Idebenone Protects against Retinal Damage and Loss of Vision in a Mouse Model of Leber’s Hereditary Optic Neuropathy

<div><p>Leber’s hereditary optic neuropathy (LHON) is an inherited disease caused by mutations in complex I of the mitochondrial respiratory chain. The disease is characterized by loss of central vision due to retinal ganglion cell (RGC) dysfunction and optic nerve atrophy. Despite progress towards a better understanding of the disease, no therapeutic treatment is currently approved for this devastating disease. Idebenone, a short-chain benzoquinone, has shown promising evidence of efficacy in protecting vision loss and in accelerating recovery of visual acuity in patients with LHON. It was therefore of interest to study suitable LHON models <i>in vitro</i> and <i>in vivo</i> to identify anatomical correlates for this protective activity. At nanomolar concentrations, idebenone protected the rodent RGC cell line RGC-5 against complex I dysfunction <i>in vitro.</i> Consistent with the reported dosing and observed effects in LHON patients, we describe that in mice, idebenone penetrated into the eye at concentrations equivalent to those which protected RGC-5 cells from complex I dysfunction <i>in vitro</i>. Consequently, we next investigated the protective effect of idebenone in a mouse model of LHON, whereby mitochondrial complex I dysfunction was caused by exposure to rotenone. In this model, idebenone protected against the loss of retinal ganglion cells, reduction in retinal thickness and gliosis. Furthermore, consistent with this protection of retinal integrity, idebenone restored the functional loss of vision in this disease model. These results support the pharmacological activity of idebenone and indicate that idebenone holds potential as an effective treatment for vision loss in LHON patients.</p></div

Pharmacokinetic analysis of idebenone in eye fluids.

<p>Concentrations of idebenone in aqueous humor (open circle) and vitreous humor (filled triangle) following once daily administration of idebenone in the diet for 21 days (A) and following single oral administration of idebenone at 60 mg/kg (B) were determined. Concentrations of idebenone in the eye fluids are expressed as ng/ml (left y axis) and nM (right y axis). For (A), sampling time was more than 8h after last dose of idebenone, the values therefore represent trough levels.</p

Dose-effect of idebenone treatment on multiple endpoints in an <i>in vivo</i> mouse model for LHON.

<p>Dose-effect of idebenone treatment on multiple endpoints in an <i>in vivo</i> mouse model for LHON.</p

Idebenone prevents rotenone-induced RGC death. Analysis of RGCs in the mouse retina 7 days after intravitreal injection of rotenone or DMSO (Sham).

<p>Mice were treated with dietary idebenone (IDE 200, 400 and 2000 mg/kg body weight) or vehicle. (A) Images of retinal slices stained with anti-Brn3a primary antibody to visualize RGCs. Scale bar = 25 µm. (B) Quantification of RGCs (RGC number/mm) following idebenone treatment and rotenone injection (n = 6 to 10 per group). Data expressed as mean ± SEM.</p

Idebenone time-dependently restores rotenone-induced loss of vision.

<p>Visual acuity was evaluated by counting the number of head movements at a velocity of 3 rpm 7 days prior injection (day -7) and 1 to 70 days after injection (day 1, 7, 21, 35, 70) of rotenone (5 mM). Mice were treated with dietary idebenone 2000 mg/kg or vehicle for the time of the experiment. (A) Schematic representation of the experimental setup. After vehicle (DMSO) injection, head movements in both directions of drum rotation (clockwise: CW; counterclockwise CCW) remain intact. Injection of rotenone into the left eye however, affects only the CW responses (dashed arrow) whereas CCW responses remain unaffected. (B) Quantification of clockwise (CW) head movement (number of head movements/2 min) following vehicle treatment and rotenone injection (vehicle + rotenone, n = 10 animals), and idebenone 2000 mg/kg treatment and rotenone injection (IDE 2000+ rotenone, n = 11 animals). Data are expressed as mean ± SEM. Statistical significance relative to vehicle + rotenone group: p≤0.05 (*); (C) Percentage of mice showing clockwise (CW) head movements for each treatment group 70 days after injection. Responder: group of mice showing head movements; Non-responder: group of mice without head movements.</p

Protective effect of idebenone against complex I inhibition in RGC-5 cells <i>in vitro</i>.

<p>(A) Scheme of the experimental design. RGC-5 cells were pre-incubated with idebenone or vehicle (DMSO) for 1 or 2 days before the complex I inhibitor rotenone was applied to the cells for 6 hours. Cell viability was then measured after 1 day post-incubation with idebenone or vehicle. (B) Treatment with rotenone caused a 59% reduction in cell viability in vehicle-treated RGC-5 cells. (C) Idebenone pre-treatment for 1 (white bars) and 2 days (black bars) significantly rescued cell viability (expressed as % rescue). Data are expressed as mean ± SD (n = 6 wells per group). Statistical significance relative to vehicle group: p≤0.05 (*), p≤0.001 (***).</p

Idebenone protects against rotenone-induced decrease in retinal thickness.

<p>For analysis of retinal thickness 7 days after intravitreal injection of rotenone or DMSO (Sham), mice were treated with dietary idebenone (IDE 200, 400 and 2000 mg/kg body weight) or vehicle. (A) Images of retinal slices stained with anti-beta 3 tubulin primary antibodies to visualize inner and outer retinal layers. The arrows indicate retinal thickness measured from the pigment epithelium to the nerve fiber layer. Scale bar = 30 µm. (B) Quantification of total retinal thickness (µm) following idebenone treatment and rotenone injection (n = 6 to 10 per group). Data are expressed as mean ± SEM.</p

Idebenone protects against rotenone-induced gliosis.

<p>For analysis of gliosis 7 days after intravitreal injection of rotenone or DMSO (Sham), mice were treated with dietary idebenone (IDE 200, 400 and 2000 mg/kg body weight) or vehicle. (A) Images of retinal slices stained with anti-GFAP primary antibody to visualize Müller glial cell projections (white arrows). White arrowheads indicate outer plexiform layer and also illustrate rotenone induced reduction in retinal thickness (i.e. distance between white arrow heads and ganglion cell layer at the top of the images). Scale bar = 15 µm. (B) Quantification of gliosis (GFAP signal based on relative fluorescence units, RFU) following idebenone treatment and rotenone injection (n = 3 to 7 per group). Data are expressed as mean ± SEM.</p

PP1α reverses CaMKIIα Thr286 phosphorylation and mediates the dephosphorylation of NR2B Ser1303 upon OGD.

<p>(<b>a</b>) Representative Western blots and corresponding quantitative analysis of CaMKIIα Thr286 phosphorylation. CaMKIIα phosphorylation significantly increases 1 min after OGD (control 1 min post-OGD, n = 8) with no significant change at 16 min (control 16 min post-OGD, n = 6) in control slices. PP1α slices do not display any change in CaMKIIα phosphorylation either 1 min (PP1α 1 min post-OGD, n = 6) or 16 min post-OGD (PP1α 16 min post-OGD, n = 7) compared to control. Phospho-protein levels were normalized to non-phosphorylated protein levels and β-actin was used as a loading control. Quantitative data for each condition were normalized to levels of non-OGD condition (control non-OGD, n = 14) from the same blot and exposure. *p<0.05. (<b>b</b>) Representative Western blots and corresponding quantitative analysis of NR2B Ser1303 phosphorylation. Increased level of phospho-NR2B in control slices 1 min after OGD (control 1 min post-OGD, n = 9), but not 16 min after OGD (control 16 min post-OGD, n = 8). PP1α expression or KN-93 treatment blocks this increase (PP1α 1 min post-OGD, n = 6; KN-93 1 min post-OGD, n = 6), but has no effect 16 min post-OGD (PP1α 16 min post-OGD, n = 8; KN-93 16 min post-OGD, n = 5). Phospho-protein levels were normalized to non-phosphorylated protein levels, and β-actin was used as a loading control. Quantitative data for each condition were normalized to levels of non-OGD condition (control non-OGD, n = 14) from the same blot and exposure. *p<0.05.</p

Blockade of Ca²⁺ rise and delayed cell death induced by OGD by ifenprodil and PP1α in CA1 pyramidal cells.

<p>(<b>a</b>) OGD-induced increase in ΔF/F (% relative to basal level) in control and NVP-AAM077-treated slices (control, n = 7; NVP-AAM077, n = 7). This increase is prevented by ifenprodil perfusion (n = 8) or by PP1α expression (n = 7). Schematic representation of a hippocampal slice showing the patch-clamp electrode filled with OGB-1 (left inset). Image of a CA1 pyramidal neuron loaded with OGB-1 with outlined region of interest (cell soma) used to calculate fluorescence (middle inset). Relative fluorescence (ΔF/F) traces of individually recorded CA1 pyramidal neurons in control slices, PP1α expressing slices, and slices treated with NVP-AAM077 or ifenprodil subjected to OGD (right inset). (<b>b</b>) Quantitative histogram illustrating the area under the curve used as a measure of intracellular Ca²⁺ overload during 4 min OGD, given by the calculation of ΔF/F integral normalized to control. *p<0.05. (<b>c</b>) Propidium iodide labeling of hippocampal slices showing massive cell death in the CA1 pyramidal cell layer (red staining) 48 h after OGD (control OGD, n = 7). Ifenprodil treatment (n = 6) and PP1α expression (n = 5) significantly reduced cell death as shown by an almost absent PI labeling after OGD. No cell death was observed in slices maintained in ACSF (control ACSF, n = 8; ifenprodil ACSF, n = 8; PP1α ACSF, n = 7). Scale bar: 400 µm. (<b>d</b>) Quantitative histogram with normalized labeling intensity. ***p<0.001.</p