Progressive optic atrophy in a retinal ganglion cell-specific mouse model of complex I deficiency

Wang, Luyu; Klingeborn, Mikael; Travis, Amanda M.; Hao, Ying; Arshavsky, Vadim Y.; Gospe, Sidney M.

doi:10.1038/s41598-020-73353-0

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Published: 01 October 2020

Progressive optic atrophy in a retinal ganglion cell-specific mouse model of complex I deficiency

Luyu Wang¹,
Mikael Klingeborn¹,
Amanda M. Travis²,
Ying Hao¹,
Vadim Y. Arshavsky^1,3 &
…
Sidney M. Gospe III¹

Scientific Reports volume 10, Article number: 16326 (2020) Cite this article

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Abstract

Optic atrophy resulting from retinal ganglion cell (RGC) degeneration is a prominent ocular manifestation of mitochondrial dysfunction. Although transgenic mice lacking the mitochondrial complex I accessory subunit NDUFS4 develop early-onset optic atrophy, severe systemic mitochondrial dysfunction leads to very early death and makes this mouse line impractical for studying the pathobiology of mitochondrial optic neuropathies. Theoretically, RGC-specific inactivation of ndufs4 would allow characterization of RGC degeneration over a longer time course, provided that RGC death from mitochondrial dysfunction is a cell-autonomous process. We demonstrate that the vesicular glutamate transporter VGLUT2 may be exploited to drive robust Cre recombinase expression in RGCs without any expression observed in directly neighboring retinal cell types. Deletion of ndufs4 in RGCs resulted in reduced expression of NDUFS4 protein within the optic nerves of Vglut2-Cre;ndufs4^loxP/loxP mice. RGC degeneration in Vglut2-Cre;ndufs4^loxP/loxP retinas commenced around postnatal day 45 (P45) and progressed to loss of two-thirds of RGCs by P90, confirming that intrinsic complex I dysfunction is sufficient to induce RGC death. The rapidly-developing optic atrophy makes the Vglut2-Cre;ndufs4^loxP/loxP mouse line a promising preclinical model for testing therapies for currently untreatable mitochondrial optic neuropathies such as Leber Hereditary Optic Neuropathy.

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Introduction

Mitochondrial dysfunction contributes to vision loss in many retinal and optic nerve disorders^1,2,3, with a small but important subset comprised by heritable mitochondrial diseases⁴. Chief among these is Leber Hereditary Optic Neuropathy (LHON), a maternally-inherited disease in which rapid degeneration of retinal ganglion cells (RGCs) results in profound vision loss, typically in previously healthy adolescents and young adults⁵. With a prevalence of 1:30,000 to 1:50,000, LHON is likely the most common human disease with a mitochondrial pattern of inheritance^6,7,8. It is caused by hypomorphic missense mutations in mitochondrial DNA (mtDNA) encoding subunits of respiratory complex I (NADH:ubiquinone oxidoreductase). This large protein complex found in the mitochondrial inner membrane is composed of seven mtDNA-encoded subunits and approximately 37 nuclear-encoded subunits⁹. Because the loss of function resulting from the mtDNA mutations associated with LHON is usually relatively mild, homoplasmy of the mutations is frequently required for optic neuropathy to develop^10,11,12, and the remaining tissues of the body are usually (although not invariably) spared from pathology^13,14. This is in stark contrast to the severe systemic mitochondrial disease Leigh syndrome, some forms of which also develop from mutations affecting complex I subunits¹⁵. In the latter cases, the mutations result in more profound dysfunction of mitochondrial respiration than in LHON¹⁶, leading to generalized weakness, hypotonia and progressive cardiac and respiratory failure, often culminating in death in early childhood. Notably, patients with Leigh syndrome also frequently manifest optic atrophy¹⁷.

There are currently no satisfactory methods of preventing or treating LHON or other mitochondrial optic neuropathies. Efforts to develop effective pharmacotherapies have been hampered, in part, by the lack of an ideal preclinical animal model for efficient drug testing. Two experimental mouse lines have been constructed which carry heritable mtDNA mutations affecting complex I and have been reported to exhibit LHON-like phenotypes. One mouse line with a homoplasmic point mutation in the complex I subunit ND6 demonstrates loss of ~ 25% of RGC axons within the optic nerve by 24 months of age¹⁸. The second model, a mouse line carrying a human mtDNA mutation of the ND4 subunit as a mitochondrial episome, demonstrates the onset of retinal electrophysiological abnormalities by 4 months and some thinning of inner retinal layers corresponding to RGCs by 7 months¹⁹.

Theoretically, a more rapidly progressive optic neuropathy would result from mutations that produce more severe complex I dysfunction than observed in these existing models. For instance, deletion of the nuclear gene ndufs4, which encodes an accessory complex I subunit mutated in some forms of Leigh syndrome, results in severe instability of complex I and decreases its enzymatic activity by > 50% in the retina and other tissues^20,21,22. Indeed, the germline ndufs4 knockout mouse (henceforth denoted ndufs4^−/−) has been reported to exhibit an early onset of RGC degeneration by postnatal day 42 (P42)²³. However, the utility of this mouse line as a model of mitochondrial optic neuropathy is greatly limited by its shortened lifespan: the mice proceed to die rapidly after P50 due to progressive encephalomyopathy²². Conditional deletion of ndufs4 within RGCs is a potential strategy to achieve efficient RGC degeneration with a less severe systemic phenotype, although this would require that complex I deficiency intrinsic to RGCs is sufficient to induce degeneration. The high energy demands of RGCs have long been believed to explain their particular susceptibility to mitochondrial dysfunction²⁴, yet it remains possible that pathology of other neighboring retinal neurons may play a critical role in inducing RGC degeneration^20,23. We used a transgenic mouse line expressing Cre recombinase within a subset of glutamatergic neurons to test the hypothesis that RGC degeneration in complex I deficiency is a cell-autonomous process and to characterize the progression of optic atrophy over multiple time points.

Results

Histological assessment of Vglut2-driven Cre recombinase expression in the retina

The vesicular glutamate transporter VGLUT2 is expressed in RGCs²⁵ and was previously shown to drive expression of Cre recombinase in virtually all RGCs in a transgenic mouse line with an IRES-Cre cassette inserted directly downstream from the Vglut2 stop codon (henceforth referred to as Vglut2-Cre)²⁶. However, Cre expression in other retinal neurons in this mouse line has not been reported. By crossing the Vglut2-Cre transgenic mouse with the Ai9 mouse line expressing the fluorescent reporter tdTomato from the Rosa26 locus in a Cre-dependent manner, we evaluated Vglut2-driven Cre expression throughout the retina. In retinal flatmounts, 96% ± 0.6% (mean ± SEM) of cells in the ganglion cell layer that labeled with the RGC marker RBPMS1²⁷ co-expressed tdTomato (Fig. 1A), and fewer than 1% of tdTomato-expressing cells failed to label for RBPMS1, indicating excellent specificity for RGCs within this retinal layer. Consistent with prior reports describing the expression of endogenous VGLUT2 in the retina^25,28, only a small minority of retinal neurons outside the ganglion cell layer demonstrated expression of Vglut2-driven Cre (Fig. 1B). Retinal cross-sections exhibited tdTomato expression within rare cells in the inner nuclear layer at its junction with the inner plexiform layer; co-labeling with RBPMS1 confirmed that these represent displaced RGCs (Fig. 1C)²⁷. Only a small fraction of photoreceptors exhibited tdTomato fluorescence, and these were found to represent a subset of cones based on co-labeling with cone arrestin [10.3% ± 1.2% of cones (mean ± SEM)] (Fig. 1D). A smaller fraction of horizontal cells also expressed tdTomato [6.4% ± 3.7% (mean ± SEM)] (Fig. 1E), consistent with endogenous VGLUT2 expression²⁵.

Prior reports have suggested that, in the germline ndufs4^−/− mouse, rod bipolar cells²⁰ and starburst amacrine cells²³ degenerate early and may contribute to the death of RGCs in this model. We co-labeled retinal cross-sections from Vglut2-Cre;Ai9 mice with PKC-α and ChAT and found no evidence of Cre expression in either rod bipolar cells or starburst amacrine cells, respectively (Fig. 1F,G). Thus, the Vglut2-Cre transgenic mouse line provides an opportunity to study the effect of genetic modification of RGCs without any confounding effects from altering directly neighboring cell types.

RGC somas and axons undergo progressive degeneration in Vglut2-Cre;ndufs4^loxP/loxP mice

Having demonstrated the high degree of specificity for Vglut2-driven Cre expression in RGCs histologically, we proceeded to establish a Vglut2-Cre;ndufs4^loxP/loxP transgenic mouse line in order to achieve RGC-specific deletion of ndufs4. The systemic phenotype of ndufs4 deletion in Vglut2-expressing glutamatergic neurons was recently described by another group which independently generated Vglut2-Cre;ndufs4^loxP/loxP transgenic mice but did not characterize the retinal phenotype²⁹. Consistent with their report, we observed that Vglut2-Cre;ndufs4^loxP/loxP mice are initially healthy but develop progressive weight loss, ataxia, and hindlimb weakness beginning around P60. As only a subset of neurons experience loss of ndufs4, these mice demonstrate a delayed and initially less severe neurological phenotype compared to germline ndufs4^−/− mice and achieve an almost two-fold increase in lifespan, with Vglut2-Cre;ndufs4^loxP/loxP mice surviving to P90 with consistency but dying shortly thereafter.

The longer lifespan of the Vglut2-Cre;ndufs4^loxP/loxP mice afforded the opportunity to assess RGC degeneration over a three-month period. For this purpose, histological analysis of Vglut2-Cre;ndufs4^loxP/loxP retinas and optic nerves was compared to two different control genotypes, ndufs4^loxP/loxP and Vglut2-Cre;nduf4^+/+, the latter serving to control for any potentially deleterious effect of the expression of Cre recombinase within RGCs. At P30, Vglut2-Cre;ndufs4^loxP/loxP mice demonstrated normal density of RGC somas in retinal flat mounts, consistent with the normal retinal development previously described in germline ndufs4^−/− mice²³ (Fig. 2A,B). To determine whether ndufs4 was successfully deleted in RGCs, we performed Western blot analysis to quantify the abundance of the protein product at P30. Because RGCs comprise < 1% of the total retinal cell population³⁰, a reduction of NDUFS4 protein expression within RGCs would not be detectable in an analysis of whole retinas from Vglut2-Cre;ndufs4^loxP/loxP mice. Therefore, the analysis was performed on lysates from optic nerves, of which RGC axons are the dominant component (Fig. 3). At P30, the NDUFS4 protein content in Vglut2-Cre;ndufs4^loxP/loxP optic nerve tissue was reduced by variable levels compared to control ndufs4^loxP/loxP littermates, with a mean reduction of 40% [95% confidence interval for residual NDUFS4: (0.45, 0.75)]. Given that oligodendrocytes, astrocytes, and endothelial cells are also resident to the optic nerve³¹, our observation was consistent with a robust decrease in NDUFS4 content within RGC axons by P30. Because RGC density is normal at this age, the observed reduction in NDUFS4 protein content is not attributable simply to RGC degeneration.

Histological analysis at later time points revealed substantial degeneration of RGCs by P60, with a roughly 40% decrease in RGC density occurring at proximal, intermediate and distal locations from the optic nerve head (Fig. 2A,B). This was observed in both male and female mice. Moreover, a profound decline in the RGC population was observed at P90, with roughly two-thirds of RGCs lost at this final time point. Theoretically, the reduction of RGC somas in Vglut2-Cre;ndufs4^loxP/loxP retinas could simply reflect down-regulation of the cell marker RBPMS1, rather than RGC degeneration. Therefore, we quantified total nuclei in the innermost retinal layers (reflecting a mixture of RGCs, amacrine cells, and astrocytes) and confirmed a reduction of nuclei in P60 Vglut2-Cre;ndufs4^loxP/loxP retinas (Supplementary Fig. S1 online). Moreover, as shown in Fig. 2C, we observed occasional cells in the ganglion cell layer of Vglut2-Cre;ndufs4^loxP/loxP retinas which stained for cleaved caspase 3 (0.75 cells per retinal cross section, compared to 0.10 cells per cross section in Vglut2-Cre;ndufs4^+/+ and 0.05 per cross section in ndufs4^loxP/loxP controls; p < 0.01 for both), indicative of a higher rate of apoptosis. There was no significant difference in RGC density between ndufs4^loxP/loxP and Vglut2-Cre;nduf4^+/+ controls at any distance from the optic nerve head for any of the time points, indicating that the degeneration of RGCs in Vglut2-Cre;ndufs4^loxP/loxP mice was a result of the inactivation of ndufs4 and not related to Cre toxicity (Fig. 2B).

The high specificity of Vglut2-driven Cre expression in RGCs suggests that RGC degeneration observed in Vglut2-Cre;ndufs4^loxP/loxP mice is a cell-autonomous process. To further explore this point, we directly compared the rate of RGC loss in Vglut2-Cre;ndufs4^loxP/loxP mice to that in germline ndufs4^−/− mice (Fig. 2D,E). At P30, RGC density was modestly but significantly decreased in ndufs4^−/− mice compared to Vglut2-Cre;ndufs4^loxP/loxP mice in the peripheral retina but not in more proximal regions. At P45, both ndufs4^−/− and Vglut2-Cre;ndufs4^loxP/loxP retinas demonstrated a 15–20% decrease in RGC density compared to control; there was no significant difference between ndufs4^−/− and Vglut2-Cre;ndufs4^loxP/loxP RGC densities at any of the distances from the optic nerve head at this time point. Later time points could not be assessed for the ndufs4^−/− mouse due to its shortened lifespan. Taken together, it would appear that mitochondrial dysfunction in other cell types has little, if any, impact on the early stages of degeneration of RGCs lacking ndufs4.

Optic nerve cross-sections were also analyzed in order to quantify the density of RGC axons as a measure of optic atrophy. Consistent with our observations of RGC soma densities in retinal flat mounts, we found no difference in RGC axon densities between Vglut2-Cre;ndufs4^loxP/loxP optic nerves and the controls at P30 (Fig. 4A,B). By P60, Vglut2-Cre;ndufs4^loxP/loxP optic nerves exhibited ~ 33% reduction in axon density. Surprisingly, no further reduction was observed at P90, despite additional loss of RGC somas in retinal flat mounts at this time point. We suspected that this reflected a latency in retrobulbar axon loss, as the primary stress to RGCs in mitochondrial optic neuropathies is believed to occur at the level of the retina²⁴. Supporting this explanation is our observation that at P45, RGC axon density was not significantly different in Vglut2-Cre;ndufs4^loxP/loxP optic nerves compared to control, despite RGC soma degeneration already occurring in the retina (compare Figs. 4B and 2E).

Evaluation of optic nerve cross-sections by electron microscopy confirmed normal morphology at P30 but lower RGC axon density and abnormal myelination patterns in P90 Vglut2-Cre;ndufs4^loxP/loxP optic nerves (Fig. 5A). Consistent with observations made in an LHON mouse model expressing mutant human ND4¹⁹, myelin sheaths were frequently thickened or even frankly redundant, with two or three discrete sheaths sometimes surrounding single axons (Fig. 5B). Some of the redundant myelin sheaths incompletely surrounded the axons, possibly representing residual myelin that originally enclosed axons having already undergone degeneration.

In vivo electroretinography responses in Vglut2-Cre;ndufs4^loxP/loxP mice

To assess light signaling function at various levels of the retina, we performed electroretinography (ERG) recordings on dark-adapted Vglut2-Cre;ndufs4^loxP/loxP mice and Vglut2-Cre;nduf4^+/+ control littermates. Previous ERG analyses of germline ndufs4^−/− mice revealed reduced a- and b-wave amplitudes, consistent with a decreased light response at the level of photoreceptor cells^22,23,32. However, since we observed Vglut2-driven Cre expression in only a minority of cones and in no rods or bipolar cells, little effect on these parameters was expected in Vglut2-Cre;ndufs4^loxP/loxP mice. Scotopic (Fig. 6A) and photopic (Fig. 6B) ERG recordings were obtained at P45, an age at which a maximal ~ 50% reduction of a- and b-wave amplitudes has already occurred in germline ndufs4^−/− mice³². As shown in Fig. 6C, a-wave amplitudes in Vglut2-Cre;ndufs4^loxP/loxP mice were indistinguishable from the Vglut2-Cre;nduf4^+/+ controls. b-wave amplitudes were slightly decreased in both scotopic (~ 15% reduction) and photopic (~ 20%) recordings, although these reductions were not statistically significant at any stimulus intensity. In contrast, oscillatory potentials were significantly decreased by ~ 30% in Vglut2-Cre;ndufs4^loxP/loxP mice compared to Vglut2-Cre;nduf4^+/+ littermates (Fig. 6C), reflecting impairment of feedback signaling in inner retinal neurons, including RGCs³³.

Vglut2-Cre;ndufs4^loxP/loxP retinas exhibit signs of neuroinflammation

It has been previously reported that RGC degeneration in the germline ndufs4^−/− mouse is accompanied by infiltration of activated myeloid cells into the inner plexiform and ganglion cell layers²³. To determine whether neuroinflammation also occurs in the setting of Vglut2-driven conditional deletion of ndufs4 from RGCs, we quantified the abundance of iba1-positive mononuclear cells on cryosections obtained from P60 Vglut2-Cre;ndufs4^loxP/loxP mice and Vglut2-Cre;nduf4^+/+ control littermates. Indeed, a greater than two-fold increase in iba1-positive cells was observed within the inner retina of Vglut2-Cre;ndufs4^loxP/loxP mice (Fig. 7A), and 96% of these cells co-labeled for CD68 (Fig. 7B), indicative of an active, phagocytic state. These cells may represent resident microglia or recruited myeloid cells, as iba1 cannot distinguish between these cell populations. Furthermore, whereas control retinas exhibited normal confinement of the intermediate filament glial fibrillary acidic protein (GFAP) to astrocytes and Müller cell end-feet in the ganglion cell layer, Vglut2-Cre;ndufs4^loxP/loxP retinas demonstrated up-regulated GFAP localizing to Müller cell radial processes, consistent with reactive gliosis (Fig. 7C). Per 100-µm segment of inner plexiform layer in retinal cross sections, 8.6 ± 3.7 (mean ± SEM) GFAP-positive radial processes were observed in Vglut2-Cre;ndufs4^loxP/loxP retinas, while none were observed in Vglut2-Cre;nduf4^+/+ controls (p = 0.02). However, unlike what we and others have observed in germline ndufs4^−/− retinas^23,32, there was no associated decrease in starburst amacrine cell abundance in the ganglion cell layer of Vglut2-Cre;ndufs4^loxP/loxP retinas (Fig. 7D), again supporting the notion that the observed RGC death is not dependent on inflammation induced by the death of neighboring cell types.

Discussion

In this study, we report that conditional deletion of ndufs4 in Vglut2-expressing neurons results in progressive loss of RGC somas and axons, with associated neuroinflammation of the inner retina. This phenomenon is entirely consistent with the phenotype observed at the end of the short lifespan of germline ndufs4^−/− mice²³, but the prolonged survival of Vglut2-Cre;ndufs4^loxP/loxP mice allows this pathology to progress substantially further.

By using a tdTomato reporter to assess the extent of Vglut2-driven Cre expression in other retinal neurons, we have addressed the lingering question of whether or not RGC degeneration in the setting of complex I deficiency is a cell-autonomous process. Prior characterizations of the germline ndufs4^−/− mouse indicated that the onset of RGC death is preceded by death of starburst amacrine cells²³ and potentially rod bipolar cells [²⁰, but see³²]. This raised the possibility that RGC degeneration was secondary to dysfunction of cells synapsing on RGCs and/or to the recruitment of inflammatory cells to the inner retina in response to death of neighboring cell types. However, Vglut2-driven Cre is not expressed in starburst amacrine cells or in rod bipolar cells in our model, nor did we observe any dropout of starburst amacrine cells by P60, when RGC degeneration was well underway. This, combined with the fact that RGC degeneration commences with similar kinetics in Vglut2-Cre;ndufs4^loxP/loxP mice as in ndufs4^−/− mice, suggests that intrinsic complex I dysfunction within RGCs is sufficient to produce optic atrophy. Future experiments assessing Cre expression and degeneration of post-synaptic neurons in the superior colliculus could rule out the possibility of retrograde trans-synaptic degeneration as a contributing mechanism to the RGC loss observed in this model.

In contrast to germline ndufs4^−/− mice, which exhibit ~ 50% reduction of a- and b-wave amplitudes³², preganglionic retinal signaling was minimally affected in ERG recordings from Vglut2-Cre;ndufs4^loxP/loxP mice. While the scotopic a-wave was completely unaffected (indicative of normal phototransduction), there was a trend of modestly reduced b-wave amplitudes in both scotopic and photopic recordings. We can only speculate on the reason for this. Reduced b-waves would typically be thought to reflect abnormal synaptic transmission between photoreceptors and bipolar cells or abnormal signaling intrinsic to bipolar cells. However, while a subset of cone photoreceptors exhibited Cre-dependent tdTomato expression, no rod photoreceptors or bipolar cells expressed Cre in Vglut2-Cre;ndufs4^loxP/loxP mice and would therefore be expected to be physiologically normal. It is conceivable that a mild reduction in b-wave amplitudes could be an indirect effect of RGC pathology and/or inner retinal inflammation, as animal models of traumatic or inflammatory optic neuropathy have exhibited variable effects on outer retinal signaling properties^34,35. The reduction in oscillatory potentials observed in ERG recordings of the conditional knockout mice is consistent with abnormal RGC function, as seen previously in mouse glaucoma models^33,36. However, oscillatory potentials derive from complicated inhibitory feedback pathways involving multiple inner retinal cell types, and we cannot rule out that our observations relate more to the presence of inner retinal inflammation than directly to RGC dysfunction/degeneration. Future electrophysiological analysis of Vglut2-Cre;ndufs4^loxP/loxP mice with pattern ERGs may better isolate the impact of complex I deficiency on RGC function and potentially allow pathology to be detected prior to the onset of overt degeneration.

Efficient testing of potential pharmacotherapies for mitochondrial optic neuropathies depends on the availability of an animal model exhibiting reliable and rapidly progressive RGC degeneration with a clinically relevant underlying pathobiology. We believe that the Vglut2-Cre;ndufs4^loxP/loxP transgenic mouse line satisfies these criteria and promises to be a valuable preclinical model for optic neuropathies directly related to complex I dysfunction, such as LHON. We show that RGCs with inactivated ndufs4 begin to die at ~ P45, with degeneration becoming profound by P90. Similar to prior characterization of the germline ndufs4^−/− mouse²³, Vglut2-Cre;ndufs4^loxP/loxP RGCs develop in normal abundance and show no axonal pathology at the time the mice initially reach maturity. The absence of congenital anomalies is an important feature of the ocular phenotype in these mice, as LHON also is associated with structurally and functionally normal optic nerves until the onset of overt vision loss, typically after patients reach puberty.

Although the shortened lifespan of Vglut2-Cre;ndufs4^loxP/loxP mice of ~ 90 days limits the duration of experiments possible in this model, the nearly two-fold increase in lifespan compared to ndufs4^−/− mice is paramount, as most of the RGC degeneration occurs outside of the lifespan of the germline knockout mouse. The fact that Vglut2-Cre;ndufs4^loxP/loxP mice already exhibit robust RGC degeneration at P60 means that the protective effect of any potential therapeutic strategy could be determined reliably at P60 and then assessed for durability over an additional month. Importantly, given that most human patients with LHON are not diagnosed until they have already developed some degree of vision loss in both eyes, this model will also provide the opportunity to test the ability of therapies to halt the progression of optic atrophy when administered after RGC degeneration has already commenced.

In addition to being a superior model to the germline ndufs4 knockout, the Vglut2-Cre;ndufs4^loxP/loxP mouse line has several potential advantages over other existing mouse models of complex I-associated optic neuropathy. One non-genetic strategy has been to expose animals to the toxin rotenone, a potent inhibitor of complex I. To reduce systemic toxicities, this may be administered by injection into the vitreous cavity or the superior colliculus to achieve complex I inhibition within RGCs^37,38. Rotenone toxicity rapidly and efficiently induces optic atrophy (producing RGC death as soon as 24 h after treatment in the case of intravitreal injections). This rapidly-induced pathology may phenocopy the acute vision loss experienced by some LHON patients better than our model does. However, rotenone injections require that an invasive procedure be performed on each animal and introduce a sudden metabolic insult to the RGCs that does not mimic the chronic metabolic stress experienced by these cells in heritable mitochondrial diseases. Another popular somatic approach is to generate an LHON-like phenotype through allotopic expression of complex I mutations in RGCs via in vivo electroporation or transduction by adeno-associated virus (AAV)^39,40. However, these manipulations are not heritable (again requiring intravitreal injection of each animal, with the accompanying risk of iatrogenic retinal damage), and the efficiency of mitochondrial import of the mutant proteins and subsequent incorporation into endogenous respiratory complexes is potentially problematic, as protein aggregation in the cytosol could potentially be harmful to RGCs, irrespective of the effect on complex I function^5,41.

The two existing heritable mouse models of mitochondrial optic neuropathy represent the most relevant comparisons for the present model. Lin et al. engineered a mouse line that is homoplasmic for a mtDNA G13997A point mutation resulting in a P25L amino acid substitution in the complex I subunit ND6¹⁸. This mouse holds the advantage of bearing a bona fide mitochondrial disease mutation seen in a family with Leigh syndrome, unlike the deletion of exon 2 in ndufs4, which has not been described in humans. However, the slow progression of optic atrophy in G13997A mice impedes large-scale testing of pharmacotherapies or gene therapies in this model, with no RGC axonal loss observed at 14 months and only ~ 25% by 24 months¹⁸ (compared to 33% by P60 in Vglut2-Cre;ndufs4^loxP/loxP mice). More recently, Yu et al. utilized a mitochondria-targeted AAV to deliver an episomally-expressed human ND4 mutation that is transmitted through the germline and results in mice that are functionally heteroplasmic for the mutation¹⁹. This model demonstrates profound RGC soma and axon loss by 22 months. Although histological assessment at younger ages has not been reported, pattern ERG recordings from this mouse suggest that the onset of RGC degeneration likely takes place at least two months later than in Vglut2-Cre;ndufs4^loxP/loxP mice. One final advantage is that, unlike a mouse model with a mitochondrial inheritance pattern, the Vglut2-Cre;ndufs4^loxP/loxP mouse line described here may be used to generate control littermates for experiments (e.g. half of the progeny from the cross of a Vglut2-Cre;ndufs4^loxP/loxP male to a ndufs4^loxP/loxP female will be ndufs4^loxP/loxP mice that do not develop optic atrophy). While we acknowledge that ndufs4 mutations have not been directly implicated in LHON, a mutation-independent therapeutic strategy that is protective of RGCs in the setting of ndufs4 inactivation would seem to have a high likelihood of efficacy in treating the much less severe complex I dysfunction seen in LHON patients.

Methods

Animals

All animal procedures were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals. The Institutional Animal Care and Use Committee of Duke University approved all experimental protocols. All mice were on a C57BL/6 genetic background and purchased from the Jackson Laboratory (Bar Harbor, ME). Transgenic mice homozygous for Vglut2-Cre (stock number 028863)⁴² were crossed with Ai9 mice (stock number 007909)⁴³ to achieve Cre-dependent tdTomato reporter expression. Homozygous Vglut2-Cre mice were also crossed with transgenic ndufs4^loxP/loxP mice in which exon 2 of ndufs4 is floxed (stock number 026963)²² in order to generate Vglut2-Cre;ndufs4^loxP/+ progeny. The F₁ mice were then crossed to produce Vglut2-Cre;ndufs4^loxP/loxP conditional knockout mice, as well as Vglut2-Cre;ndufs4^+/+ and ndufs4^loxP/loxP control littermates. Finally, heterozygous mice carrying a germline deletion of exon 2 of ndufs4 (stock number 027058)²² were crossed to generate homozygous ndufs4^−/− progeny. Animals were reared under a normal day/night cycle and all experiments were performed during the day.

Antibodies

The following antibodies were used for immunofluorescence experiments: rabbit polyclonal anti-RBPMS (1:500; Novus, NBP2-20112, Lot#130–96), rabbit polyclonal anti-cone arrestin (1:500, EMD Millipore, AB15282, Lot#3156749), mouse monoclonal anti-calbindin D28K (D-4) (1:500, Santa Cruz Biotechnology, sc-365360, Lot#3156749), mouse monoclonal anti-PKC-α H-7 (1:100; Santa Cruz, sc-8393, Lot#I1306), goat polyclonal anti-choline acetyltransferase (1:100; Millipore, AB144P, Lot#3169862), rabbit monoclonal anti-mCherry (1:500, Abcam, EPR20579, Lot#ab213511), rabbit polyclonal anti-cleaved caspase 3 (Asp175) (1:200, Cell Signaling, 9661 T, Lot#45), rabbit polyclonal anti-iba1 (1:1000; Fujifilm Wako Pure Chemicals Corp., 019–19741), rat monoclonal anti-CD68 FA-11 (1:200; BioLegend, 137002), and rabbit polyclonal anti-GFAP (D1F4Q) XP (1:200; Cell Signaling Technology, #12389). The following antibodies were used for Western blot analysis of optic nerve lysates: mouse monoclonal anti-NDUFS4 1-E-4 (1:200; Santa Cruz, sc-100567, Lot#A2918) and mouse monoclonal anti-β-actin (1:1000; Santa Cruz, sc-47778, Lot#D0615). Secondary antibodies against the appropriate species conjugated to Alexa Fluor 488, Alexa Fluor 568 (immunofluorescence experiments, 1:500 dilution) or Alexa Fluor 680 (Western blot experiments, 1:20,000 dilution) were purchased from Invitrogen. Cell nuclei were stained using DAPI (Sigma Aldrich).

Electroretinography

ERG recordings were performed on live mice using the Espion E2 system with a ColorDome Ganzfeld stimulator (Diagnosys LLC, Littleton, MA) as described previously^44,45. Mice were anesthetized via intraperitoneal injection of 100 mg/kg ketamine and 10 mg/kg xylazine following a 6-h dark adaptation. A mixture of 1% cyclopentolate-HCl and 2.5% phenylephrine was used to dilate the pupils. During recordings, eyes were lubricated with 1% carboxymethylcellulose sodium gel, and body temperature was maintained using a heated platform. Recordings were conducted simultaneously in both eyes using gold contact lens electrodes (Mayo Corp., Aichi, Japan), with stainless steel needle electrodes (Ocuscience, Henderson, NV) placed in the mouth (reference) and at the base of the tail (ground). ERG signals were sampled at 1 kHz and recorded with 0.15 Hz low-frequency and 500 Hz high-frequency cutoffs. Responses to flashes from 0.0001 to 500 cd*s/m² with 10 to 1 trials averaged and inter-flash intervals of 5–180 s were recorded in the dark. Then, flashes from 0.1 to 1000 cd*s/m² were recorded in the presence of rod-saturating light of 30 cd/m² to isolate cone responses. Following the recordings, the mice were euthanized for histological experiments.

Data from the ERG recordings were analyzed using Matlab 2019a (MathWorks). Oscillatory potentials were removed from the signals by 55 Hz fast Fourier transform (FFT) low-pass frequency filtering of the early part of the response and 2nd-order butterworth filtering of the non-photoreceptor-driven signal. The amplitude of b-waves was calculated from baseline to the peak for dimmer flashes and from the bottom of the a-wave to the b-wave peak for brighter flashes, and the amplitude of the a-wave was calculated from baseline to trough. To calculate the total energy of the oscillatory potentials, a trapezoidal numerical integration was performed on a 65 to 300 bandpass FFT for each 5^th-order butterworth filtered response³³. p-values were calculated to determine the statistical significance between the response amplitudes of Vglut2-Cre;ndufs4^loxP/loxP and control mice at each flash intensity using two-tailed t-tests in GraphPad software.

Histological techniques

For immunohistochemistry experiments, eyes were enucleated from euthanized mice and the anterior segments were removed to generate posterior eyecups, which were fixed for 1 h in 4% paraformaldehyde and then rinsed in phosphate-buffered saline solution (PBS). Retinal flatmounts were prepared by carefully dissecting retinas from the eyecups and blocking in 5% goat serum in PBS with 0.3% Triton X-100 for 2 h at room temperature, then incubating with anti-RBPMS primary antibody in block for 7 days at 4 °C. Retinas were washed and then incubated with donkey anti-rabbit Alexa Fluor 488 in block overnight at 4 °C. The retinas were then washed and placed on glass slides with the RGC layer facing up, and four radial cuts were made from the edge to the equator of each retina to achieve flattening prior to mounting. For nuclei counts, isolated fixed retinas were incubated in DAPI overnight prior to washing and mounting on slides.

Retinal cryosections were prepared by cryoprotecting fixed eyecups in 30% sucrose and then embedding them in optimal cutting temperature (OCT) medium (Tissue-Tek, Sakura Finetek). Retinal cross-sections, 14 μm in thickness (for iba1, CD68, and ChAT staining) or 20 μm in thickness (all others), were collected using a cryostat microtome (Microm HM 550, Thermo Fisher Scientific). Sections were rehydrated with PBS, blocked in 5% goat or donkey serum in PBS with 0.3% Triton X-100 for 2 h at room temperature, and then incubated in primary antibody in the same blocking solution overnight at 4 °C. Sections were washed and incubated with appropriate secondary antibody conjugated to Alexa Fluor 488 or Alexa Fluor 568 in block solution overnight at 4 °C. The sections were then washed prior to mounting.

All samples were mounted with Vectashield (Vector Laboratories, Burlingame, CA) under glass coverslips. Images were acquired using a Nikon Eclipse Ti2 inverted confocal microscope, a CFI Plan Fluor 60 × (oil) objective, and an A1 confocal scanner controlled by NIS-Elements software (Nikon, Tokyo, Japan). For quantification of retinal ganglion cell somas and inner retinal nuclei in retinal flatmounts, z-stacks through the retinal nerve fiber layer and ganglion cell layer of 45,000 µm² in area were obtained in each quadrant at locations of 0.5, 1.0, and 1.5 mm from the optic nerve head. RBPMS1-positive RGC somas and DAPI-stained nuclei were manually counted using the Cell Counter plugin for Fiji^46,47. Cell density was averaged among the four quadrants at each distance from the optic nerve head. For quantification of cells positive for calbindin, tdTomato, cleaved caspase 3, iba1, and CD68, retinal cross sections were imaged along their entire length, and the number of positive cells was quantified in 3–4 retinal sections per sample (taken through the optic nerve head), then averaged. For quantification of cells labeled for cone arrestin, ChAT, and GFAP, 45,000 µm² images were acquired at a 500-µm distance to either side of the optic nerve head for 3 sections per sample. For cone arrestin, the overall number of positive cell bodies and number positive for tdTomato were counted. For ChAT, the average number of positive cells/µm was calculated separately for the inner nuclear layer and the ganglion cell layer. For GFAP, the number of positive radial processes present at the inner nuclear layer/inner plexiform layer junction was counted.

To assess RGC axons, mouse optic nerve specimens were processed and analyzed as previously described⁴⁸. Briefly, euthanized mice were exsanguinated via transcardial perfusion with 80 mL of ice-cold PBS followed by fixation with 80 mL of 4% PFA. Optic nerves were dissected back to the optic chiasm and fixed in 2% paraformaldehyde and 2% glutaraldehyde overnight, then washed in PBS. Samples were embedded in the Embed-812 resin mixture and sectioned on an ultramicrotome (LKB Ultratome V; Leica, Paris, France) using a glass knife. Cross-sections of 0.27 µm thickness were stained with 1% methylene blue. Axon counts were obtained using the AxioSkop 2 Plus upright microscope and AxioVision SE64 Release 4.9.1 imaging software (Zeiss, Oberkochen, Germany; https://www.micro-shop.zeiss.com/en/us/system/axiovision+software/software+axiovision/axiovision+program/410130-0909-000#/). For each optic nerve, 4 images of 69.5 µm × 87.7 µm were obtained using a 100X (oil) objective. Image analysis consisted of automated RGB thresholding, as well as size and form factor exclusions, followed by manual exclusion of inappropriately identified axons. Approximately 40% of the total cross-sectional area of each optic nerve was sampled and counted. The final count was divided by the total area sampled to determine mean axon density per nerve.

The same mouse optic nerve specimens processed for light microscopy were thinly sectioned (60–80 nm) for transmission electron microscopy. Samples were collected on copper grids, counterstained with uranyl acetate and Sato’s lead, and then examined using an electron microscope (JEM-1400; JEOL, Tokyo, Japan) at 60 kV. Images were collected using a charge-coupled device camera (Orius; Gatan, Pleasanton, CA).

Western blot for NDUFS4 quantification

The optic nerves of euthanized mice were dissected and then sonicated in lysis buffer [25 mM HEPES buffer, pH 7.4, 150 mM NaCl, 5 mM MgCl₂, and protease inhibitors (Complete Mini, Roche, Indianapolis, IN) with 1% Triton X-100] and the protein concentration of each lysate was determined with a colorimetric assay (Bio-Rad). After mixing with SDS-PAGE sample buffer, optic nerve lysates from ndufs4^loxP/loxP and Vglut2-Cre;ndufs4^loxP/loxP mice were separated on 4–20% SDS-PAGE gels, transferred onto polyvinylidene fluoride (PVDF) membranes and blotted with the indicated primary antibodies overnight. Membranes were washed in 0.05% Tween X-20 and incubated with the appropriate secondary antibody for two hours at room temperature. The Odyssey CLx imaging system (LI-COR, Lincoln, NE) was used to image and quantify band intensities. 12.5 µg of total protein was loaded for each sample, as signal intensities for actin and NDUFS4 were both found to be within a linear range at this input.

Experimental design and statistical analysis

All histological experiments and in vivo electrophysiological experiments were performed on Vglut2-Cre;ndufs4^loxP/loxP mice and littermate controls with both sexes represented. A minimum of 5 retinas or optic nerves were analyzed for each genotype at each time point for quantitative histological experiments. RGC soma and optic nerve axon densities were compared between genotypes using two-tailed t-tests on GraphPad Prism version 8.00 for Windows (GraphPad Software). Data are presented as mean ± SEM. All in vivo ERG recordings were performed at P45 (10 eyes of 5 Vglut2-Cre;ndufs4^loxP/loxP mice and 12 eyes of 6 Vglut2-Cre;ndufs4^+/+ mice). The a-wave and b-wave amplitudes and oscillatory potential energy recorded at each flash intensity were compared using a two-tailed t-test.

Data availability

The data that support the findings of this study are available from the corresponding author on reasonable request.

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Acknowledgements

This work was funded by the National Eye Institute via grants EY028610 (S.M.G.), EY022959 (V.Y.A.), and Core Grant EY005722 (Duke University), as well as a Duke University School of Medicine Strong Start Award (S.M.G.), and an Unrestricted Research to Prevent Blindness Grant (Duke Eye Center). The authors would like to thank Angela Dixon for technical assistance with histological processing of samples.

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Authors and Affiliations

Department of Ophthalmology, Box 3712 Med Center, Duke University, 2351 Erwin Road, Durham, NC, 27710, USA
Luyu Wang, Mikael Klingeborn, Ying Hao, Vadim Y. Arshavsky & Sidney M. Gospe III
Department of Ophthalmology and Visual Sciences, University of Michigan, Ann Arbor, MI, 48105, USA
Amanda M. Travis
Department of Pharmacology and Cancer Biology, Duke University School of Medicine, Durham, NC, 27710, USA
Vadim Y. Arshavsky

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Luyu Wang
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Mikael Klingeborn
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Ying Hao
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Contributions

S.M.G. designed the research; L.W., M.K., Y.H., and S.M.G. performed the research; L.W., A.M.T., V.Y.A. and S.M.G. analyzed the data; L.W. and S.M.G. wrote the main manuscript text; and all authors reviewed and approved the manuscript.

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Correspondence to Sidney M. Gospe III.

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Wang, L., Klingeborn, M., Travis, A.M. et al. Progressive optic atrophy in a retinal ganglion cell-specific mouse model of complex I deficiency. Sci Rep 10, 16326 (2020). https://doi.org/10.1038/s41598-020-73353-0

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Received: 16 April 2020
Accepted: 16 September 2020
Published: 01 October 2020
DOI: https://doi.org/10.1038/s41598-020-73353-0

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