That Touch of Pink1—Toning Mitochondrial Respiration

If figuring out a role for Pink1 leaves you breathless, it may be because the protein is essential for healthy mitochondrial respiration. Mutants in Pink1, or PTEN-induced kinase 1, cause recessive Parkinson disease (PD), but exactly how is not clear. In yesterday’s online EMBO Molecular Medicine, the newest journal published by the European Molecular Biology Organization, researchers led by Bart De Strooper at the Center for Human Genetics, K.U. Leuven, Belgium, offer up a possible explanation. Their data suggest that Pink1 is essential for the proper function of mitochondrial complex I. This is the major conduit for electrons entering the respiratory chain, the primary powerhouse of the cell. By strangling respiration, Pink1 mutants limit formation of synaptic vesicles, which is highly dependent on energy, and tone down neurotransmission, the scientists argue. This picture fits with the well-known links between mitochondria and PD, which can be induced by blocking complex I. By highlighting complex I, the results might also speed the identification of Pink1 substrates, suggests Anne Murphy, University of California, San Diego, in a journal “Closeup” to accompany the paper in the print version.

Researchers have tried to pin down a function for Pink1 ever since it was genetically linked to PD in 2004 (see ARF related news story). The protein is targeted to mitochondria, and recent data from several labs converged on the idea that Pink1 regulates mitochondrial fission/fusion and is essential for proper mitochondrial morphology and dynamics (see ARF related news story and ARF news story). “Our major point is that we believe the role of mutant Pink1 is primarily on loss of complex I function. That then leads to the secondary effects that other groups have reported, such as mitochondrial fission/fusion and parkin recruitment,” said first author Vanessa Morais in an interview with ARF.

Some researchers may be hard to convince. “I agree that they don’t see morphological change, and definitely see a complex I defect and a defect in mitochondrial potential. But I have a problem with the idea that complex I is where the primary defect is,” said Leo Pallanck, University of Washington, Seattle, when interviewed by ARF. Research from Pallanck’s lab has linked Pink1 to mitochondrial fission. “If they had a clear molecular mechanism to link Pink1 to complex I, then they can make that argument. If not, then they do not know that a dozen other things are happening first,” he said. Morais noted she believes the complex I deficit is primary because she and her colleagues can detect it in the absence of any morphological changes to mitochondria—even subtle changes that might only be seen in the electron microscope.

Morais and colleagues used a variety of animal and cellular models to link Pink1 with complex I. In Pink1 mutant fruit flies the scientists found normal baseline synaptic transmission at neuromuscular junctions (NMJs), but when neurons were firing intensely there appeared a small but significant reduction in transmission compared to control. The deficit correlated with failure to mobilize the reserve pool of synaptic vesicles that support intense neurotransmission. Morais and colleagues were able to rescue reserve pool function when they expressed wild-type human Pink1 in the affected neurons or by priming them first with ATP. Both experiments point to mitochondrial respiration as a potential weakness in these animals.

The researchers looked for changes in mitochondrial morphology to explain the synaptic vesicle deficit. They saw normal morphology in Pink1 mutant flies and normal numbers of mitochondria in synaptic regions. In rodents, it was the same story. Mitochondria in fibroblasts taken from Pink1 mutant mice had normal morphology, and electron microscopy revealed the same was true in mitochondria from mouse brain and heart muscle. “The synaptic dysfunction on the other hand clearly demonstrates that a Pink1 functional deficit is already present when morphology of the mitochondria is not yet affected, strongly arguing that this defect is upstream of morphological alterations observed by others,” write the authors.

Morais and colleagues then turned to mitochondrial function as a possible explanation for the synaptic defect. They found a drop in mitochondrial membrane potential in organelles in both Pink1 mutant flies and Pink1-negative mice, which made them sensitive to calcium-dependent apoptotic stimuli. This is in keeping with a recent report that Pink1 protects against calcium-dependent programmed cell death (see ARF related news story). To test if the drop in membrane potential is due to slow flux through the respiratory transport chain, the researchers measured consumption of oxygen, the last electron acceptor in the chain. Feeding mitochondria with complex I electron donors glutamate or malate, they found lower oxygen consumption in Pink1-negative organelles compared to wild-type. In contrast, complex II or complex III/IV electron donors elicited the same amount of oxygen consumption in all mitochondria. Using direct enzymatic measurement, the scientists confirmed that the activity of complex I, but not the other respiratory chain complexes, was significantly lower in mitochondria from Pink1-negative cells compared to controls.

“I think for me the most interesting finding in their report is the synaptic phenotype they found in the Drosophila neuromuscular junction,” Jie Shen, Brigham and Women’s Hospital, Boston, told ARF. “They found a synaptic transmission defect only after a prolonged stimulation at a higher frequency—not basal conditions—and a defect in the reserve pool of synaptic vesicles, which can be rescued by ATP. Since evoked release by repeated stimulation requires more energy, their findings would be consistent with the ATP rescue and the mitochondrial defects that we and others have reported,” she said.

Shen and colleagues have previously reported mitochondrial complex I deficits and dopamine release impairment in Pink1 mutant mice (see Gautier et al., 2008). As for whether the primary deficit in Pink1 mutants centers on that complex, Shen says it is possible but she is cautious. “Our message was basically that while loss of Pink1 makes mitochondria more vulnerable, Pink1 is not essential for mitochondrial function,” she said. Her group found no difference between mitochondrial respiration in the cerebral cortex of young wild-type mice and young Pink1-negative mice. When the animals aged, the Pink1-negative mitochondria in the cortex developed deficits. In young mice, the scientists did see complex I and complex II deficits in the striatum, “likely due to more oxidative stress associated with dopamine metabolism,” said Shen.

Pallanck pointed out that the link between complex I and PD may be weaker than is generally believed. While inhibitors of the complex, such as rotenone, MPTP, and paraquat, are often used to induce PD-like symptoms in animal models, researchers led by Zhengui Xia and Richard Palmiter at the University of Washington, Seattle, showed that those same symptoms can be elicited by complex 1 toxins even in complex I knockout animals (see ARF related news story). The study suggests that the toxins may have other actions. “I found that study compelling,” said Pallanck.

While the precise role of Pink1 may be debatable, one thing stays clear: Pink1 mutations cause autosomal-recessive parkinsonism (Lesage and Brice, 2009). Interestingly, De Strooper and colleagues showed that while wild-type Pink1 can rescue complex I deficits in Pink1-negative fibroblasts, Pink1 genes carrying either of two pathogenic mutations (G309D or W437X) could not, suggesting that the complex I deficit may be important in human disease.—Tom Fagan

Comments

  1. We appreciate the comments of Dr. Pallanck on our work, but would like to add some additional information that will allow the reader to put this criticism in perspective. First, it should be noted that no molecular mechanism has been provided for the putative role of Pink1 in mitochondrial fission, and that our work provides an experimentally well-supported alternative mechanism to explain the available observations. We refer to our paper for further discussion.

    More importantly, some additional background on the experiments of Choi et al., as cited by Dr. Pallanck, will shed more light on the interpretation of these data. The animal toxin models that use mitochondrial Complex I inhibitors, such as MPTP and rotenone, to induce PD-like symptoms are widely used to study PD. Studies performed by researchers led by Zhengui Xia and Richard Palmiter have reported that upon deletion of an assembly factor of Complex I, Ndufs4, dopaminergic neurons remain sensitive to well-established Complex I mitochondrial inhibitors, arguing that dopaminergic neuron loss is not due to Complex I inhibition (Choi et al., 2008). However, the original report by Palmiter and colleagues (Kruse et al., 2008) shows clearly that tissue from Ndufs4-/- mice retains approximately 50 percent of respiration driven by Complex I substrates. This is in complete accordance with the 50 percent reduction of assembled Complex I retrieved by blue native PAGE in the tissue from these mice. Of note, rotenone completely inhibits the residual respiration of Ndufs4-/- mitochondria, indicating that this Complex I inhibitor is still working in this genetic background. The complete lack of Complex I activity in submitochondrial particles is likely a consequence of the harsh procedure (e.g., sonication) required for their preparation and in no way should be taken as a proof of lack of Complex I in these mitochondria. As correctly pointed out by the authors of the Choi et al. study, “…. Thus, one could argue that partially assembled complex I, although lacking complex I activity and the ability to generate NAD+, could still transfer electrons. This may explain the toxicity of rotenone and MPP+ in Ndufs4-/-; neurons.”

    In conclusion, these findings do not indicate that the residual and putatively unstable Complex I that is formed in these mice is not capable of being affected by these specific inhibitors. On the contrary, rotenone does inhibit respiration and therefore electron transfer at Complex I even in Ndufs4-/- mitochondria. Therefore, it is likely that rotenone and MPTP do not have Complex I independent effects on mitochondria, and that their effect on dopaminergic neuron viability is solely related to Complex I inhibition.

    View all comments by Vanessa Morais

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References

News Citations

  1. Pink Mutations Link Parkinson’s Disease to Mitochondria
  2. Pink Fission—Serving Up a Rationale for Parkinson Disease?
  3. Research Brief: A Swell Protein—Mitochondrial Fission Falls to Pink1, Again
  4. Parkinson Protector: Pink1 Regulates Calcium, Cell Survival
  5. Parkinson Pathways Branch Out in Flies and Mice

Paper Citations

  1. Gautier CA, Kitada T, Shen J. Loss of PINK1 causes mitochondrial functional defects and increased sensitivity to oxidative stress. Proc Natl Acad Sci U S A. 2008 Aug 12;105(32):11364-9. PubMed.
  2. Lesage S, Brice A. Parkinson's disease: from monogenic forms to genetic susceptibility factors. Hum Mol Genet. 2009 Apr 15;18(R1):R48-59. PubMed.

Further Reading

Primary Papers

  1. Morais VA, Verstreken P, Roethig A, Smet J, Snellinx A, Vanbrabant M, Haddad D, Frezza C, Mandemakers W, Vogt-Weisenhorn D, Van Coster R, Wurst W, Scorrano L, De Strooper B. Parkinson's disease mutations in PINK1 result in decreased Complex I activity and deficient synaptic function. EMBO Mol Med. 2009 May;1(2):99-111. PubMed.

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