Researchers have linked genes for amyotrophic lateral sclerosis and Parkinson’s disease in the same pathway, a cellular mechanism to degrade decrepit mitochondria. Scientists already knew that the PD genes parkin and PTEN-induced putative kinase 1 (Pink1) were important in this process, called mitophagy. In the October 7 Proceedings of the National Academy of Sciences online, first author Yvette Wong and senior author Erika Holzbaur of the Perelman School of Medicine at the University of Pennsylvania in Philadelphia now report that the ALS gene optineurin participates, too, Optineurin acts downstream of parkin to recruit recycling machinery to broken-down mitochondria, according to the new research.
The study adds to a growing body of research pointing at the cell’s powerhouses. “Several neurodegenerative diseases seem to impinge on mitochondria at some level or other,” said Mark Cookson of the National Institute on Aging in Bethesda, Maryland, who was not involved in the study. “[The mitochondrion] is a nexus for degeneration, but not a specific kind of degeneration.”
Optineurin in Mitophagy
As mitochondria age, they are replaced by fresh ones. To dispose of old mitochondria, the cell surrounds them in structures called autophagosomes, which ultimately fuse with lysosomes and degrade the old structures. The process starts with Pink1. It accumulates on damaged mitochondria and recruits parkin. Parkin marks the doomed organelle for destruction by ubiquitinating a variety of proteins on its surface. Scientists have not completely filled in the next steps on the road to perdition, but Holzbaur and Wong suspected optineurin might be involved, because it participates in other kinds of autophagy (Korac et al., 2013; Tumbarello et al., 2012). Optineurin mutations can cause either ALS or glaucoma, depending on the part of the protein affected, and mitochondrial turnover lags in both conditions (see May 2010 news story; Osborne et al., 2013). Notably, the ALS gene p62 (see Nov 2011 news story) encodes a protein that manages mitochondrial aggregation during mitophagy (Okatsu et al., 2010; Narendra et al., 2010).
Wong used live imaging in cervical cancer HeLa cell cultures to observe the recruitment of parkin, optineurin, and other mitophagy proteins to dysfunctional mitochondria. She treated the cells with the drug carbonyl-cyanide m-chlorophenyl-hydrazone (CCCP), which depolarizes mitochondria, causing them to fragment and initiate mitophagy. Within one hour of CCCP treatment, fluorescent parkin attached itself to the outer membrane of nearly all the mitochondria she saw. Optineurin joined parkin, but took a bit longer, indicating it came on second (see image above).
The researchers used mutants of each protein to confirm their hunch that parkin prompted optineurin recruitment. In HeLa cells expressing T-240-R parkin, a PD-linked mutant that cannot ubiquitinate, mitochondria attracted no optineurin. The ALS-linked optineurin mutant, E-478-G, which cannot bind ubiquitinated proteins, did not stick to mitochondria. Wong suspects that parkin ubiquitinates several more proteins that then recruit optineurin.
The researchers predicted that optineurin functions as an autophagy receptor, gathering autophagosome proteins. Optineurin was required to recruit LC3, a part of the autophagosome machinery, and cells without optineurin did not clear damaged mitochondria. All told, the authors concluded that the cells use optineurin to complete autophagosome formation and proceed with mitophagy.
Holzbaur acknowledged two major caveats: HeLa cells and CCCP, a powerful poison, which together make for an artificial system. She is repeating the work in primary neurons from mice overexpressing mutant human superoxide dismutase 1, a cause of familial ALS.
Giovanni Manfredi of Weill Cornell Medical College in New York, who was not involved in the study, considered the findings important. “With a little more work, it should be possible to demonstrate that [optineurin-dependent mitophagy] occurs in neurons as well,” he predicted.
The drug CCCP affects all mitochondrial at once, and hence "has rightly been criticized as hitting the cell with a sledgehammer,” said Holzbaur. In situ, cells only degrade some washed-up mitochondria at any given time. In HeLa cells, Wong repeated some experiments with the protein Mito-Killer Red. When hit with a yellow-green laser, this reporter generates reactive oxygen species that cause local mitochondrial damage. This system drew optineurin to the damaged parts of the mitochondria in a parkin-dependent manner.
Questions and Speculations
Wong’s work establishes a clear pathway, agreed Cookson and Manfredi. Teepu Siddique of the Northwestern University Feinberg School of Medicine in Chicago added that it fits nicely into the ALS literature. “It seems to follow the trajectory of defects in quality control of proteins and organelles in ALS,” wrote Siddique, who did not participate in the study. However, unanswered questions loom.
“The field struggles with how this mitophagy pathway causes neurodegeneration,” said Cookson. Manfredi speculated that when impaired mitochondria pile up, they might release free radicals or apoptotic factors that damage neurons. The mitochondria might physically get in the way of axonal transport, or fuse with younger mitochondria and disrupt their activity. Alternatively, Manfredi hypothesized that defective mitophagy might create an extra burden on the proteasome, the only cellular disposal system left.
The study raises the question of how mutations in one pathway lead to two distinct diseases. Holzbaur mused about cellular differences between the dopaminergic neurons affected in PD and the motor neurons susceptible to ALS. Motor neurons, for example, might express proteins that substitute for Pink1 and parkin, allowing their mutant version to go unnoticed. Conversely, dopaminergic neurons might express some factor protecting them from optineurin deficiency. Cookson suggested that Pink1, parkin, and optineurin have functions outside this mitophagy pathway, and that those activities might account for the different diseases associated with each gene.
Although it remains hard to explain why similar mutations cause disparate conditions, this is a recurring theme in neurodegeneration. Mutations in dynactin can cause either motor neuron disease (Puls et al., 2003), for instance, or Perry syndrome, which involves depression and parkinsonism (see Jan 2009 news story).
Mitochondrial defects, in particular, seem to cause wildly varying symptoms. For example, researchers reported that valosin-containing protein (VCP) participates in clearance of damaged mitochondria, and VCP mutations cause various combinations of myopathy, dementia, bone disease, or ALS (Kim et al., 2013; Dec 2010 news story). Similarly, this summer scientists reported that mutations in coiled-coil-helix-coiled-coil-helix domain containing 10 (CHCHD10), a nuclear-encoded mitochondrial protein, can lead to myopathy, ALS, or ALS with frontotemporal dementia (see Jun 2014 news story; Oct 2014 news story). In the case of p62, mutations can lead to bone disease as well as ALS (Laurin et al., 2002). For now, Holzbaur said, she thinks neuroscientists can agree on one thing: “It is clear that neurons do not do well with defects in mitochondrial health.”—Amber Dance
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