These data indicate that growth factor-deprived neuronal cells increase superoxide levels before release of mitochondrial release of cytochrome c
. These results are in agreement with previous studies demonstrating an asynchronous rise in superoxide levels in axotomized primary RGCs [9
]. In that study, cells could be classified into two categories: cells undergoing a superoxide burst (>50% above mean control levels), and cells remaining at control superoxide levels. The fraction of cells with an intracellular superoxide burst increased substantially after both axotomy and optic nerve crush, and dismutation of superoxide achieved by intracellularly delivered PEG–SOD significantly increased RGC survival [17
These in vitro and similar in vivo [10
] results are consistent with superoxide serving as a signaling molecule for the induction of apoptosis after axotomy. However, it is also possible that superoxide elevation is the result of apoptosis itself, e.g. cytochrome c
release and inhibition of mitochondrial electron flow leading to one-electron reduction of O2
]. In order to determine the relative timing of superoxide production and cytochrome c
release, we systematically assessed these markers in neuronal precursor cells after serum deprivation, and found that cytochrome c
release occurred only in the later stages of the superoxide burst.
The timing of superoxide-dependent apoptotic pathways is controversial. We previously showed that serum deprivation induces superoxide in neuronal RGC-5 cells, and that scavenging of reactive oxygen species is protective in this model [19
]. Axonal injury of retinal ganglion cells also specifically induces superoxide in vivo [10
], and scavenging of superoxide is neuroprotective—all indicative of superoxide as an early, pre-commitment phase in apoptosis, occurring prior to the irreversible release of cytochrome c
. In vivo imaging of intracellular RGC superoxide and annexin V binding demonstrated that the superoxide burst preceded phosphatidylserine exposure [10
]. Based on the results of the present study and previous research, we suggest that the order of events in axotomy-induced apoptosis is superoxide production, then cytochrome c
release, and finally phosphatidylserine exposure. This agrees with the ordering of events reported in a variety of models for studying apoptosis, including Hep2G cells treated with sodium selenite [20
], Jurkat T cells after addition of exogenous C(2)-ceramide [21
], HeLa cells after ultraviolet light exposure [22
], and cerebral ischemia in mice deficient in SOD-2 [11
Conversely, Cai and Jones reported an altered order of these steps in staurosporine-induced apoptosis [23
], with cytochrome c
release preceding oxidation, as measured by intracellular redox potential. Phosphatidylserine exposure was seen preceding cytochrome c
release after treatment with staurosporine, etoposide, and tumor necrosis factor in a different cell line [24
]. Others found that peroxidation of phosphatidylserine is an early oxidative marker occurring prior to release of cytochrome c
in the same model as Cai and Jones, suggesting a small but highly specific oxidative event prior very early in the cell death process [25
]. However, the inability of the antioxidant N
-acetylcysteine (NAC) to rescue cells from staurosporine-induced apoptosis by Cai and Jones more likely suggests that that process may proceed by a superoxide-independent pathway. Chauhan et al. demonstrated in multiple myeloma cells that induction of a superoxide-dependent apoptotic pathway can be rescued with NAC, preventing both cell death and the release of cytochrome c
and Smac, while NAC is not effective in preventing cell death or Smac release in a superoxide-independent (dexamethasone) pathway [26
]. While all the components of the apoptotic pathway are activated in both the superoxide-dependent and superoxide-independent pathways, it may be the sequence of these first steps that differentiates the two modalities, and the subsequent activation of “skipped” steps in one versus the other may be signs of an inherent redundancy to ensure that once initiated, apoptosis proceeds to caspase activation and eventual cell death.
Given that intracellular cytochrome c distribution is most accurately studied with immunocytochemistry, it was necessary to use a cross-sectional method for correlating superoxide levels with cytochrome c release. To do this, we delineated four distinct schemes that could account for the relative timing of increased superoxide production and cytochrome c release after serum deprivation (). Each of these four schemes would predict a different pattern of cells belonging to each of four quadrants—low superoxide/mitochondrial cytochrome c localization (quadrant I), low superoxide/cytosolic cytochrome c (quadrant II), high superoxide/mitochondrial cytochrome c (quadrant III), and high superoxide/cytosolic cytochrome c (quadrant IV).
At baseline, cells are in quadrant I, with background levels of superoxide and cytochrome c maintained within mitochondria. Our data demonstrate that at 72 h after serum deprivation, cells shift from quadrant I to quadrants II, III, and IV. This pattern indicates a timeline for apoptotic signaling consistent with cytochrome c release occurring in the latter half of the superoxide burst, after superoxide levels have peaked. If cytochrome c release were instead to precede the superoxide burst, then there should be no cells in quadrant III (high superoxide but mitochondrial cytochrome c), contrary to what was observed. Given that 22% of the cells were in quadrant III and 21% in quadrant IV (), then high superoxide levels were present in cells before and after cytochrome c release. This suggests that the superoxide burst precedes and partly overlaps cytochrome c release.
Fig. 5 Proposed scheme for the time-course of a superoxide burst and cytochrome c release. The distribution of cells among the four quadrants is consistent with the scheme where the release of cytochrome c occurs during the later portion of the superoxide burst. (more ...)
release is considered an early apoptotic commitment point. Our observation that the superoxide burst precedes cytochrome c
release is consistent with the ability of PEG–SOD to rescue primary RGCs after axotomy in culture [17
], as well as in vivo [10
]. Furthermore, treatment of cells with etoposide for 24 h resulted in cytochrome c
release without a corresponding increase in superoxide levels, consistent with etoposide inducing apoptosis in a superoxide-independent manner [16
], and confirming that cytochrome c
release alone does not induce superoxide production.
This study has inherent limitations. It is controversial whether RGC-5 cells are truly RGCs, and all that can be established with certainty is that they are neuronal precursor cells which can be differentiated to express cell-surface and morphological markers consistent with more mature neurons [15
]. We used serum withdrawal to trigger apoptosis, which deprives cells of multiple growth factors, a pathway similar but not identical to what occurs when neurons undergo axotomy-induced deprivation of target-derived factors. RGC-5 cells respond to serum deprivation by undergoing apoptosis via a cytochrome c
-dependent pathway. These results are therefore applicable to the underlying mechanism and signaling of apoptosis after serum deprivation in a neuronal cell line, but not necessarily axotomy.
A possible source of bias in this study is the selection of the threshold value for defining a superoxide burst. The number of cells in quadrants III and IV is entirely dependent on this threshold value. In our prior work with primary RGCs, a threshold value of 1.5 (indicating superoxide present at 50% greater than basal levels) was sufficient for detecting an increase in the proportion of cells undergoing a superoxide burst [9
]. In that study, however, up to 10% of RGCs from retinas not undergoing optic nerve crush had basal levels above threshold. This is likely due to inherent variability in basal superoxide production, and therefore it is not surprising that we observed a small fraction of RGC-5 cells above this level when incubated in complete medium. In the present study 7.4% of cells cultured in complete medium had superoxide levels greater than the threshold value of 1.5, consistent with results in primary RGCs. To further address this possible bias, we analyzed the data using two other threshold values (1.25 and 2.0). Although the raw percentages of cells in each quadrant shifted with different choices of threshold values, the change in distribution induced by serum deprivation was statistically independent of which threshold value was chosen.
A possible confounding factor is that the differentiating agent, staurosporine, induces apoptosis at high concentrations. Low levels of apoptosis were seen even at the concentration (316 nM) used in this study, with 12.9% of cells cultured in complete media undergoing apoptosis by 72 h, based on cytochrome c
release into the cytoplasm. On the other hand, although this is higher than what is normally seen with undifferentiated cells in complete media (approximately 5%) [28
], it still is significantly lower than the 54.7% of serum-deprived cells that released mitochondrial cytochrome c
In summary, superoxide production in serum-deprived neuronal cells occurs before cytochrome c
release, and thus is an early signal in triggering apoptosis. How superoxide is generated and how it transduces the apoptosis signal is unclear, but presumably preventing or reversing superoxide signaling before neurons become committed to an apoptotic fate would be potentially useful as a therapy for trophic factor-deprived neurons in the central nervous system [19