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Originally published In Press as doi:10.1074/jbc.M603717200 on August 18, 2006

J. Biol. Chem., Vol. 281, Issue 42, 31950-31962, October 20, 2006
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Mitochondrial Protein Nitration Primes Neurodegeneration in Experimental Autoimmune Encephalomyelitis*

Xiaoping Qi{ddagger}, Alfred S. Lewin§, Liang Sun{ddagger}, William W. Hauswirth{ddagger}§, and John Guy{ddagger}1

From the Departments of {ddagger}Ophthalmology, §Molecular Genetics and Microbiology, and Neurology, the University of Florida College of Medicine, Gainesville, Florida 32610-0284

Received for publication, April 18, 2006 , and in revised form, August 7, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The mechanisms of axonal and neuronal degeneration causing visual and neurologic disability in multiple sclerosis are poorly understood. Here we explored the contribution of mitochondria to neurodegeneration in the experimental autoimmune encephalomyelitis animal model of multiple sclerosis. Oxidative injury to the murine mitochondrion preceded the infiltration of inflammatory cells, classically heralded as the mediators of demyelination and axonal injury by transection. Nitration of mitochondrial proteins affected key subunits of complexes I and IV of the respiratory chain and a chaperone critical to the stabilization and translocation of proteins into the organelle. Oxidative products were associated with loss of mitochondrial membrane potential and apoptotic cell death. Reductions in the rate of synthesis of adenosine triphosphate were severe and even greater than those associated with disorders caused by mutated mitochondrial DNA. Mitochondrial vacuolization, swelling, and dissolution of cristae occurred in axons as early as 3 days after sensitization for experimental autoimmune encephalomyelitis. Our findings implicate mitochondrial dysfunction induced by protein inactivation and mediated by oxidative stress initiates a cascade of molecular events leading to apoptosis and neurodegeneration in experimental autoimmune encephalomyelitis that is not mediated by inflammatory cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Experimental autoimmune encephalomyelitis (EAE)2 is an autoimmune inflammatory disorder of primary central nervous system (CNS) demyelination (1). Treatments suppressing EAE have been successfully used in multiple sclerosis (MS). Still, despite their effectiveness in suppressing the inflammatory response, they appear to have minimal effects on axonal and neuronal loss that likely results in irreversible loss of function and disability (2-6). The mechanisms leading to neurodegeneration in MS are poorly understood. A leading hypothesis is that axons are transected by inflammatory cells (7). However, this does not explain the degeneration of neurons seen prior to the inflammatory cell infiltration or the progressive loss of function after the inflammatory phase has subsided (8).

Mitochondria play a key role in the pathogenesis of many neurological diseases (9-11). They are the primary source of neuronal ATP. Evidence implicating mitochondria in long-standing MS is a loss of ATPase activity (10). In addition, mitochondria are the primary source of cellular reactive oxygen species (ROS). Increased ROS activity is linked to many neurodegenerative diseases that have axonal and neuronal loss as a major feature (9). Whereas ROS have been recognized as key mediators of CNS injury in both EAE and MS, the contribution of mitochondria has only recently been recognized (9). We will show that mitochondrial dysfunction plays an important role in the neurodegeneration of the EAE animal model of MS and that this process begins much earlier than currently believed.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Induction and Scoring of EAE—Experimental autoimmune encephalomyelitis was induced in 56 DBA/1J mice by sensitization with 0.2 ml of sonicated homologous spinal cord emulsion in complete Freund's adjuvant (Difco) (12) injected subdermally into the nuchal area. Control animals (26 mice) received subdermal inoculation with Freund's adjuvant, and 30 mice were used as normal controls. Paralysis was graded on a scale of 0-5 with increasing severity of disease. Mice were humanely cared for in a veterinarian-supervised animal care facility that is fully accredited by the American Association of Laboratory Animal Science. At the prescribed interval they were euthanized by an overdose of sodium pentobarbital.

Mitochondrial Isolation and Immunodetection of ROS—Mitochondrial proteins were isolated from the optic nerves, retinas, brains, and spinal cords of 20 normal mice, 20 mice euthanized 3 days after sensitization for EAE, and 20 mice euthanized 6 days after antigenic sensitization. For comparisons to EAE, 20 normal unsensitized animals and 20 mice inoculated with Freund's adjuvant only and euthanized 3 days later served as controls. Mitochondria were isolated from excised CNS tissues (13). Briefly, this involved washing tissues in cold PBS, followed by resuspension in a buffer consisting of 50 mM Tris-HCl, 0.21 M D-mannitol, 70 mM sucrose, 0.1 M phenylmethylsulfonyl fluoride, 3 mM CaCl2, 20 mM EDTA, pH 7.5. Tissues were then manually homogenized. The homogenates were centrifuged at 1200 x g for 10 min at 4 °C. The resulting supernatant containing the mitochondrial fraction was collected and then centrifuged at 12,000 x g for 20 min at 4 °C. The pellet containing the mitochondria was washed and resuspended in buffer consisting of 50 mM Tris-HCl, 10 mM EDTA, 20% sucrose, pH 7.5, and then stored at -80 °C for later analysis.

For immunodetection, 15 µg of protein of the isolated mitochondrial pellet or cytoplasmic supernatant were separated on a 10% SDS-polyacrylamide gel and electro-transferred to a polyvinylidene fluoride membrane (Bio-Rad). For detection of oxidative stress, we immunostained the membrane with mouse monoclonal nitrotyrosine or inducible nitric-oxide synthase (iNOS) antibodies (Abcam, Cambridge, MA). For normalization of sample loading, we used a mitochondrial loading control VDAC1/Porin antibody (Abcam, Inc., Cambridge, MA). Goat anti-mouse IgG or goat anti-rabbit IgG horseradish peroxidase-conjugated secondary antibodies (Sigma) were reacted against the respective primary antibody. We detected complexes using the enhanced chemiluminescence (ECL) system (GE Healthcare).

Identification of Nitrated Mitochondrial Proteins—We excised the nitrated protein bands and digested proteins in the excised bands with trypsin in situ. The resulting peptides were extracted and analyzed by mass spectrometry by the University of Florida Biotechnology Core Laboratory. In brief, capillary reverse phase HPLC separation of protein digests was performed on a 10-cm x 75-µm inner diameter PepMap C18 column (LC Packings, San Francisco, CA) in combination with a home-built capillary HPLC system operated at a flow rate of 200 nl/min. In-line mass spectrometric analysis of the column eluate was accomplished by a quadrupole ion trap instrument (LCQ; ThermoFinnigan, San Jose, CA) equipped with a nanoelectrospray source. Fragment ion data generated by data-dependent acquisition via the LCQ were searched against the NCBI nr sequence data base using the SEQUEST (ThermoFinnigan) and Mascot (Matrix Science, Boston) data base search engines. In general, the score for SEQUEST protein identification was considered significant when dCn was equal to 0.08 or greater and the cross-correlation score was greater than 2.2. MASCOT probability-based MOWSE scores above the default significant value were considered for protein identification in addition to validation by manual interpretation of the tandem mass spectrometry data.

Immunohistochemistry—The retinas, optic nerves, spinal cords, and brains were immediately dissected out of 10 mice 3 days after antigenic sensitization for EAE, and 10 mice 6 days after EAE sensitization, for comparisons to 10 control mice 3 days after inoculation with only Freund's adjuvant and 10 normal unsensitized mice. Following washes in increasing concentrations of sucrose PBS buffer, the isolated tissues were snap-frozen and stored at -20 °C. Tissues were sectioned on a cryostat. After blocking in 5% normal goat serum for 30 min, they were then reacted with primary nitrotyrosine, iNOS, anti-macrophage, or a mouse monoclonal antibody directed against cyclic nucleotide phosphodiesterase (Abcam, Inc., Cambridge, MA) as an oligodendrocyte marker. For detection of inflammatory cells, we used a pan-macrophage antibody (Abcam, Inc., Cambridge, MA). After an overnight incubation at 4 °C, the specimens were washed in PBS followed by an overnight incubation with Cy2- or Cy3-conjugated anti-mouse secondary antibodies (Jackson ImmunoResearch). After washes, specimens were visualized by fluorescence microscopy. Quantitative analysis of iNOS induced fluorescence in EAE, and control optic nerve, retina, brain and spinal cord specimens were obtained from micrographs photographed at a magnification of x40. Color information in the RGB images was discarded, and the images were converted to black and white files. Using NIH Image software, the intensity of fluorescence for each micrograph was measured by thresholding of the fluorescent structures (white). The scale ranged from 255 (white) to 0 (black). Measurements encompassed a total area of 1.8 x 104 µm2 for each excised tissue.

Additionally, apoptotic cell death was assessed with a terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) reaction kit, according to the manufacturer's specifications (Roche Applied Science). The population of TUNEL-positive cells or TUNEL co-labeled oligodendrocytes in the optic nerve, retina, brain, and spinal cord were measured from micrographs photographed at a magnification of x40. They encompassed a total area of 1.8 x 104 µm2 for each excised tissue. Labeled cells were counted manually.

Mitochondrial Membrane Potential—For visualization of mitochondrial membrane potential, optic nerve, retina, spinal cord, and brain specimens were immediately dissected out of 10 mice 3 days after antigenic sensitization for EAE, and 10 mice 6 days after EAE sensitization, and for comparisons to 10 control mice 3 days after inoculation with only Freund's adjuvant and 10 normal, unsensitized mice. The excised tissues were placed in tissue culture medium containing 0.3 µM MitoTracker Red (Molecular Probes, Eugene, OR) in Dulbecco's modified Eagle's medium plus 10% fetal bovine serum for 20 min at 37 °C, then washed in PBS, and processed for frozen sectioning and visualization of tissue fluorescence with a Leitz fluorescence microscope.

For quantitation of membrane potential, the optic nerve, retina, brain, and spinal cord were dissected out of 6 mice sensitized for EAE 3 days earlier for comparisons to 6 normal unsensitized mice. The excised tissues were incubated in 0.3 µM MitoTracker Red (Molecular Probes, Eugene, OR) in Dulbecco's modified Eagle's medium plus 10% fetal bovine serum for 20 min at 37 °C and then washed in PBS. We quantified the mitochondrial membrane potential of the entire optic nerve from behind the eye to optic chiasm, the retina, the spinal cord, and brain using an Eclipse spectrofluorophotometer (Varian Instruments, Walnut Creek, CA). Fluorescence was normalized to protein content and measured using the DC protein assay kit (Bio-Rad).

Oxidative Phosphorylation Assay—The optic nerve, retina, brain, and spinal cord were dissected out of 6 mice sensitized for EAE 3 days earlier for comparisons to 6 normal unsensitized mice. Tissues were homogenized and resuspended in buffer (150 mM KCl, 25 mM EDTA, 0.1% bovine serum albumin, 10 mM potassium phosphate, 0.1 mM MgCl2, pH 7.4). The rate of ATP synthesis of excised tissues was measured by chemiluminescence using a modified luciferin-luciferase assay in digitonin-permeabilized tissues with the complex I substrates malate and pyruvate in real time using an Optocom I luminometer (MGM Instruments, Hamden, CT) and expressed per mg of protein. Cytoplasmic ATP synthesis was also measured after the addition of 10 ng/ml oligomycin to completely inhibit mitochondrial ATP production, thus giving the background level of ATP obtained by extramitochondrial substrate level phosphorylation.


Figure 1
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  		FIGURE 1.
Immunofluorescence microscopy of nitrotyrosine. Micrograph reveals nitrotyrosine immunofluorescence in the optic nerve excised 3 days after sensitization for EAE (A). In contrast, nitrotyrosine immunofluorescence is absent in the optic nerve of a control inoculated with Freund's adjuvant 3 days earlier (B). Three days after sensitization for EAE, nitrotyrosine immunofluorescence is present in the ganglion cell layer of the retina (C), but not in controls inoculated with the adjuvant (D). Nitrotyrosine immunofluorescence is seen in the brain 3 days after sensitization for EAE (E) but not in adjuvant-inoculated animals (F). Three days after sensitization for EAE nitrotyrosine immunofluorescence is present in the spinal cord (G) but absent in controls inoculated with the adjuvant (H).

 
Transmission and Light Microscopy—For light and transmission electron microscopy, optic nerve, retina, spinal cord, and brain specimens were dissected out of 10 mice 3 days after antigenic sensitization for EAE, and 10 mice 6 days after EAE sensitization, for comparisons to 10 normal unsensitized mice and 10 control mice inoculated with Freund's adjuvant and euthanized 3 days later. Immediately following euthanasia mice were perfused with fixative consisting of 4% paraformaldehyde and 2% glutaraldehyde in 0.1 M PBS buffer, pH 7.4. Tissues were further processed by immersion in 2.5% glutaraldehyde and then postfixed in 1% osmium tetroxide, 0.1 M sodium cacodylate-HCl buffer, pH 7.4. Tissues were then dehydrated through an ethanol series to propylene oxide, infiltrated, and embedded in epoxy resin that was polymerized at 60 °C overnight. Semithin longitudinal sections (0.5 µm to 1 µm) of the optic nerve head and retrobulbar nerve were stained with toluidine blue. In addition, ultrathin sections (90 nm) were placed on nickel grids for examination by transmission electron microscopy (model H7600; Hitachi, Tokyo, Japan) operating at 80 kV. For quantification of dissolution of mitochondrial cristae induced by early EAE, we analyzed 200 mitochondria from five animals sensitized for EAE and an equivalent number of mitochondria from five controls sensitized with the adjuvant. These 10 animals were euthanized 3 days after antigenic sensitization. Optic nerves were photographed at a magnification of x10,000. Using NIH Image software the electron density of each mitochondrion was measured by manually tracing the silhouette of the outer membrane followed by thresholding of the electron dense cristae, including the mitochondrial membrane. Densities ranged from 255 (black) to 0 (white).


Figure 2
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  		FIGURE 2.
Inflammatory cell immunofluorescence and light microscopy. Three days after sensitization for EAE immunofluorescence was not detected by utilizing a pan-macrophage antibody (A) and inflammatory cells were absent in the toluidine blue-stained optic nerve (B). In the brain, inflammatory cells were undetectable by immunofluorescence (C) or by toluidine blue staining (D). Immunofluorescence (E) and light microscopy (F) revealed inflammatory cells were absent in the spinal cord. As a positive control, optic nerve from an animal sensitized for EAE 30 days earlier was positive for inflammatory cells (arrows) detected by immunofluorescence (G) and by light microscopy (H). onh, optic nerve head; r, retina.

 
Statistical Analysis—Differences in the means between EAE and control groups were measured by Student's t test for unpaired data. Analysis of ATP synthesis and mitochondrial membrane potential was performed by analysis of variance. A difference in the means of 0.05 or less was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Nitration of CNS Tissues—We examined the optic nerves, retinas, brains, and spinal cords of 10 mice 3 days after sensitization for EAE with complete Freund's adjuvant and spinal cord emulsion for comparisons to specimens excised from 10 control mice 3 days after inoculation with only the Freund's adjuvant for ROS activity. At this early stage none of the animals exhibited any signs of paralysis from EAE (clinical stage 0). As an initial gauge of ROS activity, we used the peroxynitrite-mediated nitration of tyrosine residues that was detected with an antibody directed against nitrotyrosine. Peroxynitrite is formed by the reaction of superoxide and nitric oxide. Using immunofluorescence cryomicroscopy, we detected nitrated proteins in the optic nerve (Fig. 1A), ganglion cell layer of the retina (Fig. 1C), brain (Fig. 1E), and spinal cord (Fig. 1G) 3 days after sensitization for EAE. In contrast, the optic nerve (Fig. 1B), retina (Fig. 1D), brain (Fig. 1F), and spinal cord specimens (Fig. 1H) of adjuvant-inoculated mice were unlabeled.

We excluded inflammatory cells, classically heralded as the mediators of tissue injury in EAE and MS (14), as the source of ROS activity by reacting EAE tissues with a pan-macrophage antibody followed by immunofluorescence microscopy and by light microscopic examination of toluidine blue-stained tissues. No inflammatory cells were detected in the optic nerve (Fig. 2, A and B), brain (Fig. 2, C and D), or spinal cord (Fig. 2, E and F) of mice sensitized for EAE 3 days earlier. As a positive control we examined tissue specimens available from our other experiments that were from animals sensitized for EAE 30 days earlier. They tested positive for inflammatory cells (Fig. 2, G and H). Therefore, the origin of the nitrotyrosine immunofluorescence labeling in the 3-day EAE specimens appeared to be the CNS tissue itself.

Mitochondrial Protein Nitration—Because mitochondria are the primary source of cellular ROS, we probed them next. We isolated mitochondria from the optic nerves, retinas, brains, and spinal cords of 20 mice euthanized 3 days after sensitization for EAE and 20 mice euthanized 6 days after EAE sensitization for comparisons to controls consisting of 10 unsensitized animals and 10 mice inoculated with complete Freund's adjuvant and sacrificed 3 days later. Using immunoblotting, we then probed the mitochondrial isolates for peroxynitrite-mediated nitration of tyrosine residues with the nitrotyrosine antibody. We found several nitrated mitochondrial protein bands in the EAE central nervous system (Fig. 3A) but not in the control specimens. Next, we attempted to determine which proteins were specifically inactivated in the mitochondria of EAE animals.

Identification of Nitrated Proteins—We identified the nitrated mitochondrial proteins using in situ trypsin digests of the excised nitrated protein bands followed by mass spectroscopy (15). When the resulting peptide fingerprints were compared with the protein sequence, the highest match was for mitochondrial heat shock protein 70 (mtHsp70) (Fig. 3, B and C). Protein data base sequence analysis of the other peptide fingerprints obtained included two respiratory chain complexes. They were identified as the NADPH-ubiquinone oxidoreductase B14 subunit of complex I (NDUFA6) (Fig. 3D) and cytochrome c oxidase subunit IV (Fig. 3E). Consequently, oxidative damage to proteins in the mitochondrial respiratory chain was not uniform but affected subunits of complexes I and IV preferentially. Protein data base matches of another peptide fingerprint included the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Fig. 3F).


Figure 3
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  		FIGURE 3.
Detection and identification of nitrated proteins. A, immunoblots of CNS mitochondrial isolates revealed peroxynitrite-mediated nitration of tyrosine residues at day 3 and day 6 after sensitization for EAE but not in the control specimens (day 0). MS peptide fingerprint of excised nitrated 70-kDa band was identified as mtHsp70 (B). Amino acids identified by protein data base sequence analysis of mtHsp70 (C), NADPH-ubiquinone oxidoreductase B14 subunit (NDUFA6) of complex I (D), cytochrome c oxidase subunit IV (E), and GAPDH (F) are capitalized and underlined. ON, optic nerve.

 


Figure 4
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  		FIGURE 4.
Histograms of ATP synthesis. Bar plots of ATP synthesis in the 3-day EAE optic nerve (A), retina (B), and brain (C) were not significantly different from controls. ATP synthesis was reduced by 79% in the spinal cords of mice sensitized for EAE 3 days earlier, relative to control mice (* = p < 0.05, n = 6; mean ± S.D.) (D). NL, normal; ON, optic nerve; RET, retina.

 
Respiration Is Suppressed—To determine the impact of EAE on the primary function of mitochondria, we tested for generation of cellular ATP in six animals sensitized for EAE 3 days earlier and compared these to six unsensitized controls. The rates of mitochondrial ATP synthesis in the 3-day EAE optic nerve (Fig. 4A), retina (Fig. 4B), and brain (Fig. 4C) were not significantly different from controls. However, in the spinal cords of mice sensitized for EAE 3 days earlier the rate of mitochondrial ATP synthesis was reduced by 79%, relative to controls (Fig. 4D) (p < 0.05).

Mitochondrial Membrane Potential Is Attenuated—Next, we examined the effect of EAE on mitochondrial membrane potential in 10 mice sensitized for EAE and 10 control mice inoculated with Freund's adjuvant. Each group was euthanized 3 days later, and the excised tissues were incubated with the membrane-sensing dye MitoTracker Red and prepared for cryomicrotomy. Fluorescence microscopy of the 3-day retrobulbar optic nerve sectioned just behind the eye revealed marked attenuation of labeling in the central region of the nerve (Fig. 5A). In contrast, the optic nerve of adjuvant-inoculated controls showed normal mitochondrial labeling of the entire optic nerve cross-section (Fig. 5B). Still, fluorospectrometric measurements of the entire optic nerve from the eye to the optic chiasm obtained from six animals sensitized for EAE 3 days earlier, relative to six controls, did not reflect differences in some optic nerve cross-sections visualized by microscopy (Fig. 5C).

The cell bodies of axons comprising the optic nerve reside in the ganglion cell layer of the retina. Microscopic examination of EAE-sensitized animals revealed some loss of MitoTracker Red labeling in the ganglion cell layer (Fig. 5D), relative to adjuvant inoculation (Fig. 5E). Fluorospectrometric measurements of the entire retina failed to show any difference between EAE and controls (Fig. 5F).

In the EAE brain diminished MitoTracker Red labeling (Fig. 5G) contrasted with the normal labeling of the adjuvant-inoculated animals (Fig. 5H). Quantitative measurements of the brain failed to detect any significant difference between EAE and adjuvant-inoculated control animals (Fig. 5I).


Figure 5
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  		FIGURE 5.
Immunofluorescent micrographs and quantitation of mitochondrial membrane potential. A, loss of MitoTracker Red accumulation in mitochondria was evident in an optic nerve cross-section taken through the retrolaminar region of the nerve just behind the eye. B, fluorescence microscopy of this region shows normal mitochondrial labeling of control optic nerve. C, fluorospectrometric measurements of the entire optic nerve from the back of the eye to the optic chiasm did not reflect any differences between EAE and controls (n = 6; mean ± S.D.). D, fluorescence micrographs of retinal cross-sections showed diminished MitoTrackerRed accumulation in some ganglion cells relative to control retina (E). F, fluorospectrometric measurements of retinal flat mounts did not show any differences between EAE and controls (n = 6; mean ± S.D.). Fluorescence microscopy of a cross-section of EAE brain showed diminished accumulation of the mitochondrial membrane-sensing dye (G) relative to control brain (H). I, fluorospectrometric measurements of the brain did not show any significant differences between EAE and controls (n = 6; mean ± S.D.). Diminished MitoTracker Red labeling of the 3-day EAE spinal cord (J) contrasted with the normal labeling of adjuvant-inoculated mice (K). L, quantitative measurements of entire EAE spinal cords revealed a one-third reduction relative to normal (n = 6; mean ± S.D.). * = p < 0.05; ADJ, adjuvant-inoculated controls; ON, optic nerve; RET, retina.

 
We found the greatest loss of mitochondrial membrane potential in the EAE spinal cord. Here diminished MitoTracker Red labeling of 3-day EAE animals (Fig. 5J) contrasted with the normal labeling of adjuvant-inoculated mice (Fig. 5K). Quantitative measurements of the entire EAE spinal cords confirmed a one-third reduction relative to controls (Fig. 5L) (p < 0.05).

Apoptosis—Because loss of mitochondrial membrane potential is linked to cell death, we measured apoptosis in the cryopreserved CNS tissues of 10 mice sensitized for EAE and 10 animals sensitized with the adjuvant, and sacrificed 3 days later. We found many TUNEL-positive cells in the 3-day EAE optic nerve (Fig. 6A). TUNEL-labeled cells were found primarily in the center of the nerve, where the mitochondrial membrane potential appeared to be the lowest (Fig. 5A). Apoptotic cells were rarely seen in the nerves of adjuvant-inoculated animals (Fig. 6, B and C). In the retina of EAE-inoculated mice numerous TUNEL-positive cells were evident in the ganglion cell layer (Fig. 6D), but they were an infrequent finding in adjuvant-inoculated animals (Fig. 6, E and F). Cell bodies of axons comprising the optic nerve are located here. In the brain, TUNEL-positive cells were detected in EAE-sensitized animals (Fig. 6G), but they were infrequently seen in control brains (Fig. 6, H and I). Finally, many apoptotic cells were apparent in the EAE spinal cord (Fig. 6J) relative to adjuvant-inoculated animals in which they were a rare finding (Fig. 6, K and L).


Figure 6
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  		FIGURE 6.
Immunofluorescent micrographs and quantitation of apoptosis. Numerous TUNEL-positive cells (arrows) were seen in the 3-day EAE optic nerve (A) relative to only a few seen in adjuvant-inoculated animals (B). Counts of TUNEL-labeled cells in the EAE nerve were significantly increased relative to control (n = 10; mean ± S.D.) (C). In the retina of EAE-inoculated mice, many TUNEL-positive cells (arrows) were seen in the ganglion cell layer (D), whereas apoptotic cells were rare in adjuvant-inoculated controls (E). Counts of TUNEL-labeled cells in the EAE retina were markedly elevated relative to control (n = 10; mean ± S.D.) (F). In the brain, many TUNEL-positive cells (arrows) were detected in EAE-sensitized animals (G), relative to the few detected in controls (H). Counts of TUNEL-labeled cells in the EAE brain were marked elevated relative to control (n = 10; mean ± S.D.) (I). Finally, apoptotic cells (arrows) were apparent in the EAE spinal cord (J), but they were rare in adjuvant-inoculated controls (K). Counts of TUNEL-labeled cells in the EAE spinal cord were substantially increased relative to control (n = 10; mean ± S.D.) (L). * = p < 0.001; ADJ, adjuvant-inoculated controls; ON, optic nerve; RET, retina.

 


Figure 7
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  		FIGURE 7.
Immunofluorescent micrographs and quantitation of apoptotic oligodendrocytes. The nuclei of apoptotic cells in the 3-day EAE optic nerve were labeled red by TUNEL, and the cytoplasm was co-labeled green (arrows) by an antibody directed against oligodendrocytes (A). Control optic nerves from mice inoculated with the adjuvant revealed the green immunofluorescence of oligodendrocytes, but they were for the most part TUNEL-negative (arrows)(B). Relative to control, counts of TUNEL-positive oligodendrocytes were increased in the optic nerve (n = 10; mean ± S.D.) (C). TUNEL-positive oligodendrocytes (arrows) were evident in the EAE brain (D). Oligodendrocytes in the brain of an adjuvant-inoculated mouse were predominantly TUNEL-negative (arrows) (E). Relative to control, counts of TUNEL-positive oligodendrocytes were increased in the EAE brain (n = 10; mean ± S.D.) (F). Finally, TUNEL-positive cells in the EAE spinal cord were identified as oligodendrocytes (arrows) (G). Oligodendrocytes (arrows) in the spinal cord of an adjuvant-inoculated mouse were mostly negative for apoptosis (H). Relative to control, counts of TUNEL-positive oligodendrocytes were increased in the spinal cord (n = 10; mean ± S.D.) (I). * = p < 0.01, ADJ, adjuvant-inoculated controls; ON, optic nerve.

 
To identify the apoptotic cells, we co-labeled the specimens with an antibody directed against oligodendrocytes. We found the TUNEL-positive cells in the EAE specimens were co-labeled with the oligodendrocyte antibody (Fig. 7, A, D, and G). Relative to EAE, TUNEL-positive oligodendrocytes in the adjuvant-inoculated controls were infrequent; thus oligodendrocytes were labeled predominantly by the antibody only (Fig. 7, B, E, and H). Quantitative analysis of TUNEL-positive oligodendrocytes confirmed the apoptosis induced by EAE in the optic nerve (Fig. 7C), brain (Fig. 7F), and spinal cord (Fig. 7I). Unlike the brain and spinal cord, the optic nerve contains no neurons. They reside in the ganglion cell layer of the retina that we showed had numerous TUNEL-positive cells (Fig. 6D). Thus, apoptotic cell death in 3-day EAE tissues occurred in the absence of inflammatory cells. It involved both neurons and oligodendrocytes.

Nitric-oxide Synthase—To determine the signal for the cascade of events leading to apoptosis, we probed the cryopreserved EAE and adjuvant-inoculated specimens with an antibody against iNOS. We found iNOS labeling in the EAE optic nerves (Fig. 8A), retinal ganglion cells (Fig. 8D), brains (Fig. 8G), and spinal cords (Fig. 8J). The iNOS labeling had both a cytoplasmic and punctate pattern. MitoTracker Red staining of the same tissue sections shown in Fig. 5 suggested mitochondrial localization of some NOS activity. Adjuvant-inoculated tissues were negative for iNOS (Fig. 8, B, E, H, and K). Quantification of the intensity of iNOS immunofluorescence revealed only background tissue fluorescence in controls relative to the elevated levels in EAE (Fig. 8, C, F, I, and L). Additionally, Western blots of mitochondria isolated from the EAE spinal cord revealed iNOS immunoreactivity 3 and 6 days after antigenic sensitization but not in the control spinal cord (Fig. 8M).


Figure 8
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  		FIGURE 8.
Fluorescent micrographs, immunoblotting, and quantitation of nitric-oxide synthase. A, cytoplasmic and punctate iNOS immunofluorescence (arrows) was seen in the 3-day EAE optic nerve. B, NOS immunofluorescence was absent in control nerves. C, quantitative analysis revealed iNOS-induced fluorescence was increased in EAE nerves relative to the background levels of controls (n = 10; mean ± S.D.). D, the EAE retina appeared to have a slight increase in iNOS signal in the ganglion cell layer (arrow) relative to a control inoculated with the adjuvant (E). Quantitative analysis confirmed the increase in iNOS-induced fluorescence in EAE retina relative to the background levels of controls (n = 10; mean ± S.D.) (F). Cytoplasmic and punctate iNOS immunofluorescence (arrows) was seen in the 3-day EAE brain (G), but it was absent in adjuvant-inoculated controls (H). Quantitative analysis failed to confirm a significant increase in iNOS activity in the EAE brain relative to control (n = 10; mean ± S.D.) (I). The 3-day EAE spinal cord was positive for iNOS activity (arrows)(J), whereas a spinal cord micrograph of an adjuvant-inoculated control revealed no iNOS activity (K). Measurements of iNOS fluorescence confirmed the increase in the EAE spinal cord relative to control (n = 10; mean ± S.D.) (L). Immunoblots of spinal cord mitochondrial isolates revealed the 135-kDa iNOS band at day 3 and day 6 after sensitization for EAE but not in controls (day 0). For mitochondrial protein loading, the 31-kDa band of a VDAC1/Porin antibody is also shown (M). *, p = 0.01; ADJ, adjuvant-inoculated controls; ON, optic nerve; RET, retina; p, photoreceptor layer of the retina exhibiting autofluorescence.

 


Figure 9
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  		FIGURE 9.
Transmission electron micrographs of mitochondrial and axonal degeneration. A, 3 days after sensitization for EAE, mitochondrial vacuolization and dissolution of cristae (arrow) are evident in an axon of the optic nerve with a normal enveloping myelin sheath. The normal tubular architecture of mitochondrial cristae (arrow) in a control nerve is shown for comparison (B). Many axons of the EAE optic nerve exhibiting mitochondrial swelling and dissolution of cristae also had disruption of the axonal cytoskeleton (arrows) (C). These findings sharply contrasted with the normal tubular ultrastructure (arrows) of controls (D). a, axon.

 
Axonal Degeneration—Mitochondrial vacuolization and dissolution of cristae were evident in axons with a normal appearance to the enveloping myelin sheath, 3 days after sensitization for EAE (Fig. 9A). Mitochondria of controls exhibited the normal profile of cristae (Fig. 9B). Indicative of the dissolution of cristae in EAE, quantitative analysis revealed a 26% decrease in mitochondrial electron density with a mean of 126 ± 20 (mean ± S.D.) relative to 176 ± 26 for adjuvant-inoculated controls (p < 0.0001). In addition, many myelinated axons exhibiting mitochondrial swelling and dissolution of cristae also had disruption of axonal filaments and tubules (Fig. 9C). These findings sharply contrasted with the normal microtubular network of controls (Fig. 9D). The structural alterations in axons and mitochondria are indicative of neurodegeneration in early EAE.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Mitochondrial dysfunction induced by protein inactivation and mediated by oxidative stress primes apoptosis that leads to axonal and neuronal degeneration in the EAE animal model of MS. Our findings that mitochondrial proteins isolated from the optic nerve, retina, brain, and spinal cord were nitrated by peroxynitrite suggest that CNS mitochondria were the primary source of increased cellular ROS during early EAE. In addition, peroxynitrite reactants superoxide and nitric oxide are free radicals also capable of mediating tissue injury. Generation of mitochondrial superoxide is predominantly mediated by complex I of the mitochondrial electron transport chain (16). Nitric oxide is generated by the oxidation of L-arginine by nitric-oxide synthase (NOS). Although cytoplasmic isoforms of NOS are well recognized, there also is evidence for a mtNOS (17). Because mtNOS has some cross-reactivity with antibodies directed against the NADPH-binding site located at the C terminus (18), we were able to detect NOS activity in our mitochondrial isolates by using one of these antibodies. The punctate pattern of NOS immunostaining in CNS tissues also suggested a mitochondrial localization of NOS activity in EAE. Still, translocation of NOS from the cytoplasm into mitochondria has been demonstrated (19), and this may have accounted for our findings of mtNOS activity.

NOS activation appeared to be the signal driving the increased mitochondrial ROS activity during early EAE (20). We found that the peroxynitrite-mediated nitration of the optic nerve, retina, brain, and spinal cord began before infiltration of inflammatory cells that are generally considered to be the mediators of disease and axonal injury by transection (21). Whereas our previous work in EAE had focused on the release of ROS by inflammatory cells (22-26), here they were excluded as the source of ROS in early EAE by using both immunohistochemical markers of inflammation and by microscopic examination of the CNS. This result suggested that the source of elevated ROS activity in EAE-sensitized mice was the CNS tissue itself.

Mitochondria were not only the source of ROS activity but the targets of oxidative injury during early EAE. For protection against oxidative injury, the organelle maintains antioxidant enzyme defenses that include the manganese superoxide dismutase and glutathione peroxidase (27), which mediate detoxification of superoxide and hydrogen peroxide, respectively. However, there are no endogenous defenses against peroxynitrite that we found induced mitochondrial dysfunction. Oxidative damage to mitochondrial DNA has been suggested as the modifying factor in complex I deficiency in longstanding MS (28). Nevertheless, a recent study found complex I deficiency in MS patients was not because of alterations in the mitochondrial genome (28). Our findings here suggest protein damage is a major factor in respiratory chain dysfunction in the EAE animal model of MS. Nitration is recognized as a modifying event that can inactivate protein function (29). Using mass spectroscopy we were able to identify some of those affected during early EAE.

Protein nitration involved at least subunits of complexes I and IV of the respiratory chain, glycolysis, and a chaperone critical to the stabilization and import of proteins into mitochondria. Still, our immunoblots revealed more than five nitrated protein bands. However, only the MASCOT probability-based MOWSE scores above the default value were considered significant for protein identification. They were identified as the NDUFA6 subunit of complex I, cytochrome oxidase subunit IV, GAPDH, and mitochondrial Hsp70 chaperone. The NDUFA6 subunit is 1 of ~45 subunits comprising complex I. It is encoded by nuclear DNA and imported into the organelle after synthesis in the cytoplasm. The nuclear gene encoding it is unaffected by oxidative damage to mitochondrial DNA. The NDUFA subunits are critical components involved in assembly of the holoenzyme NADH dehydrogenase complex (30). This subunit of complex I was reported previously to be particularly susceptible to inactivation by peroxynitrite (31). Cytochrome oxidase subunit IV is a key subunit of complex IV of the respiratory chain. Although the nitration of complex IV is believed to have a lesser impact on respiration than nitration of complex I, involvement of both complexes contributes to loss of mitochondrial ATP synthesis here (31-32).

Although not directly involved in oxidative phosphorylation, mitochondrial Hsp70 is an import chaperone molecule critical to the translocation of proteins from the cytoplasm into the mitochondrial matrix. In fact, most mitochondrial proteins are nuclear encoded, synthesized on cytoplasmic ribosomes, and then imported into the mitochondria, usually directed by an N-terminal targeting presequence. Mitochondrial DNA encodes only 13 of the ~100 proteins needed for oxidative phosphorylation. Import of a host of proteins may be attenuated by peroxynitrite-mediated mtHsp70 inactivation, profoundly affecting function of the organelle. In addition to its import function, mtHsp mediates protein folding and unfolding. Loss of this function mediated by mtHsp70 causes protein aggregation and degradation within the organelle (33). Both roles of mtHsp70 likely contributed to our findings in early EAE.

The primary function of mitochondria is the generation of cellular energy. Because production of ATP is coupled to the respiratory chain, we gauged the effects of early EAE on mitochondrial bioenergetics by measuring the rate of ATP synthesis. By using the precursors malate and pyruvate as complex I-dependent substrates for oxidative phosphorylation, we found that EAE sensitization and presumably the cumulative effects of protein nitration suppressed ATP synthesis in the spinal cords of mice almost 80%. This magnitude of reduction in ATP synthesis is greater than that associated with disorders caused by missense mutations in mtDNA (34). Our studies here and those of others support peroxynitrite-mediated nitration of mitochondrial proteins as a cause of respiratory complex inactivation (35). Unlike mutations in mitochondrial DNA that alter one amino acid in a subunit of one of the five respiratory chain complexes, mitochondrial protein nitration involved at least two complexes and likely involved more because of modification of mtHsp70. Despite the magnitude of ATP loss, our animals exhibited no signs of paralysis 3 days after sensitization for EAE. In the EAE animal model, limb paralysis because of the spinal cord involvement is the most frequently used clinical measure of disease activity. It typically begins 1-2 weeks after antigenic sensitization. In patients, phenotypic expression of neurodegenerative diseases caused by mutated mtDNA may take years or decades. Clinical signs typical of the neurodegeneration of MS also appear to have a long latency period (9). Our study shows that the mitochondrial injury contributing to neurodegeneration begins very early in EAE.

Critical to mitochondrial bioenergetics is the maintenance of an electrochemical gradient. The negative membrane potential of mitochondria needed for maintenance of the gradient is created by the pumping of protons out of the organelle. This is mediated by the activity of respiratory complexes I, III, and IV (36). Consistent with inactivation of complex I and IV by nitration, we found accumulation of the membrane-sensing dye MitoTracker Red within mitochondria was reduced by one-third in our quantitative measurements of the EAE spinal cord. Using total tissue measurements of the optic nerve, retina, or brain we were unable to detect an overall change between normal and EAE tissues. Because RGCs consist of a tiny fraction of all retinal neurons, it is easy to understand why a loss of membrane potential in this retinal layer would be difficult to detect by quantitative measurements of the entire retina. In EAE, the retrobulbar portion of the optic nerve adjacent to the globe is the primary focus of demyelination in the visual system (37-38) that is also frequently affected in MS. Tissue sections of this region of the nerve revealed a loss of membrane potential that was masked by measurements of the entire nerve. Inactivation of respiratory chain complexes by peroxynitrite likely contributed to loss of membrane potential in early EAE.

Apoptosis is another important cellular function of mitochondria. As shown by others (39), we too found apoptotic neurons in the ganglion cell layer of the retina in EAE. Their axons compose the optic nerve. Unlike previous studies focusing on later stages of EAE, apoptosis of RGCs here was not caused by inflammatory cell-mediated axonal transection (7). Inflammatory cells were absent in our 3-day EAE specimens. ROS inactivation of key mitochondrial proteins played a role in the apoptosis of RGCs in our EAE studies. Knockdown of the NDUFA6 has been shown to mediate apoptosis (40). We showed this very subunit was nitrated and likely inactivated. It contributed to loss of complex I-dependent ATP synthesis in early EAE and likely to apoptosis too. This is supported by our previous experiments that knocked down gene expression of a closely related complex I subunit, NDUFA1, that resulted in apoptotic death of RGCs (41). Loss of ganglion cells has recently been linked to poor visual function in patients with optic neuritis and MS (42). In addition to its role in glycolysis, translocation of nitrosylated GAPDH to the nucleus is linked to apoptotic cell death, or necrosis with ATP depletion (43) that we showed occurred in early EAE. These factors likely play a role in the demise of RGCs and axons of the optic nerve. In addition to neurons, we detected apoptosis of myelin-forming oligodendrocytes in EAE optic nerves, brains, and spinal cords. Previously, ROS-mediated mtDNA damage was shown to induce apoptosis in cultured oligodendroglia (20). Clearly, our study suggests that peroxynitrite-mediated mitochondrial protein nitration was a key factor in apoptosis during early EAE.

Finally, we supported our findings of mitochondrial dysfunction with ultrastructural evidence of mitochondrial and axonal disruption as further proof that the neurodegenerative process starts during early EAE. We found that myelinated axons contained swollen mitochondria that exhibited disorganization and dissolution of cristae, some to the point that only a double membrane sheath identified the organelle. Although no evidence for inflammatory cell-mediated axonal transection was detected during the early stage of EAE studied here, impairments in calcium homeostasis, another important function of mitochondria, were recently detected in autopsied MS brains (21).

In summary, we have produced evidence that the pathway leading toward neurodegeneration in EAE commences much earlier than currently believed and that mitochondria are intimately involved in this degenerative process. Our previous studies of complex I knockdown proved that amelioration of apoptosis in ganglion cells of the retina and loss of axons in the optic nerve may be achieved by genetically protecting mitochondria against oxidative stress (44). Whether a similar strategy applied to EAE may help avert the demise of axons, neurons, and oligodendroglia in MS remains to be demonstrated.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants EY07982 (to J. G.) and EY12355 (to J. G.) and Research to Prevent Blindness (to J. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence and reprint requests should be addressed: Dept. of Ophthalmology, University of Florida, Box 100284, Gainesville, FL 32610-0284. Tel.: 352-846-2102; Fax: 352-392-7839; E-mail: johnguy{at}eye.ufl.edu.

2 The abbreviations used are: EAE, experimental autoimmune encephalomyelitis; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nickend labeling; CNS, central nervous system; MS, multiple sclerosis; ROS, reactive oxygen species; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NOS, nitric-oxide synthase; iNOS, inducible NOS; mt, mitochondrial; PBS, phosphate-buffered saline; HPLC, high pressure liquid chromatography; RGC, retinal ganglion cell. Back


   ACKNOWLEDGMENTS
 
We thank Mabel Wilson, for reviewing and editing the manuscript; the reviewers and editors of JBC for their helpful suggestions requesting additional in vivo functional experiments; and Dr. Stan Stevens and Scott McClung of the University of Florida ICBR protein core for mass spectroscopy technical assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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