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Keywords:

  • knockout mouse;
  • manganese superoxide dismutase (Sod2);
  • metalloporphyrin;
  • mitochondria;
  • neuronal survival;
  • superoxide

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References

The objective of this study was to determine whether metalloporphyrin catalytic antioxidants influence the survival of neuronal cultures in an in vitro model of age-related mitochondrial oxidative stress. Neuronal cultures were prepared from cerebral cortices of manganese superoxide dismutase (MnSOD or Sod2) knockout (homozygous –/–, heterozygous –/+ or wild-type +/+) mice. The ability of catalytic antioxidants, manganese tetrakis-(4-benzoic acid) porphyrin (MnTBAP) and manganese tetrakis-(N-ethyl-2-pyridyl) porphyrin (MnTE-2-PyP) to influence the survival of cultured cerebrocortical neurones from Sod2-replete (+/+) and Sod2-deficient (+/– or –/–) mice was assessed. Sod2–/– cultures showed accelerated cell death in serum-free conditions when grown in ambient oxygen. MnTBAP and MnTE-2-PyP delayed the death of Sod2–/– cultures and improved the survival of Sod2+/+ and Sod2+/– cultures in serum-free conditions. The results suggest that metalloporphyrin antioxidants can delay neuronal death resulting specifically from increased mitochondrial oxidative stress. Furthermore, Sod2-deficient neuronal cultures provide a simple model system to screen the biological efficacy of mitochondrial antioxidants.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References

Mitochondrial oxidative stress has been shown to play an important mechanistic role in the pathogenesis of age-related neurodegenerative diseases (Beal, 2000). Mitochondria are the cell's most prominent estimated source of superoxide radical production. The existence of three superoxide dismutases (SODs) in the cytoplasm (CuZnSOD or Sod1; McCord & Fridovich, 1969), mitochondria (MnSOD or Sod2; Weisiger & Fridovich, 1973) and extracellular compartment (ECSOD or Sod3; Marklund, 1984) to maintain low steady-state superoxide levels underscores the importance of superoxide in physiological and pathological processes. Generation of mice lacking each of the three compartmentalized SODs reveals that complete lack of Sod2 is lethal (Li et al., 1995), whereas mice lacking Sod1 or Sod3 are viable and do not exhibit any major phenotypes (Carlsson et al., 1995; Lebovitz et al., 1996; Reaume et al., 1996).

The overexpression of endogenous SODs in tissue culture and whole animals has provided protection against the deleterious effects of a wide range of oxidative stress paradigms (Chan et al., 1995; Huang et al., 1999). The use of SOD and catalase as therapeutic agents to attenuate free radical injury has the drawback that these natural products are large in size and therefore have limited penetration to cellular compartments. Several classes of low-molecular-weight SOD mimetics have been developed to overcome some of these limitations. Manganic porphyrins are a class of catalytic antioxidants that scavenge a wide range of reactive oxygen species such as superoxide, peroxide, peroxynitrite and lipid peroxyl radicals (Patel & Day, 1999). Work from this laboratory has shown that manganic porphyrins can protect mature neuronal cultures from excitotoxic injury by scavenging intracellular superoxide radicals (Patel et al., 1996; Li et al., 2001).

The in vivo deletion of Sod2 in the mouse dramatically shortens lifespan concomitant with a fulminating mitochondrial disease (Li et al., 1995; Melov et al., 1999). Both the shortened lifespan and mitochondrial pathology can be partially rescued by catalytic antioxidants (Melov et al., 1998, 2001). In vitro model systems that recapitulate age-related neurodegeneration and resultant mitochondrial oxidative stress may be useful screening tools to predict the biological efficacy of potential neuroprotective agents such as mitochondrial antioxidants. The goals of this study were two-fold. The first was to establish an in vitro model system in which neurodegeneration results from mitochondrial oxidative stress. The second goal was to determine if two metalloporphyrins with distinct antioxidant potencies were neuroprotective in this in vitro model of mitochondrial oxidative stress.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References

Neuronal survival of Sod2+/+, Sod2+/– and Sod2–/– cultures followed distinct temporal profiles following initial plating in low oxygen and serum-containing media and subsequent exposure to ambient oxygen and serum-free conditions. Sod2–/– cultures showed age-dependent neurotoxicity on days 2, 3 and 5 as judged by lactate dehydrogenase (LDH) release (Fig. 1) and morphological assessment. Sod2+/+ and Sod2+/– cultures remained viable in serum-free conditions until day 5 following media change (Fig. 1). Sod2+/– cultures showed a small (but statistically insignificant) increase in neurotoxicity on day 3 (compared with Sod2+/+ cultures). Overall, no significant differences were observed between the viability of Sod2+/+ and Sod2+/– cultures.

image

Figure 1. Time-course of LDH release in cortical cultures from Sod2+/+, Sod2+/– and Sod2+/– mice after plating in serum-free media and grown in ambient oxygen levels. Bars represent mean ± SEM, n= 16–20, *P < 0.05, anova.

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The ability of metalloporphyrins to influence the accelerated death of Sod2–/– cultures was assessed. Sod2–/– cultures maintained in MnTBAP or MnTE-2-PyP (1, 3 and 10 µm) for 3 days showed concentration-dependent decreases in neurotoxicity (Fig. 2). MnTE-2-PyP was more efficacious as a neuroprotective agent compared with MnTBAP. By day 5, the neuroprotective effects of MnTBAP and MnTE-2-PyP had diminished significantly (Fig. 3A).

image

Figure 2. Effect of MnTBAP and MnTE-2-PyP (1, 3 and 10 µm) on neuronal death in cortical cultures from Sod2–/– on day 3 after growth in serum-free conditions. Values are normalized to the maximal LDH released in the experiment. Points represent mean ± SEM, n= 10–16, *P < 0.05, anova.

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image

Figure 3. Effect of MnTBAP and MnTE-2-PyP on the survival of cultures from Sod2–/– (A), Sod2+/– (B) and Sod2+/+ (C) mice on day 5 after growth in serum-free conditions. Values are normalized to the maximal LDH released in each experiment and points represent mean ± SEM, n= 4–12, *P < 0.05, anova.

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Neuronal cultures from Sod2+/+ and Sod2+/– mice did not survive in non-supplemented serum-free media by day 5. Surprisingly, both MnTBAP and MnTE-2-PyP improved the survival of Sod2+/+ and Sod2+/– cultures 5 days after media change to serum-free conditions (Fig. 3B,C). MnTE-2-PyP dramatically improved neuronal survival in both Sod2+/+ and Sod2+/– cultures, an effect that was evident on day 7 following serum withdrawal (data not shown). The addition of SOD (1–100 U mL−1) or catalase (100 U mL−1) failed to influence the survival of Sod2–/– cultures on day 3 or Sod2+/+ and Sod2+/– cultures on day 5 (data not shown).

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References

Three major findings arise. First, this work provides an in vitro model system that recapitulates age-related mitochondrial oxidative stress, which may be useful for screening the biological efficacy of mitochondrial antioxidants. Secondly, metalloporphyrin catalytic antioxidants (MnTBAP and MnTE-2-PyP) delay the age-related death of neuronal cultures from Sod2–/– mice. Finally, metalloporphyrins prolonged the survival of Sod2+/+ and Sod2+/– in serum-free media. The results suggest that cell-permeable antioxidants such as metalloporphyrins may be useful for maintaining cultured cells in serum-free media.

Cell culture models derived from SOD1- and SOD2-deficient animals have provided important information regarding our understanding of mechanisms by which compartment-specific superoxide formation modulates cellular functions (Przedborski et al., 1996; Gonzalez-Zulueta et al., 1998; Huang et al., 1999). The sensitivity to ambient oxygen has previously been demonstrated in cultured Sod2–/– neurons derived from mice bred on the CD-1 background (Li et al., 1998). The experimental paradigm used here was particularly useful for testing neuroprotection because it revealed accelerated death in Sod2–/– cultures but not in Sod2+/– and Sod2+/+ cultures. The accelerated death and resultant sensitivity of Sod2–/– neurons towards ambient oxygen levels most likely occurs due to increased steady-state levels of superoxide generated normally by mitochochondrial respiration. Normally, superoxide generated in this manner is efficiently scavenged by SOD2. The complete absence of SOD2, however, would render oxidant-sensitive targets vulnerable to attack by normal levels of superoxide and thereby lead to mitochochondrial dysfunction and cell death. Surprisingly, 50% SOD2 activity was sufficient to protect cultured neurons of SOD2+/– mice from ambient oxygen levels.

The death of Sod2+/+ and Sod2+/– neurons observed on day 5 probably occurred due to growth in serum-free media devoid of supplements. Overnight incubation in a low oxygen environment during the first day followed by normoxic conditions may have also contributed to the death of these neurons.

MnTBAP and MnTE-2-PyP belong to the metalloporphyrin class of catalytic antioxidants that scavenge a wide range of reactive species such as superoxide, hydrogen peroxide, peroxynitrite and lipid peroxyl radicals and have been shown to be protective in numerous in vivo and in vitro models of oxidative stress (Patel & Day, 1999). Whereas MnTBAP and MnTE-2-PyP share a core manganic meso-porphyrin structure, they differ importantly in terms of substituent groups and cell-free antioxidant potencies. Benzoic acid-substituted MnTBAP has modest SOD activity, which is approximately 50 times lower than pyridine-substituted MnTE-2-PyP (Patel & Day, 1999). In vivo administration of MnTBAP has been shown to improve the pathology and extend the lifespan of Sod2–/– mice (Melov et al., 1998). The ability of MnTBAP and MnTE-2-PyP to protect against accelerated death of Sod2–/– cultures and prolong the survival of Sod+/+ and Sod2+/– cultures confirms the role of mitochondrial superoxide toxicity in the injury process. The inability of CuZnSOD or catalase to provide any protection in this model suggests a role for mitochondrial superoxide radicals in mediating the injury. Consistent with its higher SOD activity in comparison with MnTBAP, MnTE-2-PyP showed greater potency in protecting Sod2–/– cultures on day 3 and Sod+/– or Sod+/+ cultures at day 5. The inability of metalloporphyrins to protect Sod2–/– cultures on day 5 may be related to the vulnerability of these cultures to life in ambient oxygen, combined with prolonged serum deprivation, rather than a manifestation of drug toxicity. The latter may be ruled out because Sod2+/– and Sod+/+ cultures showed improved survival in the presence of metalloporphyrins on day 5.

In summary, this work provides a simple model system to screen the biological efficacy of mitochondrial antioxidants that may be useful in age-related pathologies. Furthermore, because maintenance of cultured cells in serum-free media often requires multiple reagents, metalloporphyrins or other catalytic antioxidants may be useful supplemental additives for maintaining cultured cells in serum-free media.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References

Tissue culture supplies were purchased from Gibco. MnTBAP and MnTE-2-PyP (Aeol 10113) were provided by Incara Pharmaceuticals. Biochemical reagents were purchased from Sigma. Adult Sod2+/– mice (C57Bl/6 background) were crossed and the presence of a vaginal plug in the females was designated as gestational day 0. Mixed neuronal and glial cultures were prepared from embryonic day 14–15 mouse cerebral hemispheres as described previously (Patel, 1996). Embryo carcasses were genotyped as described previously (Liang et al., 2001). Cells from each embryo were plated in individual 96-well dishes at a density of 200 000 cells per well. Neuronal cultures were initially plated in serum containing minimum essential medium (MEM with Earle's salts supplemented with 3 g L−1 glucose, 5% horse serum and 5% fetal calf serum) in a low oxygen environment (5% O2, 5% CO2, 90% argon) for 18 h. The presence of serum during this initial period promotes adherence of neurons to the substrate and the low oxygen levels protect Sod2-deficient neurons from ambient oxygen levels. Chamber oxygen concentration was maintained at 5% during the first day by continuously monitoring with an oxygen analyser. This oxygen concentration was based on initial studies characterizing the survival of wild-type and Sod-deficient neuronal cultures, which revealed that Sod2+/+, Sod2+/– and Sod2–/– cultures could survive for approximately 12 days in vitro in 5% oxygen levels and serum-containing media. Following this plating procedure, culture medium was replaced with serum-free non-supplemented MEM containing vehicle (0) or varying concentrations (1, 3 and 10 µm) of MnTBAP and MnTE-2-PyP and placed in an incubator with normal ambient oxygen (designated as day 0). Neurotoxicity was determined by the measurement of LDH in the supernatant media (Vassualt, 1983) and by phase-contrast microscopy 2, 3 and 5 days thereafter. LDH release has been previously characterized as a sensitive quantitative marker of neuronal injury in cortical cultures in this laboratory (Patel et al., 1996; Li et al., 2001). In the present studies, LDH release was particularly advantageous as a measure of the time-course of cell damage because small amounts of supernatant media could be sampled from the same culture wells on different days with minimal disturbance to the cells. This is difficult to achieve with other methods of assessing cell damage (e.g. fluorescent dyes). Morphological analysis by phase contrast microscopy further validated the results obtained by measurement of LDH release.

Acknowledgments

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References

I am grateful to Dr Brian Day for Sod2 mutant mice, Incara Pharmaceuticals for metalloporphyrins, and Cynthia Pedersen, Joe Milano and Lisa Calavetta for excellent technical assistance. This work was supported by grants to M.P. from NIH (NS39587) and the Maria Teresa Jones foundation of Colorado.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References
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