Choi WS, Kruse SE, Palmiter RD, Xia Z.
Mitochondrial complex I inhibition is not required for dopaminergic neuron death induced by rotenone, MPP+, or paraquat.
Proc Natl Acad Sci U S A. 2008 Sep 30;105(39):15136-41.
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The paper by Whitworth and colleagues adds to the work of Plun-Favreau suggesting that there is a relationship between the PINK1/parkin pathway and HtrA2 (aka Omi). Some of the data are very interesting, such as Fgure 4 which shows a change in processing of PINK1 in the absence of the protease Rhomboid-7. If this is confirmed, then we know at least one of the processing pathways for PINK1, which is interesting in terms of the two proposed pools of PINK1 reported in the literature. A few years ago we suggested that PINK1 might have both mitochondrial and cytoplasmic pools and that the processed form of PINK1 is especially abundant in the latter (Beilina et al., 2005). This result is confirmed in several other recent studies and now by Whitworth et al. (Fig 4B). We also know that the processed, cytosolic form is capable of protecting cells (Haque et al., 2008) and that the endogenous PINK1 protein is present at the cytoplasmic face of the organelle, as demonstrated recently by Zhou et al. (2008).
This does bring up a couple of questions that have been a concern for a while. One of the reasons that we didn’t previously emphasize the cytoplasmic pool of PINK1 (Beilina et al., 2005) is that we were worried that it might be influenced by the level of overexpression. This still might be the case—if PINK1 is at the mitochondrial surface, then when it is overexpressed, the protein might be cleaved by cytosolic proteases to promote degradation of the excess kinase. Although we don’t know what the normal target(s) for PINK1 is/are, presumably excess kinase activity would disturb cellular signaling and may be quite stressful for the cell. It remains to be clarified whether this occurs for endogenous protein at reasonable expression levels. Again, Zhou et al. show a mitochondrial localization for endogenous PINK1, including the mature form, although the kinase domain faces out to the cytoplasm. This brings out the general point of interpretation of overexpression systems, which is probably a very difficult problem for a kinase like PINK1 that is normally only expressed at very low levels in the cell. In our hands, PINK1 protein is prone to insolubility (especially when the leader peptide is present), presumably because it is not very abundant and there is no evolutionary constraint for the protein to be soluble. This means that triggering cell stress by increasing levels of expression may not be physiologically reasonable, even if it is done in vivo. I wonder what happens with stable recessive mutant versions of PINK1 in the same system. If these observations are related to normal function, then all mutant proteins that are themselves stable should have no effects. However, if the results are influenced by the artificial nature of overexpression systems, then the mutant proteins will probably worsen the situation as they tend to be slightly unfolded—the only ones that would not make things worse would be those that, like the L347P mutation found in the Filipino patients, are highly unstable.
Another helpful observation is that PINK1 knockout flies have distinct phenotypes including mitochondrial abnormalities. Examining how Omi affects this phenotype would be very interesting although there are caveats here as well. First, the reported HtrA2/Omi mutants associated with PD may in fact be rare but benign polymorphisms (Ross et al., 2008; Simon-Sanchez and Singleton, 2008, but see also Bogaerts et al., 2008), making it hard to interpret some results. Second, as Whitworth et al. say, it would be good to see this confirmed in another species where PINK1 is normally present. Some of the biology of PINK1, such as the genetic relationship with parkin, is clearly conserved across species (Exner et al., 2007). But others do appear to vary a little, such as the effects of pink1 deficiency on mitochondrial morphology (Exner et al., 2007), so it would be important to see if there is an interaction between Omi/HtrA2 and PINK1, parkin, and other components in mammalian systems. On this last point, it is interesting that using non-denaturing techniques, Van Humbeek et al. have shown that endogenous parkin is in an ~110 kDa complex and that other reported interactors from overexpression and immunoprecipitation studies are not major components. Therefore, it will be interesting to see if all of these proteins form a native supercomplex as predicted here.
Choi et al. report that rotenone induces the death of dopaminergic neurons, but surprisingly this appears to be independent of rotenone’s inhibitory actions on mitochondrial complex I activity. The most compelling data provided by the investigators in support of this conclusion is that dopaminergic neurons from Ndufs4-deficient mice, which lack any detectible mitochondrial complex I activity in cultured neurons or in purified mitochondria, remain sensitive (in fact, even more sensitive) to rotenone-induced cell death. This implies that rotenone can kill these neurons by a mechanism unrelated to its inhibition of mitochondrial complex I activity.
This is potentially a very important point. A large body of evidence implicated mitochondrial complex I dysfunction in Parkinson disease (PD). Complex I is impaired in the substantia nigra at early stages of PD. MPTP, a complex I inhibitor, kills dopaminergic neurons in experimental models, suggesting that a complex I defect in PD might play a role in the degeneration of dopaminergic neurons. On the other hand, the precise mechanism of action of MPTP has been questioned. Reassurance that the mechanism of relevance is complex I inhibition came from subsequent studies from Timothy Greenamyre’s laboratory indicating that chronic systemic infusion of rotenone in rats leads to progressive loss of dopaminergic neurons. However, the current study now also calls into question the mechanism of action of rotenone in that model.
Still, the issue is far from settled. Todd Sherer and colleagues in Dr. Greenamyre’s laboratory have demonstrated that human neuroblastoma cells expressing a rotenone-insensitive NADH dehydrogenase are completely protected against rotenone-induced cell death, indicating that the site of action for rotenone-induced cell death (in these human neuroblastoma cells) is mitochondrial complex I (Sherer et al., 2003). The reason for the discrepancies between the current data and the prior study of Sherer and colleagues is unclear. Choi et al. provide a scholarly discussion regarding issues of potential importance for interpreting their results. One point of concern is that 37 percent of total oxygen consumption measured in whole-brain fragments from neonatal Ndufs4-deficient mice is rotenone sensitive, suggesting that there may be substantial residual complex I activity in these mice. Though assays of complex I function did not reveal detectible complex I activity in cultured cells or in purified mitochondria from the Ndufs4-deficient mice, it remains possible that a residual complex I activity was present but undetected with their assays. In any case, this study represents an important note of caution. Though many lines of data implicate a role for mitochondrial dysfunction in PD, alternative explanations are possible, and future studies are needed to clarify the mechanism(s) of toxicity of rotenone and the precise role of mitochondrial complex I dysfunction in PD.
This is interesting work. The data presented indicate 24-hour 2.5-10 nanomolar rotenone, 48-hour 5-10 micromolar MPP+, and 24-hour 25-50 micromolar paraquat exposures kill mouse embryonic mesencephalic neuron cultures that lack the complex I Ndufs4 subunit. The data also suggest toxicity under these conditions may not require complex I substrate-induced oxygen consumption. How rigorously Ndufs4 knockout and the other experimental parameters used in this work model human idiopathic PD in general, and the human idiopathic PD complex I defect specifically, is unclear. At this time it is probably reasonable to interpret these data within a very narrow context.
With great interest, I read the paper by Choi et al. and an earlier paper describing the NDUFS4 knockout mice. The results from Choi et al. are very clear. Midbrain neuronal cultures from NDUFS4 KO or mitochondria isolated from these neurons do not have any complex I activity, as measured in two independent assays. Deleting the NDUFS4 gene in mice, as mutations of the gene does in humans, obliterates complex I activity in cultured neurons. Previous work from Tim Greenamyre’s group suggested that the complex I-inhibiting activity of rotenone is the key to the selective toxicity of rotenone on dopaminergic neurons. However, NDUFS4-deficient DA neurons are not resistant to rotenone or MPP+, another PD toxin that inhibits complex I. These striking results from Zhengui Xia and colleagues suggest that complex I inhibition is not the reason for rotenone’s selective toxicity on DA neurons. Our previous study has shown that the microtubule-depolymerizing effect of rotenone underlies its selective toxicity on DA neurons (Ren et al., 2005). Microtubule depolymerization blocks vesicular transport and results in accumulation of vesicles in the soma. Since oxidation of leaked monoamines produces a large amount of reactive oxygen species, constitutive leakage of neurotransmitter from the accumulated vesicles causes the selective death of monoaminergic neurons, including DA neurons (Ren and Feng, 2007). Non-monoaminergic neurons are spared because their neurotransmitters cannot be oxidized. An easy experiment to pin down whether complex I inhibitors still have toxicity on NDUFS4-deficient DA neurons is to use amytal or piericidin A, which do not impact microtubules. Both rotenone and MPP+ have been shown to disrupt microtubule function and are thus not good agents to dissect the involvement of complex I in the death of DA neurons.
The results of Choi et al. challenge the notion that complex I inhibition plays a significant role in Parkinson disease. In the absence of complex I activity, cultured midbrain DA neurons from NDUFS4 KO mice do not exhibit elevated oxidative stress compared to DA neurons from wild-type controls. Since respiration and ATP generation apparently are normal, it appears that NDUFS4-deficient neurons may utilize succinate and complex II to bypass the failed complex I. The in vivo situation in the NDUFS4 KO mice is more complicated, where ~37 percent of complex I activity remains. This is consistent with the greatly reduced complex I holoenzyme on blue native gel electrophoresis. Yet the number of TH+ neurons is normal when the NDUFS4 is selectively deleted in TH+ neurons in the conditional knockout mice. Thus, even with 73 percent inhibition of complex I in NDUFS4 knockout mice, DA neurons can still live fine. The conditional knockout mice behave normally as far as nine months. More results from these mice would shed greater insights on the role of complex I in the degeneration of DA neurons and Parkinson disease. Fundamentally, complex I inhibition does not appear to answer why DA neurons are selectively degenerated. The unique combination of morphological and neurochemical features seem to render nigral DA neurons much more vulnerable to microtubule-depolymerizing agents including rotenone (Feng, 2006).