dutta et al mitochondrial dysfunction in ms annneurol06

7/28/2019 Dutta Et Al Mitochondrial Dysfunction in MS AnnNeurol06

1/12

Mitochondrial Dysfunction as a Cause ofAxonal Degeneration in Multiple Sclerosis

PatientsRanjan Dutta, PhD,1 Jennifer McDonough, PhD,1 Xinghua Yin, MD,1 John Peterson, PhD,1

Ansi Chang, MD,1 Thalia Torres, BS,1 Tatyana Gudz, PhD,1 Wendy B. Macklin, PhD,1 David A. Lewis, MD,3

Robert J. Fox, MD,2 Richard Rudick, MD,2 Karoly Mirnics, MD,3 and Bruce D. Trapp, PhD1

Objective: Degeneration of chronically demyelinated axons is a major cause of irreversible neurological disability inmultiple sclerosis (MS) patients. Development of neuroprotective therapies will require elucidation of the molecularmechanisms by which neurons and axons degenerate. Methods: We report ultrastructural changes that support Ca2 -mediated destruction of chronically demyelinated axons in MS patients. We compared expression levels of 33,000 char-acterized genes in postmortem motor cortex from six control and six MS brains matched for age, sex, and postmorteminterval. As reduced energy production is a major contributor to Ca2 -mediated axonal degeneration, we focused onchanges in oxidative phosphorylation and inhibitory neurotransmission. Results: Compared with controls, 488 transcriptswere decreased and 67 were increased (p < 0.05, 1.5-fold) in the MS cortex. Twenty-six nuclear-encoded mitochondrial

genes and the functional activities of mitochondrial respiratory chain complexes I and III were decreased in the MSmotor cortex. Reduced mitochondrial gene expression was specific for neurons. In addition, pre-synaptic and postsyn-aptic components of GABAergic neurotransmission and the density of inhibitory interneuron processes also were de-creased in the MS cortex. Interpretation: Our data supports a mechanism whereby reduced ATP production in demy-elinated segments of upper motor neuron axons impacts ion homeostasis, induces Ca2 -mediated axonal degeneration,and contributes to progressive neurological disability in MS patients.

Ann Neurol 2006;59:478 489

Rapid communication between neurons requires en-ergy and the insulation of axons by discontinuous seg-ments of myelin. Voltage-gated Na channels produce

nerve impulses and are concentrated at the nodes ofRanvier,1 the short unmyelinated axon segment be-tween individual myelin internodes. The nerve impulserapidly jumps from node to node by a process calledsaltatory conduction. Multiple sclerosis (MS) is aninflammatory, demyelinating disease of the centralnervous system (CNS) that destroys myelin, oligoden-drocytes, axons, and neurons.2 Pathologically, demyeli-nation predominates during early stages of MS. Neu-rological disability associated with demyelinatinglesions is initially reversible because of a variety ofadaptive changes in the MS brain. As part of these

changes, Na channels are distributed diffusely alongthe surface of demyelinated axons,3 resulting in slowbut effective nerve communication. This also increases

the energy demands of neuronal communication andrenders the demyelinated axon more susceptible to hy-poxic/ischemic damage (for review, see Stys4).

After an initial stage (commonly 10 15 years) ofrelapses and remissions (RRMS), most MS patientsenter a course of irreversible and continuous neuro-logical decline, termed secondary progressive multiplesclerosis (SPMS).2 During SPMS, new inflammatorybrain lesions substantially decrease with age,5 butneurological decline continues due in part to degen-eration of chronically demyelinated axons.6 To iden-tify neuronal gene changes that may contribute to ax-

From the 1Department of Neurosciences, Lerner Research Institute;2Mellen Center for Multiple Sclerosis Treatment and Research,Cleveland Clinic Foundation, Cleveland, OH; and 3Department ofPsychiatry, University of Pittsburgh School of Medicine, Pittsburgh,PA.

Received Aug 16, 2005, and in revised form Oct 6. Accepted forpublication Oct 6, 2005.

R.D. and J.M. contributed equally to this study.

This article includes supplementary materials available via the inter-net at http://www.interscience. wiley.com/jpages/0364-5134/supp-mat

Published online Jan 3, 2006, in Wiley InterScience(www.interscience.wiley.com). DOI: 10.1002/ana.20736

Address correspondence to Dr Trapp, Department of Neuro-sciences/NC30, Lerner Research Institute, Cleveland Clinic Foun-dation, 9500 Euclid Avenue, Cleveland, OH 44195.E-mail: [email protected]

Current address for Dr McDonough: Oak Clinic for MS Researchat Kent State University, Kent, OH.

Current address for Dr Gudz: Department of Neurobiology, Med-ical University of South Carolina, Charleston, SC.

478 2006 American Neurological AssociationPublished by Wiley-Liss, Inc., through Wiley Subscription Services


2/12

onal degeneration in chronic MS patients, weconducted an unbiased comparison of mRNA expres-

sion changes in nonlesioned motor cortex from six con-trol and six MS patients obtained by rapid autopsy usingthe human U133 microarray platform (Affymetrix,Santa Clara, CA) containing more than 33,000 well-characterized genes. Previous studies have profiled geneexpression in demyelinated lesions79 and normal-appearing white matter from MS patients.10,11 To ourknowledge, this is the first study of non-lesion cerebralcortex from MS patients using the complete set ofU133 arrays.

In MS cortex, we detected significant reductions ingene products specific for the mitochondrial electron

transport chain and demonstrated that cortical mito-chondria have a diminished capacity to exchange elec-trons in respiratory chain complex I and III. In addi-tion, pre-synaptic and postsynaptic components ofinhibitory neurotransmission and the density of inhib-itory interneuron processes were decreased in MS mo-tor cortex samples. We propose that these changes neg-atively impact on ion homeostasis in demyelinatedsegments of upper motor neuron axons and contributeto axonal degeneration and progressive neurologicaldisability in MS patients.

Patients and Methods

TissueMS brain tissues were obtained from The Cleveland Clinic.Patients or their relatives provided informed consent in theform of an advanced directive as part of an institutional re-view boardapproved protocol. Control brain tissues wereobtained from the University of Pittsburgh, Center for theNeuroscience of Mental Disorders Brain Bank and have beencharacterized previously by microarray analysis.12 MS andcontrol donors were matched for age and sex and postmor-tem interval (PMI; p 0.05; Table). MS patients were se-verely disabled (Expanded Disability Status Scale [EDSS]score, 7.0) at the time of death. Tissues belonging to MSand control cases used for microarray analysis, real-time

(RT) polymerase chain reaction (PCR) analysis, and West-ern blots are described in the Table. Blocks of motor cortexwere removed at autopsy and frozen at 70C. To ensurethe absence of demyelination and other pathologies, werecut and immunostained frozen sections (14m thick) fromeach block for myelin proteolipid protein and major histo-compatibility class (MHC) II antigens as described previ-ously.13 Sections were also cut for isolating RNA, protein,or mitochondria-enriched fractions. Paraformaldehyde-fixedblocks were obtained from the contralateral hemisphere fromeach brain and used for immunocytochemical and in situ hy-bridization studies.

Table. Details of Patients Examined in this Study

PatientNo.

Age(yr)/Sex

PostmortemInterval (hr)

Type ofMultipleSclerosis

Durationof Disease

(yr)EDSSScore Cause of Death

MS1 64/M 4.0 PP 13 8.0 MS2 48/F 5.0 SP 26 9.0 MS3 56/M 3.5 SP 32 9.5

MS4 47/F 6.5 SP 31 9.0 MS5 79/F 4.5 SP 51 9.0 MS6 45/M 15.5 SP 19 7.0 MS7 54/M 5.0 SP 15 9.5 MS8 51/F 5.0 SP 25 9.0 MS9 57/F 5.0 SP 39 9.5 MS10 52/M 4.8 SP 25 7.5 C1 62/M 7.2 Ruptured abdominal

aortic aneurysmC2 46/F 15.0 Cardiac tamponadeC3 60/M 9.5 Atherosclerotic car-

diovascular diseaseC4 54/F 8.0 Atherosclerotic car-

diovascular diseaseC5 60/F 9.5 Mitral valve prolapse

C6 62/M 21.0 AspirationC7 53/M 12.0 Ischemic heart diseaseC8 52/F 13.5 Polymicrobial sepsis

Samples M1 to M6 and Control C1 to C6 were used for microarray analysis. Mean age and postmortem interval (HR) for multiple sclerosis(56.5 5.3; 6.5 1.8) and control (57.3 2.5; 11.7 2.1) patients was statistically similar in samples used for the microarray analysis.Samples MS 1-10 were used for electron microscopy analysis; real-time polymerase chain reaction analysis was done using samples MS1MS6and C1C4, C7, C8 (control C7 and C8 were included to supplement inadequate sample recovery from sample C5 and C6). Western blotanalysis was performed using samples C1, C4, C5, C7, C8, M1, M3, M4, and M5. EDSS Expanded Disability Status Scale (possible range,1-10, with a higher score indicating a greater degree of disability); PP primary progressive; SP secondary progressive.

Dutta et al: Mitochondrial Dysfunction in MS 479


3/12

Electron Microscopy of Spinal Cord AxonsSpinal cords from 10 MS patients (MS110 in the Table)

were obtained by rapid autopsy, cut into 2cm segments, andplaced in 4% paraformaldehyde for 48 hours. A 5mm-thickslice then was taken from each end of the block, placed in4% paraformaldehyde and 2.5% glutaraldehyde for 1 week,and then processed to Epon embedding (EMbed 812 kit,Electron Microscopy Sciences, Hatfield, PA) by standard

procedures. Areas containing demyelinated lesions, identifiedin 1m-thick sections, were sectioned for ultrastructuralanalysis, stained with lead citrate and uranyl acetate, and ex-amined in a Phillips electron microscope. Equal numbers ofmyelinated and demyelinated axons were photographed, andthe ultrastructural integrity of axoplasm was evaluated. Thepercentage of axons in each group was divided into those

with and without intact neurofilaments, and the resultingdifferences were analyzed using the Students t-test.

Microarray Procedures and Statistical AnalysisCortical gray matter was separated from subcortical whitematter by scoring motor cortex blocks at the white matter/

gray matter juncture with a scalpel prior to cutting 60m-thick sections in a cryostat. RNA was isolated from pooledcortical sections using RNAgents Total RNA Isolation Sys-tem protocol (Promega, Madison, WI). RNA integrity and28S/18S ratios were determined with an Agilent 2100 Bio-analyzer (Agilent Technologies, Palo Alto, CA). BiotinylatedcRNA probes (Enzo Diagnostics, Farmingdale, NY) weregenerated from 8g total RNA and successfully hybridizedto Test 3 arrays and then to Affymetrix Human GenomeU133A/U133B arrays according to the manufacturers in-structions (Affymetrix, Santa Clara, CA). Microarray dataimages were analyzed using Microarray Analysis Suite soft-

ware 5.0 (Affymetrix). Univariate and principle componentanalysis determined intensity distribution and eliminated

sample outliers. Microarray probe level normalization oflog2-transformed data were performed using RMA14 andsubjected to statistical significance using a two-tailed group-

wise t-test assuming equal variance. In the initial microarrayexperiment genes were considered differentially expressed ifthey showed 1.5-fold change (average log2 ratio, |0.585|)and p 0.05 across replicates of the two tissue types. Todetect whether selection of genes were caused by chance andnot inherent differences, we performed false discovery rate(FDR) analysis using a permutation test that has been vali-dated in previously published studies.15,16 Details of theanalysis are in online Supplemental Data II. Data were visu-alized using Genespring version 5.0 (Silicon Genetics, Red-

wood City, CA). Expressed sequence tags that met the in-clusion criteria were BLAST searched against publicdatabases for sequence homology. Functional annotations ofthe altered transcripts were carried out using EASE (Expres-sion Analysis Systemic Explorer; available at http://david.niaid.nih.gov/david/ease.htm) which validated occurrence ofeach biological category within a queried group of genes byusing Fishers exact test.17

Quantitative Real-time Polymerase Chain ReactionRNA from the six MS samples and six control samples wasDNase-treated (Qiagen, Valencia, CA) and 2g was reverse-

transcribed using random primers with Stratagene reversetranscriptase (Stratagene, La Jolla, CA). Gene-specific PCR

was performed (See Supplemental Data I for primer se-quences) using the SYBR Green I kit and a Roche Lightcy-cler (Roche Diagnostics, Indianapolis, IN) and standardizedto 18S RNA (Ambion, Austin, TX). Each sample was run intriplicate. Mean fold change was calculated between controland MS RNA and compared by the Students t-test.

Western BlottingMitochondrial-enriched fractions were isolated from homog-enates of total gray matter as described previously.18 Equalamounts of protein (10g) were loaded on 4-12% NuPageBis-Tris gels in MES buffer (Invitrogen, Carlsbad, CA) andblotted according to the manufacturers instructions. Threeseparate blots were performed for each protein and exposedonto biomax XAR films (Kodak, Rochester, NY). Films werescanned, and average intensity was quantitated with Scion-Image (NIH, Bethesda, MD) and compared by the Studentst-test.

Mitochondrial Electron Transport Chain ActivityActivities of mitochondrial respiratory chain complexes I, III,and IV were measured as described.19,20 In brief,mitochondria-enriched fractions were obtained by differentialcentrifugation from sections from six MS blocks and sixmatched control blocks belonging to three sets of patientsand controls. To allow free access of substrates to the com-plexes, freeze-thawed mitochondrial membranes were dis-rupted with 1% sodium cholate, and the activities of indi-vidual complexes were determined using appropriate electrondonors and acceptors. Enzyme activity was corrected fornonenzymatic reduction of acceptors, using appropriate in-hibitors. All measurements were performed in duplicate andstandardized to protein concentration in at least three inde-

pendent experiments. Data were analyzed by Students t-test.

ImmunocytochemistryAll tissues used in this study were immunostained for prote-olipid protein and MHC class II antigens as described pre-viously.13 Blocks with evidence of cortical demyelination orother pathologies were excluded. For quantitative analysis ofimmunocytochemistry, motor cortex was embedded in par-affin and sectioned at thickness of 10m. Multiple sectionsfrom four MS brains and two control brains were incubated

with antibodies specific for parvalbumin or mitochondrial-encoded complex IV subunit I protein (cytochrome c oxidasesubunit I). The percentage of cortical area occupied by parv-

albumin stained processes and the area of upper motor neu-ron perikarya occupied by mitochondria were quantified us-ing Photoshop 6.0 (Adobe, San Jose, CA). These data wereanalyzed by the Students t-test.

AntibodiesNADH dehydrogenase (ubiquinone) 1 alpha subcomplex 6(NDUFA6), succinate dehydrogenase complex subunit A(SDHA), low molecular mass ubiquinone-binding protein(QP-C), mitochondrial-encoded complex IV subunit I pro-tein and porin-specific antibodies were obtained from Mo-lecular Probes (Eugene, OR). MHC class II (HLA-DR)

480 Annals of Neurology Vol 59 No 3 March 2006


4/12

antigen-specific antibody was purchased from Dako (Carpin-teria, CA). Glutamic acid decarboxylase 1, parvalbumin, ac-tin, proteolipid protein, and GABA A1 receptor-specificantibodies were obtained from Chemicon (Temecula, CA).

In Situ HybridizationParaffin-embedded sections from control and MS motor cor-tex were hybridized with riboprobes specific to NADH de-

hydrogenase (ubiquinone) 1 subcomplex 6 (NDUFA6,complex I), low molecular mass ubiquinone-binding protein(QP-C, complex III), ATP synthase, H transporting, mi-tochondrial F0 complex, subunit e (ATP 51, complex V),and proteolipid protein, which was used as a control. 35S-dUTP (Perkin-Elmer, Boston, MA)labeled riboprobes weregenerated using T3/T7 polymerase and the in-vitro tran-scription kit (Ambion, Austin, TX; primer sequences are ap-pended in Supplemental Data I). Both the sense and anti-sense riboprobes containing 5 105 cpm were placed onidentical sections and hybridized overnight at 60C. For cel-lular resolution of mRNA expression, sections were dippedin autoradiographic NTB2 emulsion (Kodak), stored at 4Cfor 4 days, developed, counterstained with hematoxylin andeosin, and examined by dark-field and bright-field micros-copy. Bright-field images were used to quantify mRNAabundance. For mitochondrial probes, grains per neuronalperikaryal area were quantified for 55 to 130 upper motorneurons in sections from both control and MS motor corti-ces. For proteolipid protein mRNA, total grains per oligo-dendrocyte perikaryal area were quantified. Statistical signif-icance was determined by the Students t-test.

Results

Ultrastructure of Demyelinated AxonsDegeneration of chronically demyelinated axons has

been proposed as a mechanism that drives continuousirreversible neurological disability in chronic MS pa-tients.6 To elucidate possible mechanisms of this ax-onal degeneration, we analyzed myelinated and demy-elinated axons in the spinal cords of 10 severelydisabled (EDSS score, 7.0) MS patients (see Table)by electron microscopy. Specimens obtained by ourrapid autopsy protocol retained significant ultrastruc-tural integrity as indicated by preservation of myelinmembranes, cytoplasmic organelles, and extracellularspaces (Fig 1A). Greater than 95% of the myelinatedaxons had normal-appearing axoplasm with well-preserved microtubules, mitochondria, and neurofila-

ments with appropriate side-arm extensions (see Fig1B). Fifty percent of the demyelinated axons had sim-ilarly well-preserved axoplasm (see Fig 1C). Because ofreduced side-arm extension, however, neurofilamentspacing was less than that found in myelinated axons.The remaining 50% of demyelinated axons had abnor-mal axoplasm with reduced organelle content and vary-ing degrees of neurofilament fragmentation (see Fig1D). Because the myelinated and demyelinated axonswere from the same sections, we conclude that theseaxoplasmic changes are inherent to 50% of the demy-

elinated axons and not caused by artifacts induced byautolysis or tissue processing. These observations sup-port the possibility that half of the demyelinated axonsin spinal cord lesions from chronic MS patients haveactivated Ca2-dependent enzymes that are known tofragment neurofilaments.21

Microarray Comparisons

Non-lesion motor cortex samples from six MS patientsand six individuals without neurological disease wereobtained at autopsy (see Table) and compared on Af-fymetrix U133A and U133B microarrays. Since ourgoal was to examine neuronal gene changes that maycontribute to degeneration of chronically demyelinatedaxons, we studied tissue from MS patients with severedisability (EDSS score, 7.0). No outliers were de-tected in the arrays using univariate, principal compo-nent, or correlation coefficient (0.96 0.91 for con-trols; 0.960.75 for MS samples) analysis. Transcriptchanges separated control and MS samples into sepa-

rate clusters (Fig 2A). Among the 555 significantly al-tered transcripts, 488 were decreased and 67 were in-creased in the MS samples (p 0.05, 1.5-fold). Astandardized MIAME/MGED-format22 description ofthe data set is included in Supplemental Data II. Per-mutation testing estimated FDR of 5.9% across thecomparison of control and MS samples (see Fig 2B).This indicated that approximately 95% of the tran-script alterations were caused by true biological differ-ences between the MS and control samples and not bychance. Altered transcripts were classified usingEASE into gene ontologybased biological processes.

Transcripts related to oxidative phosphorylation(p 0.00049), synaptic transmission (p 0.0080)cellular transport (p 0.0085) MHC related(p 0.000013), antigen presentation (p 0.00029),antigen processing (p 0.00035) and translationalinitiation (p 0.0060) were among the major signif-icant biological groups that were altered in MS motorcortex (complete list of classification attached as Sup-plemental Data II). We focus here on the analysis ofbiological categories of oxidative phosphorylation andsynaptic transmission.

Reduced Mitochondrial Function in Upper

Motor NeuronsOxidative phosphorylation is catalyzed by five largemultiprotein complexes that are encoded by both nu-clear and mitochondrial DNA. Among the 119 nuclear-encoded mitochondrial electron transport chain geneson the arrays, 103 were called present in all samplesand 26 were significantly (p 0.05) decreased in MSsamples (Fig 3A). Decreased transcripts are listed inFigure 3A and include 10 of 36 for complex I, 0 of 7for complex II, 4 of 11 for complex III, 7 of 24 forcomplex IV, and 5 of 25 for complex V. Transcript



5/12

decreases were confirmed by quantitative RT PCR (seeFig 3A) and extended to protein for a subset of thesegenes (see Fig 3B). Levels of complex II 70kDa protein

SDHA were similar in control and MS samples. Thedecrease in mitochondrial electron transport gene ex-pression appear to be functionally relevant as the activ-

Fig 1. Ultrastructure of myelinated and demyelinated axons from spinal cords of chronic multiple sclerosis patients. The electronmicrograph in panel A contains four axons (Ax1Ax4) at the edge of a demyelinated lesion: each displays varying degree of ultra-structural integrity. The myelinated axon (Ax1) has normal-appearing axoplasm (B) with intact and appropriately oriented neuro-

filaments. Axoplasm of demyelinated Ax2 (C) is also intact, but neurofilament spacing is significantly reduced. Neurofilaments indemyelinated Ax 3 (D) are fragmented and barely detectable in demyelinated Ax4. Less than 5% of the myelinated axons and 50%of demyelinated axons analyzed had abnormal axoplasm (E). Scale bars; A 2m, BD 200nm. *p 0.00000001.



6/12

ities of respiratory chain complex I and III were signif-icantly decreased by 61% and 40%, respectively(compared with control), in mitochondria-enrichedfractions isolated from postmortem MS cortex (see Fig3C). Complex IV activity was not changed signifi-

cantly. Mitochondrial density was similar in upper mo-tor neuronal cell bodies in MS and control tissue asquantified by staining for the mitochondrial encodedcomplex IV subunit 1 protein (see Fig 3D), levels ofporin (see Fig 3B), and normal mRNA levels for 75%

Fig 2. Altered transcripts in multiple sclerosis motor cortices. Hierarchical clustering of transcripts that are significantly altered (p 0.05; 1.5-fold) in control and multiple sclerosis microarray comparisons (A). Dual clustering (condition and gene expression) usingPearsons correlation (similarity 0.001) arranged samples in columns and altered transcripts in rows according to expression level.Control (C1C6) and multiple sclerosis (MS1MS6) motor cortex data grouped separately supporting disease-related gene expression

patterns. Moreover, discernable clusters of upregulated (red) and downregulated genes (blue) were generally consistent across all mul-tiple sclerosis samples. Transcripts obtained in our experimental outcome depicted true biological discovery (B). False discovery rate(FDR) was assessed using 10 random permutations of the multiple sclerosis and control samples (FDR110, shown in top panel).Each group contained three multiple sclerosis and three control samples. FDR analysis was performed with varying stringency (p 0.050.01; fold change, 1.52.0) to determine optimum cutoff criteria (middle panel). The first row represents the number of dif-

ferentially expressed probe sets with varying stringency ofp-value and fold change. The second row (FDR #) corresponds to the me-dian number of probe sets reaching significance across the permutation analyses, whereas the third row represents the percentage of

FDR in relationship to the experimental findings. Note that for p 0.05 and 1.5-fold. the average FDR was 5.9%, suggestingthat most of the differentially expressed probe sets represent data that could not be obtained by chance. Finally, the results were plot-ted with p-value/fold cutoff (x axis) with percentage of false discovered (left y axis) transcripts and number of true (right y axis)transcripts across the comparison. Although increasing the stringency (p-value 0.01/fold 2.0) reduced the median FDR (33 to 1), italso eliminated many of the putatively true and significant gene expression changes (555 to 16).



7/12

Fig 3. Mitochondrial electron transport chain gene transcripts are decreased in multiple sclerosis motor cortex. Twenty-six electrontransport chain transcripts are decreased significantly (p 0.05) in multiple sclerosis motor cortex (A). These include NDUFB7(NADH dehydrogenase ubiquinone 1 beta subcomplex), NDUFB8 (NAD dehydrogenase ubiquinone 1 beta subcomplex 8),NDUFB2 (NADH dehydrogenase ubiquinone 2), NDUFS2L (NADH-ubiquinone oxidoreductase 39kDa protein), NDUFV2(NADH dehydrogenase ubiquinone flavoprotein 2), NDUFS2 (NADH dehydrogenase ubiquinone Fe-S protein 2), NDUFA6(NADH dehydrogenase ubiquinone 1 subcomplex 6), NDUFS4 (NADH dehydrogenase ubiquinone Fe-S protein 4), NDUFB1(NADH dehydrogenase ubiquinone 1 subcomplex 1), QP-C (low molecular mass ubiquinone binding protein), UQCRC2(ubiquinol-cytochromec reductase core protein II), UQBP (ubiquinone binding protein), UQCR (ubiquinol-cytochrome c reduc-tase), COX5B (cytochrome c oxidase subunit 5B), COX7B (cytochrome c oxidase subunit 7B), COX7C (cytochrome c oxidase sub-unit 7C), COX17 (cytochrome c oxidase assembly protein 17), COX5A (cytochrome c oxidase subunit 5A), COX11 (cytochrome coxidase assembly protein), ATP5I (ATP synthase, H transporting, F0 complex subunit e), ATP5 (ATP synthase, H transporting,F0 complex subunit b), ATP5J (ATP synthase, H transporting, F0 complex subunit F6) ATP5G3 (ATP synthase, H transporting,F0 complex subunit c). Expression levels of NDUFS4, QP-C, COX5A, and ATP5G3 mRNA decreased when measured by real-time polymerase chain reaction. Mean fold changeSEM, *p 0.05. Western blots of mitochondrial proteins NDUFA6 (complex

I) and QP-C (complex III) also show significant decrease in multiple sclerosis motor cortex, whereas complex II protein, SDHA II,was unchanged (B). Protein levels were normalized to porin. Fold changes represent the average of three separate blots. *p 0.01.

Activity of electron transport complexes I and III are decreased in mitochondrial-enriched fractions from motor cortex of multiplesclerosis patients (C). Complex I function, as assayed as NADH-ferricyanide reductase activity, was decreased in multiple sclerosismotor cortex by 61%. Complex III function, as assayed by decylcoubiquinol-ferricytochrome c oxidoreductase activity, was decreasedby 40%. Complex IV function was similar. (black bars) control; (gray bars) multiple sclerosis motor cortex. *p 0.006. Confocalimages of upper motor neurons immunostained with antibodies specific for complex IV/subunit 1 depict similar percentage of neuro-nal cell body area occupied by mitochondrial in control (left side panel D) and multiple sclerosis (right side panel D). Scale bars10m.



8/12

of the nuclear-encoded mitochondrial genes. Our datatherefore support mitochondrial dysfunction in themotor cortex of MS patients due to dysfunction of spe-cific genes and not due to a deficiency in the numberof mitochondria themselves.

To investigate whether mitochondrial transcriptswere reduced in upper motor neurons, we performedquantitative in situ hybridization for several mitochon-drial transcripts (Fig 4). Localization of proteolipidprotein mRNA served as control. When viewed bydark-field microscopy, mitochondrial mRNAs werehighly enriched in upper motor neurons in both con-trol (see Fig 4A, C) and MS (see Fig 4B, D) sections,whereas proteolipid protein mRNAs were enriched inglial cells (see Fig 4E, F). The densities of silver grainsrepresenting mitochondrial mRNA were significantlyreduced in upper motor neurons in MS tissue sectionswhile proteolipid protein mRNA densities were similar(see Fig 4G). Thus, the mitochondrial mRNA tran-script reductions detected in microarrays and by RT

PCR occur predominately in neurons. Based on silvergrain densities, mitochondrial mRNA reductions ap-peared to occur in most upper motor neurons ana-lyzed. Thus, reduced mitochondrial gene expression isunlikely to occur only in neurons that may havetransected axons. Only 6 of the 26 nuclear-encoded

mitochondrial genes (see Fig 3A) were significantly al-tered in white matter lesions (a tissue with axonopathy)from MS patients (Peterson JW, Supplemental Data I).Furthermore, none of these mitochondrial genes weresignificantly decreased in microarray comparisons ofnormal-appearing white matter representing primarilythe glial cell population (Supplemental Data I,11).These data suggest mitochondrial gene expressiontherefore is not globally altered in brains from MS pa-tients. Collectively, our data support reduced nuclear-encoded mitochondrial genes expression in upper mo-tor neurons in the motor cortex of patients with MS.

Decreased Inhibitory NeurotransmissionApproximately 25% of the neurons in the cerebral cor-tex utilize the inhibitory neurotransmitter GABA tomodulate the firing of motor and other neurons.23 Themajority of GABA receptors on the surface of corticalneurons consist of subunits 1, 3, and 2.24,25

mRNA encoding GABA A 1 (1.84, p 0.041)

and 3 (1.52, p 0.003) receptor subunits weresignificantly decreased in microarrays from MS cortex(Fig 5A). The 2 subunit was also reduced on the mi-croarrays but did not reach statistical significance(1.46, p 0.073). mRNA encoding GABA A recep-tor associated protein (GABRAP), which modulates

Fig 4. Mitochondrial transcripts are decreased in neurons from the motor cortex of multiple sclerosis patients. Distribution of mito-chondrial complex I (NDUFA6) mRNA in sections from control (A, C) and multiple sclerosis motor cortex (B, D). Mitochondrialtranscripts were highly enriched in neurons. Silver grain densities were determined for at least fifty upper motor neurons (layersVVI) in sections from three multiple sclerosis and two control blocks and expressed as grains/m2 soma area (G). Levels of mRNAencoding proteins specific for respiratory chain complex I (NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 6), III (low mo-lecular mass ubiquinone-binding protein), and V (ATP synthase, H transporting, mitochondrial F0 complex subunit e) were sig-nificantly decreased in multiple sclerosis motor neurons. Dark-field images of proteolipid protein mRNA in sections from control (E)and multiple sclerosis (F) motor cortex. Proteolipid protein mRNA densities were similar in oligodendrocytes in multiple sclerosisand control cerebral cortex sections (G). Scale bars, A, B, E, F 25m; C, D 10m. p 0.0002, *p 0.00002.



9/12

GABA receptor function,

26

was decreased 1.45-fold(p 0.022). Reductions in select transcripts were con-firmed by quantitative RT PCR using the RNA fromthe same six MS and six control samples (see Fig 4A).All changes as measured by RT PCR were slightlylarger than changes observed by microarray compari-sons and mRNA encoding GABA A receptor 2 wassignificantly reduced (see Fig 5A). GABA A1 recep-tor protein was decreased 3.8-fold (p 0.032) inWestern blot analysis (see Fig 5B).

Four transcripts related to presynaptic inhibitory in-terneuron function were significantly decreased in mi-croarrays of MS motor cortices. mRNA encoding

GAD67, the enzyme that synthesizes GABA, was de-creased 1.9-fold (p 0.029) in microarrays and 4.4-fold (p 0.05) by RT PCR (see Fig 4A). GAD67protein was decreased 4.4-fold (p 0.005) by West-ern blot (see Fig 5B). Parvalbumin, a Ca2-bindingprotein, expressed by a subpopulation of inhibitory in-terneurons27 was decreased 2.7-fold by microarrays and4-fold by RT PCR. We quantified the percentage ofcortical area stained for parvalbumin in tissue sectionsfrom control (see Fig 5C) and MS (see Fig 3D) motorcortex and found 30% reduction in parvalbumin-

stained processes in MS motor cortex (see Fig 5E).Two other inhibitory interneuron transcripts, cholecys-tokinin (CCK) (1.57, p 0.018) and tachykinin(TAC1; 1.76, p 0.023) were also decreased in MSsamples (see Fig 5A). Neurotransmitter-related changesappear to be specific for inhibitory neurotransmissionas levels of glutamate receptor subunit mRNAs weresimilar in control and MS samples (see Fig 5A). Col-lectively, our data suggest that inhibitory neurotrans-mission is reduced at both pre-synaptic and postsynap-tic levels in the motor cortex of chronic MS patients.

Discussion

Degeneration of chronically demyelinated axons is amajor cause of the continuous, irreversible neurologicaldisability that occurs in the chronic stages of MS. In acohort of MS patients similar to that investigated here,axonal loss in chronically demyelinated spinal cord le-sions averaged 68%.28 We show here that approxi-mately 50% of the remaining demyelinated axon pro-files in chronic lesions of MS have pathological changesthat suggest Ca2-activated protease activity includingfragmented neurofilaments, depolymerized microtu-bules, and decreased organelle content.21,29 We con-

Fig 5. Inhibitory neurotransmission transcripts are decreased. GABA system genes were significantly decreased in samples from multi-ple sclerosis motor cortex (A; upper portion). Transcript levels of GABA receptor subunits GABR1 (gamma-aminobutyric acid re-ceptor alpha 1), GABRAP (GABA receptor associated protein), GAD67 (glutamate decarboxylase 1), GABR3 (gamma-aminobutyric acid receptor A beta 3), GABR2 (gamma-aminobutyric acid receptor A gamma 2), PARV (parvalbumin), CCK(cholecystokinin), and TAC1 (tachykinin) were significantly decreased in multiple sclerosis samples. Transcript changes associated

with GABRA1, GABRA3, GAD67, and PARV were confirmed by quantitative real-time polymerase chain reaction. Mean foldchange SEM, **p 0.01. Levels of excitatory neurotransmitter transcripts were similar in control and multiple sclerosis motorcortex (A, lower portion). Transcript levels for glutamate receptor genes AMPA1 (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor 1), NMDA2A (N-methyl D-aspartate receptor 2A), AMPA3 (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor 3), AMPA2 (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor 2), and EEA3(kainate receptor subunit 3) are shown. Panel B shows Western blots for GABAergic proteins in multiple sclerosis and control sam-

ples. GABR1 was decreased 3.8-fold and GAD67 was decreased 4.4-fold in multiple sclerosis motor cortex samples when comparedwith matched controls. Protein levels were standardized to levels of-actin. Relative fold changes were calculated from three sepa-rate blots using Scion Image (NIH, Bethesda, MD). *p 0.001. Processes of inhibitory interneurons are decreased in multiplesclerosis motor cortex. Control (C) and multiple sclerosis (D) motor cortex sections were stained with parvalbumin antibodies. The

percentage of cortical area occupied by parvalbumin-positive processes was quantified and significantly reduced in multiple sclerosissections (E). Scale bars 10m.



10/12

clude, based on global transcript profiles, biochemicalanalysis, and morphological studies of motor cortexsamples, that motor neurons in chronic MS patientshave significantly impaired mitochondrial function anddecreased inhibitory innervation. These data supportincreased excitability of upper motor neurons that havea reduced capacity to produce ATP.

The objective of microarray comparisons is to deci-pher inherent biological differences keeping technicalvariations (noise, artifacts, false-positives) to a mini-mum. To maximize statistical validity of our microar-ray data, we performed rapid autopsies, 6 by 6 com-parisons, FDRs, and RT PCR (for select transcripts).Being more sensitive, RT PCR almost always producedgreater differences than microarray comparisons. Thebiological relevance of the transcript changes were ex-tended to protein expression levels as determined bywestern blots and cell specificity by immunohistochem-istry and in situ hybridization. Our data demonstratethe value of an unbiased search of the whole genome as

changes in neuronal mitochondrial gene expression andinhibitory neurotransmission have not been reported inprevious studies of MS pathogenesis.

Our data are consistent with the hypothesis that amismatch between energy demand and reduced supplyof ATP causes degeneration of chronically demyeli-nated axons in MS patients. The energy demand ofnerve conduction is increased by the diffuse distribu-tion of voltage-gated Na channels along the demyeli-nated axolemma3,30 and possibly by increased firing ofupper motor neurons due to decreased inhibitory in-put. Both of these changes increase Na influx into the

axon, which is normally exchanged for extracellular K

by the Na/K ATPase in a rapid and energy-dependent manner.31 We speculate that this exchangeis impaired in chronically demyelinated axons becausethe upper motor neuron supplies it with dysfunctionalmitochondria. Increased axoplasmic Na concentra-tions will reverse the energy-independent Na/Ca2

exchanger and exchange axoplasmic Na for extracel-lular Ca2.32 Chronic increases in axoplasmic Ca2

concentrations depolymerize microtubules33 and acti-vate proteases that fragment neurofilaments,4,29 as wasobserved in the electron microscopic studies reportedhere. Cytoskeletal pathology and reduced ATP produc-

tion decrease axonal transport, reduce axoplasmic or-ganelle density, and eventually cause axonal degenera-tion.

The reduction in respiratory chain complex I (61%)and III (40%) activities in mitochondrial preparationsfrom the motor cortex of MS patients (see Fig 3C)indicates significant neuronal mitochondrial dysfunc-tion and by inference decreased ATP production in de-myelinated axons. Because the Na/K ATPase uti-lizes approximately 50% of available CNS energy,31 itis likely that its function is impaired in chronically de-

myelinated axons in chronic MS brains. The ionic cas-cade described above has been unequivocally docu-mented as the cause of myelinated axon degenerationin experimental models of CNS white matter hypoxiaand ischemia (for review see Stys4). Because myelinatedaxons in chronic MS spinal cord displayed little evi-dence of Ca2-mediated pathology, demyelination ap-pears to be a dominant factor in producing the axonalpathology documented in Figure 1.

Previous studies have reported reduced mitochon-drial respiratory chain complex I activity in chronic ac-tive lesions from MS patients34 and attributed it to ox-idative damage of mitochondrial DNA by nitricoxide.35 When applied to myelinated axons in-vitro,nitric oxide causes conduction block and these axonsdegenerate when they are activated.36 In our microar-ray comparisons, levels of nitric oxide (NO)synthesiz-ing enzymes were similar between control and MS cor-tex. Because neuronal nitric oxide synthase (nNOS)activity and NO production is Ca2 dependent,37 it is

possible that NO and reduced mitochondrial gene ex-pression may act in concert to reduce energy produc-tion in chronically demyelinated axons.

We demonstrated both reduced GABA-related genetranscripts and density of inhibitory interneuron pro-cesses in the motor cortex samples from MS patients.We have shown previously38 that activated microglia inthe cerebral cortex of MS patients physically associatewith neuronal perikarya and proximal dendrites. In ro-dents, similar targeting of neurons by activated micro-glia can be initiated by nerve transection,39 demyeli-nation,40 delayed-type hypersensitivity, and systemic

lipopolysaccharide administration.

41

Furthermore,these microglia separate pre-synaptic and postsynapticspecializations and thereby remove what is predomi-nately inhibitory interneuron input.41 It is possible thatthis microglial response is initially neuroprotective asreducing inhibitory innervation of motor neurons in-creases firing of synaptic NMDA (N-methyl-D-aspartate)receptors and inhibits cell death pathways by triggeringcAMP-response element binding protein (CREB) activ-ity and brain-derived neurotrophic factor (BDNF) ex-pression.42,43 In the context of chronic demyelination,however, decreased inhibitory innervation of corticalneurons may cause axonal degeneration by increasing

the firing of demyelinated axons that have higher thannormal energy demands and an impaired ability toproduce ATP.

Although little is known about the molecular regu-lation of mitochondrial genes in neurons, neuronal mi-tochondrial gene expression can be regulated by neuro-nal functional activity.44 Functional magnetic resonanceimaging (MRI) studies of MS patients have describedreduced cortical energy consumption after inflamma-tory white matter lesions at sites that contain their ax-onal projections.45 Experimental inhibition of sensory



11/12

input to the human cortex rapidly reduces corticalGABA but not glutamate levels as detected by mag-netic resonance spectroscopy (MRS).46 Because thehallmark of MS disease progression is altered neuronalfunction, it is reasonable to suggest that decreased neu-ronal mitochondrial gene expression is a downstreamevent of demyelination and possibly reduced inhibitoryinnervation. Coordinated regulation of genes involvedin similar functions occurs by shared transcriptionalregulatory motifs or RNA stability sequences.47,48 Un-derstanding the mechanisms that regulate nuclear-encoded mitochondrial genes in upper motor neuronsmay lead to therapeutics that increase demyelinated ax-onal ATP production. A variety of ionotrophic andmetabotrophic receptors can be activated by reverse op-eration of the Na transporters leading to further over-load of cellular Ca2.49 The Na channel blockersphenytoin and flecainide have been shown to reduceaxonal degeneration and neurological disability in ro-dent models of MS.50,51 Our data hence provide fur-

ther rationale for modifiers of neurotransmitter systemNa channel and/or Na/Ca exchanger blockers ascandidates for neuroprotective therapeutics in chronicstages of MS.

Additional InformationPrimers used for RT PCR and in situ hybridizationstudies and fold changes of mitochondrial transcriptscompared between control, normal-appearing whitematter, and white matter lesions are included in Sup-plemental Data I. Details of the microarray experimen-tation and analysis and biological classification of al-

tered transcripts are enclosed as Supplemental Data II.

This work was supported by the NIH (National Institute on Neu-rological Disorders and Stroke, NS35058, B.D.T., NS38667,B.D.T.) and a National Multiple Sclerosis Society postdoctoral fel-lowship (J.M.).

We thank Dr G. Kidd for valuable input, S. Foells for technicalhelp, and S. De Stefano for editorial assistance.

References1. Pedraza L, Huang JK, Colman DR. Organizing principles of

the axoglial apparatus. Neuron 2001;30:335344.2. Noseworthy JH, Lucchinetti C, Rodriguez M, et al. Multiple

sclerosis. N Engl J Med 2000;343:938952.3. Craner MJ, Newcombe J, Black JA, et al. Molecular changes in

neurons in multiple sclerosis: altered axonal expression ofNav1.2 and Nav1.6 sodium channels and Na/Ca2 ex-changer. Proc Natl Acad Sci USA 2004;101:81688173.

4. Stys PK. White matter injury mechanisms. Curr Mol Med2004;4:109126.

5. Rudick RA, Goodman A, Herndon RM, et al. Selecting relaps-ing remitting multiple sclerosis patients for treatment: the casefor early treatment. J Neuroimmunol 1999;98:2228.

6. Trapp BD, Ransohoff RM, Fisher E, et al. Neurodegenerationin multiple sclerosis: relationship to neurological disability.Neuroscientist 1999;5:48 57.

7. Whitney LW, Becker KG, Tresser NJ, et al. Analysis of geneexpression in multiple sclerosis lesions using cDNA microarrays.Ann Neurol 1999;46:425 428.

8. Tajouri L, Mellick AS, Ashton KJ, et al. Quantitative and qual-itative changes in gene expression patterns characterize the ac-tivity of plaques in multiple sclerosis. Brain Res Mol Brain Res2003;119:170183.

9. Lock C, Hermans G, Pedotti R, et al. Gene-microarray analysisof multiple sclerosis lesions yields new targets validated in au-

toimmune encephalomyelitis. Nat Med 2002;8:500 508.10. Graumann U, Reynolds R, Steck AJ, et al. Molecular changes

in normal appearing white matter in multiple sclerosis are char-acteristic of neuroprotective mechanisms against hypoxic insult.Brain Pathol 2003;13:554573.

11. Lindberg RL, De Groot CJ, Certa U, et al. Multiple sclerosis asa generalized CNS diseasecomparative microarray analysis ofnormal appearing white matter and lesions in secondary pro-gressive MS. J Neuroimmunol 2004;152:154167.

12. Mirnics K, Middleton FA, Marquez A, et al. Molecular char-acterization of schizophrenia viewed by microarray analysis ofgene expression in prefrontal cortex. Neuron 2000;28:5367.

13. Trapp BD, Peterson J, Ransohoff RM, et al. Axonal transectionin the lesions of multiple sclerosis. N Engl J Med 1998;338:278285.

14. Irizarry RA, Hobbs B, Collin F, et al. Exploration, normaliza-tion, and summaries of high density oligonucleotide array probelevel data. Biostatistics 2003;4:249 264.

15. Mirnics K, Korade Z, Arion D, et al. Presenilin-1-dependenttranscriptome changes. J Neurosci 2005;25:15711578.

16. Lazarov O, Robinson J, Tang YP, et al. Environmental enrich-ment reduces Abeta levels and amyloid deposition in transgenicmice. Cell 2005;120:701713.

17. Hosack DA, Dennis G Jr, Sherman BT, et al. Identifying bio-logical themes within lists of genes with EASE. Genome Biol2003;4:R70.

18. Triepels RH, Hanson BJ, van den Heuvel LP, et al. Humancomplex I defects can be resolved by monoclonal antibody anal-ysis into distinct subunit assembly patterns. J Biol Chem 2001;

276:88928897.19. Krahenbuhl S, Chang M, Brass EP, et al. Decreased activities ofubiquinol:ferricytochrome c oxidoreductase (complex III) andferrocytochrome c:oxygen oxidoreductase (complex IV) in livermitochondria from rats with hydroxycobalamin[c-lactam]-induced methylmalonic aciduria. J Biol Chem 1991;266:2099821003.

20. Birch-Machin MA, Turnbull DM. Assaying mitochondrial re-spiratory complex activity in mitochondria isolated from humancells and tissues. Methods Cell Biol 2001;65:97117.

21. Stys PK, Jiang Q. Calpain-dependent neurofilament breakdownin anoxic and ischemic rat central axons. Neurosci Lett 2002;328:150154.

22. Brazma A, Hingamp P, Quackenbush J, et al. Minimum infor-mation about a microarray experiment (MIAME)toward

standards for microarray data Nat Genet 2001;29:365371.23. Hendry SH, Schwark HD, Jones EG, et al. Numbers and pro-

portions of GABA-immunoreactive neurons in different areas ofmonkey cerebral cortex. J Neurosci 1987;7:15031519.

24. Sarto I, Wabnegger L, Dogl E, et al. Homologous sites ofGABA(A) receptor alpha(1), beta(3) and gamma(2) subunits areimportant for assembly. Neuropharmacology 2002;43:482491.

25. Fritschy JM, Brunig I. Formation and plasticity of GABAergicsynapses: physiological mechanisms and pathophysiological im-plications. Pharmacol Ther 2003;98:299323.

26. Wang H, Bedford FK, Brandon NJ, et al. GABA(A)-receptor-associated protein links GABA(A) receptors and the cytoskele-ton. Nature 1999;397:6972.



12/12

27. Hashimoto T, Volk DW, Eggan SM, et al. Gene expressiondeficits in a subclass of GABA neurons in the prefrontal cortexof subjects with schizophrenia. J Neurosci 2003;23:63156326.

28. Bjartmar C, Kidd G, Mork S, et al. Neurological disability cor-relates with spinal cord axonal loss and reduce N-acetyl aspar-tate in chronic multiple sclerosis patients. Ann Neurol 2000;48:893901.

29. Jiang Q, Stys PK. Calpain inhibitors confer biochemical, butnot electrophysiological, protection against anoxia in rat optic

nerves. J Neurochem 2000;74:21012107.30. Waxman SG, Craner MJ, Black JA. Na channel expressionalong axons in multiple sclerosis and its models. Trends Phar-macol Sci 2004;25:584591.

31. Ames A III. CNS energy metabolism as related to function.Brain Res Brain Res Rev 2000;34:4268.

32. Stys PK, Waxman SG, Ransom BR. Na-Ca2 exchanger me-diates Ca2 influx during anoxia in mammalian central nervoussystem white matter. Ann Neurol 1991;30:375380.

33. OBrien ET, Salmon ED, Erickson HP. How calcium causesmicrotubule depolymerization. Cell Motil Cytoskeleton 1997;36:125135.

34. Lu F, Selak M, OConnor J, et al. Oxidative damage to mito-chondrial DNA and activity of mitochondrial enzymes inchronic active lesions of multiple sclerosis. J Neurol Sci 2000;

177:95103.35. Sarti P, Giuffre A, Barone MC, et al. Nitric oxide and cyto-

chrome oxidase: reaction mechanisms from the enzyme to thecell. Free Radic Biol Med 2003;34:509520.

36. Kapoor R, Davies M, Blaker PA, et al. Blockers of sodium andcalcium entry protect axons from nitric oxide-mediated degen-eration. Ann Neurol 2003;53:174180.

37. Yun HY, Dawson VL, Dawson TM. Neurobiology of nitricoxide. Crit Rev Neurobiol 1996;10:291316.

38. Peterson JW, Bo L, Mork S, et al. Transected neurites, apopto-tic neurons and reduced inflammation in cortical MS lesions.Ann Neurol 2001;50:389 400.

39. Gehrmann J, Matsumoto Y, Kreutzberg GW. Microglia: intrin-sic immuneffector cell of the brain. Brain Res Brain Res Rev1995;20:269287.

40. Diestel A, Aktas O, Hackel D, et al. Activation of microglialpoly(ADP-ribose)-polymerase-1 by cholesterol breakdown prod-ucts during neuroinflammation: a link between demyelinationand neuronal damage. J Exp Med 2003;198:17291740.

41. Wujek J, Yin X, Fujii Y, et al. Activated microglia target neu-rons and strip synapses in an animal model of multiple sclerosis.Soc Neurosci Abstr 2003;529:11.

42. Hardingham GE, Fukunaga Y, Bading H. ExtrasynapticNMDARs oppose synaptic NMDARs by triggering CREB

shut-off and cell death pathways. Nat Neurosci 2002;5:405414.

43. Hardingham GE, Bading H. The Yin and Yang of NMDA re-ceptor signalling. Trends Neurosci 2003;26:8189.

44. Liu S, Wong-Riley M. Disproportionate regulation of nuclear-and mitochondrial-encoded cytochrome oxidase subunit pro-teins by functional activity in neurons. Neuroscience 1995;67:197210.

45. Blinkenberg M, Rune K, Jensen CV, et al. Cortical cerebralmetabolism correlates with MRI lesion load and cognitive dys-function in MS. Neurology 2000;54:558564.

46. Levy LM, Ziemann U, Chen R, et al. Rapid modulation ofGABA in sensorimotor cortex induced by acute deafferentation.Ann Neurol 2002;52:755761.

47. Beer MA, Tavazoie S. Predicting gene expression from se-quence. Cell 2004;117:185198.

48. Wilusz CJ, Wilusz J. Bringing the role of mRNA decay in thecontrol of gene expression into focus. Trends Genet 2004;20:491497.

49. Stys PK. General mechanisms of axonal damage and its preven-tion. J Neurol Sci 2005;233:313.

50. Hains BC, Saab CY, Lo AC, et al. Sodium channel blockadewith phenytoin protects spinal cord axons, enhances axonalconduction, and improves functional motor recovery after con-tusion SCI. Exp Neurol 2004;188:365377.

51. Bechtold DA, Kapoor R, Smith KJ. Axonal protection usingflecainide in experimental autoimmune encephalomyelitis. AnnNeurol 2004;55:607616.


dutta et al mitochondrial dysfunction in ms annneurol06

Documents