differential sensitivity of oligodendrocytes and motor neurons to reactive nitrogen species:...

*Department of Biological Sciences, University of Alabama in Huntsville, Huntsville, Alabama, USA

�Cherokee Labsystems, Huntsville, Alabama, USA

�Burke Medical Research Institute, Weill Cornell Medical College, White Plains, New York, USA

Nitric oxide (NO) is a free radical gas that at normalphysiological concentrations is essential for many cellularprocesses, such as neurotransmission, differentiation, andsignal transduction (Peunova and Enikolopov 1995; Stamleret al. 1997; Stuehr 1999; Packer et al. 2003). These phys-iological processes are regulated by NO produced at steadystate concentrations from �50 to �500 nM (Clough et al.1998; Huk et al. 1998; Pacher et al. 2007). Neurons produceNO at 33 nM concentrations during normal physiologicalfunctions (low flux NO) (Leonard et al. 2001).

In excessive amounts, NO is toxic and plays a role inParkinson’s disease, Alzheimer’s disease, amyotrophic lateralsclerosis (ALS), CNS injury, and in multiple sclerosis (MS)(Kawase et al. 1996; Hall et al. 1998; Huk et al. 1998;Panahian and Maines 2001; Cassina et al. 2002; Ischiropo-ulos and Beckman 2003; Vaziri et al. 2004; Pacher et al.2007). In pathological conditions, NO can increase to 10times the concentrations seen before the insult (Clough et al.1998; Huk et al. 1998; Pacher et al. 2007). Activated

microglial and astrocytes can produce NO at steady stateconcentrations as high as 1 lM (high flux NO) (Tominagaet al. 1994; Kawase et al. 1996; Hall et al. 1998; Stuehr1999; Pacher et al. 2007).

Received June 22, 2008; revised manuscript received January 8, 2009;accepted January 9, 2009.Address correspondence and reprint requests to Amy Bishop, PhD,

Department of Biological Sciences, Shelby Center, University ofAlabama in Huntsville, Huntsville, AL 35899, USA.E-mail [email protected] used: 3NY, 3-nitrotyrosine; ALS, amyotrophic lateral

sclerosis; BSA, bovine serum albumin; CM, conditioned media; DETA-NONOate, (z)-1-[2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate]; DMEM, Dulbecco’s modified Eagle’s medium; FBS,fetal bovine serum; HO1, heme-oxygenase-1; IAR, induced adaptiveresistance; MAP2, Microtubule-associated protein 2; MBP, myelin basicprotein; MS, multiple sclerosis; NO, nitric oxide; RNS, reactive nitrogenspecies; spermine-NONOate, N-[4-[1-(3-aminopropyl)-2-hydroxy-2-ni-trosohydrazino]butyl]-1,3-propanediamine; ZnPPIX, zinc protoporphy-rin IX.

Abstract

Depending on its concentration, nitric oxide (NO) has benefi-

cial or toxic effects. In pathological conditions, NO reacts with

superoxide to form peroxynitrite, which nitrates proteins

forming nitrotyrosine residues (3NY), leading to loss of protein

function, perturbation of signal transduction, and cell death.

3NY immunoreactivity is present in many CNS diseases,

particularly multiple sclerosis. Here, using the high flux NO

donor, spermine-NONOate, we report that oligodendrocytes

are resistant to NO, while motor neurons are NO sensitive.

Motor neuron sensitivity correlates with the NO-dependent

formation of 3NY, which is significantly more pronounced in

motor neurons when compared with oligodendrocytes,

suggesting peroxynitrite as the toxic molecule. The

heme-metabolizing enzyme, heme-oxygenase-1 (HO1), is

necessary for oligodendrocyte NO resistance, as demon-

strated by loss of resistance after HO1 inhibition. Resistance

is reinstated by peroxynitrite scavenging with uric acid further

implicating peroxynitrite as responsible for NO sensitivity.

Most importantly, differential sensitivity to NO is also present

in cultures of primary oligodendrocytes and motor neurons.

Finally, motor neurons cocultured with oligodendrocytes, or

oligodendrocyte-conditioned media, become resistant to NO

toxicity. Preliminary studies suggest oligodendrocytes release

a soluble factor that protects motor neurons. Our findings

challenge the current paradigm that oligodendrocytes are the

exclusive target of multiple sclerosis pathology.

Keywords: heme-oxygenase-1, motor neurons, nitric oxide,

nitrotyrosine, oligodendrocytes, peroxynitrite.

J. Neurochem. (2009) 109, 93–104.

JOURNAL OF NEUROCHEMISTRY | 2009 | 109 | 93–104 doi: 10.1111/j.1471-4159.2009.05891.x

� 2009 The AuthorsJournal Compilation � 2009 International Society for Neurochemistry, J. Neurochem. (2009) 109, 93–104 93

At high fluxes, NO, released during CNS pathology, ismore likely to react with oxygen species to form reactivenitrogen species (RNS), such as peroxynitrite (ONOO·),which damage a variety of macromolecules, includingproteins (Tamir et al. 1993; Beckman 1996; Estevez et al.1998, 2000; Cassina et al. 2002; Ischiropoulos and Beckman2003; Pacher et al. 2007). Peroxynitrite-dependent nitrationof tyrosine residues, forming 3-nitrotyrosine (3NY), disruptsprotein structure and function, thereby interrupting oraltering cell signaling (Bishop et al. 2004; Bishop et al.2006; Cassina et al. 2002; Ischiropoulos and Beckman 2003;Estevez et al. 1998, 2000; Pacher et al. 2007). Nitrotyrosineis found in the CNS of patients with spinal injury, ALS,Parkinson’s disease, Alzheimer’s disease, and MS, and isconsidered a footprint for peroxynitrite mediated damage inthe cell (Estevez et al. 1998, 2000; Ischiropoulos andBeckman 2003; Prat and Antel 2005; Jack et al. 2007;Pacher et al. 2007). In particular, in MS, progression andseverity is tightly associated with levels of RNS in the CSFand blood serum (Giovannoni et al. 1997, 1998).

The accepted hypothesis for MS pathogenesis is that itexclusively affects white matter with the demyelination ofaxons occurring first, followed by axonal injury, withsubsequent damage to neurons (Silber and Sharief 1999).However, growing evidence indicates that axonal injury is animportant and early event in MS which may occur beforedysfunction of the oligodendrocytes and myelination (Fer-guson et al. 1997; Trapp et al. 1999; Bitsch et al. 2000; DeStefano et al. 2001; Peterson et al. 2001; Werner et al.2001). How important, if at all, is initial axonal injury in theMS pathology is still unclear (Ferguson et al. 1997; Trappet al. 1999; Bitsch et al. 2000; De Stefano et al. 2001;Peterson et al. 2001; Werner et al. 2001).

Because oligodendrocytes and axons, as well as theirinteractions, are targets for the pathological process seen inMS, and NO is involved in MS pathology, we investigatedthe NO sensitivity of both oligodendrocytes and motorneurons alone, and in coculture. For this study, we used morephysiologically relevant (lower) NO doses than we did in ourprevious studies of NO sensitivity in motor neurons (Bishopet al. 1999, 2004; Bishop and Cashman 2003; Bishop andAnderson 2005), and dissected the mechanisms of NOtoxicity and resistance in both cell types. Our mechanisticstudies of these two cell types, in isolation and together,provide support for the hypothesis that axonal damage playsan early role in MS pathology.

Materials and methods

Vertebrate animalsThe use of rats were conformed to all institutional and government

regulations. Our animal facility has an experienced animal care

technician. The protocol, with measures to prevent pain and

suffering, was approved by our Institutional Animal Care and Use

Committee (assurance number 07-001-R). Embryonic day 15 timed

pregnant Sprague–Dawley rat females were killed with carbon

dioxide and sterilized by ethanol wash. The uteri were removed and

the embryos harvested for spinal cords.

Primary motor neuron isolationThe spinal cords (cervical and thoracic portions) were isolated from

each embryo as per the protocol of Dr Alvaro Estevez (Bishop et al.1999, 2004; Estevez et al. 1998, 2000; Schnaar and Schnaffner,

1981). The meninges layers were dissected away and the dorsal

roots removed. The ventral part of the spinal cord was dissected

away, minced, and separated by bovine serum albumin (BSA)

gradient followed by Optiprep gradient into fractions enriched for

motor neurons. This was repeated several times to ‘enrich’ further

for motor neurons (Bishop et al. 1999, 2004) followed by

immunopanning (Estevez et al. 1998, 2000). The motor neurons

were plated on flasks coated with a mixture of laminin and poly-D-

lysine at a density of 2 · 106 cells per flask and cultured under

37�C, 5% CO2 in minimal Eagle’s Medium supplemented with D-

glucose, L-glutamine, and 5% fetal bovine serum (FBS) (Estevez

et al. 1998, 2000; Bishop et al. 1999, 2004).

Primary oligodendrocyte isolationPrimary oligodendrocytes were isolated from adult female

Sprague–Dawley Rats as described (Johnson et al. 2004). The

spinal cord (ventral to sacral) was isolated, placed in cold

Liebovitz’s L-15 medium, then transferred to a dish containing

0.25% trypsin with 50 lg/mL Dnase I in Hanks. The minced cord

was incubated at 37�C for 1 h in trypsin, and centrifuged at 400–

500 g for 5 min at 4�C. The supernatant was removed and replaced

with L-15 medium containing 10% FBS and 50 lg/mL Dnase. The

cord fragments were triturated in the L-15 medium. The resultant

cell suspension was added to a Percoll gradient, then centrifuged at

30 000 g for 30 min at 4�C. The myelin layer was removed, the

oligodendrocyte cell fraction was transferred to a tube containing

2% FBS L-15, and then plated in specialized media as described

(Johnson et al. 2004).

Tissue culture: cell lines

NSC34D cell lineThe NSC34D cell line, a further differentiated version of the NSC34

cell line used in our previous studies, was used in our studies

detailed here. NSC34 cells are a fusion of primary mouse spinal cord

motor neurons with spinal neuroblastoma cells that, upon terminal

differentiation with retinoic acid or serum reduction (10–2%),

become NSC34D. The NSC34Ds provide a homogenous line that

have the characteristics of differentiated motor neurons, such as the

expression of NMDA receptors, but with the very limited ability to

divide (Cashman et al. 1992; Matsumoto et al. 1995; Eggett et al.2000). Cells were grown in 1 : 1 Dulbecco’s modified Eagle’s

medium (DMEM) without sodium pyruvate/HAMS F12 mixture,

1% non-essential amino acids (NEAA), and 2% FBS as described

(Eggett et al. 2000).

MO3.13 cells (human oligodendrocyte model)The oligodendrocyte MO3.13 cell line was used as an oligoden-

drocyte model. MO3.13 is an immortalized human-human hybrid

Journal Compilation � 2009 International Society for Neurochemistry, J. Neurochem. (2009) 109, 93–104� 2009 The Authors

94 | A. Bishop et al.

cell line created from the fusion of a 6-thioguanine resistant mutant

of the human rhabdomyosarcoma with adult human oligodendro-

cytes (McLaurin et al. 1995). These cells differentiate upon serum

deprivation (10–2%) and have properties of mature primary

oligodendrocyte, such as the expression of myelin basic protein

(MBP), CNPase, proteolipid protein, and O1 protein (Buntinx et al.2003; Li et al. 2002; McLaurin et al. 1995). Cells were grown in a

humidified 5% CO2 environment and plated in a 1 : 1 DMEM

without sodium pyruvate/HAMS F12 mixture, 1% non-essential

amino acids (NEAA), and 2% FBS.

Cocultures of NSC34D and MO3.13 cellsMixed culture plates of oligodendrocytes and motor neurons were

plated (at about a 1 : 3 ratio) and maintained in DMEM/HAMS F12

mixture supplemented with 2% heat inactivated-FBS.

Tissue culture primary cells

OligodendrocytesCells were plated in 12-well plates with 1 mL O-medium which was

comprised of a variety of factors that maintained differentiated

oligodendrocytes and selected against fast growing cells as

described (Johnson et al. 2004). The oligos were maintained at

37�C with 5% CO2 for 6–7 days.

Motor neuronsCells were incubated in neurobasal media with factors conducive to

motor neurons, as described (Estevez et al. 1998), and maintained at

37�C with 5% CO2 for 2–3 days.

Cell survival assayCell viability was indicated primarily by Trypan blue exclusion,

intact morphology, and neurite outgrowth. Many cells that died, not

only rounded up on the plate, but lifted off and became ‘floaters,’

> 99% of which did not exclude Trypan blue (Bishop et al. 1999).Lack of Trypan blue exclusion was used in early experiments as the

means to detect dead cells. Eventually, we relied on cell counts as

live cells remained attached to plates and were not rounded (Bishop

et al. 1999, 2004). Percent cell survival was calculated by dividing

post-NO treatment total live cell counts by pre-NO treatment total

live cell counts and multiplying by 100%.

ImmunocytochemistryPrimary cells were grown in media designated for that cell type.

Cells were fixed with 4% p-formaldehyde for 10 min, washed with

phosphate-buffered saline (PBS), and then permeabilized with a

0.1–0.25% Triton X-100 solution. After being rinsed with PBS, cells

were blocked with a 4% BSA solution in 0.1% phosphate buffered

saline with tween (PBST), rinsed with PBS, and then incubated with

the appropriate antibody.

Labeling and detection of motor neurons: MAP2 stainingMicrotubule-associated protein 2 (MAP2) is a critical marker for

mature CNS neurons (Herzog and Weber 1978; Brugg and Matus

1991). Primary motor neurons were incubated overnight at 4�C with

rabbit anti-MAP2 antibody [1 : 500; Chemicon (Millipore), Bill-

erica, MA, USA], washed with PBS, and incubated for 1 h with

goat-anti-rabbit Alexa Fluor 488 IgG (1 : 1000; Molecular Probes,

Eugene, OR, USA). Fluorescing antibodies were detected using

broadband filters (Chroma Series 41028). Oligodendrocytes do not

stain green with MAP2 while neurons do stain green.

Labeling and detection of oligodendrocytes: MBP stainingPrimary oligodendrocytes were stained with a marker for mature

oligodendrocytes, MBP (Baumann and Pham-Dinh 2001). Primary

oligodendrocytes were incubated with mouse monoclonal anti-MBP

antibody (1 : 1000; Covance, Berkeley, CA, USA), rinsed with PBS

and incubated for 1 h with goat-anti-mouse Alexa Fluor 488 IgG2b

secondary antibody (1 : 1000; Molecular Probes). Fluorescing

antibodies were detected using broadband filters (Chroma Series

41028).

Note that for negative controls, we used the MAP in a plate of

oligodendrocytes and the MBP on the neurons to control for non-

specific staining. We also used only the secondary Ab on plates to

control for non-specific staining. In all cases, the frame was black,

with no green labeled morphology.

Nitric oxide treatment protocolsMotor neurons and oligodendrocytes were plated at the same cell

density, fed the same volume of media at the same temperature

and pH and treated with a range of NO doses. For treatment at

subtoxic ‘physiological’ NO dose, the compound (z)-1-[2-amino-

ethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate] DETA-

NONOate with a half-life of 16 h was used (Bishop et al. 1999,2004). Spermine-NONOate, N-[4-[1-(3-aminopropyl)-2-hydroxy-2-

nitrosohydrazino]butyl]-1,3-propanediamine, which has a half-life

of �40 min, was used for the cytotoxic ‘pathological’ NO

challenges (Bishop et al. 1999, 2004). Both NO donors, partic-

ularly DETA-NONOate, release NO with predictable and easily

controlled kinetics based on pH and temperature 37�C (Bishop

et al. 1999, 2004; Bouton and Demple 2000; Estevez et al. 1998,2000; Raoul et al. 2002). The donors were kept on ice at pH 10 to

prevent NO release, which was commenced with the addition of

the donors to the pH 7.4 37�C media. NO donors release rates

have been determined experimentally (Beckman et al. 1996;

Bishop et al. 1999, 2004; Bouton and Demple 2000; Estevez

et al. 1998, 2000; Raoul et al. 2002) and in our lab (Fig. 1a).

After treatment for the prescribed amount of time (usually 1 h),

the media was changed repeatedly to eliminate the NO donor. We

used spent donor cocktail for the untreated cells as a negative

control, and it was found to not release NO, nor kill cells. We

then measured damage parameters 24 h after NO treatment as this

was found to be the onset of maximal damage as determined by

our previous studies in motor neurons (Bishop et al. 1999, 2004;Bishop and Anderson 2005; Bishop et al. 2006).

Additional experiments included treatment of the oligodendro-

cytes with cytotoxic NO challenge, coupled with 20 lM zinc

protoporphyrin (ZnPPIX), an heme-oxygenase-1 (HO1) inhibitor

(Yang et al. 2001; Akins et al. 2004) or coupled with 10 lM uric

acid, an efficient peroxynitrite scavenger (Hooper et al. 2000;

Pacher et al. 2007). To control for any toxicity of each test agent,

some cells were incubated with the test agent alone.

Nitric oxide assayNitric oxide release as a function of time was confirmed with a

colorimetric NO assay kit that is based on the Greiss Reaction

(Active Motif Nitric Oxide Analysis Kit, Active Motif Inc.,

� 2009 The AuthorsJournal Compilation � 2009 International Society for Neurochemistry, J. Neurochem. (2009) 109, 93–104

NO resistance of oligodendrocytes and motor neurons | 95

Fig. 1 Differential NO sensitivity of human oligodendrocyte (MO3.13)

cells and differentiated motor neuron (NSC34D) cells. (a) The graph is

the comparison between the kinetics of NO release from different NO

donors: DETA-NONOate, spermine-NONate and spent donors as a

control. (b) These are micrographs of NSC34D for cell count and

changes in morphology (100· magnification). The first row of panels

are a dose–curve of DETA-NONOate: spent donor, a dose that re-

leases 2 pm/s, and a 10-fold higher dose. The second row of panels is

a dose–curve of spermine-NONOate: spent spermine-NONoate, 1/10

the ‘high NO’ dose, and the high NO dose that releases 110 pm/s. (c)

The graph is a quantification of (b) with percentage cell survival on the

y axis and NO flux on the x axis. Percentage cell survival is a per-

centage of a control of untreated cells. The SEM between experiments

(n = 4) was calculated. Significance between high flux cell survival of

cells exposed to DETA-NONOate versus spermine-NONoate is

p < 0.001, designated with an asterisk. (d) The graph is the average of

all the experiments (n = 3) with a comparison of NO sensitivity of

NSC34D versus NSC34. The percentage cell survival is on the y axis

and NO flux is the x axis. Percentage cell survival is a percentage of a

control of untreated cells. The SEM was determined. There was no

significant difference between the experimental groups. (e) These

panels are micrographs of cells (100· magnification) organized into an

MO3.13 row and a NSC34D row. The 60-min dose of NO is organized

into columns. UT is untreated-treated with spent donor, Low NO is

2 pmol/s NO flux, High NO is 110 pmol/s NO flux. Cell death was

assayed 24 h post-treatment. (f) The graph is the average of all the

treatments (n = 6) with percentage cell survival on the y axis and NO

flux on the x axis. Percentage cell survival is a percentage of a control

of untreated cells. The SEM between experiments was calculated, and

significance between high flux cell survival of oligodendrocytes versus

motor neurons is p < 0.001, designated by an asterisk. The percent-

age cell survival of untreated and High NO-challenged oligodendro-

cytes does not differ significantly.



Carlsbad, CA, USA) utilizing nitrate reductase as described (Bishop

et al. 1999, 2004). NO concentrations were then calculated based on

the read values and a standard curve (repeated in triplicate for each

experiment), predicted curves from past studies (Beckman et al.1996; Bishop et al. 1999, 2004; Bouton and Demple 2000; Estevez

et al. 1998, 2000; Raoul et al. 2002), studies in our lab (Fig. 1a),

and reported as a flux (pmol/s) which is calculated from total NO

released during the 1 h of treatment.

Western blot/immunoblotProtein samples were quantified, using the BIORAD Protein Assay,

for equal protein loading of the gels (BIORAD Laboratories,

Hercules, CA, USA). For immunoblotting, cell extracts were loaded

on sodium dodecyl sulfate–polyacrylamide gel electrophoresis, run,

and transferred onto Nytran blots as described (Bishop et al. 1999,2004). The blots were washed, blocked with BSA, incubated with

rabbit primary anti-3NY (1 : 1000), a kind gift from Dr Alvaro

Estevez., and developed with a colorimetric secondary antibody.

For positive control, we used peroxynitrite treated albumin and

molecular weight markers, both of which exhibited the 3NY

formation. For negative controls, we washed the blot with just

secondary Ab and found no staining.

Immunoblot analysisBlots from western blot analysis were analyzed using the UN-SCAN

IT gel densitometry software (Silk Scientific, Orem, UT, USA).

Blots were stained with Ponceau to control for even loading and

uniform transfer.

Statistical analysisExperiments were repeated a minimum of four times. For any data

point involving cell counting, a minimum of 200 cells were counted

from at least five randomly chosen fields. Cell survival was

calculated by dividing post-NO treatment total cell counts by pre-

NO treatment total cell counts, and multiplying by 100%. The mean

of the data points were taken, and the SEM was calculated. The data

were analyzed by a two-tailed t test and significance (p value) was

calculated. A value of p < 0.01 was determined to be significant.

Results

Physiologically relevant nitric oxide dosesWe assayed the release rate of NO from NO donors, tochoose pre-treatment and challenge doses that were closer tothe range of NO concentrations seen by cells duringphysiological and pathological conditions respectively(Tominaga et al. 1994; Kawase et al. 1996; Clough et al.1998; Hall et al. 1998; Stuehr 1999; Pacher et al. 2007). ForNO donors, we used a final concentration of 1 lM DETA-NONOate which donates only NO (Dickhout et al. 2005) ata lower flux rate of 2 pmol/s (low dose) (Fig. 1a). For thechallenge NO dose, we used 10 lM final concentrationspermine-NONOate, which donates NO at a higher flux(Cornish et al. 2002) of 110 pmol/s (high dose) (Fig. 1a).

In light of the fact that high flux NO seen inpathological situations produces RNS with resultant 3NY

formation (Tamir et al. 1993, 1996; Cornish et al. 2002),and cell death (Estevez et al. 1998, 2000; Ischiropoulosand Beckman2003; Prat and Antel 2005; Jack et al. 2007;Pacher et al. 2007) we wanted to ask if spermine-NONOate, unlike DETA-NONOate, does indeed showgreater toxicity. We exposed NSC34D (motor neuron cellline) to a dose–response curve of DETA versus spermineand found a significant difference of cell survival at thehighest dose (104% ± 10 vs. 16% ± 8 (n = 4), p < 0.001)(Fig. 1b and c). Thus, NO doses detailed in Fig. 1 mimicphysiological versus pathological NO-mediated conditions(Pacher et al. 2007), and are valid tools to study cellularNO resistance mechanisms.

For studies with motor neurons and oligodendrocytes, weutilized primary cells and recognized model cell lines. Inparticular, for our motor neurons, we used NSC34D which isterminally differentiated NSC34,both of which are acceptedmodels of motor neurons (Bishop et al. 1999; Bishop andCashman 2003; Bishop et al. 2004; Bishop and Anderson2005; Bishop et al. 2006; Cashman et al. 1992; Durhamet al. 1993; Eggett et al. 2000; Matsumoto et al. 1995). Morethan 99% of NSC34D cells are positive for the motor neuronmarkers NMDA receptors, which would presumably makethe NSC34D cells more NO sensitive than the NSC34 cells(Cashman et al. 1992; Eggett et al. 2000; Bishop et al.2004). However, we have found no increase in NO sensitivityat the high NO doses (110 pm/s) in NSC34D when comparedwith NSC34 (12 ± 5 vs. 13 ± 19; n = 3) (Fig. 1d), thus bothmotor neuron lines exhibit NO sensitivity which indicates thateither NSC34 or NSC34D is an acceptable model of motorneurons for our studies of NO sensitivity (Cashman et al.1992; Bishop et al. 1999, 2004; Eggett et al. 2000).

Comparison of NO sensitivity of oligodendrocytesand motor neuronsNSC34D and MO3.13 (terminally differentiated oligoden-drocyte cell line where > 90% express MBP) were exposedto a range of NO fluxes for 1 h, and cell death was assayed24 h later. Untreated cells were exposed to spent NO donoras a control. Even at low NO fluxes (2–13 pmol/s), motorneurons were significantly more NO sensitive than wereoligodendrocytes (percentage cell survival of 61 ± 9% vs.82 ± 11%, n = 8, p < 0.001) (Fig. 1e and f). This differentialsusceptibility was more evident at high NO fluxes(110 pmol/s) where motor neurons survival was minimaland oligodendrocytes were resistant (12 ± 5% vs. 115 ± 7%,n = 8, p < 0.001) (Fig. 1e and f). Dead cells that remainedattached to the plate were rounded with no neurites/processesand did not exclude Trypan blue. Of the cells that lifted fromthe plate > 99% did not exclude Trypan blue (Bishop et al.1999, 2004) (Fig. 1e). Thus, the MO3.13 cells, unlikeNSC34D cells, were completely unaffected by cytotoxicdoses of NO, thereby expressing differential sensitivity to adirect NO challenge in the flux range we used (Fig. 1e and f).



Differential NO resistance in primary motor neurons andoligodendrocytesWe asked if this differential NO sensitivity can be verified inprimary rat oligodendrocyte and motor neuron cultures. Theprimary motor neurons were stained for motor neuronspecific proteins (MAP2), assayed for purity, and were�80% pure (Fig. 2a). The primary oligodendrocytes werestained for MBP and determined to be �65% pure (Fig. 2a).The oligodendrocyte morphology changed profoundly uponfixation and staining, but was still good enough for thepurposes of assaying purity, as we were merely countingimmunoreactive cells. For the experiments, we did not fixthe cells.

We found that primary oligodendrocytes were resistant totreatment with the low dose of NO (2–13 pm/s DETA-NONOate) (69 ± 7%, n = 4), while the primary motorneurons were significantly (p < 0.001) more sensitive(33 ± 2%, n = 4) (Fig. 2b and c). When the treatment dosewas 110 pm/s NO (administered by spermine-NONOate), wefound that primary oligodendrocytes were still resistant(67 ± 1%, n = 4), while motor neurons were quite sensitive(1 ± 1%, n = 4, p < 0.001). For the micrograph in Fig. 2b,we chose one of the few fields of HNO (High NO) motorneurons that contained cells, rather than debris, to illustratethe decay in morphology. At the highest flux of NO there wasa change in oligodendrocyte morphology, suggesting someNO sensitivity, but not nearly as much as seen in the primarymotor neurons (Fig. 2b and c).

Heme-oxygenase-1-dependent mechanism for differentialNO resistance on the two cell typesWe have found in previous studies that, in motor neurons,HO1 is important for induced adaptive resistance (IAR) – aphenomenon where pre-treatment with low dose of NO lendsNO resistance to normally quite NO sensitive motor neurons(Bishop et al. 2004; Bishop et al. 2006; Fung-Yet et al.1999; Kitamura et al. 2003). Here, we use lower NO dosesthat are within the physiological range (2 pmol/s) for the pre-treatment dose to induce resistance to a more physiologicallyrelevant challenge dose (110 pmol/s) and found that yes, IARcan still be demonstrated in motor neurons (IAR 72% ± 5 vs.High NO alone 9 ± 4, n = 4, p < 0.001) (Fig. 3a and b). Weutilized a more specific HO1 inhibitor, ZnPPIX (Yang et al.2001; Akins et al. 2004) and found that IAR in motorneurons is abrogated by the inhibition of HO1 activity(72 ± 5 vs. 30 ± 14, n = 4, p < 0.001) (Fig. 3a and b).

With our previous studies in motor neurons (Bishopet al. 1998; Bishop et al. 2004; Bishop et al. 2006) and theabove study in mind, we asked if the constitutive resistanceexhibited by the oligodendrocytes was dependent on HO1.We found that yes, NO resistance to cytotoxic challengeexhibited by the oligodendrocytes is abrogated by theaddition of 20 lM ZnPPIX (compare 115% ± 7, n = 9, vs.31% ± 10, n = 6, p < 0.001) (Fig. 3c and d). We controlled

for possible toxicity of the ZnPPIX by incubating cellswith the HO1 inhibitor alone, and found little toxicity(Fig. 3c and d). This profound ZnPPIX-mediated decreasein NO resistance oligodendrocytes indicates that HO1activity is needed for the oligodendrocyte constitutive NOresistance.

Fig. 2 Viability of pure primary oligodendrocytes and pure primary

motor neurons as a function of NO flux. (a) The first row is micro-

graphs of primary motor neurons with the first panel phase contrast,

the second and third panel fluorescence images of cells labeled for the

neuronal marker MAP2. For the lower row, which contains primary

oligodendrocytes, the first panel is phase contrast, while the second

panel is fixed cells under phase contrast, with the third panel fluo-

rescence images of cells labeled with oligo maker, myelin basic pro-

tein, MBP. The percentage purity = (number of MBP positive cells/

total cells) · 100. Non-fixed cells were used for all experiments. (b)

These are photomicrographs of primary motor neurons and primary

oligodendrocytes treated with low dose NO and then high dose of NO.

Note, most of the fields of motor neurons treated with high NO fluxes

were empty. We chose a field with neurons to show the decay of

morphology. (c) Cell survival was quantified; the values represent the

mean ± SEM of n = 4 performed in triplicate. The significance

(p < 0.001) was determined between cell survival after high flux NO

challenge of oligodendrocytes versus motor neurons and designated

by an #.



Involvement of peroxynitrite in nitric oxide toxicity andresistanceIn light of the many studies which indicate that much of NOtoxicity seen in MS, ALS, or spinal injury, is because ofprotein nitration with subsequent formation of 3NY residuesby the RNS, peroxynitrite (Estevez et al. 1998; Ischiropoulosand Beckman 2003; Jack et al. 2007; Pacher et al. 2007; Pratand Antel 2005) we asked whether the NO toxicity we see isbecause of peroxynitrite. For our studies we utilized uricacid, which is a specific peroxynitrite scavenger (Hooperet al. 2000), to determine if it ameliorated NO sensitivity inthe motor neurons. Motor neuron NO sensitivity wasabrogated by the addition of 10 lM uric acid (9% ± 4 vs.67% ± 19, n = 4, p < 0.001) indicating that a significantportion of the NO toxicity seen in motor neurons is becauseof peroxynitrite(Fig. 3a and b).

We asked if the oligodendrocyte resistance was, in fact, toperoxyntrite, by investigating if the NO-sensitive HO1-inhibited oligodendrocytes could be rescued with addition ofuric acid. When these cells were incubated with the 10 lMuric acid before challenge, NO-mediated cell death wasprevented (31% ± 10, n = 6, vs. 91% ± 7, n = 4, p < 0.003)(Fig. 3c and d). In fact, incubation of the HO1-inhibitedoligodendrocytes with uric acid before cytotoxic NO treat-ment restored them to the percentage cell survival ofoligodendrocyctes that were incubated with the HO1 inhib-itor alone (91% ± 7, n = 4, vs. 90% ± 10, n = 6), with nosignificant difference, indicating that the cell saving effect ofuric acid was > 99% (Fig. 3c and d) Thus, we can concludethat > 99% of the toxicity seen in NO-challenged HO1-inhibited oligodendrocytes is because of peroxynitrite.

Involvement of 3-nitrotyrosine formation in nitric oxidesensitivityIn addition to the pharmacological evidence, we wanted todetermine, in motor neurons, if the cytotoxic NO doses weused for our experiments produced peroxynitrite as indi-cated by the intracellular formation of 3NY. Motor neuronswere treated, lysed, and analyzed by western blot andprobed for 3NY formation, with nitrated albumin as apositive control and untreated lysate as a negative control(Fig. 4). We used the densitometry software on the wholelane, rather than on individual bands, as a source ofcomparison (Fig. 4). In motor neurons challenged with highdose NO alone we see an increase in 3NY formation, andin IAR we see a mitigation of 3NY formation at thesephysiologically relevant doses (46% above untreated vs.31% above untreated). When IAR is abrogated by theaddition of the HO1 inhibitor ZnPPIX we see a concomitantincrease in 3NY formation (34% vs. 89% 3NY increase)linking HO1 activity to 3NY inhibition. In motor neuronschallenged with high dose NO, we see more 3NY formationwhich is abrogated by the addition of the peroxynitritescavenger, uric acid (46% increase in 3NY levels above

untreated control vs. 10% increase in 3NY formation aboveuntreated controls, n = 3).

We asked if the differential sensitivity seen in oligoden-drocytes versus motor neurons is reflected in differences in3NY formation in response to cytotoxic NO challenge, hencefurther suggesting that the NO resistance seen in oligoden-drocytes is to peroxynitrite rather than NO per se (Fig. 4cand d). Motor neuron lysates were loaded on one half of thegel and the oligodendrocyte lysates were loaded on the otherhalf. In motor neurons, high NO doses (110 pm/s) do yieldsignificantly increased 3NY formation (�8-fold) (n = 4)above UT, indicating that NO challenged results in intracel-lular 3NY formation, thereby implicating intracellular per-oxynitrite formation (Fig. 4c and d). In fact, there is anincrease in 3NY formation of at least �4-fold (n = 4) as aresult of high dose NO challenge in motor neurons, whencompared with that in oligodendrocytes (Fig. 4c and d). Insome particular bands, there is a 10-fold increase in 3NY inNO challenged motor neurons when compared with NOchallenged oligodendrocytes. Clearly, Fig. 4c and otherwesterns indicate that the NO challenge yields peroxynitriteand that oligodendrocytes somehow mitigate the peroxyni-trite mediated 3NY formation in response to NO challenge.

Mitigation of motor neuron NO sensitivity upon coculturewith oligodendrocytesAs oligodendrocytes are NO-resistant at the fluxes we used,and motor neurons are quite NO sensitive we asked if theoligodendrocytes can bestow their NO resistance upon motorneurons, or vise versa. We cocultured the two cell types andthe ratio at which both cell types exhibited optimal healthwas �66% motor neurons and �33% oligodendrocytes(Fig. 5a). It was found that in coculture the oligodendrocytesmyelinate motor neuron axons as indicated morphologicallyby Nodes of Ranvier (Fig. 5a). Although this is chiefly amorphological detail, it further legitimizes the coculture as aneffective model to study the interplay between motor neuronsand oligodendrocytes. We observed the cultures in phasecontrast, and then stained the cultures with a fluorescentantibody specific to neurons, MAP2. More than 99% of theneurons in the culture expressed the neuron specific antibody,while none of the oligodendrocytes expressed the neuronspecific antibody. Thus, the green cells seen in the coculturesin Fig. 5 were neurons. For the oligodendrocyte cultures, asthey exhibited no green cells, we checked the phase contrastto make sure we were at an NO dose (50 pmol/s) where therewas no oligodendrocyte loss.

We exposed pure motor neurons, pure oligodendrocytes,and cocultures to NO, and asked if motor neurons coculturedwith oligodendrocytes were more resistant to NO than weremotor neurons alone. Motor neurons in coculture weresignificantly more resistant (109% ± 5, n = 4, vs. 53% ± 2,n = 4, p £ 0.001) (Fig. 5b and c). Clearly, the oligodendro-cytes bestow upon the neurons their native resistance. We



(a) (c)

(b) (d)

Fig. 3 NO resistance is turned off by HO1 inhibitor and involves

peroxynitrite. (a) Photomicrographs of motor neurons, all at 100·magnification, are organized in panels: UT, cells treated with spent NO

donor; UA, cells treated with 10 lM uric acid alone; induced adaptive

resistance (IAR), cells pre-treated with low dose NO (2 pm/s) followed

2 h later by high dose NO (�100 pm/s); high dose NO (�100 pm/s);

IAR + ZnPPIX, IAR in the presence of 20 lM ZnPPIX; high dose

NO + UA. (b) The cell survival data are organized along the same

labeling as seen in (a). The cell survival data were quantified, SEM

determined, and p < 0.001 was determined to be a significant differ-

ence between high NO and high NO with uric acid designated by an

asterisk. The # is the p < 0.001 significant difference between IAR and

IAR + ZnPPIX. (c) Photomicrographs of oligodendrocytes, all at 100·magnification, are organized in panels: UT, cells treated with spent NO

donor; ZnPPIX, cells treated with the 20 lM HO1 inhibitor alone; UA,

cells treated with 10 lM uric acid alone; the High NO panel,�100 pm/s

NO; HNO + ZnPPIX, cells challenged with high dose NO (110 pm/s) in

the presence of the HO1 inhibitor; and finally, the HNO + ZnPPIX +

UA, cells challenged with high dose NO (�110 pm/s) in the presence

of the HO1 inhibitor + uric acid. (d) The cell survival data are

organized along the same labeling as seen in (c). The cell survival

data were quantified, and SEM determined. There was significant

difference (p < 0.001) between HNO, HNO + ZnPPIX, and HNO +

ZnPPIX + uric acid, designated by an asterisk.



then asked if the oligodendrocytes merely shield the motorneurons from NO or if they secrete a factor that protectsneurons. We incubated one set of motor neurons with mediaconditioned by oligodendrocytes and another set of neuronswith neuron conditioned media (CM). We challenged bothsets of motor neurons with NO and found that there wassignificant protection exerted by the oligodendrocyte CM asindicated by the increase in percentage cell survival(41% ± 0.1 vs. 80% ± 0.4, n = 4, p £ 0.001) (Fig. 5d ande). This protection was attenuated by incubation of the oligoCM with the HO1 inhibitor, ZnPPIX (64% ± 5) andaugmented by the incubation of the oligo CM with theprotease, trypsin (96% ± 6) indicating that the secreted factormay be HO1, or a protein with HO1 like activity.

Discussion

With this study, we have established that motor neurons aremore sensitive to NO than are oligodendrocytes, that thisdifferential NO resistance seen in oligodendrocytes isbecause of resistance to peroxynitrite rather than to NOper se, and that the NO sensitivity seen in motor neurons is,again, to peroxynitrite rather than NO. In addition, we foundthat the resistance seen in oligodendrocytes is HO1 depen-dent. Most importantly, we have found that oligodendrocytescan imbue motor neurons with NO resistance when cocul-tured with neurons and that oligodendrocytes secrete anunknown neuron protective factor that possibly could beHO1 itself or a protein with HO1 activity. These results addto the current findings of prominent researchers in the MSfield and suggest directions for further study – one of whichis isolation and characterization of the secreted factor.

Nitric oxide is a proven common denominator in spinalinjury, ALS, and MS (Beckman et al. 1996; Giovannoniet al. 1997, 1998; Ischiropoulos and Beckman 2003). In ALSthere is substantial motor neuron death with concomitantaccumulation of NO and other RNS with such reliability thatthese NO metabolites (3NY proteins) are markers for thedisease (Ischiropoulos and Beckman 2003; Reiter et al.2006). These same RNS have been detected in patients withMS where metabolites of NO, nitrate and nitrite, and free3NY and 3NY proteins are found in the CSF of MSindividuals, the concentrations of which are correlated withthe severity and duration of the disease (Giovannoni et al.1997, 1998; Liu et al. 2001; Acar et al. 2003; Bizzozeroet al. 2005). Therefore, our studies of NO sensitivity inoligodendrocytes and motor neurons, are apropos to MS, andoffer further directions for research.

Although MS has been considered a disease predominantlyaffecting white matter brain tissue with demyelination first,followed by axonal injury and damage to neurons (Silber andSharief 1999), we may have to re-examine this currentparadigm of MS etiology in light of our findings of increasedNO sensitivity of motor neurons when compared witholigodendrocytes. In one study, amyloid precursor protein, asensitive marker of axonal damage, was found within acuteMS brain lesions. In fact, several investigators have data thathas led them to the conclusion that axonal damage inducesMS, with demyelination occurring after (Ferguson et al.1997; Bitsch et al. 2000; De Stefano et al. 2001).

Several important studies address NO sensitivity ofoligodendrocytes (Prat and Antel 2005) or motor neurons(Estevez et al. 1998; Ischiropoulos and Beckman 2003; Jacket al. 2007) individually. Our study of NO sensitivity in bothcell types, in direct comparison and in coculture, is animportant step towards elucidating which cell type starts thecascade of demyelination and cell death in MS. Ourobservations from our direct comparison of motor neuronshaving significantly more NO sensitivity than oligodendro-

Fig. 4 MO3.13 NO resistance is turned off by HO1 inhibitor and in-

volves peroxynitrite. (a) Western blot from motor neuron cell lysates. 1.

MW, molecular weight; 2. UA, 10 lM uric acid; 3. HNO, high dose NO

(110 pm/s); 4. HNO + UA, high dose NO + uric acid; 5. IAR, low dose

NO (2 pm/s) followed 2 h later by high dose NO (110 pm/s); 6.

IAR + ZnPPIX, IAR protocol in the presence of HO1 inhibitor (20 lM).

(b) Quantification of data from (a) and other western blots. Xfold in-

crease is from 3NY total from untreated (treated with spent NO do-

nors). (c)1. MW, molecular weight; 2, 3NY, nitrated albumin; 3,

Untreated MO3.13; 4, low dose NO MO3.13; 5, high NO MO3.13; 6,

high NO MO3.13; 7, high NO NSC34D; 8, high NO NSC34D; 9, low

NO NSC34D; 10, untreated NSC34D. (d) Quantification of data from

(c) and other western blots. Xfold increase is from 3NY total from

untreated (treated with spent NO donors).



cytes, and the fact that oligodendrocytes lend NO resistanceto motor neurons in coculture, lead one to imagine ascenario, in MS, where the excess NO released overwhelmsthe protective effects of oligodendrocytes, or even turnsoligodendrocytes against neurons, causing neuronal death

with subsequent demyelination. In normal CNS functions,the interplay between the axons of neurons and oligoden-drocytes is critical for maturation of oligodendrocytes.Disturbance of this cellular relationship possibly leads toMS pathology (Silber and Sharief 1999).

(a)

(b)

(c)

(d)

(e)

Fig. 5 Cocultures of motor neurons and oligodendrocytes. (a) A

micrograph of the cocultured motor neurons and oligodendrocytes,

with oligodendrocytes myelinating motor neuron axons as indicated

morphologically by Nodes of Ranvier, which are indicated by white

arrows. These are all at 320· magnification. (b) Micrographs of cells

(magnification 320·), all labeled with MAP2 (a neuron specific protein).

The green cells were exclusively motor neurons. The top row is un-

treated: oligodendrocytes, motor neurons, and cocultures. The bottom

row is high dose NO (50 pm/s which is half of toxic dose in other

experiments): oligodendrocytes, motor neurons, and cocultures.

(c) This is a quantification of the data from the micrographs in (a). SEM

(n = 4) was calculated. Comparison of the two groups: NO challenged

motor neurons versus NO challenged motor neurons cocultured with

oligodendrocytes, was made and found to be significant (p < 0.001) as

indicated by asterisk. (d) A micrograph of motor neurons untreated,

challenged with high NO (110 pm/s), untreated in the presence of oligo

conditioned media (OLCM), challenged with high NO in the presence

of OLCM, challenged in the presence of OLCM treated with the HO1

inhibitor (20 lM ZnPPIX), or challenged in the presence of OLCM

incubated with 10% trypsin. (e) This is a quantification of (d) and is the

average of at least (n = 4) experiments. SEM was calculated and the

difference in percentage cell survival between NO challenged neurons

with and without OLCM was significant (p £ 0.001), designated by an

asterisk. The difference between NO challenged neurons with OLCM

versus NO challenged neurons with ZnPPIX-treated OLCM was sig-

nificant, designated by an #. The difference between NO challenged

cells with OLCM and NO challenged cells with OLCM treated with 10%

trypsin was significant, designated by a +.



Finally, our finding of an HO1-mediated mechanism foroligodendrocyte NO-resistance, which can be bestowed uponmotor neurons in coculture, offers a possible therapeutictarget for mitigation of axonal injury seen in MS.

Acknowledgements

We gratefully acknowledge the expertise of Dr Neil R. Cashman and

his kind gift of the NSC34 cells and the expertise of Dr Bruce

Demple. I would also like to acknowledge the support of NASA and

Dr Robert R. Richmond (NASA Biology Directorate), the Louis B.

Stokes Alliance for Minority Participation (LSAMP), and NINDS

NIH AREA R15 grant.

References

Acar G., Idiman F., Idiman E., Kirkali G., Cakmakci H. and Ozakbas S.(2003) Nitric oxide as an activity marker in multiple sclerosis.J. Neurol. 250, 588–592.

Akins R. Jr, McLaughlin T., Boyce R., Gilmour L. and Gratton K. (2004)Exogenous metalloporphyrins alter the organization and functionof cultured neonatal rat heart cells via modulation of heme oxy-genase activity. J. Cell. Physiol. 201, 26–34.

Baumann N. and Pham-Dinh D. (2001) Biology of oligodendrocyte andmyelin in the mammalian central nervous system. Physiol. Rev. 81,871–927.

Beckman J. S. (1996) Oxidative damage and tyrosine nitration fromperoxynitrite. Chem. Res. Toxicol. 9, 836–844.

Beckman J. S., Estevez A. G., Crow J. P. and Barbeito L. (2001)Superoxide dismutase and the death of motoneurons in ALS.Trends Neurosci. 24, S15–S20.

Bishop A. and Anderson J. (2005) NO Signaling in the CNS: From thePhysiological to the Pathological. Toxicology (Special Issue) NitricOxide, Cell Signalling and Death 208, 193–205.

Bishop A. and Cashman N. R.. (2003) Induced adaptive resistance tooxidative stress in the CNS: discussion of possible mechanismsand their therapeutic potential. Curr. Drug Metab. 4, 171–184.

Bishop A., Marquis J. C., Cashman N. R. and Demple B. (1999)Adaptive resistance to nitric oxide in motor neurons. Free Radic.Biol. Med. 26, 978–986.

Bishop A., Fung-Yet S., Perrella M J., Lee A. M., Cashman N. R. andDemple B. (2004) Decreased resistance to nitric oxide in motorneurons of HO-1 null mice. BBRC 325, 3–9.

Bishop A., Gooch R. and Anderson J. (2006) Induced adaptiveresistance to nitrooxidative stress in the CNS: therapeutic implica-tions. Central Nervous System Agents in Medicinal Chemistry(Formerly Curr. Med. Chem.) 6, 281–291(11).

Bitsch A., Schuchardt J., Bunkowski S., Kuhlmann T. and Bruck W.(2000) Acute axonal injury in multiple sclerosis. Correlation withdemyelination and inflammation. Brain 123, 1174–1183.

Bizzozero O. A., DeJesus G., Bixler H. A. and Pastuszyn A. (2005)Evidence of nitrosative damage in the brain white matter ofpatients with multiple sclerosis. Neurochem. Res. 30, 139–149.

Bouton C. and Demple B. (2000) Nitric oxide-inducible expressionof heme oxygenase-1 in human cells. Translation-independentstabilization of the mRNA and evidence for direct action of nitricoxide. J. Biol. Chem. 275, 32688–32693.

Brugg B. and Matus A. (1991) Phosphorylation determines the bindingof microtubule-associated protein 2 (MAP2) to microtubles inliving cells. J. Cell Biol. 114, 735–743.

Buntinx M., Vanderlocht J., Hellings N., Vandenabeele F., Lambrichts I.,Raus J., Ameloot M., Stinissen P. and Steels P. (2003) Character-

ization of three human oligodendroglial cell lines as a model tostudy oligodendrocyte injury: morphology and oligodendrocyte-specific gene expression. J. Neurocytol. 32, 25–38.

Cashman N. R., Durham H. D., Blusztajn J. K., Oda K., Tabira T., ShawI. T., Dahrouge S. and Antel J. P. (1992) Neuroblastoma · spinalcord (NSC) hybrid cell lines resemble developing motor neurons.Dev. Dynamics 194, 209–221.

Cassina P., Peluffo H., Pehar M., Martinez-Palma L., Ressia A.,Beckman J. S., Estevez A. G. and Barbeito L. (2002) Peroxy-nitrite triggers a phenotypic transformation in spinal cordastrocytes that induces motor neuron apoptosis. J. Neurosci. Res.67, 21–29.

Clough G. F., Bennett A. R. and Church M. K. (1998) Measurement ofnitric oxide concentration in human skin in vivo using dermalmicrodialysis. Exp. Physiol. 83, 431–434.

Cornish A. S., Jijon H., Yachimec C. and Madsen K. L. (2002) Perox-ynitrite enhances the ability of salmonella Dublin to invade T84monolayers. Shock 18, 93–96.

De Stefano N., Narayanan S., Francis G. S., Arnaoutelis R., TartagliaM. C., Antel J. P., Matthews P. M. and Arnold D. L. (2001)Evidence of axonal damage in the early stages of multiple sclerosisand its relevance to disability. Arch. Neurol. 58, 65–70.

Dickhout J. G., Hossain G. S., Pozza L. M., Zhou J., Lhotak S. andAustin R. C. (2005) Vascular endothelium: implications in ath-erogenesis. Arterioscler. Thromb. Vasc. Biol. 25, 2623–2629.

Durham H. D., Dahrouge S. and Cashman N. R. (1993) Evaluation ofthe spinal cord neuron X neuroblastoma hybrid cell line NSC-34as a model for neurotoxicity testing. Neurotoxicology 14, 387–395.

Eggett C. J., Crosier S., Manning P., Cookson M. R., Menzies F. M.,McNeil C. J. and Shaw P. J. (2000) Development and characteri-sation of a glutamate-sensitive motor neuron cell line. J. Neuro-chem. 74, 1895–1902.

Estevez A. G., Spear N., Manuel S. M., Radi R., Henderson C. E.,Barbieto L. and Beckman J. S. (1998) Nitric oxide and superoxidecontribute to motor neuron apoptosis induced by trophic factordeprivation. J. Neurosci. 18, 923–931.

Estevez A. G., Sampson J. B., Zhuang Y.-X., Spear N., Richardson G. J.,Crow J. P., Tarpey M. M., Barbeito L. and Beckman J. S. (2000)Liposome-delivered superoxide dismutase prevents nitric oxidedependent motor neuron death induced by trophic factor with-drawal. Free Radic. Biol. Med. 28, 437–446.

Ferguson B., Matyszak M. K., Esiri M. M. and Perry V. H. (1997)Axonal damage in acute multiple sclerosis lesions. Brain 120,393–399.

Fung-Yet S., Perrella M. A., Layne M. D., Hsieh C. M., Maemura K.,Kobzik L., Weisel P., Christou H., Kourembanas S. and Lee M.(1999) Hypoxia induces severe right ventricular dilation and in-farction in heme oxygenase-1 null mice. J. Clin. Invest. 103, 23–29.

Giovannoni G., Heales S. J., Silver N. C., O’Riordan J., Miller R. F.,Land J. M., Clark J. B. and Thompson E. J. (1997) Raised serumnitrate and nitrite levels in patients with multiple sclerosis.J. Neurol. Sci. 145, 77–81.

Giovannoni G., Heales S. J., Land J. M. and Thompson E. J. (1998) Thepotential role of nitric oxide in multiple sclerosis. Mult. Scler. 4,212–216.

Hall E. D., Oostveen J. A. and Gurney M. E. (1998) Relationship ofmicroglial and astrocytic activation to disease onset and progres-sion in a transgenic model of familial ALS. Glia 23, 249–256.

Herzog W. and Weber K. (1978) Fractionation of brain microtubule-associated proteins (isolation of two different which stimulatetubulin polymerization in vitro). Eur. J. Biochem. 98, 1–8.

Hooper D. C., Scott G. S., Zborek A., Mikheeva T., Kean R. B.,Koprowski H. and Spitsin S. V. (2000) Uric acid, a peroxynitrite



scavenger, inhibits CNS inflammation, blood–CNS barrier per-meability changes, and tissue damage in a mouse model of MS.FASEB J. 14, 691–698.

Huk I., Brovkovych V., Nanobash Vili J., Weigel G., Neumayer Ch.,Partyka L., Patton S. and Malinski T. (1998) Bioflavonoid quer-cetin scavenges superoxide and increases nitric oxide concentrationin ischaemia-reperfusion injury: an experimental study. Br. J. Surg.85, 1080–1085.

Ischiropoulos H. and Beckman J. S. (2003) Oxidative stress and nitrationin neurodegeneration: cause, effect, or association? J. Clin. Invest.111, 163–169.

Jack C., Antel J., Bruck W. and Kuhlmann T. (2007) Contrasting po-tential of nitric oxide and peroxynitrite to mediate oligodendrocyteinjury in multiple sclerosis. Glia 55, 926–934.

Johnson M. I., Bunge R. P. and Wood P. M. (2004). Primary CellCultures for the Study for Myelination. Protocols for Neural CellCulture, 3rd edition, pp. 95–115. Human Press, Totowa.

Kawase M., Kinouchi H., Kato I., Akabane A., Kondo T., Arai S.,Fujimura M., Okamoto H. and Yoshimoto T. (1996) Induciblenitric oxide synthase following hypoxia in rat cultured glial cell.Brain Res. 738, 319–322.

KitamuraY., IshidaY., TakataK. et al. (2003)Hyperbilirubinemia protectsagainst focal ischemia in rats. J. Neurosci. Res. 71, 544–550.

Leonard C. S., Michaelis E. K. and and Mitchell K. M. (2001) Activity-dependent nitric oxide concentration dynamics in the laterodorsaltegmental nucleus in vitro. J. Neurophysiol. 86, 2159–2172.

Li W., Maeda Y., Ming X., Cook S., Chapin J., Husar W. and Dowling P.(2002) Apoptotic death following Fas activation in human oligo-dendrocyte hybrid cultures. J. Neurosci. Res. 69, 189–196.

Liu J. S., Zhao M. L., Brosnan C. F. and Lee S. C. (2001) Expression ofinducible nitric oxide synthase and nitrotyrosine in multiple scle-rosis lesions. Am. J. Pathol. 158, 2057–2066.

Matsumoto A., Yoshino H., Yuki N., Hara Y., Cashman N. R., HandaS. and Miyatake T. (1995) Ganglioside characterization of a cellline displaying motor neuron-like phenotype: GM2 as a possiblemajor ganglioside in motor neurons. J. Neurol. Sci. 131, 111–118.

McLaurin J., Trudel G. C., Shaw I. T., Antel J. P. and Cashman N. R.(1995) A human glial hybrid cell line differentially expressinggenes subserving oligodendrocyte and astrocyte phenotype.J. Neurobiol. 26, 283–293.

Pacher P., Beckman J. S. and Liaudet L. (2007) Nitric oxide and per-oxynitrite in health and disease. Physiol. Rev. 87, 315–424.

Packer M. A., Stasiv Y., Benraiss A., Chmieinicki E., Grinberg A.,Westphal H., Goldman S. A. and Enikolopov G. (2003) Nitricoxide negatively regulates mammalian adult neurogenesis. Proc.Natl Acad. Sci. USA 100, 9566–9571.

Panahian N. and Maines M. (2001) Site of injury-directed induction ofheme oxygenase-1 and -2 in experimental spinal cord injury:differential functions in neuronal defense mechanisms? J. Neuro-chem. 76, 539–554.

Peterson J. W., Bo L., Mork S. et al. (2001) Transected neurites,apoptotic neurons, and reduced inflammation in cortical multiplesclerosis lesions. Ann. Neurol. 50, 389–400.

Peunova N. and Enikolopov G. (1995) Nitric oxide triggers a switch togrowth arrest during differentiation of neuronal cells. Nature 375,68–73.

Prat A. and Antel J. (2005) Pathogenesis of multiple sclerosis. Demye-linating diseases. Curr. Opin. Neurol. 18, 225–230.

Raoul C., Estevez A. G., Nishimune H., Cleveland D. W., deLapeyriereO., Henderson C. E., Haase G. and Pettmann B. (2002) Moto-neuron death triggered by a specific pathway downstream of Faspotentiation by ALS-linked SOD1 mutations. Neuron 35, 1067–1083.

Reiter T. A., Pang B., Dedon P. C. and Demple B. (2006) Resistance tonitric oxide-induced necrosis in heme oxygenase-1 overexpressingpulmonary epithelial cells associated with decreased lipid peroxi-dation. J. Biol. Chem. 281, 36603–36612.

Schnaar R. L. and Schaffner A. E. (1981) Separation of cell types fromembryonic chicken and rat spinal cord: characterization of moto-neuron enriched fractions. J. Neurosci. 1, 204–217.

Silber E. and Sharief M. K. (1999) Axonal degeneration in the patho-genesis of multiple sclerosis. J. Neurol. Sci. 170, 11–18.

Stamler J. S., Jia L., Eu J. P., Mcmahon T. J., Demchenko I. T., Bon-aventura J., Gernert K. and Piantadosi C. A. (1997) Blood flowregulation by S-nitrosohemoglobin in the physiological oxygengradient. Science 276, 2034–2037.

Stuehr D. J. (1999) Mammalian nitric oxide synthases. Biochim.Biophys. Acta 1411, 217–230.

Tamir S., Lewis R. S., de Rojas Walker T., Deen W. M., Wishnok J. S.and Tannenbaum S. R. (1993) The influence of delivery rate on thechemistry and biological effects of nitric oxide. Chem. Res. Toxi-col. 6, 895–899.

Tamir S., Burney S. and Tannenbaum S. R. (1996) DNA damage bynitric oxide. Chem. Res. Toxicol. 9, 821–827.

Tominaga T., Sato S., Ohnishi T. and Ohnishi S. T. (1994) Electronparamagnetic resonance (EPR) detection of nitric oxide producedduring forebrain ischemia of the rat. J. Cereb. Blood Flow Metab.14, 715–722.

Trapp B. D., Ransohoff R. and Rudick R. (1999) Axonal pathology inmultiple sclerosis: relationship to neurologic disability. Curr. Opin.Neurol. 12, 295–302.

Vaziri N. D., Lee Y. S., Lin C. Y., Lin V. W. and Sindhu R. K. (2004)NAD(P)H oxidase, superoxide dismutase, catalase, glutathioneperoxidase and nitric oxide synthase expression in subacute spinalcord injury. Brain Res. 995, 76–83.

Werner P., Pitt D. and Raine C. S. (2001) Multiple sclerosis: alteredglutamate homeostasis in lesions correlates with oligodendrocyteand axonal damage. Ann. Neurol. 50, 169.

Yang G., Nguyen X., Ou J., Rekulapelli P., Stevenson D. K. and DenneryP. A. (2001) Unique effects of zinc protoporphyrin on HO-1induction and apoptosis. Blood 97, 1306–1313.



differential sensitivity of oligodendrocytes and motor neurons to reactive nitrogen species:...

Documents