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. 2008 Sep;83(3):373-87.
doi: 10.1016/j.ajhg.2008.08.013. Epub 2008 Sep 4.

Optimized Allotopic Expression of the Human Mitochondrial ND4 Prevents Blindness in a Rat Model of Mitochondrial Dysfunction

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Free PMC article

Optimized Allotopic Expression of the Human Mitochondrial ND4 Prevents Blindness in a Rat Model of Mitochondrial Dysfunction

Sami Ellouze et al. Am J Hum Genet. .
Free PMC article

Abstract

Mitochondrial diseases due to mutations in mitochondrial DNA can no longer be ignored in most medical areas. With prevalence certainly higher than one in 6000, they probably represent the most common form of metabolic disorders. Despite progress in identification of their molecular mechanisms, little has been done with regard to therapy. We have recently optimized the allotopic expression for the mitochondrial genes ATP6, ND1, and ND4 and obtained a complete and long-lasting rescue of mitochondrial dysfunction in the human fibroblasts in which these genes were mutated. However, biosafety and benefit to mitochondrial function must be validated in animal models prior to clinical applications. To create an animal model of Leber Hereditary Optic Neuropathy (LHON), we introduced the human ND4 gene harboring the G11778A mutation, responsible of 60% of LHON cases, to rat eyes by in vivo electroporation. The treatment induced the degeneration of retinal ganglion cells (RGCs), which were 40% less abundant in treated eyes than in control eyes. This deleterious effect was also confirmed in primary cell culture, in which both RGC survival and neurite outgrowth were compromised. Importantly, RGC loss was clearly associated with a decline in visual performance. A subsequent electroporation with wild-type ND4 prevented both RGC loss and the impairment of visual function. Hence, these data provide the proof-of-principle that optimized allotopic expression can be an effective treatment for LHON, and they open the way to clinical studies on other devastating mitochondrial disorders.

Figures

Figure 1
Figure 1
Efficacy of Gene Transduction for In Vivo ELP (A) Immunofluorescence analyses with antibodies to BRN3a and GFP of cryostat retinal sections from animals electroporated with the GFP vector. Six animals were sacrificed 15, 25, 50, and 75 days after the intervention. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bars represent 50 μm. (B) Numerical evaluation of data presented in (A). Four independent sections per animal were counted for BRN3a-positive cells in controls (RGC-GFP); in electroporated animals the number of cells labeled with both BRN3a and GFP antibodies was estimated (RGC-GFP+). Mean ± SEM for six control rats and animals euthanized 15, 25, 50, or 75 days after in vivo ELP are shown. (C) The steady-state levels of endogenous and hybrid ND4 mRNAs were determined in purified RGCs from animals subjected to in vivo ELP with either mutant ND4 (RGCs + G11778A-ND4), wild-type ND4 (RGCs + WT-ND4), or the empty vector (RGCs + GFP) by RT-PCR. Upper panel: Semiquantitative estimations were made after 25, 30, and 35 cycles of PCR amplification with 60 μg of RNA as a starting material. Densitometric analyses were performed with signals revealed by electrophoresis (1/10 of the RT-PCR reaction) for three independent RNA extractions and two RT-PCRs per RNA. Bar graphs represented ratios ± SEM of either endogenous mtDNA ND4 (mtND4) or β-actin signals to COX10-ND4 signal obtained with RNA purified from RGCs expressing either mutant (G11778A-ND4) or wild-type ND4 (WT-ND4). Bottom panel: Relative amounts of β-actin, COX10-ND4, IGF1, SDH1, mtND4 and mtATP6 mRNAs were evaluated after 30 cycles of PCR amplification of the reverse-transcribed products from 60 μg of RNAs obtained from RGCs expressing either mutant ND4 (RGCs + G11778A-ND4), wild-type ND4 (RGCs + WT-ND4), or the empty vector (RGCs + GFP). Bar graphs represent ratios ± SEM of COX10-ND4, IGF1, SDH1, mtND4, and mtATP6 signals to β-actin signal. RT-PCR experiments were performed twice for three independent RNA extractions. Specific oligonucleotides used for each gene are summarized in Table S1.
Figure 2
Figure 2
Ex Vivo Properties of RGCs Isolated from Electroporated Eyes (A) RGCs were purified as previously described. After 10 days of culture, cells were visualized by indirect immunofluorescence with antibodies against NF200 or ATP synthase α-subunit. RGCs were labeled essentially within their neurites; a clear difference in their lengths was noticed between cells purified from eyes expressing mutant ND4 (G11778A-ND4) and those expressing wild-type ND4. The merged image of immunofluorescence revealed a significant colocalization of both NF200 and ATP synthase-α signals in cells examined. The scale bar represents 25 μm (magnification of 200×). A high-power view (magnification of 630×) featuring one of the cells immunolabeled with the antibodies is also presented for each condition; scale bars represent 8 μm. (B) The subcellular localization of the allotopically expressed ND4 proteins was examined by indirect immunofluorescence with an antibody against the FLAG epitope (ND4-FLAG) in RGCs cultured for 10 days, and this was compared to signal obtained with the antibody against NF200. The merged image of immunofluorescence shows a significant colocalization of the fusion ND4 and NF200 proteins in the cytosol and along neuritic processes. The scale bar corresponds to 25 μm (magnification of 200×). A high-power view (magnification of 630×) featuring immunolabeled cells is also presented for each condition; scale bars represent 8 μm. In both (A) and (B), arrows indicate either RGC somas lacking neuritic extensions or isolated neurites that were not associated with RGC nuclei. These signals have been often found in eyes electroporated with mutant ND4, indicating its deleterious effect on RGC survival ex vivo. (C) A retinal ganglion cell purified from eyes electroporated with mutant ND4, which developed long neuritic processes, is shown. This RGC did not immunostain with anti-FLAG antibody, indicating that it did not express mutant ND4. The bar corresponds to 25 μm (magnification of 200×). In the bottom of the image, a RGC with two short neurites is also shown, this cell was immunolabeled with anti-FLAG antibody, indicating the expression of mutant ND4. Scale bars represent 25 μm. A high-power view (magnification of 630×) featuring the two RGCs immunolabeled with the antibodies is also presented; scale bars represent 8 μm.
Figure 3
Figure 3
In Vivo Expression of Mutant ND4 (A) Immunofluorescence analyses with antibodies against the NF200 protein and the FLAG epitope were performed on cryostat retinal sections from animals electroporated with either mutant (G11778A ND4) or the wild-type ND4 gene and sacrificed 25 days later. The image illustrates cells strongly immunostained with NF200 in the GCL, indicating that they were RGCs. The signal was essentially visualized along cell axons and around the nuclear envelops but was excluded from the nucleus (DAPI). As we observed in cultured RGCs (Figure 2) ND4 fusion proteins presented the same subcellular distribution than NF200, illustrated by the significant colocalization of both Flag and NF200 signals (MERGED). A magnification of 630× is shown; the scale bar corresponds to 10 μm. (B) The effect of in vivo ELP on RGC viability was assessed by immunostaining with BRN3a for labeling ganglion cell nuclei and with GFP for labeling electroinduced cells. The number of RGC-positive cells were estimated with merged images of BRN3a and DAPI stainings. It clearly appeared that the number of RGCs was strongly reduced when the eyes expressed mutant ND4 (G11778A ND4) in comparison to when eyes were electroporated with wild-type ND4. Retinal sections shown correspond to animals sacrificed 25 days after in vivo ELP. Scale bars represent 50 μm (magnification of 200×). GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; and ONL, outer nuclear layer. (C) Retinas were assessed for TUNEL labeling after in vivo ELP with either mutant (G11778A ND4) or wild-type ND4 vector. The frequency of TUNEL-positive cells was greater in the GCL of retinas expressing mutant ND4 (G11778A ND4) than in retinas expressing its wild-type counterpart (wild-type ND4). The intensity of staining and the number of cells displaying advanced apoptotic features (arrows) was increased in retinas expressing mutant ND4. Some cells showed preferential staining of chromatin in the proximity of the nuclear envelop, a feature of early-stage apoptosis (arrows). Rarely, few TUNEL-positive cells were evidenced in the INL of animals electroporated with mutant ND4, suggesting that amacrine cells expressing mutant ND4 could undergo apoptosis. Scale bars represent 25 μm (magnification of 400×). GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; and ONL, outer nuclear layer.
Figure 4
Figure 4
NF200-Labeled Axons in Whole-Mmount Retinas (A) Immunostaining with NF200 in whole-mount retinas from animals subjected to a unique ELP clearly showed many fewer NF200-positive axons when animals were electroinduced with mutant ND4 (G11778A-ND4) than when both retinas expressed either the empty vector (GFP) or the wild-type gene (WT-ND4). (B) Retinal flat-mount labeled with NF200 from a control animal (13 weeks old) was compared to the labeling obtained in animals electroporated twice. It clearly appeared that when the second ELP was performed with wild-type ND4 there was good axon preservation (G11778A-ND4 + WT-ND4), whereas relatively poor preservation was seen in the GFP-treated retina (G11778A-ND4 + GFP), similar to the one observed when a unique ELP was performed with mutant ND4 (A). A magnification of 200× is shown. ON, optic nerve.
Figure 5
Figure 5
Assessment of Visual Function via Optomotor Tests of Long Evans Rats (A) Head-tracking responses of ten controls (8 and 12 weeks old) compared with rats electroporated in their right eyes with the wild-type ND4 gene (B). The three grating frequencies of 0.125, 0.25, and 0.5 cyc/deg are shown in both clockwise (arrows revolving in clockwise direction) and counterclockwise (arrows revolving in counterclockwise direction) directions of motion. Visual thresholds in these animals did not depend on the direction of rotation given that no significant difference was measured in the three grating frequencies monitored. Moreover, the scores were comparable in all the rats assessed, indicating that electroporation with wild-type ND4 (B) did not compromise visual acuity. (C) Optomotor tracking in ten animals subjected to in vivo ELP with mutant ND4. In vivo ELP in the right eye with mutant ND4 strongly diminished tracking when the grating motion was counterclockwise but had no effect on clockwise tracking. According to a Student's t test, a significant difference was measured in the three grating frequencies monitored, indicating a link between mutant ND4 expression and the loss of visual function. (D) Ten animals were subjected to a second ELP with wild-type ND4 14 days after mutant ND4 expression in their right eyes. Head-tracking responses in the counterclockwise motion were significantly better in these animals than in those subjected to a single ELP with mutant ND4. For instance, comparative analyses of means tracking in the 0.125 cyc/deg grating frequency of clockwise and counterclockwise responses were not statically different according to a Student's t test (p = 0.1). For animals subjected to ELP, all the assessments were performed 25 and 35 days after the intervention, and values presented are the means ± SEM of these two assessments.

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