NO contributes to excitotoxic neuronal cell death (Huang et al., 1994
; Dawson et al., 1996
), which can result in neuronal deficits in a variety of brain regions after stroke or the development other neurodegenerative conditions. Recent evidence suggests an essential role for the stress-activated protein kinase p38 in excitotoxic neuronal cell death in cultured cerebellar neurons (Kawasaki et al., 1997
; Cao et al., 2004
) and forebrain and hippocampal neurons (Legos et al., 2002
), and in vivo in retinal neurons (Manabe and Lipton, 2003
) and cerebral ischemia (Legos et al., 2001
). NO species are known to activate p38 in nonneuronal cells, but this reportedly depends on a TAB1-mediated p38 autophosphorylation mechanism (Ge et al., 2002
) that is not required for glutamate-evoked neuronal p38 activation (unpublished data). Information on the effect of NO on neuronal p38 is limited—in cerebellar granule neurons, an activation of p38 after a 3-h incubation with NO donor has been reported (Lin et al., 2001
). Because glutamate activates p38 within 2 min and apoptosis is complete within 3 h in these cells (Cao et al., 2004
), the significance of this result has been unclear. To consider the possible relationship between NO and p38, we first investigated in more detail the time course of p38 activation and its requirement for the cell death process. Addition of glutamate to cerebellar granule neuron cultures leads to a transient but strong activation of p38 at 5 min, with little elevation above basal level at 30 min and no detectable increase for the next 3 h ( A). Pyknosis is an early indicator of this form of cell death, preceding loss of membrane integrity by several hours (Cao et al., 2004
). Cell death, assessed by pyknosis, is already detectable 60 min after glutamate addition and is complete within 3 h ( B). SB203580, at a concentration that selectively inhibits p38 in intact cerebellar granule neurons (Coffey et al., 2000
; Cao et al., 2004
), is known to prevent this pyknosis. Adding the inhibitor 30 min before the addition of glutamate strongly prevents the pyknosis, but if the inhibitor is added 30 min after glutamate exposure, the pyknosis is indistinguishable from that in controls ( C). These data suggest that the early transient increase of p38 activity is important for glutamate-induced pyknosis.
Figure 1. Glutamate-induced neuronal death requires the early phase of p38 activation. (A) Phospho-p38 levels in cerebellar granule neuron extracts prepared at times indicated after exposure to 50 μM glutamate. Phosphorylated p38 levels increase rapidly (more ...)
We subsequently examined the effect of nNOS inhibitors on p38 activation 5 min after glutamate addition, when phospho-p38 levels are at their highest. 7-Nitroindazole, which does not discriminate between NOS isoforms (with selectivity ratios of 0.9–1.4-fold; for review see Alderton et al., 2001
), and N
-arginine, which selectively targets nNOS (Zhang et al., 1997
), both substantially and significantly reduced the glutamate-induced rapid increase in p38 activation loop phosphorylation observed at 5 min (). After this, the glutamate-induced pyknosis was also significantly diminished, as expected (). Both of these inhibitors are competitive with arginine, and thus the effects of the inhibitors are completely reversed by the presence of excess arginine in the culture medium ().
Figure 2. Inhibitors of NOS and nNOS reduce glutamate-induced p38 activation and pyknosis. (A) Immunoblot revealing phospho-p38 levels in cerebellar granule neuron extracts prepared at times indicated, in the presence of carrier (DMSO) or the pan-NOS– and (more ...)
If NO species are genuinely involved in glutamate-induced p38 activation, then NO should, like glutamate, activate p38 within minutes of addition. Therefore, an NO donor was added to cerebellar granule neuron cultures and immunoblotting with phospho-p38 antibody was performed on cell lysates prepared at times after addition of NO donor, as shown (). We used the NONOate diethylamine/NO adduct (Dea/NO), which degrades with a t1/2
of 2.1 min (at 37°C), to release NO. Dea/NO at 250 or 10 μM induced a substantial increase in phospho-p38 level ( [top and middle] and B). The lower concentration is only threefold greater than the amount used in a recent study to supply a physiologically relevant amount of NO sufficient to enhance long-term potentiation without effects on basal synaptic transmission (Bon and Garthwaite, 2003
). The activation of p38 by Dea/NO appears to occur by a direct effect on the neurons and not by indirect stimulation of glutamate release, because the NMDA receptor antagonist MK801 does not prevent it (unpublished data). It has been suggested that peroxynitrite (ONOO−
) may mediate the neurotoxic actions of NO. Therefore, we also tested this, at a concentration reported to activate p38 in 293 cells (Ge et al., 2002
); once again, a rapid p38 activation loop phosphorylation was detected ( [bottom] and B). If concentrations of NO donor sufficient to activate p38 are of relevance to p38-mediated death by glutamate, then the donors should induce rapid pyknosis in a manner similar to that of glutamate. Pyknosis of cells was measured between 30 and 180 min after addition of donor, revealing that Dea/NO indeed induces a rapid pyknosis dependent on the amount of donor added ( C). The pan-caspase inhibitor zVAD-fmk failed to inhibit p38-dependent pyknosis induced by glutamate (Cao et al., 2004
). Pyknosis induced by NO and ONOO−
is similar in that zVAD-fmk fails to prevent it as well (unpublished data).
Figure 3. NO species activate neuronal p38 and induce rapid pyknosis. (A) Application of NO donors, as shown, to neurons increases levels of phospho-p38, with a time course similar to that induced by glutamate. The pan-p38 blot indicates equal loading of samples. (more ...)
Based on the experiments just described, we concluded that glutamate-induced activation of p38 and the subsequent death of cerebellar granule neurons require activity of nNOS and can be reproduced with NO donors. PSD95 ablation/dissociation has been shown to be neuroprotective (Sattler et al., 1999
; Aarts et al., 2002
), but as described in the Introduction, it may have additional effects. Because hypotheses exist concerning the mechanism by which this protein mediates coupling of NMDA receptors to nNOS and the consequent sensitization of the enzyme to glutamate-mediated calcium influx, we developed a construct expected to bind PSD95 in a manner identical to, and therefore competitive with, that of endogenous nNOS. The domain structures of full length nNOS and the NH2
-terminal fragment we used are shown in A. This NH2
-terminal fragment contains the nNOS PDZ domain and the adjacent β finger, both of which are required for binding PSD95-PDZ2 (Christopherson et al., 1999
; Tochio et al., 2000a
), and we therefore named it nNOS-PBD (PSD95-binding domain). Transfection with GFP and GST fusion constructs leads to expression of a protein running at ~70 kD ( B and not depicted). To investigate whether this construct is able to selectively bind PSD95-PDZ2, we cotransfected GST-tagged nNOS-PBD into COS7 cells with GFP-tagged PSD95-PDZ1, PSD95-PDZ2, or PSD95-PDZ3, or GFP alone. Pull-down of nNOS-PBD with immobilized glutathione revealed that a selective interaction had formed within intact cells with PDZ2 but not PDZ1, PDZ3, or unfused GFP ( C), and that this interaction was sufficiently stable to be detected after cell lysis and multiple washing steps.
Figure 4. nNOS-PBD selectively and stably interacts with the PSD95-PDZ2 domain. (A) Domain map of nNOSα (1,433 aa in length). The PDZ and β finger (βƒ) domains have been reported to interact with the NMDA receptor scaffold PSD95, (more ...)
The purpose of producing the nNOS-PBD construct was to prevent glutamate-induced activation of p38 and the resulting cell death. Therefore, we investigated whether the construct was capable of doing this. Cerebellar neuron cultures were cotransfected with empty vector or nNOS-PBD together with GST-tagged p38. Thus, we were able to selectively recover p38 from transfected neurons with glutathione immobilized on beads. In the presence of empty vector, the recovered p38 exhibited a large increase in activation loop phosphorylation in response to glutamate. However, the presence of cotransfected nNOS-PBD substantially reduced the p38 activation (). Subsequently, neurons were transfected with nNOS-PBD or empty vector together with GFP marker plasmid so that the transfected cells could be identified. Glutamate induced pyknosis in ~50% of neurons in the presence of empty vector, but the presence of nNOS-PBD greatly reduced this response ().
Figure 5. nNOS-PBD inhibits glutamate-evoked activation of p38α and subsequent pyknosis. (A) Neurons were cotransfected with p38α and nNOS-PBD or empty vector (pCMV), as indicated. Cells were stimulated with glutamate or control, p38α was (more ...)
Next, we considered whether nNOS-PBD inhibits glutamate-induced p38 activation and cell death by acting upstream or downstream of NO. Neurons were challenged with NO donor as in , and p38 from transfected cells showed a large increase in phosphorylation that was not prevented by cotransfection with PBD (). NO donor–induced pyknosis was also not prevented by transfection with nNOS-PBD ( C).
Figure 6. nNOS-PBD does not prevent NO-evoked activation of p38α or subsequent pyknosis. (A) Neurons were cotransfected with p38α and nNOS-PBD or empty vector (pCMV), as indicated. Cells were stimulated with 10 μM Dea/NO as NO donor (as (more ...)
The nNOS-PBD construct was designed to selectively interfere with the NMDA receptor–PSD95 complex. Thus, it was important to evaluate whether it perturbed the general properties of the NMDA receptors, other than nNOS-dependent p38 activation. Patch-clamp recordings of whole-cell currents induced by the rapid application of NMDA applied via a U-tube showed that cells transfected with either nNOS-PBD or vector control were indistinguishable ( A). We detected no significant differences in induced current amplitude ( B, left) or time to peak current ( B, right). Capacitance was also unchanged (unpublished data), which suggests that no gross alterations in cell structure were induced. NMDA receptor activity leads to calcium influx into the cell. However, the actual changes in cytoplasmic free calcium also depend on the calcium-handling machinery of the cell. We measured the calcium response of cells transfected with nNOS-PBD or vector control by cotransfecting a fluorescence resonance energy transfer (FRET)–based calcium reporter “precocious cameleon,” or YC2.12 (Nagai et al., 2002
). Once again, the nNOS-PBD– and vector-transfected cells were indistinguishable ( C).
Figure 7. nNOS-PBD does not perturb NMDA receptor electrophysiological characteristics or NMDA-induced calcium response. (A) Whole-cell currents recorded from neurons transfected with GFP-tagged nNOS-PBD or pEGFP-C1 vector as control, after the rapid (U-tube) application (more ...)
Together, these data support the proposal that the nNOS-PBD construct reduces glutamate-induced p38 activation and pyknosis by acting upstream of NO production, without causing general perturbation of either the electrophysiological characteristics of the NMDA receptor or the downstream signaling pathways. This suggests that a protein sequence that interacts with PSD95 in the same way as nNOS may be sufficient to confer significant neuroprotection via inhibition of glutamate-evoked p38 activation. Conversely, it would be expected that the PSD95-PDZ2 domain, which binds nNOS sequences ( C), would also be capable of conferring neuroprotection, whereas PDZ3 would not. PDZ1 may also be expected to be neuroprotective via interaction with the COOH termini of NMDA receptor subunits, but this interaction can be anticipated to dissociate PSD95 from the receptor complex and, therefore, to nonspecifically affect all aspects of NMDA receptor–PSD95 function. It is possible that the free PDZ2 domain may also act in this way, as PDZ2 is able to interact in vitro and in yeast with COOH-terminal peptide sequences derived from several NMDA receptor subunits (Niethammer et al., 1996
), but those NMDA receptors that associate via PSD95 to nNOS may do so only via NMDA receptor interaction with PDZ1, because PDZ2 mediates the interaction with nNOS (Christopherson et al., 1999
). We investigated the ability of glutamate to evoke pyknosis of cells transfected with the constructs shown in C; i.e., either PSD95-PDZ1, PSD95-PDZ2, PSD95-PDZ3, or empty epitope vector (unfused GFP). Both PDZ2 and PDZ1 conferred neuroprotection, but PDZ3 failed to protect ().
Figure 8. Free PDZ1 and PDZ2 domains inhibit glutamate-induced death, but PDZ3 has no effect. Neurons were transfected with the GFP-tagged PSD95-PDZ domains described in the C legend, or empty vector, and then treated with glutamate or not treated. Pyknosis (more ...)