Physiologically relevant NO doses
We assayed the release rate of NO from NO donors, to choose pretreatment and challenge doses that were closer to the range of NO concentrations seen by cells during physiological and pathological conditions respectively (Clough et al.,1998
, Hall et al.,1998
, Kawase et al., 1996
, Pacher et al., 2007
, Tominaga et al.,1994
). For NO donors, we used a final concentration of 1uM DETA-NONOate which donates only NO (Dickhout et al., 2005
) at a lower flux rate of 2 pmoles/s ( low dose) (). For the challenge NO dose, we used 10uM final concentration spermine-NONOate, which donates NO at a higher flux (Cornish et al., 2002
) of 110 pmoles/s (high dose) ().
In light of the fact that high flux NO seen in pathological situations produces RNS with resultant 3NY formation (Cornish et al., 2002 Tamir et al., 1993
), and cell death (Estevez et al., 1998 2000
, Ischiropoulos & Beckman,2003
, Jack et al., 2007
, Pacher et al.,2007
, Prat & Antel et al., 2005
) we wanted to ask if spermine-NONOate, unlike DETA-NONOate, does indeed show greater toxicity. We exposed NSC34D (motor neuron cell line) to a dose response curve of DETA versus spermine and found a significant difference of cell survival at the highest dose (104%+/−10 vs 16%+/−8 (n=4),p<.001) (). Thus, NO doses detailed in mimic physiological versus pathological NO-mediated conditions (Pacher et al., 2007
), and are valid tools to study cellular NO resistance mechanisms.
For studies with motor neurons and oligodendrocytes, we utilized primary cells and recognized model cell lines. In particular, for our motor neurons, we used NSC34D
which is terminally differentiated NSC34,both of which are accepted models of motor neurons (Bishop et al , 1999
,2006, Cashman et al., 1992
, Durham et al.1993
, Eggett et al., 2000
, Matsumoto et al.,1995
). More than 99% of NSC34D
cells are positive for the motor neuron markers NMDAR, which would presumably make the NSC34D cells more NO sensitive than the NSC34 cells (Bishop et al., 2004
, Cashman et al.1992
, Egget et al., 2000
). However, we have found no increase in NO sensitivity at the high NO doses (110pm/s) in NSC34D
as compared to NSC34, (12+/−5 vs 13+/−19 ; n=3)(), thus both motor neuron lines exhibit NO sensitivity which indicates that either NSC34 or NSC34D is an acceptable model of motor neurons for our studies of NO sensitivity (Bishop et al., 1999
, Cashman et al., 1992
, Egget et al., 2000
Comparison of NO sensitivity of oligodendrocytes and motor neurons
NSC34D and MO3.13 (terminally differentiated oligodendrocyte cell line where >90% express myelin basic protein) were exposed to a range of NO fluxes for one hour, and cell death was assayed twenty four hours later. Untreated cells were exposed to spent NO donor as a control. Even at low NO fluxes (2–13pmoles/s), motor neurons were significantly more NO sensitive than were oligodendrocytes (% cell survival of 61 ± 9% vs 82 ± 11%, n=8, p<.001) (). This differential susceptibility was more evident at high NO fluxes (110 pmoles/s) where motor neurons survival was minimal and oligodendrocytes were resistant (12 ± 5% vs 115 ± 7%, n=8, p<.001)(). Dead cells that remained attached to the plate were rounded with no neurites/processes and did not exclude trypan blue. Of the cells that lifted from the plate >99% did not exclude trypan blue (Bishop et al.,1999
) (). Thus, the MO3.13 cells, unlike NSC34D cells, were completely unaffected by cytotoxic doses of NO, thereby expressing differential sensitivity to a direct NO challenge in the flux range we used ().
Differential NO resistance in primary motor neurons and oligodendrocytes
We asked if this differential NO sensitivity can be verified in primary rat oligodendrocyte and motor neuron cultures. The primary motor neurons were stained for motor neuron specific proteins (MAP2), assayed for purity, and were ~80% pure (). The primary oligodendrocytes were stained for myelin basic protein and determined to be ~65% pure (). The oligodendrocyte morphology changed profoundly upon fixation and staining, but was still good enough for the purposes of assaying purity, since we were merely counting immunoreactive cells. For the experiments, we did not fix the cells.
Viability of pure primary oligodendrocytes and pure primary motor neurons as a function of NO flux
We found that primary oligodendrocytes were resistant to treatment with the low dose of NO (2–13pm/s DETA NONoate) (69± 7%, n=4), while the primary motor neurons were significantly (p<.001) more sensitive (33 ± 2%, n=4) (). When the treatment dose was 110pm/s NO (administered by spermine-NONOate), we found that primary oligodendrocytes were still resistant (67 ± 1 %, n=4), while motor neurons were quite sensitive (1 ± 1%, n=4,p<.001). For the micrograph in , we chose one of the few fields of HNO motor neurons that contained cells, rather than debris, to illustrate the decay in morphology. At the highest flux of NO there was a change in oligodendrocyte morphology, suggesting some NO sensitivity, but not nearly as much as seen in the primary motor neurons ().
HO1-dependent mechanism for differential NO resistance on the two cell types
We have found in previous studies that, in motor neurons, HO1 is important for induced adaptive resistance-a phenomenon where pretreatment with low dose of NO lends NO resistance to normally quite NO sensitive motor neurons (Bishop et al., 1998, 2004
, 2006, Fung et al., 1999, Kitamura et al., 2003
). Here we use lower NO doses that are within the physiological range (2pmoles/s) for the pretreatment dose to induce resistance to a more physiologically relevant challenge dose (110pmoles/s) and found that yes, IAR can still be demonstrated in motor neurons (IAR 72%+/−5 vs High NO alone 9+/−4, n=4, p<.001) (). We utilized a more specific HO1 inhibitor, zinc protoporphyrin (ZnPPIX) (Akins et al., 2004
, Yang et al., 2001
) and found that IAR in motor neurons is abrogated by the inhibition of HO1 activity (72+/−5 vs 30+/−14, n=4, p<.001) ().
NO resistance is turned off by HO1 inhibitor and involves peroxynitrite
With our previous studies in motor neurons (Bishop et al., 1998, 2004
, 2006) and the above study in mind, we asked if the constitutive resistance exhibited by the oligodendrocytes was dependent on HO1. We found that yes, NO resistance to cytotoxic challenge exhibited by the oligodendrocytes is abrogated by the addition of 20uM ZnPPIX (compare 115% +/− 7(n=9) vs 31%+/−10 (n=6) p<.001) (). We controlled for possible toxicity of the ZnPPIX by incubating cells with the HO1 inhibitor alone, and found little toxicity (). This profound ZnPPIX-mediated decrease in NO resistance oligodendrocytes indicates that HO1 activity is needed for the oligodendrocyte constitutive NO resistance.
Involvement of peroxynitrite in NO toxicity and NO resistance
In light of the many studies which indicate that much of NO toxicity seen in MS, ALS or spinal injury, is due to protein nitration with subsequent formation of nitrotyrosine residues (3NY) by the RNS, peroxynitrite, (Esteves, 1998, Ischiropoulos & Beckman, 2003
, Jack et al., 2007
,Pacher et al., 2007
, , Prat & Antel 2005
) we asked whether the NO toxicity we see is due to peroxynitrite. For our studies we utilized uric acid, which is a specific a peroxynitrite scavenger (Hooper et al., 2000
) to determine if it ameliorated NO sensitivity in the motor neurons. Motor neuron NO sensitivity was abrogated by the addition of 10uM uric acid (9%+/.4 vs 67%+/-19, n=4,p<.001) indicating that a significant portion of the NO toxicity seen in motor neurons is due to peroxynitrite().
We asked if the oligodendrocyte resistance was, in fact, to peroxyntrite, by investigating if the NO-sensitive HO1-inhibited oligodendrocytes could be rescued with addition of uric acid. When these cells were incubated with the 10uM uric acid before challenge, NO-mediated cell death was prevented (31%+/−10 (n=6) vs 91%+/−7 (n=4)p<.003)(). In fact, incubation of the HO1-inhibited oligodendrocytes with uric acid before cytotoxic NO treatment restored them to the % cell survival of oligodendrocyctes that were incubated with the HO1 inhibitor alone (91%+/−7 (n=4) vs 90%+/− 10 (n=6)), with no significant difference, indicating that the cell saving effect of uric acid was >99% () Thus we can conclude that >99% of the toxicity seen in NO-challenged HO1-inhibited oligodendrocytes is due to peroxynitrite.
Involvement of 3NY formation in NO sensitivity
In addition to the pharmacological evidence, we wanted to determine, in motor neurons, if the cytotoxic NO doses we used for our experiments produced peroxynitrite as indicated by the intracellular formation of 3NY. Motor neurons were treated, lysed, and analyzed by Western Blot and probed for 3NY formation, with nitrated albumin as a positive control and untreated lysate as a negative control (). We used the densitometry software on the whole lane, rather than on individual bands, as a source of comparison (). In motor neurons challenged with high dose NO alone we see an increase in 3NY formation, and in IAR we see a mitigation of 3NY formation at these physiologically relevant doses (46% above untreated versus 31% above untreated). When IAR is abrogated by the addition of the HO1 inhibitor ZnPPIX we see a concomitant increase in 3NY formation (34% 3NY increase vs 89% 3NY increase) linking HO1 activity to 3NY inhibition. In motor neurons challenged with high dose NO we see more 3NY formation which is abrogated by the addition of the peroxynitrite scavenger, uric acid (46% increase of 3NY levels above untreated control vs 10% increase of 3NY formation above untreated controls, n=3).
MO3.13 NO resistance is turned off by HO1 inhibitor and involves peroxynitrite
We asked if the differential sensitivity seen in oligodendrocytes versus motor neurons is reflected in differences in 3NY formation in response to cytotoxic NO challenge, hence further suggesting that the NO resistance seen in oligodendrocytes is to peroxynitrite rather than NO per se (). Motor neuron lysates were loaded on one half of the gel and the oligodendrocyte lysates were loaded on the other half. In motor neurons, high NO doses(110pm/s) do yield significantly increased 3NY formation (~8 fold) (n=4) above UT, indicating that NO challenged results in intracellular 3NY formation, thereby implicating intracellular peroxynitrite formation (). In fact, there is an increase of 3NY formation of at least ~4 fold (n=4) as a result of high dose NO challenge in motor neurons, as compared to that in oligodendrocytes (). In some particular bands there is a 10 fold increase of 3NY in NO challenged motor neurons as compared to NO challenged oligodendrocytes. Clearly, and other westerns indicate that the NO challenge yields peroxynitrite, and that oligodendrocytes somehow mitigate the peroxynitrite mediated 3NY formation in response to NO challenge.
Mitigation of motor neuron NO sensitivity upon coculture with oligodendrocytes
Since oligodendrocytes are NO-resistant at the fluxes we used, and motor neurons are quite NO sensitive we asked if the oligodendrocytes can bestow their NO resistance upon motor neurons, or vise versa. We cocultured the two cell types and the ratio at which both cell types exhibited optimal health was ~66% motor neurons and ~33% oligodendrocytes (). It was found that in coculture the oligodendrocytes myelinate motor neuron axons as indicated morphologically by Nodes of Ranvier (). Although this is chiefly a morphological detail, it further legitimizes the coculture as an effective model to study the interplay between motor neurons and oligodendrocytes. We observed the cultures in phase contrast, and then stained the cultures with a fluorescent antibody specific to neurons, MAP2. More than 99% of the neurons in the culture expressed the neuron specific antibody, while none of the oligodendrocytes expressed the neuron specific antibody. Thus the green cells seen in the cocultures in were neurons. For the oligodendrocyte cultures, since they exhibited no green cells, we checked the phase contrast to make sure we were at an NO dose (50pmoles/s) where there was no oligodendrocyte loss.
Cocultures of motor neurons and oligodendrocytes
We exposed pure motor neurons, pure oligodendrocytes ,and cocultures to NO, and asked if motor neurons cocultured with oligodendrocytes were more resistant to NO than were motor neurons alone. Motor neurons in coculture were significantly more resistant (109%+/−5 (n=4) vs 53% +/− 2, n=4, p<.001) (). Clearly the oligodendrocytes bestow upon the neurons their native resistance. We then asked if the oligodendrocytes merely shield the motor neurons from NO or if they secrete a factor that protects neurons. We incubated one set of motor neurons with media conditioned by oligodendrocytes and another set of neurons with neuron conditioned media. We challenged both sets of motor neurons with NO and found that there was significant protection exerted by the oligodendrocyte conditioned media as indicated by the increase in % cell survival (41%+/−0.1 vs 80%+/−0.4, n=4, p<.001) (). This protection was attenuated by incubation of the oligo CM with the HO1 inhibitor, ZnPPIX,(64%+/−5) and augmented by the incubation of the oligo CM with the protease, trypsin (96%+/−6) indicating that the secreted factor may be HO1, or a protein with HO1 like activity.