The protein parkin just got a demotion. While it once was considered essential for disposal of worn-out mitochondria, a paper in the August 20 Nature proposes that while parkin does amplify the process of mitophagy, its partner PINK1 is perfectly capable of doing the job all on its own. Both PINK1 and parkin mutations cause familial Parkinson’s and are potential therapeutic targets. Based on the new results, drug discovery research could prioritize PINK1, suggested study co-first author Lesley Kane of the National Institute of Neurological Disorders and Stroke in Bethesda, Maryland. Moreover, Kane and colleagues in the laboratory of senior author Richard Youle cemented a previously reported link between mitophagy and two amyotrophic lateral sclerosis genes. They are the kinase TBK1 and its substrate optineurin (OPTN), which recruits the autophagosome to damaged mitochondria.
Improper disposal of old or damaged mitochondria contributes to both ALS and Parkinson’s (see Jul 2015 news; Aug 2013 news). Scientists are investigating how normal mitophagy occurs in order to eventually understand how disease perturbs it.
Researchers initially pictured healthy mitophagy as a linear pathway, wrote Noriyuki Matsuda and Keiji Tanaka of the Tokyo Metropolitan Institute of Medical Science in a commentary accompanying the Nature paper. In this scenario, damaged mitochondria accumulate PINK1, which phosphorylates parkin, which in turn ubiquitinates proteins on the organelle’s outer membrane. The ubiquitin signal brings to the scene proteins called autophagy receptors. These bind autophagy mediators such as LC3, which coat the membranes that gobble the unwanted mitochondria and become the autophagosome (see Feb 2010 news; Kazlauskaite et al., 2014).
However, recently researchers reported that PINK1 phosphorylates not only parkin, but also directly phosphorylates ubiquitin, too, and this action brings parkin to the mitochondrial surface (Shiba-Fukushiima et al., 2014; Okatsu et al., 2015; Koyano et al., 2014; Kane et al., 2014). Last year, scientists also discovered that optineurin acted downstream of parkin, indicating it could be one of the autophagy receptors (see Oct 2014 news). Youle’s lab now adds a loop-the-loop to that linear pathway, which relegates parkin to the role of optional player (see image above right). When it’s there, parkin does ubiquitinate mitochondrial proteins that then act as substrates for PINK1. But it is not necessary. PINK1 can also start mitophagy all by itself. In the latter case, the occasional ubiquitination of mitochondrial proteins, due to natural protein turnover, provides enough substrate for PINK1 to phosphorylate and get mitophagy going.
If something goes wrong with mitophagy, cells fill up with sickened mitochondria. To probe the components of the disposal process, the researchers created a basic cell system. Kane, with co-first authors Danielle Sliter of NINDS and Michael Lazarou, who has since moved to Monash University in Melbourne, Australia, began with HeLa cells. These make PINK1, but not parkin, so the authors added that gene for their experiments. Using gene editing (see Sep 2014 news), they knocked out all five known autophagy receptors, TAX1BP1, NDP52, NBR1, p62, and OPTN, so they could add each one back individually to see how it affected mitophagy. They called these quintuple knockouts pentaKOs.
The authors damaged the mitochondrial respiratory chain in the cells with a combination of oligomycin and antimycin A. In normal cells, this induces mitophagy. As indicators for mitophagy, they measured mitochondrial protein and DNA in the cells, quantifying the inner mitochondrial membrane protein cyclooxygenase II (COXII) by western blotting and the mitochondrial chromosome directly by immunofluorescence for DNA. If COXII and mitochondrial DNA were present, Kane and colleagues concluded that the cell had failed to dispose of the organelle; if those markers were gone, they assumed mitophagy was working. Other researchers have used mitochondrial DNA to assess whether the organelle remains intact (Wong and Holzbaur, 2014), and Youle’s group chose COXII as their protein marker because it resides inside the organelle and is encoded by mitochondrial DNA, Kane said. “It is hard to say that it is absolute proof of mitophagy,” she conceded, adding that is why the authors used two separate measures.
In normal cells, the drug cocktail destroyed much of the COXII and mitochondrial DNA within 24 hours, confirming active mitophagy. Not so in the pentaKOs. When the authors added autophagy receptors back individually, only NDP52 and OPTN, and to a lesser extent TAX1BP1, restored proper mitophagy (see image above). Therefore, these must be the autophagy receptors that are specific for phosphorylated ubiquitin chains on damaged mitochondria. Though the receptors have redundant functions in these cell cultures, they are expressed differentially throughout the body, with OPTN prevalent in neurons, Kane said.
Why were only certain autophagy receptors involved in mitophagy? The authors surmised it might have to do with the unique kind of phosphorylation, on serine 65, that PINK1 adds to ubiquitin molecules (Wauer et al., 2015). It may call out specifically to only these receptors. To test whether ubiquitin phosphorylated at Ser65 would be sufficient to recruit OPTN and NDP52, the researchers created HeLa cells in which a PINK1 construct was inducibly recruited to healthy mitochondria. They left parkin out of this experiment because it would overload the mitochondria with ubiquitin chains, Kane said. Rather, they relied on occasionally ubiquitinated outer-membrane proteins labeled for natural turnover as potential substrates for PINK1 Ser65 phosphorylation. When they dispatched PINK1 to the mitochondria, it did recruit OPTN and NDP52 on its own, whereas a deactivated PINK1 incapable of phosphorylation did not, demonstrating that PINK1 needs to phosphorylate a substrate to do so. To confirm that the substrate is ubiquitin, the authors expressed mutant OPTN and NDP52 missing their ubiquitin-binding domains; these could not find the mitochondria.
This indicated PINK1 phosphorylates ubiquitin and recruits autophagy receptors, all in the absence of parkin. But can the cell complete mitophagy without parkin? To find out, the authors turned to the fluorescent, pH-sensitive mitochondrial marker mKeima, which changes color when the mitochondria land in acidic lysosomes. Sorting cells by color, they observed that 3 to 5 percent of cells executed mitophagy with just OPTN and PINK1. Adding parkin raised the proportion of cells to nearly 24 percent. Thus, PINK1 can work alone, but parkin adds a big boost.
Youle and colleagues suspected that OPTN and NDP52 would work by recruiting LC3, but they were surprised. The mitochondria of double mutants missing both autophagy receptors still collected some LC3, but pentaKOs did not. The authors concluded that other receptors such as p62 might pull in LC3, but not induce mitophagy on their own.
Then what were OPTN and NDP52 needed for? In the NDP52/OPTN double knockouts, recruitment of other parts of the autophagy machinery, e.g., WIPI1 and DFCP1, was subpar. The PINK1 pathway leading to OPTN and NDP52 must somehow recruit these other components of the complete autophagosome, Kane said.
“This thorough study emphasizes an indispensable role for PINK1 in generating phospho-ubiquitin,” commented Helene Plun-Favreau of University College London, who was not involved in the work. “Parkin, on the other hand, seems to amplify the signal … as opposed to being indispensable for mitophagy.”
Dispensable, but still important. “Familial Parkinson’s disease can be caused by mutations in either PINK1 or parkin,” Matsuda and Tanaka wrote in their commentary. “Parkin clearly cooperates with PINK1 and has an important role in PINK1-mediated mitophagy under physiological conditions.”
And what of the ALS link? Since OPTN mutations cause familial ALS (see May 2010 news), the study authors probed how disease-linked mutations might affect mitophagy. ALS-linked OPTN mutants were not recruited to mitochondria and did not support mitophagy. The ALS gene TBK1 phosphorylates OPTN, activating it (see Feb 2015 news; Mar 2015 news). Cells lacking NDP52, which relied only on OPTN for mitophagy, could not destroy mitochondria if TBK1 was also missing. This confirmed that optineurin needs the kinase to degrade mitochondria.
The work suggests that OPTN and TBK1’s role in ALS involves mitophagy, and that mitophagy might be a point of overlap between the degeneration of motor neurons in ALS and that of dopaminergic neurons in Parkinson’s, commented Tim Harris of Biogen in Cambridge, Massachusetts. Harris did not participate in the study. Kane cautioned that more work would be necessary to confirm that the whole PINK1/parkin pathway contributes to ALS pathogenesis in people.
“Mitophagy is an extremely important process as far as neurodegeneration is concerned. We need to understand more about it,” Harris said. Youle’s paper on HeLa cells is a starting point, he said, but the same system should be studied in neurons. Kane responded, “We feel these cells are a good basic tool. We completely agree that future studies need to include not just neurons, but dopaminergic neurons.” She noted that a body of work in neurons and mice already indicates the PINK1/parkin pathway turns on in relevant cells (see Jan 2008 news; Pickrell et al., 2015).
1. Lazarou M, Sliter DA, Kane LA, Sarraf SA, Wang C, Burman JL, Sideris DP, Fogel AI, Youle RJ. The ubiquitin kinase PINK1 recruits autophagy receptors to induce mitophagy. Nature. 2015 Aug 20;524(7565):309-14. Epub 2015 Aug 12. [PubMed].
2. Matsuda N, Tanaka K. Cell biology: Tagged tags engage disposal. Nature. 2015 Aug 20;524(7565):294-5. Epub 2015 Aug 12. [PubMed].
To view commentaries, primary articles and linked stories, go to the original posting on Alzforum.org here.
Copyright © 1996–2016 Biomedical Research Forum, LLC. All Rights Reserved.