November 2005
Volume 46, Issue 11
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Retina  |   November 2005
Expression of the Opa1 Mitochondrial Protein in Retinal Ganglion Cells: Its Downregulation Causes Aggregation of the Mitochondrial Network
Author Affiliations
  • Satomi Kamei
    From the Institut National de la Santé et de la Recherche Médicale (INSERM) U583, Physiopathologie et Thérapie des Déficits Sensoriels et Moteurs, Institut des Neurosciences de Montpellier, Hôpital Saint Eloi, Montpellier, France; the
  • Murielle Chen-Kuo-Chang
    From the Institut National de la Santé et de la Recherche Médicale (INSERM) U583, Physiopathologie et Thérapie des Déficits Sensoriels et Moteurs, Institut des Neurosciences de Montpellier, Hôpital Saint Eloi, Montpellier, France; the
  • Chantal Cazevieille
    From the Institut National de la Santé et de la Recherche Médicale (INSERM) U583, Physiopathologie et Thérapie des Déficits Sensoriels et Moteurs, Institut des Neurosciences de Montpellier, Hôpital Saint Eloi, Montpellier, France; the
  • Guy Lenaers
    From the Institut National de la Santé et de la Recherche Médicale (INSERM) U583, Physiopathologie et Thérapie des Déficits Sensoriels et Moteurs, Institut des Neurosciences de Montpellier, Hôpital Saint Eloi, Montpellier, France; the
  • Aurélien Olichon
    Centre National de la Recherche Scientifique (CNRS) UMR5088, Laboratoire de Biologie Cellulaire et Moléculaire du Contrôle de la Prolifération (LBCMCP), Université Paul Sabatier, Toulouse, France; and the
  • Pascale Bélenguer
    Centre National de la Recherche Scientifique (CNRS) UMR5088, Laboratoire de Biologie Cellulaire et Moléculaire du Contrôle de la Prolifération (LBCMCP), Université Paul Sabatier, Toulouse, France; and the
  • Gautier Roussignol
    INSERM U661/CNRS UMR 5203, Institut de Génomique Fonctionnelle, Montpellier, France.
  • Nicole Renard
    From the Institut National de la Santé et de la Recherche Médicale (INSERM) U583, Physiopathologie et Thérapie des Déficits Sensoriels et Moteurs, Institut des Neurosciences de Montpellier, Hôpital Saint Eloi, Montpellier, France; the
  • Michel Eybalin
    From the Institut National de la Santé et de la Recherche Médicale (INSERM) U583, Physiopathologie et Thérapie des Déficits Sensoriels et Moteurs, Institut des Neurosciences de Montpellier, Hôpital Saint Eloi, Montpellier, France; the
  • Adeline Michelin
    From the Institut National de la Santé et de la Recherche Médicale (INSERM) U583, Physiopathologie et Thérapie des Déficits Sensoriels et Moteurs, Institut des Neurosciences de Montpellier, Hôpital Saint Eloi, Montpellier, France; the
  • Cécile Delettre
    From the Institut National de la Santé et de la Recherche Médicale (INSERM) U583, Physiopathologie et Thérapie des Déficits Sensoriels et Moteurs, Institut des Neurosciences de Montpellier, Hôpital Saint Eloi, Montpellier, France; the
  • Philippe Brabet
    From the Institut National de la Santé et de la Recherche Médicale (INSERM) U583, Physiopathologie et Thérapie des Déficits Sensoriels et Moteurs, Institut des Neurosciences de Montpellier, Hôpital Saint Eloi, Montpellier, France; the
  • Christian P. Hamel
    From the Institut National de la Santé et de la Recherche Médicale (INSERM) U583, Physiopathologie et Thérapie des Déficits Sensoriels et Moteurs, Institut des Neurosciences de Montpellier, Hôpital Saint Eloi, Montpellier, France; the
Investigative Ophthalmology & Visual Science November 2005, Vol.46, 4288-4294. doi:https://doi.org/10.1167/iovs.03-1407
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      Satomi Kamei, Murielle Chen-Kuo-Chang, Chantal Cazevieille, Guy Lenaers, Aurélien Olichon, Pascale Bélenguer, Gautier Roussignol, Nicole Renard, Michel Eybalin, Adeline Michelin, Cécile Delettre, Philippe Brabet, Christian P. Hamel; Expression of the Opa1 Mitochondrial Protein in Retinal Ganglion Cells: Its Downregulation Causes Aggregation of the Mitochondrial Network. Invest. Ophthalmol. Vis. Sci. 2005;46(11):4288-4294. https://doi.org/10.1167/iovs.03-1407.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. Mutations in the mitochondrial dynamin-related GTPase OPA1 cause autosomal dominant optic atrophy (ADOA), but the pathophysiology of this disease is unknown. As a first step in functional studies, this study was conducted to evaluate the expression of Opa1 in whole retina and in isolated retinal ganglion cells (RGCs) and to test the effects of Opa1 downregulation in cultured RGCs.

methods. Opa1 mRNA isoforms from total retina and from RGCs freshly isolated by immunopanning were determined by RT-PCR. Protein expression was examined by immunohistochemistry and Western blot with antibodies against Opa1 and cytochrome c, and the mitochondrial network was visualized with a mitochondrial marker. Short interfering (si)RNA targeting OPA1 mRNAs were transfected to cultured RGCs and mitochondrial network phenotypes were followed for 15 days, in comparison with those of cerebellar granule cells (CGCs).

results. Opa1 expression did not predominate in rat postnatal RGCs as found by immunohistochemistry and Western blot analysis. The pattern of mRNA isoforms was similar in whole retina and RGCs. After a few days in culture, isolated RGCs showed fine mitochondrial punctiform structures in the soma and neurites that colocalized with cytochrome c and Opa1. Opa1 knockdown in RGCs induced mitochondrial network aggregation at a higher rate than in CGCs.

conclusions. Results suggest that the level of expression and the mRNA isoforms do not underlie the vulnerability of RGCs to OPA1 mutations. However, aggregation of the mitochondrial network induced by the downregulation of Opa1 appears more frequent in RGCs than in control CGCs.

Autosomal dominant optic atrophy (ADOA; Kjer type; Mendelian Inheritance in Man [MIM] 165500; National Center for Biotechnology Information, Bethesda, MD) 1 is the most frequent form of hereditary optic neuropathy, with a prevalence ranging between 1 in 12,000 2 3 and 1 in 50,000. 4 The disease appears with an insidious onset of variable visual loss, caecocentral visual field scotoma, tritanopia, and symmetric optic atrophy visible as a temporal pallor of the optic disc, 5 6 7 8 without any extraocular symptoms. Histopathology has shown a decrease in the number of retinal ganglion cells (RGCs) that predominates in the central retina and a loss of myelin and nerve tissue within the optic nerve, optic chiasm, and optic tracts, 9 10 suggesting that the disorder is a primary degeneration of RGCs with ascending optic atrophy. 10  
We and others have identified OPA1, a mitochondrial dynamin-related guanosine triphosphatase (GTPase) and have found that mutations in the OPA1 gene cause ADOA. 11 12 13 14 15 16 17 18 19 20 21 OPA1 spans more than 90 kb and is composed of 31 exons, 22 including those coding the GTPase domain (exons 8-15), the central dynamin domain (exons 16-26), and the basic N-terminal leader sequence (exons 1-3) necessary for its mitochondrial localization. 23 24 Consistent with this localization, OPA1 transcripts are ubiquitous. 11 12 However, tissue-specific expression of OPA1 isoforms produced by alternative splicing may underlie some specific neuronal requirements for OPA1 functions. 25  
Mitochondria exist as small isolated particles or as extended filaments or clusters that can convert from one form to the other. The predominance of either form shapes the so-called mitochondrial network and is determined by the balance between mitochondrial fission and fusion events. 26 These events are controlled by two subfamilies of dynamin-related GTPases acting on the outer membrane, 27 and the yeast dynamin-related protein Msp1/Mgm1, 28 29 orthologous to OPA1, acting on the inner membrane. Msp1/Mgm1 is also essential for the maintenance of mitochondrial DNA and the mitochondrial network. 29 30 31 Similar functions of OPA1 are suspected 32 and indeed, in monocytes from patients with ADOA, mitochondria were abnormally aggregated. 11 Recent studies have demonstrated that OPA1 is located in the mitochondrial inner membrane space (IMS) mainly anchored to the cristae inner membrane (IM). 32 33 In HeLa cells, knockdown of OPA1 using RNA interference (short interfering [si]RNA) induced mitochondrial network fragmentation and IM perturbation, suggesting a role for OPA1 in the mitochondrial fusion process. 34  
An important point is that only RGCs seem affected by OPA1 mutations despite the ubiquitous OPA1 expression. Possible hypotheses include a predominant expression of OPA1 in RGCs, a specific pattern of OPA1 isoforms in RGCs, and a particular mitochondrial network or specific requirements for OPA1 function in RGCs. Little is known about the mitochondrial network structures and OPA1 expression in RGCs. To address these questions, we first analyzed the Opa1 expression, the mitochondrial distribution, and the pattern of Opa1 isoforms, in retina and purified RGCs from rats. Second, as a prerequisite for functional investigations, we examined the effect of downregulation of Opa1 expression on the mitochondrial network of purified retinal ganglion cells (RGCs) in comparison to cerebellar granule cells (CGCs). 
Materials and Methods
Care and use of animals followed the animal welfare guidelines of the Institut National de la Santé et de la Recherche Médicale (INSERM) under the approval of the Ministère Français de l’Agriculture et de la Forêt and was in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Chemicals and Reagents
Affinity-purified anti-OPA1 antibody was obtained as previously described 32 and anti-Thy1.1 antibody (T11D7e) was kindly provided by Frank W. Pfrieger (immunopanning) and David Hicks (immunofluorescence). 35 Anti-cytochrome c antibody (6H2.B4) was purchased from BD-Pharmingen (San Diego, CA); Alexa 488 anti-mouse IgG, Alexa 488 anti-rabbit IgG, Alexa 594 anti-rabbit IgG and rhodamine-conjugated phalloidin from Molecular Probes (Eugene, OR); cy-3-conjugated goat anti-mouse IgM from Jackson ImmunoResearch Laboratories (West Grove, PA); affinity-purified anti-mouse IgM and affinity-purified anti-rabbit IgG from Rockland (Gilbertsville, PA); anti-rat macrophage antiserum from Accurate Chemical and Scientific Corp. (Westbury, NY); papain from Worthington Biochemical (Freehold, NY); and ovomucoid from Roche Biochemicals (Basel, Switzerland); recombinant human brain-derived neurotrophic factor (BDNF) and rat ciliary neurotrophic factor (CNTF) from Peprotech (Rocky Hill, NJ); and B27, enzyme-inhibiting (Neurobasal) medium, phosphate-buffered saline (PBS), Earle’s balanced salt solution (EBSS), sodium pyruvate, and normal goat serum from Invitrogen-Gibco (Grand Island, NY). Unless noted, all other reagents were obtained from Sigma-Aldrich (St. Louis, MO). 
Cell Cultures
Retinal ganglion cells (RGCs) from 5- to 8-day-old Wistar rats were isolated and purified by the two-step immunopanning procedure. 35 36 They were plated at a 20,000 RGCs/cm2 density on 12-mm glass coverslips in 24- or 6-well plates and cultured as previously described. 36 37 To evaluate the purity of RGCs, 5-day cultures were immunolabeled with mouse IgM anti-rat Thy1.1 (1:10) for 10 minutes at 37°C, followed by incubation with cy-3-conjugated goat anti-mouse IgM (1:200) for 1 hour at room temperature, fixation with 4% paraformaldehyde (PFA) in PBS for 15 minutes, and staining with Hoechst dye (0.5 μg/mL) for 15 minutes. 
Culture of cerebellar neurons were prepared from postnatal day 6 to 7 mice, as described. 38 The cultures contained 95% granular neurons. Dissociated neurons were plated at a density of 0.75 to 1.25 × 105 cells/glass coverslip and maintained for 3 weeks in culture. 
Mitochondrial Network Analysis and Opa1 Expression
Rat eyes (3–4-week-old) were frozen after fixation by aortic perfusion with 4% PFA-PBS and consecutive overnight incubation at 4°C in the same solution. Eyes were then cut in 14-μm sections with a cryostat (Leica, Deerfield, IL). Cells cultured on glass coverslips were fixed in 4% PFA-PBS for 15 minutes at room temperature. Cultured cells and cryosections were preincubated for 60 minutes at room temperature with PBS containing 0.1–0.3% Triton X-100 (Merck, Darmstadt, Germany) and 10% normal goat serum. The specimens were incubated overnight at 4°C with the rabbit polyclonal anti-OPA1 (1/10) followed or not by a subsequent incubation with the mouse monoclonal anti-cytochrome c (1:200, 2 hours, room temperature). They were then incubated for 90 minutes with the corresponding fluorescent dye-conjugated IgG (i.e., Alexa 594 goat anti-rabbit [1:2000]) or Alexa-488 goat anti-mouse [1:2000]) and counterstained with Hoechst (0.5 μg/mL, 15 minutes, room temperature). To examine and quantify mitochondrial phenotypes, cells were directly stained in culture using 150 nM of a mitochondrial marker (CMX MitoTracker Red; Molecular Probes) for 30 minutes at 37°C, fixed with 4% PFA in culture medium, permeabilized with PBS containing 0.2% Triton X-100 for 10 minutes, and stained with Hoechst, as described earlier. To examine Opa1 expression in RGCs with siRNA treatment, after first incubation with anti-OPA1, cultures were incubated with rhodamine-conjugated phalloidin (1 U/mL) and Alexa 488 goat anti-rabbit (1:2000), and counterstained with Hoechst. Samples were examined under an epifluorescence or a confocal microscope (Bio-Rad, Hercules, CA). 
RNA Extraction and RT-PCR
Total RNA from retina and brain of 20 day-old rats and from isolated RGCs was extracted (RNeasy; Qiagen, Valencia, CA) according to the manufacturer’s instructions. First-strand cDNA synthesis was performed with reverse transcriptase (Super Script II; Invitrogen, NL), with 0.33 to 1 μg RNA and 50 picomoles of random primers (Promega, Madison, WI). As a control, reverse transcription of RNA from brain tissue was performed without reverse transcriptase. cDNA was PCR-amplified under standardized conditions as follows: denaturation at 94°C for 3 minutes followed by 35 cycles of 30 seconds at 94°C, 30 seconds at 58°C, 2 minutes 30 seconds at 72°C, and a final elongation at 72°C for 10 minutes. For alternatively spliced transcripts, rat Opa1 was amplified using primers located in exons 3 (ME3S: 5′-GTGACTATAAGTGGATTGTGCCT-3′) and 9 and 10 (MK5AS: 5′- CACTCAGAGTCACCTTAACTGG-3′). All PCR products were separated by electrophoresis through 2% agarose gels. Each PCR fragment was purified with a kit (Qiaquick; Qiagen) and sequenced on a capillary sequencer (Applied Biosystems, Foster City, CA). 
siRNA Preparation and Transfection
To design target-specific siRNAs, we selected an AA(N19)UU sequence (5′-AAGUCAUCAGUCUGAGCCAGGUU-3′) at position c.1811-1833 of the rat Opa1 open reading frame (GenBank accession AY510274) that is shared by all Opa1 isoforms. 25 34 39 The specificity of this sequence was verified by BLAST search. The 23-nt sense and antisense strands with 2-base overhangs were chemically synthesized by Dharmacon Research (Lafayette, CO) in deprotected and desalted form. Premade siRNA (Scramble II; Dharmacon), the target of which is not present in mammalian cells, was used as a negative control. 
Transfection of siRNAs into RGCs and CGCs was performed at culture day 7 (TransMessenger transfection reagent; Qiagen). 40 According to the manufacturer’s instructions, 0.8 or 2.0 μg siRNA per well (24- or 6-well plates) was condensed (Enhancer R; Qiagen) and preincubated with 4 or 10 μL of transfection reagent (TransMessenger; Qiagen). Then, 300 or 900 μL culture medium was combined with this mixture, and the entire solution was added directly to the cells. The mixture in culture wells was changed to a normal culture medium after 4 hours’ incubation. After transfection, cells were cultured for 5 to 15 days and then subjected to quantification of phenotypes using the mitochondrial marker and Hoechst, immunofluorescence, cell counting, and Western blot analysis. Results are based on four independent siRNA transfection experiments. 
Western Blot Analysis
Immediately after panning isolation, RGCs and other retinal cells (nonadherent cells in anti-thy1.1 panning step) were harvested and washed twice in ice-cold PBS. Equal amounts of both cell categories were solubilized in 60 μL of Laemmli sample buffer, run in 8% SDS-PAGE, and transferred to a nitrocellulose membrane (Invitrogen, Groningen, The Netherlands). The milk-blocked membrane was then incubated overnight at 4°C with the anti-OPA1 antibody (1:2000) or anti-β actin antibody (1:1000; clone C4: Chemicon, Temecula, CA), subjected to a horseradish-peroxidase–conjugated anti-rabbit IgG (1:5000; Roche Diagnostics, Mannheim, Germany), and revealed with an enhanced chemiluminescence Western blot analysis kit (Applied Biosystems) or by the alkaline phosphatase technique. Polyacrylamide gels were also fixed with 40% methanol and 10% acetic acid for 30 minutes at room temperature and incubated in 0.25 mg/mL Coomassie blue in 10% acetic acid for 1 hour. RGCs treated with siRNA were trypsinized with 0.125% trypsin, washed twice in ice-cold PBS, and subjected to the same procedure. 
Statistics
All results are presented as the mean ± SE. Statistical significance was determined by analysis of variance (ANOVA) and the Tukey post hoc test. P < 0.05 was considered to be statistically significant. 
Results
Purity and Yield of Isolated RGCs
By using the sequential immunopanning method, we isolated approximately 20,000 to 40,000 RGCs per retina, accounting for 15% to 30% of the RGCs in a P8 rat retina. 35 Approximately 96% of the cells after 5 days in culture were labeled with Thy1.1 (Fig. 1)and hence were identified as RGCs. 
Expression of Opa1 in Rat Postnatal RGCs
One possibility to the vulnerability of RGCs to OPA1 mutations is that OPA1 would be predominantly expressed in RGCs. To address this question, we examined Opa1 expression by using an antibody against recombinant OPA1 in rat retinal sections. Opa1 staining was seen in ganglion cell layer (GCL), inner plexiform layer (IPL), outer plexiform layer (OPL), photoreceptor (PR) inner segments (IS) and pigment epithelium (PE). In each of these layers, cytochrome c staining was also found. However, cytochrome c expression was stronger than that of Opa1 in IS and PE, whereas in other layers, including the GCL, it was similar (Figs. 2A 2B 2C 2D) . The specificity of the anti-OPA1 antibody was confirmed by control experiments with polyclonal rabbit IgG (1.25 μg/mL) in place of the primary anti-OPA1 antibody, which showed no fluorescence (Fig. 2E) . The Opa1 expression level was also analyzed by Western blot in isolated RGCs in comparison with other retinal cells (Fig. 2F)and actin expression (Fig. 2G) . Opa1 was present in equal amounts in both cell categories, as two doublets of 92- and 86-kDa fragments, as previously found. 32  
mRNA Isoforms in Rat RGCs
In human and mouse OPA1, exons 4b and 5b, together with exon 4, generate eight isoforms by alternative splicing. 25 41 Another hypothesis regarding the vulnerability of RGCs to OPA1 mutations is that RGCs express a particular pattern of OPA1 isoforms. To test this, we purified total RNA from rat brain, retina, and RGCs and analyzed the whole Opa1 cDNA by RT-PCR. The open reading frame was found to be 2883 bp in size encoding a 960-amino-acid protein with 84% identity with the human sequence (GenBank accession number AB011139; http://www.ncbi.nlm.nih.gov/Genbank; provided in the public domain by the National Center for Biotechnology Information, Bethesda, MD). Use of ME3S and MK5AS primers resulted in the amplification of six distinct fragments (Fig. 3) , as in humans and the mouse. 25 41 Rat transcripts from brain, retina, and RGCs exhibited similar isoform patterns with a predominance of the one lacking exons 4b and 5b, as found in the corresponding human tissues. 
Mitochondrial Network and Opa1 Distribution in Cultured RGCs
We next examined the mitochondrial distribution of isolated rat RGCs every day for 5 days after seeding in culture. At day 1, round or punctiform structures stained with the mitochondrial marker (MitoTracker Red; Molecular Probes) were found abundantly throughout the cytoplasm (Fig. 4A)but in lesser amounts in neurites, although heavy staining was seen in branch points and growth cones. As days passed, the staining became finer and more diffuse, with less punctiform structures (Figs. 4B 4C) . These structures were also strongly labeled with the anti-OPA1 antibody in colocalization with the mitochondrial protein cytochrome c, indicating that they were indeed mitochondria expressing Opa1 (Figs. 4D 4E 4F)
Downregulation of Opa1 by siRNA Treatment
To inhibit endogenous expression of Opa1 we designed an Opa1 siRNA that matches a sequence situated downstream of the GTPase coding sequence from rat and mouse and analyzed its effect in comparison with a control siRNA (Scramble II; Dharmacon) in RGCs and in CGCs. In RGCs, 10 days after siRNA treatment, the inhibitory effect on the endogenous Opa1 production was analyzed by Western blot. It showed a dramatic reduction of the various forms of Opa1 in cultures treated with the cognate siRNA compared with those treated with the control siRNA, with equal amounts of total protein loaded on the gel (Fig. 5C) . Immunofluorescence labeling of Opa1 siRNA-treated cells showed that 30% to 60% of RGCs had none or drastically reduced amounts of Opa1, whereas cells treated with the control siRNA showed strong Opa1 labeling (Figs. 5A 5B) . Similarly, in the CGCs, treatment with the Opa1 siRNA drastically downregulated the amounts of Opa1 after 15 days compared with the mock-treated cells, as observed by Western blot (Fig. 5I)and immunofluorescence (Figs. 5G 5H)
Downregulation of Opa1 Leads to Aggregation of the Mitochondrial Network
In parallel, the evolution of the mitochondrial morphology was monitored in cultures labeled with a mitochondrial marker (MitoTracker Red; Molecular Probes) and compared with that in CGCs. In RGCs, before siRNA transfection (day 7 in cultures), two phenotypes of the mitochondrial network were observed. Phenotype I featured moderate staining with fine and evenly spaced punctiform structures throughout the cytoplasm and neurites, as seen in control-treated RGCs (Fig. 5D) , whereas phenotype II was characterized by strong and irregular staining in the cytoplasm with clustered structures in neurites, as seen in Opa1 siRNA-treated RGCs (Fig. 5E) . After siRNA transfection, the two categories of mitochondrial network phenotypes were counted every 5 days during 15 days, and the ratio of the number of cells with phenotype II divided by the number of cells with phenotype I was calculated (Figs. 5F) . In RGCs, at day 0, phenotype I predominated in both control and Opa1 siRNA-treated cultures and remained as such in control siRNA-treated cultures for the following 15 days. In contrast, the ratio drastically and significantly (P < 0.05 at days 10 and 15) increased in a time-dependent manner in Opa1 siRNA-treated cultures, indicating predominance of the aggregated phenotype II (Fig. 5F) . These differences were not due to cell death, as cell densities (7000/cm2) did not vary significantly throughout the experiment (not shown). In CGCs, phenotypes from control-treated cultures showed mainly diffuse, fine, regular staining (Fig. 5J) , and there was strong punctiform staining, more frequently seen in Opa1 siRNA-treated cultures (Fig. 5K) , resembling respectively the phenotypes I and II found in RGC cultures. The ratio between these phenotypes, as defined for the RGCs, was followed after transfection of the siRNA for 15 days (Fig. 5L) . In both the control and Opa1 siRNA-treated cultures, this ratio evolved, as the amount of cells presenting a strong punctiform phenotype increased with time. In Opa1-siRNA treated cells, however, this ratio evolved faster and became significantly different from control data at days 10 and 15 (Fig. 5L) . Finally, comparing the RGCs and the CGCs kinetics revealed that the changes of mitochondrial network induced by the loss in Opa1 were much more rapid and drastic in RGCs than in CGCs. 
Discussion
The purpose of this study was to evaluate the expression of Opa1 in whole retina and isolated RGCs as well as the effect of Opa1 downregulation in cultured RGCs, which may relate more directly to the pathophysiology of ADOA than other tissues or cell lines. 11 12 32 34 42 43 We purified (96% of total cell content) and maintained RGCs up to 3 weeks in culture, enough to perform various molecular and immunocytochemical analyses. 
It is a common consensus that ADOA is due to the primary degeneration of RGCs, with ascending axonal degeneration leading to optic atrophy. The fact that only RGCs seem affected by OPA1 mutations despite the ubiquitous OPA1 expression suggests that RGCs are particularly vulnerable to mitochondrial membrane disorder. We first tested two hypotheses for this vulnerability. One hypothesis is that OPA1 is predominantly expressed in RGCs. In the rat retina, we observed Opa1 expression in all retinal layers, except in the nuclear layers and PR outer segments, which contain tiny amounts of cytoplasm and therefore very few mitochondria. Except for PR IS that showed relatively weak Opa1 expression compared with that of cytochrome c, Opa1 labeling paralleled that of cytochrome c, in particular in RGCs, indicating that the protein was not predominantly expressed in these cells. This result agrees with that of a former study performed in the adult mouse retina 44 but surprisingly not with observations performed on mouse and rat postnatal retinas. 45 We confirmed by Western blot analysis that similar amounts of Opa1 protein are present in total extracts from purified RGCs or from the whole retina, from 5- to 8-day-old rats. Another pathophysiological hypothesis could be that the pattern of Opa1 mRNA isoforms is particular to RGCs, as isoforms may be sublocalized to various mitochondrial compartments 43 with various and specific functional specializations. In fact, Opa1 transcripts amplified by RT-PCR from total retina and from isolated RGCs demonstrated similar patterns. Considering that the number of RGCs is less than 0.5% of that of the total retinal cell content, 35 this result indicates that neither the patterns nor the abundance of Opa1 mRNA isoforms or protein variants is specific to RGCs and therefore they could not explain the vulnerability of postnatal RGCs to OPA1 mutations. 
Mitochondria play a major role in the energy generation necessary for cellular activities. Several pieces of work have been recently reported from the viewpoint of mitochondrial distribution in RGCs and their axons. In the optic nerve, mitochondrial enzyme activity was found to distribute in intraretinal nonmyelinated fibers, including the lamina cribrosa, but not in myelinated retrolaminar fibers. 46 47 In another study, Wang et al. 48 demonstrated the accumulation of mitochondria in varicosities of the intraretinal ganglion cell axons in human and nonhuman primates, suggesting that these varicosities may be functional sites that serve local high-energy demands for signal transmission in the unmyelinated fibers. Altogether, RGCs may need a particular mitochondrial network to maintain the efficient transmission of action potentials along the intraretinal unmyelinated portions of their axons. We therefore examined the mitochondrial network and Opa1 expression in RGCs isolated from neonatal rats. At early stages of culture, the RGC mitochondrial network was found as abundant round or punctuated structures in the soma and in lesser amounts in neurites. Anti-Opa1 immunofluorescence labeling confirmed the presence of Opa1, colocalizing with mitochondrial protein cytochrome c, in discrete cytoplasmic structures. We observed that the mitochondrial network clustered in the branch points and growth cones, especially in the first few days of culture, as previously described in dorsal root ganglion cells. 49 Later, they spread out along the neurite length as finer and lesser punctiform structures, in agreement with the suggestion that clustered mitochondria may be storage pools of mitochondria that can be mobilized to provide energy for axonal transport during neuronal regeneration. 49 In addition, clustered mitochondria themselves may be able to supply the energy needed for further neurite outgrowth. 
To test the response of RGCs to the downregulation of the endogenous Opa1, we knocked down Opa1 expression by using siRNA and compared it with the response of CGCs treated in the same way. We found that the mitochondrial network was modified from an evenly spread form to an aggregated pattern similar to that seen in monocytes from patients with ADOA 11 and in sympathetic neurons deprived in neuronal growth factor (NGF). 50 In a surprising finding, more RGCs with an aggregated mitochondrial network accumulated than the CGCs with this phenotype. Thus, RGCs may be more vulnerable to Opa1 deprivation than other type of neurons. It is possible that mitochondrial network remodeling is especially frequent in RGCs, or that reduced expression of Opa1 impairs metabolic pathways that are critical to these cells. Overall, the observed mitochondrial aggregation may represent a preapoptotic state 51 that could throw the sensitivity to various apoptotic stimuli out of balance. This could account for the loss of RGCs in patients with ADOA and consequently lead to a progressive decrease in visual acuity through their life. Further functional studies are needed to answer these questions. 
 
Figure 1.
 
Five-day culture of RGCs isolated by two-step immunopanning. (A) Cells were immunolabeled with anti-Thy1.1 antibody (red) and counterstained with Hoechst dye (blue). Cells expressed Thy1.1 indicating that they all were RGCs. (B) Phase-contrast micrograph showing the morphology of cultured RGCs. Scale bar, 30 μm.
Figure 1.
 
Five-day culture of RGCs isolated by two-step immunopanning. (A) Cells were immunolabeled with anti-Thy1.1 antibody (red) and counterstained with Hoechst dye (blue). Cells expressed Thy1.1 indicating that they all were RGCs. (B) Phase-contrast micrograph showing the morphology of cultured RGCs. Scale bar, 30 μm.
Figure 2.
 
Rat retinal sections were immunolabeled with antibody to (A) Opa1 and (B) cytochrome c, counterstained with (D) Hoechst dye, and visualized with a confocal microscope. (A) Opa1 was expressed in the GCL, IPL, OPL, PR IS, and PE, in which (B) cytochrome c staining was also found. (C) Merged micrograph demonstrating that Opa1 expression was weaker than that of cytochrome c in PRs and PE, whereas in other layers it was similar. (D) Hoechst staining showed each nuclear layer of retina. (E) Control staining with polyclonal rabbit IgG in place of the primary anti-OPA1 antibody showed no fluorescence. INL, inner nuclear layer, ONL, outer nuclear layer, OS, outer segment of PR. Scale bar, 30 μm. (F) Immunoblots of RGC (lane 1) and other retinal cell (lane 2) extracts incubated with anti-OPA1 antibody showing two double bands (92 and 86 kDa) in equivalent amounts in both cell categories. (G) Stripping and reincubation of the same immunoblot with an anti-β actin antibody shows that equivalent amounts of protein were loaded in both lanes.
Figure 2.
 
Rat retinal sections were immunolabeled with antibody to (A) Opa1 and (B) cytochrome c, counterstained with (D) Hoechst dye, and visualized with a confocal microscope. (A) Opa1 was expressed in the GCL, IPL, OPL, PR IS, and PE, in which (B) cytochrome c staining was also found. (C) Merged micrograph demonstrating that Opa1 expression was weaker than that of cytochrome c in PRs and PE, whereas in other layers it was similar. (D) Hoechst staining showed each nuclear layer of retina. (E) Control staining with polyclonal rabbit IgG in place of the primary anti-OPA1 antibody showed no fluorescence. INL, inner nuclear layer, ONL, outer nuclear layer, OS, outer segment of PR. Scale bar, 30 μm. (F) Immunoblots of RGC (lane 1) and other retinal cell (lane 2) extracts incubated with anti-OPA1 antibody showing two double bands (92 and 86 kDa) in equivalent amounts in both cell categories. (G) Stripping and reincubation of the same immunoblot with an anti-β actin antibody shows that equivalent amounts of protein were loaded in both lanes.
Figure 3.
 
Detection of Opa1 isoforms in several rat tissues. RT-PCR of Opa1 RNA from rat retina (lane 1), purified RGCs (lane 2), brain (lane 3), and RT product from brain without reverse transcriptase (lane 4) between exons 3 and 9, using ME3S and MK5AS primers, resulted in the amplification of six distinct fragments. Left: fragment sizes. The isoform pattern of Opa1 in retina and RGCs was similar.
Figure 3.
 
Detection of Opa1 isoforms in several rat tissues. RT-PCR of Opa1 RNA from rat retina (lane 1), purified RGCs (lane 2), brain (lane 3), and RT product from brain without reverse transcriptase (lane 4) between exons 3 and 9, using ME3S and MK5AS primers, resulted in the amplification of six distinct fragments. Left: fragment sizes. The isoform pattern of Opa1 in retina and RGCs was similar.
Figure 4.
 
Representative confocal micrographs of the mitochondrial distribution and morphology, and Opa1 expression in purified rat RGCs 1 to 5 days after seeding in culture. (A) In 1-day cultures, round or punctiform structures stained with a mitochondrial marker were abundant in the cytoplasm and were present in lower amounts in neurites, with clustering in branch points and growth cones (arrows). (B) In 2-day cultures, punctiform marker staining spread and (C) at 5 days became more diffuse. Double immunolabeling with antibody against (D) OPA1 and (E) mitochondrial protein cytochrome c was performed in 5-day cultures. (F) Merged micrograph demonstrated that these proteins mainly colocalized. Scale bar, 10 μm.
Figure 4.
 
Representative confocal micrographs of the mitochondrial distribution and morphology, and Opa1 expression in purified rat RGCs 1 to 5 days after seeding in culture. (A) In 1-day cultures, round or punctiform structures stained with a mitochondrial marker were abundant in the cytoplasm and were present in lower amounts in neurites, with clustering in branch points and growth cones (arrows). (B) In 2-day cultures, punctiform marker staining spread and (C) at 5 days became more diffuse. Double immunolabeling with antibody against (D) OPA1 and (E) mitochondrial protein cytochrome c was performed in 5-day cultures. (F) Merged micrograph demonstrated that these proteins mainly colocalized. Scale bar, 10 μm.
Figure 5.
 
Phenotypes of RGCs and CGCs treated with Opa1 or control siRNAs. (AF) RGCs and (GL) CGCs. Immunofluorescence of RGCs treated with control (A) or Opa1 (B) siRNA and incubated with anti-OPA1 antibodies; Western blot (C) using OPA1 antibodies or actin on corresponding extracts (lane 1: control; lane 2: Opa1 siRNA). Phenotypes observed with mitochondrial marker of RGCs treated with control (D) or Opa1 (E) siRNA define the strong irregular and fine punctiform phenotypes and the evolution of ratio between them during a 15-day kinetic (F). *Significant at P < 0.05. Opa1 immunofluorescence performed on CGCs treated with control (G) or Opa1 (H) siRNA, using OPA1 antibodies and Western blot analysis using OPA1 or actin antibodies on corresponding extracts show the extinction of Opa1 expression (I; lane 1: control; lane 2: Opa1 siRNA). Phenotypes observed with mitochondrial marker of CGCs treated with control (J) or Opa1 (K) siRNA and the corresponding ratio at days 0, 5, 10, and 15 after transfection (L). *Significant at P < 0.05. Scale bar: (A, B, D, E) 20 μm; (G, H, J, K) 10 μm.
Figure 5.
 
Phenotypes of RGCs and CGCs treated with Opa1 or control siRNAs. (AF) RGCs and (GL) CGCs. Immunofluorescence of RGCs treated with control (A) or Opa1 (B) siRNA and incubated with anti-OPA1 antibodies; Western blot (C) using OPA1 antibodies or actin on corresponding extracts (lane 1: control; lane 2: Opa1 siRNA). Phenotypes observed with mitochondrial marker of RGCs treated with control (D) or Opa1 (E) siRNA define the strong irregular and fine punctiform phenotypes and the evolution of ratio between them during a 15-day kinetic (F). *Significant at P < 0.05. Opa1 immunofluorescence performed on CGCs treated with control (G) or Opa1 (H) siRNA, using OPA1 antibodies and Western blot analysis using OPA1 or actin antibodies on corresponding extracts show the extinction of Opa1 expression (I; lane 1: control; lane 2: Opa1 siRNA). Phenotypes observed with mitochondrial marker of CGCs treated with control (J) or Opa1 (K) siRNA and the corresponding ratio at days 0, 5, 10, and 15 after transfection (L). *Significant at P < 0.05. Scale bar: (A, B, D, E) 20 μm; (G, H, J, K) 10 μm.
The authors thank Frank W. Pfrieger and Daniela Mauch for technical support in two-step immunopanning; Nicole Lautredou-Audouy for assistance in confocal imaging; and Matthieu J. Guitton, Olivier Payet, and Jérôme Ruel for help with statistical analysis. 
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Figure 1.
 
Five-day culture of RGCs isolated by two-step immunopanning. (A) Cells were immunolabeled with anti-Thy1.1 antibody (red) and counterstained with Hoechst dye (blue). Cells expressed Thy1.1 indicating that they all were RGCs. (B) Phase-contrast micrograph showing the morphology of cultured RGCs. Scale bar, 30 μm.
Figure 1.
 
Five-day culture of RGCs isolated by two-step immunopanning. (A) Cells were immunolabeled with anti-Thy1.1 antibody (red) and counterstained with Hoechst dye (blue). Cells expressed Thy1.1 indicating that they all were RGCs. (B) Phase-contrast micrograph showing the morphology of cultured RGCs. Scale bar, 30 μm.
Figure 2.
 
Rat retinal sections were immunolabeled with antibody to (A) Opa1 and (B) cytochrome c, counterstained with (D) Hoechst dye, and visualized with a confocal microscope. (A) Opa1 was expressed in the GCL, IPL, OPL, PR IS, and PE, in which (B) cytochrome c staining was also found. (C) Merged micrograph demonstrating that Opa1 expression was weaker than that of cytochrome c in PRs and PE, whereas in other layers it was similar. (D) Hoechst staining showed each nuclear layer of retina. (E) Control staining with polyclonal rabbit IgG in place of the primary anti-OPA1 antibody showed no fluorescence. INL, inner nuclear layer, ONL, outer nuclear layer, OS, outer segment of PR. Scale bar, 30 μm. (F) Immunoblots of RGC (lane 1) and other retinal cell (lane 2) extracts incubated with anti-OPA1 antibody showing two double bands (92 and 86 kDa) in equivalent amounts in both cell categories. (G) Stripping and reincubation of the same immunoblot with an anti-β actin antibody shows that equivalent amounts of protein were loaded in both lanes.
Figure 2.
 
Rat retinal sections were immunolabeled with antibody to (A) Opa1 and (B) cytochrome c, counterstained with (D) Hoechst dye, and visualized with a confocal microscope. (A) Opa1 was expressed in the GCL, IPL, OPL, PR IS, and PE, in which (B) cytochrome c staining was also found. (C) Merged micrograph demonstrating that Opa1 expression was weaker than that of cytochrome c in PRs and PE, whereas in other layers it was similar. (D) Hoechst staining showed each nuclear layer of retina. (E) Control staining with polyclonal rabbit IgG in place of the primary anti-OPA1 antibody showed no fluorescence. INL, inner nuclear layer, ONL, outer nuclear layer, OS, outer segment of PR. Scale bar, 30 μm. (F) Immunoblots of RGC (lane 1) and other retinal cell (lane 2) extracts incubated with anti-OPA1 antibody showing two double bands (92 and 86 kDa) in equivalent amounts in both cell categories. (G) Stripping and reincubation of the same immunoblot with an anti-β actin antibody shows that equivalent amounts of protein were loaded in both lanes.
Figure 3.
 
Detection of Opa1 isoforms in several rat tissues. RT-PCR of Opa1 RNA from rat retina (lane 1), purified RGCs (lane 2), brain (lane 3), and RT product from brain without reverse transcriptase (lane 4) between exons 3 and 9, using ME3S and MK5AS primers, resulted in the amplification of six distinct fragments. Left: fragment sizes. The isoform pattern of Opa1 in retina and RGCs was similar.
Figure 3.
 
Detection of Opa1 isoforms in several rat tissues. RT-PCR of Opa1 RNA from rat retina (lane 1), purified RGCs (lane 2), brain (lane 3), and RT product from brain without reverse transcriptase (lane 4) between exons 3 and 9, using ME3S and MK5AS primers, resulted in the amplification of six distinct fragments. Left: fragment sizes. The isoform pattern of Opa1 in retina and RGCs was similar.
Figure 4.
 
Representative confocal micrographs of the mitochondrial distribution and morphology, and Opa1 expression in purified rat RGCs 1 to 5 days after seeding in culture. (A) In 1-day cultures, round or punctiform structures stained with a mitochondrial marker were abundant in the cytoplasm and were present in lower amounts in neurites, with clustering in branch points and growth cones (arrows). (B) In 2-day cultures, punctiform marker staining spread and (C) at 5 days became more diffuse. Double immunolabeling with antibody against (D) OPA1 and (E) mitochondrial protein cytochrome c was performed in 5-day cultures. (F) Merged micrograph demonstrated that these proteins mainly colocalized. Scale bar, 10 μm.
Figure 4.
 
Representative confocal micrographs of the mitochondrial distribution and morphology, and Opa1 expression in purified rat RGCs 1 to 5 days after seeding in culture. (A) In 1-day cultures, round or punctiform structures stained with a mitochondrial marker were abundant in the cytoplasm and were present in lower amounts in neurites, with clustering in branch points and growth cones (arrows). (B) In 2-day cultures, punctiform marker staining spread and (C) at 5 days became more diffuse. Double immunolabeling with antibody against (D) OPA1 and (E) mitochondrial protein cytochrome c was performed in 5-day cultures. (F) Merged micrograph demonstrated that these proteins mainly colocalized. Scale bar, 10 μm.
Figure 5.
 
Phenotypes of RGCs and CGCs treated with Opa1 or control siRNAs. (AF) RGCs and (GL) CGCs. Immunofluorescence of RGCs treated with control (A) or Opa1 (B) siRNA and incubated with anti-OPA1 antibodies; Western blot (C) using OPA1 antibodies or actin on corresponding extracts (lane 1: control; lane 2: Opa1 siRNA). Phenotypes observed with mitochondrial marker of RGCs treated with control (D) or Opa1 (E) siRNA define the strong irregular and fine punctiform phenotypes and the evolution of ratio between them during a 15-day kinetic (F). *Significant at P < 0.05. Opa1 immunofluorescence performed on CGCs treated with control (G) or Opa1 (H) siRNA, using OPA1 antibodies and Western blot analysis using OPA1 or actin antibodies on corresponding extracts show the extinction of Opa1 expression (I; lane 1: control; lane 2: Opa1 siRNA). Phenotypes observed with mitochondrial marker of CGCs treated with control (J) or Opa1 (K) siRNA and the corresponding ratio at days 0, 5, 10, and 15 after transfection (L). *Significant at P < 0.05. Scale bar: (A, B, D, E) 20 μm; (G, H, J, K) 10 μm.
Figure 5.
 
Phenotypes of RGCs and CGCs treated with Opa1 or control siRNAs. (AF) RGCs and (GL) CGCs. Immunofluorescence of RGCs treated with control (A) or Opa1 (B) siRNA and incubated with anti-OPA1 antibodies; Western blot (C) using OPA1 antibodies or actin on corresponding extracts (lane 1: control; lane 2: Opa1 siRNA). Phenotypes observed with mitochondrial marker of RGCs treated with control (D) or Opa1 (E) siRNA define the strong irregular and fine punctiform phenotypes and the evolution of ratio between them during a 15-day kinetic (F). *Significant at P < 0.05. Opa1 immunofluorescence performed on CGCs treated with control (G) or Opa1 (H) siRNA, using OPA1 antibodies and Western blot analysis using OPA1 or actin antibodies on corresponding extracts show the extinction of Opa1 expression (I; lane 1: control; lane 2: Opa1 siRNA). Phenotypes observed with mitochondrial marker of CGCs treated with control (J) or Opa1 (K) siRNA and the corresponding ratio at days 0, 5, 10, and 15 after transfection (L). *Significant at P < 0.05. Scale bar: (A, B, D, E) 20 μm; (G, H, J, K) 10 μm.
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