NADH-dehydrogenase type-2 suppresses irreversible visual loss and neurodegeneration in the EAE animal model of MS

Abstract

To address mitochondrial dysfunction that mediates irreversible visual loss and neurodegeneration of the optic nerve in the experimental autoimmune encephalomyelitis (EAE) animal model of multiple sclerosis (MS), mice sensitized for EAE were vitreally injected with self-complementary adenoassociated virus (scAAV) containing the NADH-dehydrogenase type-2 (NDI1) complex I gene that quickly expressed in mitochondria of almost all retinal ganglion cells (RGCs). Visual function assessed by pattern electroretinograms (PERGs) reduced by half in EAE showed no significant reductions with NDI1. Serial optical coherence tomography (OCT) revealed significant inner retinal thinning with EAE that was suppressed by NDI1. Although complex I activity reduced 80% in EAE was not improved by NDI1, in vivo fluorescent probes indicated mitochondrial oxidative stress and apoptosis of the EAE retina were reduced by NDI1. Finally, the 42% loss of axons in the EAE optic nerve was ameliorated by NDI1. Targeting the dysfunctional complex I of EAE responsible for loss of respiration, mitochondrial oxidative stress and apoptosis may be a novel approach to address neuronal and axonal loss responsible for permanent disability that is unaltered by current disease modifying drugs for MS that target inflammation.

Figure 1

Ocular expression of NDI1. Retinal flat mounts were performed at 1 month after injection of NDI1 and evaluated for (a) DAPI, (b) Thy 1.2, (c) NDI1, and (d) NDUFB6 with (e) a–d of this same tissue merged. These retinal flat mounts were examined at higher magnification for (f) DAPI, (g) Thy 1.2, (h) NDI1, (i) NDUFB6, and (j) f–i of this same tissue merged. Longitudinal sections of this infected retina were examined for (k) DAPI, (l) Thy 1.2, (m) NDI1, (n) NDUFB6, and (o) k–n merged. The boxed insets show the RGC layer at higher magnification with a colocalization profile plot for all channels over a 25 µm long dotted line. (p) A bar plot of NDI1 delivered by triple mutant (Y444F+Y500F+Y730F) scAAV labeled all Thy 1.2 cells by 7 days. (q) One month after scAAV injections, immunoblots were probed for NDI1. (r) Reverse transcriptase PCR was performed for NDI1 derived transcripts. Scale bars: 100 µm and 10 µm for whole mounts, 25 µm and 10 µm for LS. n = 6 for IF, n = 7 for NDI1 and GFP counting data, n = 12 for western blots, n = 12 for reverse transcriptase PCR, number of repetitions = 3 for each experiment, ***P = 0.0001–0.0009. INL, inner nuclear layer; ON, optic nerve; ONL, outer nuclear layer; RE, retina; RGC, retinal ganglion cell layer; scAAV, self-complementary adenoassociated virus.

Figure 2

Visual function. Pattern electroretinogram analysis of unsensitized controls vitreally injected with scAAV-cherry (Con/mCh), EAE mice vitreally injected with scAAV-mCherry (EAE-mCh) and EAE mice vitreally injected with scAAV-NDI1 (EAE-NDI1) were performed at 1 MPI (n = 18, 17, and 15, respectively), 3 MPI (n = 15, 16, and 15, respectively), and 6 MPI (n = 10, 16, and 15, respectively). (a) Box-whisker plots of PERG amplitudes, (b) averaged PERG waveforms, and (c) box-whisker plots for PERG latency are plotted. Number of repetitions = 3, for each group and time point, *P = 0.05-0.01; **P = 0.001–0.009; ***P = 0.0001–0.0009. EAE, experimental autoimmune encephalomyelitis; MPI, month(s) post injection; ms, milliseconds; PERG, pattern electroretinogram; scAAV, self-complementary adenoassociated virus.

Figure 3

Serial in vivo optical coherence tomography (OCT) imaging. Serial high-resolution spectral domain OCT (SD-OCT) imaging was performed at 3 MPI of (a) unsensitized mCherry-injected controls with (b) three-dimensional (3D) thickness map, 6 months after injection in (c) 2D and (d) 3D (n = 11, no repetition). At 3 months, the inner retinal thinning (e) in the EAE-mCherry cross section and (f) in the 3D map are shown. By 6 months, worsening thinning on (g) cross sectional imaging and (h) 3D map are shown (n = 7, no repetition). The optic nerve head is seen on the (i) cross sectional image and (j) 3D map and at 6 months on the (k) retinal cross section and (l) 3D map (n = 17, no repetition). Box-whisker plots of the OCT data compared the groups at (m) 3 and 6 MPI. P < 0.05; *P = 0.05-0.01; **P = 0.001–0.009; ***P = 0.0001–0.0009. EAE, experimental autoimmune encephalomyelitis; MPI, month(s) post injection.

Figure 4

Optic nerve ultrastructure. Six months after intravitreal AAV injections transmission electron microscopy shows (a) a mononuclear inflammatory cell (IC) and axonal loss in the EAE-mCherry optic nerve, with (b) an axon exhibiting electron dense aggregations characteristic of ongoing degeneration (arrow). With NDI1 treatment, (c) a mononuclear cell (IC) and (d) preservation of optic nerve axons (ax) are shown. (e) A bar plot of optic nerve axon counts is shown. n = 12 for each group, no repetition, **P = 0.0058. Scale bar = 2 µm. AAV, adenoassociated virus; Ax, axon; EAE, experimental autoimmune encephalomyelitis; IC, inflammatory cell.

Figure 5

Respiration. (a) Complex I + III, complexes I or III activities were performed and plotted (n = 7 for each group, number of repetitions = 3). Three months after EAE sensitization and scAAV injections, complex I + III, complexes I and III activities were performed. (b) Values and (c) percent reductions of EAE relative to the 1 month control values are shown. n = 6, number of repetitions = 3. ****P < 0.001; **P < 0.007. EAE, experimental autoimmune encephalomyelitis; scAAV, self-complementary adenoassociated virus.

Figure 6

Oxidative stress. One hour after intravitreal injection of DHE, CSLO images were performed of the optic nerve head and retinal nerve fiber layer of the right eyes (a, infrared) and (b, red-free) treated with (c) NDI1 (green barrier filter), (d) DHE-induced fluorescence (red barrier filter) and with the (e) red and green filters merged. CSLO imaging of the left eye injected with (f) scAAV-GFP (infrared), (g) red-free, (h) GFP (green), (i) superoxide (red), and (j) merged. One day after imaging, a bar plot of retinal flat mount fluorescent intensity of superoxide levels in (k) EAE mice injected with scAAV-GFP or scAAV-NDI1 relative to retinas of unsensitized controls injected with scAAV-GFP is plotted. (l) Confocal microscopy of longitudinally sectioned retinas has superoxide (red) (arrows) in scAAV-GFP–injected eyes. (m) The signal (red) with NDI1 treatment is shown. (n) A bar plot of fluorescence intensities of CM-H₂DCFDA–stained retinas is shown. ***P < 0.001; **P < 0.005; *P = 0.01. n = 10 for each group, no repetition. CSLO, confocal scanning laser ophthalmoscope; DHE, dihydroethidium; EAE, experimental autoimmune encephalomyelitis; GCL, ganglion cell layer; ONL, outer nuclear layer; INL, inner nuclear layer; scAAV, self-complementary adenoassociated virus.

Figure 7

Apoptosis. Immunofluorescence of cleaved caspase 3 and Thy 1.2 in longitudinal retinal cryosections of mice sensitized for EAE 6 months earlier whose eyes were injected with scAAV-mCherry and stained for (a) DAPI. (b) Cleaved caspase 3 is shown in the ganglion cell layer where (c) Thy 1.2 colocalized with (d) apoptotic RGCs. The insets show higher magnification of the boxed RGCs. The retina from NDI1-rescued mice with (e) DAPI, but no (f) cleaved caspase 3 in (g) Thy 1.2–labeled (h) RGCs. (i) A bar plot shows the percentage of TUNEL-labeled cells to DAPI-labeled cells in the RGC layer of GFP-injected eyes versus NDI1-rescued eyes. Error bars represent standard error. n = 3 for each group, number of repetitions = 3. **P = 0.001. Scale bars = 25 µm. EAE, experimental autoimmune encephalomyelitis; RGC, retinal ganglion cell layer; ONL, outer nuclear layer; OS, left eye control; OD, right eye rescued; INL, inner nuclear layer; scAAV, self-complementary adenoassociated virus.

Figure 8

RGC ultrastructure. (a) Transmission electron microscopy performed on the retinas of EAE animals injected with scAAV-cherry show condensation of nuclear chromatin (arrow) and loss of cells in the RGC layer. (b) EAE animals treated with scAAV-NDI1 with RGCs with the characteristic elliptical and lighter nucleus are shown. (c) With NDI1 an apoptotic cell is shown (arrow) adjacent to normal appearing RGCs. The IPL of NDI1-treated eyes of EAE mice with increased cystic spaces is shown. Scale bar = 10 µm. n = 12 for each group, no repetition. EAE, experimental autoimmune encephalomyelitis; IPL, inner plexiform layer; RGC, retinal ganglion cell; scAAV, self-complementary adenoassociated virus; V, vitreous.