancy of OPA1 expression between mouse and human retina is clearly due to the species difference. A significant staining of OPA1 in inner segment of photoreceptor was observed in this study. Double staining results indicate OPA1 protein is expressed in the inner segment of rod photoreceptor, which is well aligned with the rod outer segment, marked by rhodopsin. As OPA1 is a mitochondrial protein, the expression of OPA1 in the mitochondria-rich inner segment suggests OPA1 protein may play a role in the mitochondrial function of the retinal neurons. Inconsistent results of OPA1 expression in the mammalian optic nerve have been reported in literature (Aijaz et al., 2004; Ju et al., 2005; Pesch et al., 2004). In our study, OPA1 is expressed both in the prelaminar and laminar portion of the adult human optic nerve and mouse optic nerve (data not shown). Thus, we propose that RGCs express OPA1 protein in the soma and the protein extends along the axons within the nerve fiber layer, to the optic nerve. Within the optic nerve, it is expressed all the way, from the prelaminar portion, through the lamina cribrosa to the retrolaminar myelinated portion. The expression of OPA1 is co-localized with the expression of neurofilament protein, but not with the expression of GFAP, suggesting the OPA1 is distributed within the axonal tract but not in the astrocytes. The precise function of OPA1 protein in the retina is not completely clear. The inner segment of photoreceptors, the nerve fiber layer of retina and optic axons within the optic nerve, are all mitochondria rich regions of the eye. In this study, we also clearly observed that OPA1 protein is expressed in the mitochondria rich region of retinal neurons. Taken together, these observations support the scenario that OPA1
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protein has a specific role in the mitochondrial function of both inner and outer retinal neurons. Based on the results described here, the role of OPA1 protein in the mitochondrial function of retinal neurons, and the mechanism how it induces RGC degeneration in autosomal dominant optic atrophy is worth further investigation. Acknowledgements This work was supported by grants of Taipei Veterans General Hospital ((VGH 93-327), and National Science Council (NSC 92-2314-B-075-046; NSC 93-2314-B-075-003) to M.-Y.Y, and VGH-UST (VGHUST 94-P1-13) to A.-G.W. References Aijaz, S., Erskine, L., Jeffery, G., Bhattacharya, S.S., Votruba, M., 2004. Developmental expression profile of the optic atrophy gene product: OPA1 is not localized exclusively in the mammalian retinal ganglion cell layer. Invest. Ophthalmol. Vis. Sci. 45, 1667e1673. Alexander, C., Votruba, M., Pesch, U.E.A., Thiselton, D.L., Mayer, S., Moore, A., Rodriguez, M., Kellner, U., Leo-Kottler, B., Auburger, G., Bhattacharya, S.S., Wissinger, B., 2000. OPA1, encoding a dynaminrelated GTPase, is mutated in autosomal dominant optic atrophy linked to chromosome 3q28. Nat. Genet. 26, 211e215. Baris, O., Delettre, C., Amati-Bonneau, P., Surget, M.O., Charlin, J.F., Catier, A., Derieux, L., Guyomard, J.L., Dollfus, H., Jonveaux, P., Ayuso, C., Maumenee, I., Lorenz, B., Mohammed, S., Tourmen, Y., Bonneau, D., Malthiery, Y., Hamel, C., Reynier, P., 2003. Fourteen novel OPA1 mutations in autosomal dominant optic atrophy including two de novo mutations in sporadic optic atrophy. Hum. Mutat. 21, 656. Bonneau, D., Souied, E., Gerber, S., Rozet, J.M., D’Haens, E., Journel, H., Plessis, G., Weissenbach, J., Munnich, A., Kaplan, J., 1995. No evidence of genetic heterogeneity in dominant optic atrophy. J. Med. Genet. 32, 951e953. Delettre, C., Lenaers, G., Griffoin, J.M., Gigarel, N., Lorenzo, C., Belenguer, P., Pelloquin, L., Grosgeorge, J., Turc-carel, C., Parret, E., Astarie-Dequeker, C., Lasquellec, L., Arnaud, B., Ducommun, B., Kaplan, J., Hamel, C., 2000. Nuclear gene OPA1, encoding a mitochondrial dynamin-related protein, is mutated in dominant optic atrophy. Nat. Genet. 26, 207e210. Delettre, C., Griffoin, J.M., Kaplan, J., Dollfus, H., Lorenz, B., Faivre, L., Lenaers, G., Belenguer, P., Hamel, C.P., 2001. Mutation spectrum and splicing variants in the OPA1 gene. Hum. Genet. 109, 584e591. Delettre, C., Lenaers, G., Pelloquin, L., Belenguer, P., Hamel, C.P., 2002. OPA1 (Kjer type) dominant optic atrophy: a novel mitochondrial disease. Mol. Genet. Metab. 75, 97e107. Eiberg, H., Kjer, B., Kjer, P., Rosenberg, T., 1994. Dominant optic atrophy (OPA1) mapped to chromosome 3q region. I. Linkage analysis. Hum. Mol. Genet. 3, 977e980. Elenius, V., 1991. Rod thresholds in dominantly inherited juvenile optic atrophy. Ophthalmologica 202, 208e212. Frank, S., 2006. Dysregulation of mitochondrial fusion and fission: an emerging concept in neurodegeneration. Acta. Neuropathol. (Berl.) 111, 93e100. Griparic, L., van der Wel, N.N., Orozco, I.J., Peters, P.J., van der Bliek, A.M., 2004. Loss of the intermembrane space protein Mgm1/OPA1 induces swelling and localized constrictions along the lengths of mitochondria. J. Biol. Chem. 279, 18792e18798. Herlan, M., Vogel, F., Bornhovd, C., Neupert, W., Beichert, A.S., 2003. Processing of Mgm1 by the Thomboid-type protease Pcp1 is required for maintenance of mitochondrial morphology and of mitochondrial DNA. J. Biol. Chem. 278, 27781e27788. Johnston, P.B., Gaster, R.N., Smith, V.C., Tripathi, R.C., 1979. A clinicopathologic study of autosomal dominant optic atrophy. Am. J. Ophthalmol. 88, 868e875.
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Johnston, R.L., Seller, M.J., Behnam, J.T., Burdon, M.A., Spalton, D.J., 1999. Dominant optic atrophy. Refining the clinical diagnostic criteria in light of genetic linkage studies. Ophthalmology 106, 123e128. Jonasdottir, A., Eiberg, H., Kjer, B., Kjer, P., Rosenberg, T., 1997. Refinement of the dominant optic atrophy locus (OPA1) to a 1.4-cM interval on chromosome 3q28-3q29, within a 3-Mb YAC contig. Hum. Genet. 99, 115e120. Ju, W.K., Misaka, T., Hkushnareva, Y., Nakagomi, S., Agarwal, N., Kubo, Y., Lipton, S.A., Bossy-Wetzel, E., 2005. OPA1 expression in the normal rat retina and optic nerve. J. Comp. Neurol. 488, 1e10. Kjer, P., Jensen, O.A., Klinken, L., 1983. Histopathology of the eye, optic nerve and brain in a case of dominant optic atrophy. Acta. Ophthalmol. 61, 300e312. Kline, L.B., Glaser, J.S., 1979. Dominant optic atrophy. The clinical profile. Arch. Ophthalmol. 97, 1680e1686. Lee, Y.J., Jeong, S.Y., Karbowski, M., Smith, C.L., Youle, R.J., 2004. Roles of the mammalian mitochondrial fission and fusion mediators Fis1, Drp1, and Opa1 in apoptosis. Mol. Biol. Cell. 15, 5001e5011. Lunkes, A., Hartung, U., Magarino, C., Rodriguez, M., Palmero, A., Rodriguez, L., Heredero, L., Weissenbach, J., Weber, J., Auburger, G., 1995. Refinement of the OPA1 gene locus on chromosome 3q28-q29 to a region of 2-8 cM, in one Cuban pedigree with autosomal dominant optic atrophy type Kjer. Am. J. Hum. Genet. 57, 968e970. McQuibban, G.A., Saurya, S., Freeman, M., 2003. Mitochondrial membrane remodeling regulated by a conserved rhomboid protease. Nature 423, 537e541. Misaka, T., Miyashita, T., Kubo, Y., 2002. Primary structure of a dynaminrelated mouse mitochondrial GTPase and its distribution in brain, subcellular localization, and effect on mitochondrial morphology. J. Biol. Chem. 277, 15834e15842.
Olichon, A., Baricault, L., Gas, N., Guillou, E., Valette, A., Belenguer, P., Lenaers, G., 2003. Loss of OPA1 perturbates the mitochondrial inner membrane structure and integrity, leading to cytochrome c release and apoptosis. J. Biol. Chem. 278, 7743e7746. Pesch, U.E., Leo-Kottler, B., Mayer, S., Jurklies, B., Kellner, U., ApfelstedtSylla, E., Zrenner, E., Alexander, C., Wissinger, B., 2001. OPA1 mutations in patients with autosomal dominant optic atrophy and evidence for semidominant inheritance. Hum. Mol. Genet. 10, 1359e1368. Pesch, U.E.A., Fries, J.E., Bette, S., Kalbacher, H., Wissinger, B., Alexander, C., Kohler, K., 2004. OPA1, the disease gene for autosomal dominant optic atrophy, is specifically expressed in ganglion cells and intrinsic neurons of the retina. Invest. Ophthalmol. Vis. Sci. 45, 4217e4225. Puomila, A., Huoponen, K., Mantyjarvi, M., Hamalainen, P., Paananen, R., Sankila, E.M., Savontaus, M.L., Somer, M., Nikoskelainen, E., 2005. Dominant optic atrophy: correlation between clinical and molecular genetic studies. Acta. Ophthalmol. Scand. 83, 337e346. Smith, D.P., 1972. Diagnostic criteria in dominantly inherited juvenile optic atrophy. A report of three new families. Am. J. Optom. Physiol. Optics. 49, 183e200. Toomes, C., Marchbank, N.J., Mackey, D.A., Craig, J.E., NewburyEcob, R.A., Bennett, C.P., Vize, C.J., Desai, S.P., Black, G.C., Patel, N., Teimory, M., Markham, A.F., Inglehearn, C.F., Churchill, A.J., 2001. Spectrum, frequency and penetrance of OPA1 mutations in dominant optic atrophy. Hum. Mol. Genet. 10, 1369e1378. Votruba, M., Moore, A.T., Bhattacharya, S.S., 1997. Genetic refinement of dominant optic atrophy (OPA1) locus to within a 2 cM interval of chromosome 3q. J. Med. Genet. 34, 117e121. Votruba, M., Fitzke, F.W., Carter, A., Bhattacharya, S.S., Moore, A.T., 1998. Clinical features in affected individuals from 21 pedigrees with dominant optic atrophy. Arch. Ophthalmol. 116, 315e358.
OPA1 expression in the human retina and optic nerve An-Guor Wang a,b,c, Ming-Ji Fann c,d,e, Hsin-Yi Yu a,c, May-Yung Yen a,b,* a
Department of Ophthalmology, Taipei Veterans General Hospital, Taipei, Taiwan b Faculty of Medicine, National Yang-Ming University, Taipei, Taiwan c Institute of Neuroscience, National Yang-Ming University, Taipei, Taiwan d Faculty of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei, Taiwan e Brain Research Center, University System of Taiwan, Taipei, Taiwan Received 22 March 2006; accepted in revised form 9 June 2006 Available online 18 July 2006
Abstract Mutations in the optic atrophy type 1 (OPA1) gene give rise to human autosomal dominant optic atrophy. The purpose of this study is to investigate OPA1 protein expression in the human retina and optic nerve. A rabbit polyclonal antiserum was generated using a fusion protein covering amino acids 647 to 808 of the human OPA1 protein as the immunogenic antigen. Western blot and immunofluorescence staining were performed to examine OPA1 expression in the human retina and optic nerve. In human retina, we found that OPA1 expression was clearly present in retinal ganglion cells and photoreceptors. OPA1 immunoreactivity was also present in the nerve fiber layer, inner plexiform layer and outer plexiform layer. However, OPA1 protein was not detected in the choline acetyltransferase-positive, calretinin-positive, and calbindinpositive amacrine cells, nor in the calbindin-positive horizontal cells. In the human optic nerve, expression of OPA1 was present in the axonal tract that was labeled with neurofilament specific antibody. In conclusion, expression of OPA1 gene is present in the mitochondria-rich regions of the retina and optic nerve. This suggests that OPA1 protein might be involved in the functioning of the mitochondria that are present in both inner and outer retinal neurons. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: dominant optic atrophy; mitochondria; OPA1
1. Introduction Autosomal dominant optic atrophy (DOA) is the most common form of hereditary optic neuropathy and has a prevalence of 1:12,000 to 1:50,000 (Delettre et al., 2002); the disease has an insidious onset in early childhood. It is characterized by progressive loss of visual acuity, bilateral atrophy of the optic nerve, central visual field defects and color vision deficits (Johnston et al., 1999; Kline and Glaser, 1979; Votruba et al., 1998). This disease shows a highly variable manifestation, with the patient’s visual acuity usually between 6/9 and 6/60; but the acuity may be as good as 6/6 or as poor as light * Corresponding author. Department of Ophthalmology, Taipei Veterans General Hospital, No. 201, Section 2, Shih-Pai Road, Taipei 11217, Taiwan. Tel.: þ886 2 2875 7325; fax: þ886 2 2876 1351. E-mail address: [email protected] (M.-Y. Yen). 0014-4835/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.exer.2006.06.004
perception. There is considerable variation in the visual function among patients, even within the same family (Johnston et al., 1999; Votruba et al., 1998). Evidence from electrophysiological (Elenius, 1991; Smith, 1972) and histopathological studies (Johnston et al., 1979; Kjer et al., 1983) shows that the fundamental underlying defect is degeneration of the retinal ganglion cells (RGC) and this is followed by ascending optic atrophy. The gene responsible for DOA has been mapped to human chromosome 3q28-qter, and named Optic Atrophy Type 1 (OPA1) (Bonneau et al., 1995; Eiberg et al., 1994; Jonasdottir et al., 1997; Lunkes et al., 1995; Votruba et al., 1997). The OPA1 gene spans more than 70 kb, is composed of 31 exons, and encodes a dynamin-related guanosine triphosphatase (GTPase) with 960 amino acids (Alexander et al., 2000; Delettre et al., 2000). Ninety-eight different mutations of the OPA1 gene have been identified in DOA patients (Alexander
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et al., 2000; Baris et al., 2003; Delettre et al., 2000, 2001; Pesch et al., 2001; Puomila et al., 2005; Toomes et al., 2001). Although precise function of the OPA1 protein is not completely clear, the fact that its protein sequence is similar to those of the yeast Msp1 and Mgm1p proteins and evidence gathered from perturbation experiments suggest that it may act as a factor maintaining the integrity and function of mitochondria within cells (for review see Frank, 2006). It has been demonstrated that cells overexpressing wild-type or mutant forms of the OPA1 protein exhibit fragmented mitochondria clustering near the nucleus (Griparic et al., 2004). Loss of OPA1 expression in cultured cells by small interfering RNA leads to swelling and stretch of mitochondria at the first stage and dispersal of fragmented mitochondria throughout the cytosol at the later stage, which is followed by cytochrome c release and apoptotic events (Griparic et al., 2004; Lee et al., 2004; Olichon et al., 2003). It is likely that OPA1 regulates the processes of mitochondrial fusion and fission. OPA1 is ubiquitously expressed in all human tissue when they are examined by Northern blot (Alexander et al., 2000; Delettre et al., 2000). The highest transcript level is found in retina, followed by the brain, testis, heart and skeletal muscle (Alexander et al., 2000). In the mammalian retina, OPA1 protein has been reported to be expressed in the RGC, amacrine cells, horizontal cells, the inner plexiform layer (IPL), and the outer plexiform layer (OPL) (Aijaz et al., 2004; Ju et al., 2005; Pesch et al., 2004). It was also found that OPA1 is expressed in the axonal mitochondria of the optic nerve (Ju et al., 2005). Nevertheless, inconsistent OPA1 expression was reported by different research groups. Aijaz et al. (2004) observed OPA1 in the RGC layer and inner nuclear layer (INL) of mouse and human retina. Ju et al. (2005) reported that OPA1 immunoreactivity is present in RGC and horizontal cells in adult rat retina. Pesch et al. (2004) described staining of OPA1 protein not only in RGC, but also in starburst amacrine cells and horizontal cells of the adult mouse and rat retina. Thus, whether OPA1 is expressed in the amacrine cells or horizontal cell of mammalian retina remains controversial. In the optic nerve, Ju et al. (2005) found that OPA1 protein is expressed in the entire optic nerve of adult rat. However, there was no detectable immunoreactivity in the optic nerve of the adult rat in the study by Pesch et al. (2004). In human tissue, Aijaz et al. (2004) observed that OPA1 is expressed exclusively in the myelinated region beyond the lamina cribrosa in the optic nerve. Thus, it is not clear whether OPA1 is expressed in the prelaminar portion of mammalian optic nerve. Furthermore, investigation of OPA1 expression in human ocular tissues has been rarely carried out (Aijaz et al., 2004). To understand the exact cellular expression pattern of OPA1 in normal human retina and to explore whether OPA1 expresses in the unmyelinated prelaminar zone of human optic nerve, we generated a polyclonal antiserum against OPA1 protein and examined the cellular expression pattern of OPA1 in the retina and optic nerve of adult human eyes in conjunction with various other immunological markers.
2. Materials and methods All experiments were performed according to institutional protocol guidelines. Experiments involving human tissues conformed to the guidelines set forth in the Declaration of Helsinki for the use of human tissue in research. The Institutional Review Board of the Taipei Veterans General Hospital approved the use of the donated eyeballs for this research. Three donated eyeballs used in immunofluorescent staining were obtained from three donors with no history of ocular disease (41-year-old male, 69-year-old male and 78-year-old male). Human retinal protein used in Western blot was obtained from another eye of the 41-year-old donor. 2.1. Reverse transcription Human brain RNA was purchased from Clontech (Palo Alto, CA, USA). Three micrograms of RNA in 10 ml was denatured at 75 C for 5 min. Reverse transcription was performed in a total volume of 20 ml containing 50 mM Trise HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 0.5 mM dNTP, 0.5 mg oligo(dT), 20 U of RNasin (Promega, Madison, WI, USA), and 100 U of Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA) for 1.5 h at 50 C. Samples were then stored at 20 C until use. 2.2. Preparation of fusion protein The cDNA (495 bp) encoding amino acid 647 to amino acid 808 of human OPA1 was amplified by PCR. Amplification of cDNA was conducted in a thermal cycler (Perkin Elmer 9700) with a 25 ml mixture containing 1 ml of adult human retina cDNA, 1 PCR buffer, 0.5 U high-fidelity Taq DNA polymerase (Roche, Mannheim, Germany), 0.25 mM dNTP, and one set of primers (100 nM). The forward primer was 50 GGAACTTTTAACACCACAGTG-30 and the reverse primer was 50 -ATAAGCTGGGTGCTCCTCAT-30 . The following conditions were used for the PCR: 35 cycles of denaturation at 94 C for 15 s; annealing at 55 C for 30 s, extension at 68 C for 30 s and finally, termination extension at 68 C for 5 min. The PCR products were then digested with BamHI and XhoI restriction enzymes and cloned into pET29a vector, which had been digested with the same enzymes. Clones that contained the correct insert were selected and sequenced to confirm the insert. Fusion protein was induced by 1 mM IPTG and the protein purified by passing through a metal chelating column following manufacturer’s protocol (Novagen, Darmstadt, Germany). 2.3. Antibodies To generate OPA1 polyclonal antiserum, the fusion proteins were injected into an adult New Zealand rabbit. Booster injections were given subsequently. Sera were collected every week from the fourth week after first injection and their titers were analyzed by dot blot assay. The resulting antiserum against
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OPA1 was used at a 1:200 dilution for immunofluorescence staining and at a 1:6000 dilution for Western blotting. Other primary antibodies were anti-cytochrome c monoclonal antibody (1:500 dilution, BD PharMingen, San Jose, CA, USA), mouse anti-neurofilament 200 kDa monoclonal antibody (1:500 dilution), goat anti-ChAT polyclonal antibodies (1:100 dilution), mouse anti-calretinin monoclonal antibody (1:100 dilution), mouse anti-rhodopsin monoclonal antibody (1:300 dilution) (Chemicon International, Temecula, CA, USA), anti-glial fibrillary acidic protein monoclonal antibody (1:200 dilution, SigmaeAldrich, Saint Louis, MO, USA), and mouse anti-calbindin monoclonal antibody (1:450 dilution, Swant, Bellinzona, Switzerland). A hybridoma clone that secretes an IgM monoclonal antibody against Thy-1 was purchased from American Type Culture Collection (Manassas, VA, USA), and twofold-diluted conditioned medium was used for immunofluorescence staining. The secondary antibodies used were Alexa Fluor 488 goat anti-rabbit antibodies (at 1:600 dilution, Molecular Probes, Eugene, OR, USA), Rhodamine (TRITC)-conjugated goat anti-mouse IgG antiserum and Cy3-conjugated donkey antigoat IgG antiserum (the latter two both at 1:600 dilution, Jackson ImmunoResearch, West Grove, PA, USA), FITC-conjugated goat anti-mouse IgM antiserum (at 1:600 dilution, Bethyl, Montgomery, TX, USA), and rhodamine-conjugated goat anti-rabbit antibodies (at 1:600 dilution, Chemicon International). 2.4. Subcellular localization of OPA1 HeLa cells were cultured at 37 C with Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum in a 5% CO2 incubator. Immunolabeling was performed overnight at 4 C in phosphate-buffered saline (PBS) containing 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4, and 5% bovine serum albumin (BSA) using anti-OPA1 antiserum and anti-cytochrome c monoclonal antibody as the primary antibodies. Cells were next incubated with the matched secondary antibodies for 2 h. Each step was preceded by washing in PBS. Specimens were analyzed using a Nikon Diaphot inverted fluorescence microscope (E300, Nikon, Tokyo, Japan) equipped with a MicroMax cool CCD (Princeton Instrument, Trenton, NJ, USA). Image acquisition was carried out by MetaMorph software (Universal Image Corp., Downingtown, PA, USA) with individual filter sets for each channel and photomicrographs were assembled using Adobe PhotoShop (Adobe Systems, San Jose, CA, USA). 2.5. Immunofluorescence staining of human retina and optic nerve For the human tissue sections, the posterior segments of three normal donated eyeballs were processed after removal of the corneal buttons for cornea transplantation. These posterior segments were fixed in 4% paraformaldehyde/PBS solution at 4 C overnight. The eyeballs were then transferred to
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30% sucrose/PBS overnight. The eyeballs were then divided into small pieces, submerged in OCT compound, and frozen using dry ice. Sections were cut 12 mm thick and collected on SuperFrost slides (Fisher Scientific, Pittsburgh, PA, USA). Sections were stored at 20 C until use. Sections were fixed with 4% paraformaldehyde for 20 min, then pretreated with 0.1% trypsin for 10 min at room temperature. They were then incubated with 0.2% Triton X-100 in PBS for 10 min, and then with 5% BSA in PBS for 60 min at room temperature. Sections were incubated in the primary antibodies, diluted in 5% BSA/PBS, overnight at 4 C. Finally, they were incubated with the matched secondary antibodies for 2 h at room temperature. Each step was preceded by washes in PBS. To stain the nucleus, the sections were incubated with 0.16 mg/ml 40 ,6-diamidino-2-phenylindole (DAPI) for 15 min at room temperature. The sections were examined by fluorescence microscopy. 2.6. Western blot of OPA1 protein For the human tissue, retina was dissected out from the posterior segments of a normal donated eyeball under balanced salt solution after removal of the corneal buttons for cornea transplantation. Mouse tissues were obtained from adult Balb/C mice. Tissues were homogenized in 10 volumes of lysis buffer. Samples (50 mg of protein) of tissue or recombinant proteins were loaded with an equal volume of 2 loading buffer (125 mM TriseHCl, pH 6.8; 4% SDS; 20% glycerol and 10% b-mercaptoethanol), boiled for 5 min and resolved by SDSepolyacrylamide gel (5% for tissue protein, 10% for recombinant protein) electrophoresis. Gels were electroblotted onto Immobilon-P membranes (Millipore, Bedford, MA, USA) in 25 mM Tris HCl, pH 8.3 containing 192 mM glycine, 20% methanol. Membranes were blocked in PBS/0.1% Tween (PBST) containing 10% fat-free dry milk (Carnation) for 1 h. Primary antisera were added overnight in PBST with 1% dry milk. After washing, HRP-conjugated goat anti-rabbit antiserum (Bethyl, Montgomery, TX, USA, 1:4,000) was incubated for 1 h. Immunoreactive bands were detected by enhanced chemiluminescence (ECL, Amersham Pharmacia, Buckinghamshire, UK) followed by exposure to Hyperfilm MP (Amersham Pharmacia). 3. Results 3.1. Characterization of the OPA1 protein A rabbit polyclonal antiserum was raised against a peptide corresponding to amino acids 647 to 808 (peptide OPA1647e808) of the predicted human OPA1 protein. The specificity of this antiserum (anti-OPA1) was first verified by Western blotting. The antiserum recognized a 24-kDa band but no other bacterial proteins when total proteins from the Escherichia coli strain over-expressing the peptide OPA1647e808 were analyzed (Fig. 1A). Pre-incubation of the antiserum with purified OPA1647e808 peptide completely abolishes the 24-kDa band (right panel, Fig. 1A), suggesting that the
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Fig. 1. (A) A Western blot of OPA1 protein. Anti-OPA1 antiserum recognized a 24-kDa band during the total proteins from E. coli over-expressing the OPA1647e808 peptide. Preincubation of the antiserum with purified peptide OPA1647e808 completely abolishes the 24-kDa band. (B) In various tissues, the molecular weight of the recognized protein was about 97 kDa. This 97-kDa band was present abundantly in the human retina, mouse retina, and mouse brain. There was also another band with an approximate molecular weight of 111 kDa present in the mouse tissues that was recognized by the anti-OPA1 antiserum. The expression of the OPA1 bands was blocked by preincubation of the anti-OPA1 antiserum with purified peptide OPA1647e808, which indicates the specificity of the anti-OPA1 antiserum. (C) Immunocytochemistry of OPA1 protein. HeLa cells were stained with anti-OPA1 antiserum (green), anti-cytochrome c antibody (red), and DAPI (blue). The expression of OPA1 in HeLa cell was co-localized with the expression of cytochrome c (C1e4), and this signal could be blocked by preincubation of anti-OPA1 antiserum with the peptide OPA1647e808 (C5e8). Scale bar in C is 10 mm.
antiserum recognizes OPA1647e808 specifically. We then used this antiserum to examine expression of OPA1 in human retina, mouse retina and mouse brain by Western blotting. A major band of approximately 97 kDa was detected in all the tissues analyzed. Another band with molecular weight of 111 kDa was also detected in the mouse tissues (Fig. 1B). The 111-kDa band may represent the un-processed immature OPA1 protein as the predicted size of OPA1 protein is 111 kDa. The 97-kDa band is likely the mature form of the OPA1 protein, as suggested by previous studies (Aijaz et al., 2004; Herlan et al., 2003). No protein was detected in the Western blot when the anti-OPA1 antiserum was pre-incubated with peptide OPA1647e808 (Fig. 1B, right panel) or without addition of the primary antiserum (data not shown), indicating that the reactivity against these two bands by the anti-OPA1 antiserum was specific. Previous studies have shown that expression of OPA1 occurs in the mitochondria. Thus, we examined whether the anti-OPA1 antiserum that we had generated could also detect OPA1 proteins in the mitochondria. HeLa cells, which express
OPA1 endogenously, were incubated with anti-OPA1 antiserum. A typical tubular interconnected mitochondrial network stained by anti-cytochrome c antibody was revealed by the anti-OPA1 antiserum (Fig. 1C). In a control experiment, no staining signal was observed when cells were incubated with anti-OPA1 antiserum in the presence of the peptide OPA1647e808 (Fig. 1C). A negative control, without addition of the anti-OPA1 antiserum, did not detect any immunoreactivity in the HeLa cells (data not shown). Taken together, these results indicate that the anti-OPA1 antiserum used in this study recognizes OPA1 proteins specifically. 3.2. OPA1 in human retina To determine the precise cellular location of OPA1 expression in the adult human retina and optic nerve, sections were incubated with the anti-OPA1 antiserum together with various retinal cell-specific markers. When the primary antibody was omitted, as a negative control for the immunofluorescence staining, there was no signal detected by the secondary
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antibodies in the human retina and optic nerve (data not shown). The OPA1 staining signal in the sections was competed out when the anti-OPA1 antiserum was pre-incubated with peptide OPA1647e808 (data not shown). Sections from peripapillary retina and optic nerve were examined from the prepared blocks such that a thick layer of optic nerve fibers overlying the RGC could be observed. We found that the expression of OPA1 was abundantly detected in the nerve fiber layer (NFL) and RGC (Fig. 2A). OPA1 immunoreactivity was also observed in the inner plexiform layer (IPL), the outer
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plexiform layer (OPL) and inner segment of photoreceptor (Fig. 2A). Under high power, the expression of OPA1 was co-localized with 200-kDa neurofilament protein in the nerve fiber layer (Fig. 2B). When the sections were double stained with GFAP, a glial cell marker, immunoreactivity of OPA1 was present in the whole thickness of the nerve fiber layer, while GFAP was stained as thin strands within the axonal tracts and did not co-localize with OPA1 (Fig. 2B). In the RGC layer, we used Thy-1 protein as the marker for RGC and the results showed that Thy-1-positive cells expressed
Fig. 2. OPA1 expression in human retina. Normal retina of an adult human was stained with anti-OPA1 antiserum (green), DAPI (blue), and various antibodies (red). (A) The expression of OPA1 was distributed in the nerve fiber layer (NFL) and RGC layer (GCL), inner plexiform layer (IPL), outer plexiform layer (OPL), and inner segment (IS) of photoreceptor of retina. (B) In the NFL, the expression of OPA1 protein was co-localized with the expression of neurofilament protein (NF) in the axonal tracts (B1e3), while OPA1 expression was not co-localized with GFAP, which is expressed as thin fibers intermingled among the axonal tracts (B4e6). (C) In the RGC layer, OPA1 immunoreactivity was detected in the RGCs (GC), which was labeled by anti-Thy-1 antibody (C1e3). Displaced amacrine cells (AC) in the RGC layer were labeled with the antibody against choline acetyltransferase (ChAT), but not the OPA1 antiserum (C4e6). (D) Amacrine cells in the inner nuclear layer (INL) were labeled with anti-calretinin antibody and anti-calbindin antibody, but were negative for OPA1 expression (D1e6). Horizontal cells (HC) labeled by calbindin were also negative for OPA1 expression (D4e6). (E) There was OPA1 immunoreactivity in the outer nuclear layer (E2e3, E5e6). The inner segment (IS) of photoreceptor is also labeled by anti-OPA1 antiserum (E2e3, E5e6). The signal is aligned with the rod outer segment labeled by antirhodopsin antibody (E4e6), but not well co-localized with cone cells marked by calbindin (E1e3). Scale bar in A is 100 mm, and in B is 25 mm.
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OPA1 protein (arrows in Fig. 2C). To examine the possibility of OPA1 expression in the amacrine cells, we used anti-OPA1 antiserum along with antibodies against choline acetyltransferase (ChAT), calbindin, and calretinin. In the RGC layer, displaced amacrine cells (marked by ChAT) did not express OPA1 protein (Fig. 2C). Amacrine cells in the superficial inner nuclear layer expressed calretinin and/or calbindin, but did not express OPA1 protein (Fig. 2D). Horizontal cells, marked by calbindin in the inner nuclear layer, did not express OPA1 protein (Fig. 2D). In the outer retina, OPA1 expression was present in the outer nuclear layer (Fig. 2E). The inner segment of rod photoreceptors was also labeled by the anti-OPA1 antiserum. The signal was aligned with, but not co-localized to the outer segment marked by anti-rhodopsin antibody, which labels rod cells (Fig. 2E). Moreover, the expression of OPA1 protein did not co-localized with calbindin, a cone cell marker (Fig. 2E). A similar pattern of OPA1 expression was observed in retinas of all three of the donated normal human eyeballs studied here. 3.3. OPA1 in human optic nerve head We next determined whether OPA1 is expressed in the human optic nerve. Longitudinal sections of adult human optic nerve stained with the anti-OPA1 antiserum and showed intense OPA1 immunoreactivity. To examine the precise expression pattern of OPA1, sections were double stained with antibodies against GFAP (an astrocyte marker) and against 200-kDa neurofilament protein (an axonal marker). In a lowpower field, OPA1 protein expressed in the prelaminar and
lamina cribrosa regions of the optic nerve head and in the optic nerve beyond the lamina cribrosa (Fig. 3A). At higher magnification of the prelaminar portion (Fig. 3B), the staining of OPA1 protein showed a filamentous pattern and OPA1 was co-localized with 200-kDa neurofilament protein (Fig. 3B). However, OPA1 did not co-localized with GFAP protein, which was manifested as multiple parallel thin fibers along the axons (Fig. 3B). In the lamina cribrosa (LC), expression of OPA1 was in the axonal fibers, which were marked by 200-kDa neurofilament protein (Fig. 3C). Again, the expression of OPA1 was not co-localized with GFAP (Fig. 3C). 4. Discussion OPA1 mutations are responsible for DOA, the most common form of hereditary optic neuropathy (Alexander et al., 2000; Delettre et al., 2000, 2002; Eiberg et al., 1994; Jonasdottir et al., 1997; Votruba et al., 1997). However, the expression pattern of OPA1 protein in the human retina and optic nerve is not well characterized. To assess the expression of OPA1 in the human retina and optic nerve, we first prepared an antiOPA1 antiserum and used this antiserum to visualize the distribution of OPA1 protein. In comparison to previous studies that utilized antisera against synthesized short peptides of OPA1 to detect its expression in mammalian retinas, an antiserum against a larger fusion protein covering amino acid 647 to amino acid 808 of human OPA1 was used in this study. This was in expectation that antibodies thus generated would show greater binding and higher specificity against OPA1. Two bands with apparent molecular weights of 111 kDa and
Fig. 3. OPA1 expression in human optic nerve. Normal optic nerve of adult human was stained with anti-OPA1 antiserum (green), anti-GFAP antibody and/or antineurofilament antibody (red), and DAPI (blue). (A) OPA1 protein was expressed in a filamentous pattern from the prelaminar region (PL), through lamina cribrosa (LC), to the myelinated optic nerve (ON) beyond the lamina cribrosa. There were small blood vessels (BV) in the prelaminar portion of the optic nerve head. (B) In the high-power fields of the prelaminar region, the expression of OPA1 protein was co-localized with neurofilament protein (B1e3), but not with GFAP (B4e6). (C) In the lamina cribrosa, OPA1 expresses in the axonal tracts between the glial trabeculae of the cribriform plate and co-localized with neurofilament protein (C1e3). The expression of GFAP does not co-localize with the expression of OPA1 (C4e6). Scale bar in A is 100 mm, and in B is 50 mm.
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97 kDa were detected in mouse tissue suggesting that the antiserum recognizes unprocessed OPA1 (predicted size of 111 kDa) and mature OPA1 (the signal-sequence cleaved form with a predicted size of 99 kDa). In the previous studies, two bands have also been detected by antisera against short peptides (McQuibban et al., 2003; Misaka et al., 2002). Several studies have reported OPA1 expression in the mammalian retina. We noted that discrepancies concerning OPA1 expression in retinal interneuron exist among these previous studies (Aijaz et al., 2004; Ju et al., 2005; Pesch et al., 2004). As our antiserum also recognizes mouse OPA1 (Fig. 1B), mouse retina was examined with this antiserum. We found that OPA1 expression is in mouse calretinin-positive amacrine cell, starburst amacrine cell, and horizontal cell, in addition to RGC and photoreceptors (data not shown). In human retina, we observed OPA1 immunoreactivity in RGC and photoreceptors. The detection of OPA1 immunoreactivity in the human photoreceptors has not been reported previously. Nevertheless, we did not observe OPA1 immunoreactivity in the calretinin-positive or calbindin-positive amacrine cells, nor in the ChAT-positive starburst amacrine cells of the human retina. Furthermore, we did not observe OPA1 expression in calbindin-positive horizontal cells. Thus, amacrine cells and horizontal cells, both involved in the lateral signal processing, do not express OPA1 protein in human retina based on our study. Since we used the same antiserum in two species, the discrepancy of OPA1 expression between mouse and human retina is clearly due to the species difference. A significant staining of OPA1 in inner segment of photoreceptor was observed in this study. Double staining results indicate OPA1 protein is expressed in the inner segment of rod photoreceptor, which is well aligned with the rod outer segment, marked by rhodopsin. As OPA1 is a mitochondrial protein, the expression of OPA1 in the mitochondria-rich inner segment suggests OPA1 protein may play a role in the mitochondrial function of the retinal neurons. Inconsistent results of OPA1 expression in the mammalian optic nerve have been reported in literature (Aijaz et al., 2004; Ju et al., 2005; Pesch et al., 2004). In our study, OPA1 is expressed both in the prelaminar and laminar portion of the adult human optic nerve and mouse optic nerve (data not shown). Thus, we propose that RGCs express OPA1 protein in the soma and the protein extends along the axons within the nerve fiber layer, to the optic nerve. Within the optic nerve, it is expressed all the way, from the prelaminar portion, through the lamina cribrosa to the retrolaminar myelinated portion. The expression of OPA1 is co-localized with the expression of neurofilament protein, but not with the expression of GFAP, suggesting the OPA1 is distributed within the axonal tract but not in the astrocytes. The precise function of OPA1 protein in the retina is not completely clear. The inner segment of photoreceptors, the nerve fiber layer of retina and optic axons within the optic nerve, are all mitochondria rich regions of the eye. In this study, we also clearly observed that OPA1 protein is expressed in the mitochondria rich region of retinal neurons. Taken together, these observations support the scenario that OPA1
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protein has a specific role in the mitochondrial function of both inner and outer retinal neurons. Based on the results described here, the role of OPA1 protein in the mitochondrial function of retinal neurons, and the mechanism how it induces RGC degeneration in autosomal dominant optic atrophy is worth further investigation. Acknowledgements This work was supported by grants of Taipei Veterans General Hospital ((VGH 93-327), and National Science Council (NSC 92-2314-B-075-046; NSC 93-2314-B-075-003) to M.-Y.Y, and VGH-UST (VGHUST 94-P1-13) to A.-G.W. References Aijaz, S., Erskine, L., Jeffery, G., Bhattacharya, S.S., Votruba, M., 2004. Developmental expression profile of the optic atrophy gene product: OPA1 is not localized exclusively in the mammalian retinal ganglion cell layer. Invest. Ophthalmol. Vis. Sci. 45, 1667e1673. Alexander, C., Votruba, M., Pesch, U.E.A., Thiselton, D.L., Mayer, S., Moore, A., Rodriguez, M., Kellner, U., Leo-Kottler, B., Auburger, G., Bhattacharya, S.S., Wissinger, B., 2000. OPA1, encoding a dynaminrelated GTPase, is mutated in autosomal dominant optic atrophy linked to chromosome 3q28. Nat. Genet. 26, 211e215. Baris, O., Delettre, C., Amati-Bonneau, P., Surget, M.O., Charlin, J.F., Catier, A., Derieux, L., Guyomard, J.L., Dollfus, H., Jonveaux, P., Ayuso, C., Maumenee, I., Lorenz, B., Mohammed, S., Tourmen, Y., Bonneau, D., Malthiery, Y., Hamel, C., Reynier, P., 2003. Fourteen novel OPA1 mutations in autosomal dominant optic atrophy including two de novo mutations in sporadic optic atrophy. Hum. Mutat. 21, 656. Bonneau, D., Souied, E., Gerber, S., Rozet, J.M., D’Haens, E., Journel, H., Plessis, G., Weissenbach, J., Munnich, A., Kaplan, J., 1995. No evidence of genetic heterogeneity in dominant optic atrophy. J. Med. Genet. 32, 951e953. Delettre, C., Lenaers, G., Griffoin, J.M., Gigarel, N., Lorenzo, C., Belenguer, P., Pelloquin, L., Grosgeorge, J., Turc-carel, C., Parret, E., Astarie-Dequeker, C., Lasquellec, L., Arnaud, B., Ducommun, B., Kaplan, J., Hamel, C., 2000. Nuclear gene OPA1, encoding a mitochondrial dynamin-related protein, is mutated in dominant optic atrophy. Nat. Genet. 26, 207e210. Delettre, C., Griffoin, J.M., Kaplan, J., Dollfus, H., Lorenz, B., Faivre, L., Lenaers, G., Belenguer, P., Hamel, C.P., 2001. Mutation spectrum and splicing variants in the OPA1 gene. Hum. Genet. 109, 584e591. Delettre, C., Lenaers, G., Pelloquin, L., Belenguer, P., Hamel, C.P., 2002. OPA1 (Kjer type) dominant optic atrophy: a novel mitochondrial disease. Mol. Genet. Metab. 75, 97e107. Eiberg, H., Kjer, B., Kjer, P., Rosenberg, T., 1994. Dominant optic atrophy (OPA1) mapped to chromosome 3q region. I. Linkage analysis. Hum. Mol. Genet. 3, 977e980. Elenius, V., 1991. Rod thresholds in dominantly inherited juvenile optic atrophy. Ophthalmologica 202, 208e212. Frank, S., 2006. Dysregulation of mitochondrial fusion and fission: an emerging concept in neurodegeneration. Acta. Neuropathol. (Berl.) 111, 93e100. Griparic, L., van der Wel, N.N., Orozco, I.J., Peters, P.J., van der Bliek, A.M., 2004. Loss of the intermembrane space protein Mgm1/OPA1 induces swelling and localized constrictions along the lengths of mitochondria. J. Biol. Chem. 279, 18792e18798. Herlan, M., Vogel, F., Bornhovd, C., Neupert, W., Beichert, A.S., 2003. Processing of Mgm1 by the Thomboid-type protease Pcp1 is required for maintenance of mitochondrial morphology and of mitochondrial DNA. J. Biol. Chem. 278, 27781e27788. Johnston, P.B., Gaster, R.N., Smith, V.C., Tripathi, R.C., 1979. A clinicopathologic study of autosomal dominant optic atrophy. Am. J. Ophthalmol. 88, 868e875.
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Olichon, A., Baricault, L., Gas, N., Guillou, E., Valette, A., Belenguer, P., Lenaers, G., 2003. Loss of OPA1 perturbates the mitochondrial inner membrane structure and integrity, leading to cytochrome c release and apoptosis. J. Biol. Chem. 278, 7743e7746. Pesch, U.E., Leo-Kottler, B., Mayer, S., Jurklies, B., Kellner, U., ApfelstedtSylla, E., Zrenner, E., Alexander, C., Wissinger, B., 2001. OPA1 mutations in patients with autosomal dominant optic atrophy and evidence for semidominant inheritance. Hum. Mol. Genet. 10, 1359e1368. Pesch, U.E.A., Fries, J.E., Bette, S., Kalbacher, H., Wissinger, B., Alexander, C., Kohler, K., 2004. OPA1, the disease gene for autosomal dominant optic atrophy, is specifically expressed in ganglion cells and intrinsic neurons of the retina. Invest. Ophthalmol. Vis. Sci. 45, 4217e4225. Puomila, A., Huoponen, K., Mantyjarvi, M., Hamalainen, P., Paananen, R., Sankila, E.M., Savontaus, M.L., Somer, M., Nikoskelainen, E., 2005. Dominant optic atrophy: correlation between clinical and molecular genetic studies. Acta. Ophthalmol. Scand. 83, 337e346. Smith, D.P., 1972. Diagnostic criteria in dominantly inherited juvenile optic atrophy. A report of three new families. Am. J. Optom. Physiol. Optics. 49, 183e200. Toomes, C., Marchbank, N.J., Mackey, D.A., Craig, J.E., NewburyEcob, R.A., Bennett, C.P., Vize, C.J., Desai, S.P., Black, G.C., Patel, N., Teimory, M., Markham, A.F., Inglehearn, C.F., Churchill, A.J., 2001. Spectrum, frequency and penetrance of OPA1 mutations in dominant optic atrophy. Hum. Mol. Genet. 10, 1369e1378. Votruba, M., Moore, A.T., Bhattacharya, S.S., 1997. Genetic refinement of dominant optic atrophy (OPA1) locus to within a 2 cM interval of chromosome 3q. J. Med. Genet. 34, 117e121. Votruba, M., Fitzke, F.W., Carter, A., Bhattacharya, S.S., Moore, A.T., 1998. Clinical features in affected individuals from 21 pedigrees with dominant optic atrophy. Arch. Ophthalmol. 116, 315e358.