Mutant NADH dehydrogenase subunit 4 gene delivery to mitochondria by targeting sequence-modified adeno-associated virus induces visual loss and optic atrophy in mice

Abstract

Purpose: Although mutated G11778A NADH ubiquinone oxidoreductase subunit 4 (ND4) mitochondrial DNA (mtDNA) is firmly linked to the blindness of Leber hereditary optic neuropathy (LHON), a bona fide animal model system with mutated mtDNA complex I subunits that would enable probing the pathogenesis of optic neuropathy and testing potential avenues for therapy has yet to be developed.

Methods: The mutant human ND4 gene with a guanine to adenine transition at position 11778 with an attached FLAG epitope under control of the mitochondrial heavy strand promoter (HSP) was inserted into a modified self-complementary (sc) adeno-associated virus (AAV) backbone. The HSP-ND4FLAG was directed toward the mitochondria by adding the 23 amino acid cytochrome oxidase subunit 8 (COX8) presequence fused in frame to the N-terminus of green fluorescent protein (GFP) into the AAV2 capsid open reading frame. The packaged scAAV-HSP mutant ND4 was injected into the vitreous cavity of normal mice (OD). Contralateral eyes received scAAV-GFP (OS). Translocation and integration of mutant human ND4 in mouse mitochondria were assessed with PCR, reverse transcription-polymerase chain reaction (RT-PCR), sequencing, immunoblotting, and immunohistochemistry. Visual function was monitored with serial pattern electroretinography (PERG) and in vivo structure with spectral domain optical coherence tomography (OCT). Animals were euthanized at 1 year and processed for light and transmission electron microscopy.

Results: The PCR products of the mitochondrial and nuclear DNA extracted from infected retinas and optic nerves gave the expected 500 base pair bands. RT-PCR confirmed transcription of the mutant human ND4 DNA in mice. DNA sequencing confirmed that the PCR and RT-PCR products were mutant human ND4 (OD only). Immunoblotting revealed the expression of mutant ND4FLAG (OD only). Pattern electroretinograms showed a significant decrement in retinal ganglion cell function OD relative to OS at 1 month and 6 months after AAV injections. Spectral domain optical coherence tomography showed optic disc edema starting at 1 month post injection followed by optic nerve head atrophy with marked thinning of the inner retina at 1 year. Histopathology of optic nerve cross sections revealed reductions in the optic nerve diameters of OD versus OS where transmission electron microscopy revealed significant loss of optic nerve axons in mutant ND4 injected eyes where some remaining axons were still in various stages of irreversible degeneration with electron dense aggregation. Electron lucent mitochondria accumulated in swollen axons where fusion of mitochondria was also evident.

Conclusions: Due to the UGA codon at amino acid 16, mutant G11778A ND4 was translated only in the mitochondria where its expression led to significant loss of visual function, loss of retinal ganglion cells, and optic nerve degeneration recapitulating the hallmarks of human LHON.

Figure 1

MTS AAV-capsid localizes within mitochondria where the delivered wild-type human ND4 is translated in cultured cells. A: Illustration of the COX8 MTS fused in frame with GFP and inserted into the VP2 capsid of AAV. B: Illustration of the mitochondrial heavy strand promoter (HSP) driving expression of the mitochondrial mutant human ND4 subunit of complex I to which is appended a FLAG epitope. C: Fluorescence microscopy of live cultured cells infected with the COX8GFP VP2 MTS AAV revealed punctate and perinuclear expression of GFP suggestive of mitochondrial localization. D: MitoTracker Green staining of mitochondria surrounds nuclei labeled with DAPI. E: Transmission electron microscopy shows silver enhanced GFP immunogold inside mitochondria as well as within the nucleus. F: ND4FLAG immunogold is evident only within the mitochondria. Abbreviations m=mitochondria and n=nucleus.

Figure 2

Mutant human G11778A ND4 transmission in mice. A: RT–PCR of RNA extracted from retinal mitochondria (RM), optic nerve mitochondria (OM), retinal nuclear (RN), or optic nerve nuclear (ON) debris, retinal cytoplasm (RC), and optic nerve cytoplasmic (OC) fractions of experimental right eyes had the expected 500 bp band for ND4FLAG that was absent in RNA extracted from control left eyes infected with scAAV-GFP. B: A sequencing chromatograph shows the corresponding DNA sequence to be that of the mutant human ND4 where the base adenine (A; arrows) has replaced guanine (G). C: One of the sequences, SEQmutND41-OM, is aligned to the wild-type human ND4 (ND4mito) showing this G to A transition (arrows). It also reveals the sequence of the mouse ND4 (mitoND4mouse) confirmed that the PCR products were indeed mutant human G11778A ND4, further supporting that exogenous ND4 was imported into retinal and optic nerve mitochondria by a mitochondria-targeted AAV where it was transcribed. D: Immunoblotting of isolated optic nerve and retinal mitochondria showed that the MTS-targeted AAV directed the synthesis of mutant human ND4FLAG in the experimental eyes, but the control eyes injected with GFP were negative for FLAG. ND4 was overexpressed in experimental eyes relative to the endogenous ND4 of GFP injected control eyes. Expression of NDUFS4, a nuclear encoded complex I subunit, is shown for housekeeping. E: Amino acid sequence of ND4 with the start methionine (met) shows that the TGA codon is a termination sequence for protein synthesis in the cytoplasm, but specifies the amino acid tryptophan for synthesis within the mitochondrial ribosomes. F: Illustrating that full-length ND4 with 340 amino acids can be expressed only within mitochondria.

Figure 3

Wild-type human ND4 expression in RGCs does not cause visual loss in mice. Confocal immunofluorescence micrographs show DAPI stained nuclear layers of the murine retina infected 7 weeks earlier with MTS scAAV containing wild-type normal human ND4FLAG (A). The RGC layer and dendrites extending into the inner nuclear layer (INL) are labeled with an antibody against Thy1.2. The outer nuclear layer (ONL) is not stained by Thy1.2 (B). RGCs expressing human ND4FLAG are labeled by an anti-FLAG antibody (C) that colocalizes to Thy1.2 and has a perinuclear distribution characteristic of mitochondria surrounding the DAPI-stained RGC nuclei (D). A scatterplot (E) and a plot of representative PERG waveform (F) show that 7 weeks after AAV injections there were no differences in amplitude between the eyes injected with wild-type human mitochondrial (m) ND4 relative to the eyes that received no ocular gene injections.

Figure 4

Mutant human ND4 expression induces visual loss in mice. A: At 1 month post injection, a scatterplot of a root-mean-square (RMS) PERG amplitude shows a significant loss of visual function of the right eyes injected with the mutant human ND4 packaged with the MTS scAAV relative to the left eyes injected with GFP packaged with standard scAAV (p=0.0352). B: Six months post injection, this decrement between experimental and control eyes was even more significant (p=0.0182). C: Representative waveforms show normal amplitude 6 months after scAAV-GFP injection but loss (D) of PERG signal with MTS scAAV mutant ND4 injection. E: A barplot shows an increasing reduction of the ratio of the right eye to left eye amplitudes with 1-month (1M) and 6-month (6M) intervals post injection relative to baseline values (BL) obtained before AAV injections.

Figure 5

SD-OCT imaging of optic disc edema and optic atrophy. A: Spectral domain optical coherence tomography (SD-OCT) of right eyes injected with the mutant human ND4 packaged with the MTS scAAV revealed swelling of the optic nerve head (arrows) commencing at 1 month post injection. A focal increase in the thickness of the RGC and the inner plexiform layer (IPL) is apparent just to the right of the swollen optic nerve head. B: The control eye injected with scAAV-GFP showed the normal anatomy of the mouse optic nerve head. C: One year post injection, optic nerve head atrophy was apparent in the mutant ND4 injected eyes. D: The contralateral GFP injected control eyes maintained normal optic nerve head anatomy. E: One year post injection, focal thinning with loss of the inner retina was also apparent in an experimental eye, but this finding was not seen in any of the control eyes (F). RGC=retinal ganglion cell layer; IPL=inner plexiform layer.

Figure 6

Histopathology of RGC loss. Light microscopy of the eyes of mice euthanized a year post AAV injections confirmed the OCT findings of inner retinal loss induced by injection of the mutant ND4 MTS AAV showing marked focal thinning of the inner retina at low (A) and higher (B) magnifications. C: While experimental eyes of other animals did not exhibit such severe inner retinal atrophy, the more characteristic loss of cells in the ganglion cell layer was evident. Control eyes injected with scAAV-GFP showed no loss of the inner retina layers (D and E) or cells in the RGC layer (F). ON=optic nerve, RGC=retinal ganglion cell layer, INL=inner nuclear layer, and ONL=outer nuclear layer.

Figure 7

Histopathology of optic nerve atrophy. Relative to the normal optic nerves of contralateral control eyes that received scAAV-GFP (A, B), the hallmark optic nerve atrophy of LHON was prominent in mouse eyes injected with MTS scAAV mutant ND4 that appeared thinner than the contralateral experimental eyes (C, D). E: A scatterplot with quantile boxes shows quantitation of the optic nerve head areas of COX8 MTS delivered mutant (G11778A) ND4 were smaller than those of controls injected with scAAV-GFP. The lowest line of the quantile plot represents the tenth percentile and the highest line the 90th percentile. The bottom of the quantile box represents the 25th percentile and the top of the box the 75th percentile, with the median value in the middle of the box. Median values are slightly different from the means. F: A scatterplot with quantile boxes shows axonal loss in MTS AAV mutant ND4 injected eyes relative to axon counts in scAAV-GFP injected eyes. Values for nine of the mutant ND4 injected nerves were below the 25th percentile for control optic nerves injected with scAAV-GFP.

Figure 8

Ultrastructure of optic nerve axonal loss. A: One year after injection of scAAV-GFP, transmission electron micrographs disclosed optic nerve axons (a) of various sizes enveloped by myelin sheaths. Astroglial cell processes (g) coursed between fibers of the optic nerve. B: In this same animal, the opposite eye injected with the MTS scAAV mutant ND4 had a marked decrease in axonal density. Many empty spaces (e) were present where axons were apparently lost in these atrophic optic nerves and degenerating axonal profiles were evident (d). C: In a different animal, the optic nerve of the control eye injected with scAAV-GFP had normal axons (a) with the only empty space the lumen of a blood vessel (L). D: In this animal, the opposite eye injected with the MTS scAAV mutant ND4 had a marked decrease in axonal density. Many empty spaces (e) were present where axons were lost. a=optic nerve axon, e=empty space, g=astroglial process, d=degenerating axon, L=lumen.

Figure 9

Mitochondria accumulate and fuse. A: Optic nerves from mutant ND4 MTS AAV injected eyes exhibited some large-caliber axons (a) with the accumulation of mitochondria (arrow) of varying diameters. Electron-dense aggregations indicative of irreversible axonal degeneration were also evident (double arrows). B: In some swollen axons, two mitochondria were seen fusing (arrows). Irreversible axonal degeneration with intra-axonal electron dense aggregations was evident in other axons (d). C: In another axon, three mitochondria (arrows) appeared to fuse.