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An alternative Ca²⁺-dependent mechanism of neuroprotection by the metalloporphyrin class of superoxide dismutase mimetics

National Research Council, Institute for Biological Sciences, Synaptic Pathophysiology Group, Ottawa, ON, Canada

¹ Correspondence: National Research Council, Institute for Biological Sciences, Synaptic Pathophysiology Group, Montreal Road Campus, Building M-54, Ottawa, ON, Canada K1A 0R6. E-mail: joe.tauskela{at}nrc-cnrc.gc.ca

SPECIFIC AIMS

Metalloporphyrins are known as superoxide dismutase (SOD) mimetics, with SOD activity resulting from "funneling" of the negatively charged superoxide toward a redox-sensitive Mn metal within the porphine core. The aim of this study was to systematically evaluate if neuroprotection by major classes of metalloporphyrins against in vitro cerebral ischemia in cultured rat cortical neurons correlates with metalloporphyrin catalytic antioxidant scavenging ability and, if not, to determine the mechanism of neuroprotection.

PRINCIPAL FINDINGS

1. Neuroprotection by major classes of metalloporphyrins (those possessing meso-substituted imidazolium, pyridinium, and phenyl rings) against excitotoxic insults inversely correlates with SOD activity
The data show that high SOD active Mn^IIIporphyrins meso-substituted with N,N'-dimethylimidazolium or N-alkylpyridinium groups did not protect neurons against oxygen-glucose deprivation (OGD). We confirmed that Mn^IIITBAP, which possesses slight SOD activity, protected against OGD (Table 1 ). Replacing the Mn in Mn^IIITBAP with one of several redox-insensitive metals, resulting in SOD inactive metalloTBAPs, nevertheless improved neuroprotection against OGD, with IC₅₀s increasing in the following order: CO-Ru^II Co^II Cu^II Ni^I < Zn^II < Mn^III (Table 1) . A recent generation ß-octabromo analog of Mn^IIITBAP, Mn^IIIBr₈-TBAP, was reported to more potently protect cortical neuron cultures against OGD than Mn^IIITBAP, ostensibly as a result of a higher SOD mimetic activity than Mn^IIITBAP. However, we found that a control SOD inactive analog, Cu^IIBr₈TBAP (25 µM), completely protected against OGD.

Excessive postsynaptic NMDA receptor activation underlies neurotoxicity by OGD, so we evaluated if these neuroprotective trends extended to NMDA toxicity. Metalloporphyrins that protected against OGD also protected against NMDA toxicity. Exceptions to this finding were the moderately SOD active Mn^III meso-tetrakis(N-methylpyridynium-4-yl)porphyrin (Mn^IIITM-PyP(4)) or Fe^IIITM-PyP(4), which protected cultures against NMDA exposure but not against OGD, although so did an SOD inactive control, Zn^IITM-PyP(4). Hence, an undefined deleterious effect was exerted during OGD, which counteracted the NMDA-protective propensities of metalloTM-PyP(4) analogs. For all other neuroprotective metalloporphyrins, the IC₅₀ for protection was generally higher against NMDA than against OGD. Neuroprotection against NMDA toxicity likely did not result from metalloTBAP dissociation, based on a lack of protection by free metals or the metal-free TBAP.

In view of the efficacy of metalloTBAPs to offer neuroprotection, structure-neuroprotection relationships were obtained with other, primarily phenyl-substituted derivatives to further resolve features required for protection against NMDA. Compounds demonstrating protection were the ß-substituted Zn^IIcoproporphyrin, Cu^II meso-tetra(2,3,5,6-tetrafluoro-4-N(CH₃)₃⁺-phenyl)porphyrin, the peroxynitrite decomposition catalyst, Fe^IIImeso-tetrakis(4-sulfonatophenyl)porphyrin (Fe^IIITSPP), as well as its control, Zn^IITSPP. Ineffective compounds were the ß-substituted Co^IIphthalocyaninetetracarboxylate, Zn^IImeso-tetrakis(4-hydroxyphenyl)porphyrin, Mn^IIITSPP and Zn^II- or Co^II-meso-tetra(2,3,5,6-tetrafluoro-4-N(CH₃)₃⁺-phenyl)porphyrin. Since no one structural feature was required for neuroprotection, considerable complexity exists in the interplay between features responsible for neuroprotection or toxicity. Taken together, neuroprotection against direct or indirect excitotoxic insults inversely correlated with SOD activity of metalloporphyrins.

Mn^IIITBAP has been reported to protect cultured cortical neurons against paraquat, an intracellular superoxide generator, by an SOD-based mechanism. We found that Mn^IIITBAP, Zn^IITBAP, or an NMDA receptor antagonist protected neurons against paraquat. Suppression of indirect NMDA receptor-mediated toxicity may underlie protection by metalloTBAPs and an NMDA receptor antagonist against paraquat, not direct scavenging of superoxide.

2. Metalloporphyrins protect neurons against OGD by suppressing a postsynaptic NMDA receptor-mediated Ca²⁺ rise, thereby indirectly preventing neurotoxic mitochondrial Ca²⁺ accumulation
OGD or NMDA exposure induces a neurotoxic increase in intracellular Ca²⁺ (Ca²⁺_i), which was suppressed by neuroprotective metalloporphyrins (see Table 1 for the effect of metalloTBAPs on OGD-induced Ca²⁺_i rises). This finding can account for why protection was observed by exposing cultures to metalloporphyrins during, but not after, NMDA exposure. Moreover, the IC₅₀s for suppression of toxicity and Ca²⁺_i rises by metalloporphyrins generally paralleled each other, although IC₅₀s were slightly higher for Ca²⁺_i suppression. Excessive Ca²⁺ influx via the NMDA receptor increases mitochondrial Ca²⁺ uptake, eventually resulting in cytosolic delayed Ca²⁺ dysregulation, caused by abrupt mitochondrial depolarization and mitochondrial Ca²⁺ depletion, irreversibly committing neurons to die. Further experiments suggest that metalloporphyrins prevented mitochondrial Ca²⁺ uptake during NMDA exposure in the same rank order as exhibited for neuroprotection and that this suppression was as an indirect consequence of lower upstream cytosolic Ca²⁺ loading.

3. Metalloporphyrins are not cell permeable and are not NMDA receptor antagonists
The data show that metalloporphyrins were unable to quench intracellular fluorescence of fluo-4 loaded neurons when added after extended NMDA receptor activation despite substantial quenching by a positive control, MnCl₂. This suggests that entry of metalloporphyrins into neurons likely does not underlie neuroprotection. NMDA receptor antagonism likely does not account for neuroprotection, since NMDA receptor whole-cell currents were suppressed to a minor extent by several neuroprotective metalloporphyrins. For instance, 100 µM Zn^IITBAP and 400 µM Mn^IIITBAP significantly suppressed NMDA currents by 19 ± 2 and 6± 3%, respectively, but NMDA-mediated increases in Ca²⁺_i levels were suppressed by 87 ± 4 and 89 ± 2%. Surprisingly, Fe^IIITSPP (50 µM) significantly augmented currents by 11 ± 1% despite suppressing Ca²⁺_i by 97 ± 3%.

Neuroprotective metalloporphyrins suppress spontaneous synaptic activity, but a nonprotective SOD mimetic has the opposite effect
Neuroprotective metalloporphyrins do not block NMDA whole-cell currents (which represent somal or extrasynaptic NMDA receptor activation), yet suppress an NMDA-induced rise in Ca²⁺_i (extrasynaptic plus synaptic NMDA receptors), so we considered the possibility that only neuroprotective metalloporphyrins block synaptic NMDA receptor activation. Mn^IIITBAP, Zn^IITBAP, or tetrodotoxin (TTX), but not MK-801, completely suppressed spontaneous Ca²⁺ spiking (Fig. 1 ), suggesting that presynaptic release of Ca²⁺ is also inhibited by these metalloporphyrins. This ability would not influence protection efficacy of a metalloporphyrin against NMDA toxicity but would improve protection against OGD, possibly accounting for the lower IC₅₀ observed against OGD compared with NMDA toxicity. Targeting of the synaptic NMDA receptor subpopulation by Mn^IIITBAP and Zn^IITBAP may account for whole-cell currents (representing somal NMDA receptor activation) being relatively unaffected by these metalloTBAPs.

View larger version (38K): [in this window] [in a new window]   Figure 1. Differential effect of metalloporphyrins on spontaneous Ca2+ spiking in cortical cultures. Spontaneous Ca2+ spiking was monitored in fluo-4 loaded cortical cultures before and during application of MnIIITBAP (100 to 400 µM), ZnIITBAP (12.5–50 µM), MnIIITE-PyP(2) (200 µM), MK-801 (10 µM), and tetrodotoxin (TTX; 1 µM).

In contrast, the nonprotective high SOD active, Mn^IIImeso-tetrakis(N-ethylpyridinium-2-yl)porphyrin (Mn^IIITE-PyP(2)) increased the frequency of spiking by 50%. Hence, neuroprotection by metalloporphyrins against excitotoxic insults correlates with an ability to suppress spontaneous Ca²⁺ spiking.

CONCLUSIONS

This study challenges the conventional view that metalloporphyrins protect cultured cortical neurons in models of cerebral ischemia by acting as intracellular SOD mimetics or catalytic antioxidants. In every instance when an SOD-active metalloporphyrin protected, so did the respective SOD inactive control. Features that imparted high SOD activity or other antioxidant abilities, such as a Mn metal core or meso-substitution with N,N'-dimethylimidazolium or N-alkylpyridinium groups, did not result in neuroprotection against OGD or NMDA (Fig. 2 ). In contrast, features imparting slight or negligible antioxidant ability, such as a nonredox sensitive metal or meso-substituted phenyl rings bearing ortho-substituted carboxylates (metalloTBAPs) or other anionic groups, were neuroprotective. Neuroprotection by metalloporphyrins also does not correlate with other antioxidant capabilities such as scavenging of hydrogen peroxide, nitric oxide, or peroxynitrite or with an ability to inhibit lipid peroxidation.

View larger version (23K): [in this window] [in a new window]   Figure 2. Schematic representation of the neuroprotective mechanism by metalloporphyrins. The structure of the metalloporphyrin molecule is shown (box in upper left corner), with the type of metal, meso-substituted ring, isomerization status, ligand charge, or ß-pyrrole ligand listed, which result in a compound with low or high SOD activity. Neuroprotection inversely correlates with SOD activity of a metalloporphyrin. Exposing neurons to different stimuli (paraquat, OGD, NMDA, and spontaneous synaptic activity) converged to a common mediator, which was a rise in Ca2+i levels caused by activation of the NMDA receptor. Only neuroprotective metalloporphyrins suppressed an NMDA-induced rise in Ca2+i levels, albeit without directly blocking the NMDA receptor. No evidence could be found for neuronal entry of metalloporphyrins, suggesting a site of action at the level of the neuronal plasma membrane.

Exposing neurons to a number of different stimuli (paraquat, OGD, NMDA, and spontaneous synaptic activity) converged to a common mediator, which was an NMDA-induced rise in Ca²⁺_i levels (Fig. 2) . It is at this level that metalloporphyrins act. Metalloporphyrins suppressed OGD-, NMDA-, or synaptic activity-induced rises in Ca²⁺_i levels in the same general rank order as observed for neuroprotection. The inability of neuroprotective metalloporphyrins to suppress NMDA whole-cell currents (representing activation of somal NMDA receptors), despite blocking an NMDA-induced rise in Ca²⁺_i levels (in which extrasynaptic and synaptic NMDA receptors are activated), suggested targeting of a subpopulation of nonsomal NMDA receptors. The finding that only neuroprotective metalloporphyrins suppressed spontaneous Ca²⁺ spiking is consistent with targeting of the synaptic NMDA receptor subpopulation. Taken together, a model is suggested in which metalloporphyrins, acting at the plasma membrane, protect neurons against OGD by suppressing postsynaptic NMDA receptor-mediated Ca²⁺ rises, thereby indirectly preventing neurotoxic mitochondrial Ca²⁺ accumulation.

This understanding led to the identification of a new set of neuroprotective analogs (some structural components are identified in Fig. 2 ) that would not have been considered based on the traditional view of metalloporphyrin action. Though neuroprotective in a manner not originally intended, SOD inactive metalloporphyrins may represent promising therapeutic agents in diseases such as cerebral ischemia, in which Ca²⁺ toxicity is implicated. Future synthetic work may have to take into account whether oxidative- or Ca²⁺-based stress is to be the target; indeed, perhaps compounds designed to merge both of these targets may represent an important direction.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-3795fje; doi: 10.1096/fj.05-3795fje

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