Mitochondria in dopaminergic neurons go haywire in Parkinson’s disease, ramping up oxidative stress and eventually killing the cells. New evidence puts α-synuclein at the scene of the crime. In the June 8 Science Translational Medicine, researchers led by Timothy Greenamyre at the University of Pittsburgh report that oligomeric forms of α-synuclein bind the mitochondrial receptor TOM20. In rat and culture models, this interaction gummed up protein import into the organelle, eventually causing cellular respiration to flag and reactive oxygen species to rise. Knockdown of α-synuclein, or overexpression of TOM20, prevented mitochondrial problems. Importantly, the authors found α-synuclein and TOM20 bound together in tissue from Parkinson’s brains as well, suggesting the same process occurs in human disease. “Now we know α-synuclein damages mitochondria through a specific mechanism. That raises the possibility we could target it therapeutically,” Greenamyre told Alzforum.
Other researchers appreciated the insight the data provide into basic disease mechanisms of Parkinson’s. “This identifies another key target for α-synuclein that falls in with a set of pathways genetically linked to the disease, and opens up new avenues for investigation,” said Benjamin Wolozin at Boston University. Julie Andersen at the Buck Institute for Research on Aging, Novato, California, noted how this addresses unanswered questions. “Previous studies have linked increases in α-synuclein to mitochondrial dysfunction, but the mechanisms has been elusive until now.”
Much of the previous research on α-synuclein has focused on its effects on autophagy and vesicle recycling (see Jun 2012 news; Apr 2013 news; Feb 2014 news). Meanwhile, a parallel line of research linked Parkinson’s risk genes such as parkin and PINK1 to dysfunction and improper disposal of mitochondria (see Mar 2014 news; Oct 2014 news; Sep 2015 news). Some studies tied the two together, reporting an association between excess α-synuclein and mitochondrial stress, but it was not clear which came first, or what the mechanisms might be (see Hsu et al., 2000; Betarbet et al., 2006; Zaltieri et al., 2015).
Greenamyre was intrigued by the fact that when α-synuclein interacts with membranes, it can assume a helical shape (see Davidson et al., 1998). He noted this shape resembles the structure of the mitochondrial targeting signal (MTS) found on proteins destined for the organelle. Proteins containing an MTS tag bind TOM20 and its partner TOM22, triggering import into mitochondria. Greenamyre wondered if α-synuclein might bind TOM20 as well.
To test this idea, joint first authors Roberto Di Maio and Paul Barrett examined dopaminergic neurons from the brains of two rat Parkinson’s models, one produced by administering the poison rotenone, the other by viral overexpression of α-synuclein in the substantia nigra. The authors used a proximity ligation assay to detect the interaction of α-synuclein and TOM20. In this technique, antibodies against the two proteins of interest carry probes that hybridize when in close contact to generate a fluorescent signal. In both PD models, the authors saw an eightfold surge of α-synuclein and TOM20 binding over control levels. At the same time, levels of Ndufs3, a mitochondrial complex I protein, dropped, suggesting a disruption in the normal flow of proteins into mitochondria. Knocking down α-synuclein in rotenone-treated rats by about one-third abolished the α-synuclein-TOM20 interaction and restored normal mitochondrial import (see image above).
What forms of α-synuclein cause problems? To answer this, the authors turned to in vitro assays using purified, recombinant α-synuclein that they modified using various protocols. In isolated mitochondria and in dopaminergic cell cultures, monomeric, nitrated, or fibrillar α-synuclein did not bind TOM20 and had no effect on mitochondrial import. On the other hand, α-synuclein that had been oligomerized in vitro, phosphorylated at site S129, or exposed to dopamine glommed onto TOM20 and cut mitochondrial import in half. After exposure to these forms of α-synuclein that blocked protein import, TOM20 ceased to bind its partner TOM22, as seen in another proximity-ligation assay. When TOM20-TOM22 binding failed, mitochondrial respiration dropped by one-third, and protein oxidation in mitochondria doubled.
The authors wondered what the toxic species of α-synuclein had in common. They did not see any obvious differences in secondary structure between toxic and non-toxic species. Except for the fibrillar form, all appeared to exist in a largely unfolded state, with only small amounts of helical structure. However, in all three preparations of the toxic species, about one-quarter to one-third of the synuclein was trimers and tetramers, as judged by size on non-denaturing SDS gels. Non-toxic preparations, on the other hand, comprised mostly monomers, dimers, and high-molecular-weight material. Small oligomers appear to be the culprits in mitochondrial dysfunction, the authors concluded.
The authors believe these toxic species are unlikely to be the physiological tetramers described by Dennis Selkoe at Brigham and Women’s Hospital in Boston and others (see Apr 2015 conference news). Unlike those labile forms, the toxic variety are highly stable, even resisting boiling, Greenamyre told Alzforum. He plans to analyze their structure further, and is also examining how genetic variants of α-synuclein that cause PD interact with mitochondria, and what forms they assume.
How relevant are the findings to human disease? The authors examined postmortem sections from the substantia nigra of five PD brains and four controls. As in the animal models, they found about eightfold more α-synuclein-TOM20 complexes using the proximity ligation assay, and 50 percent less mitochondrial Ndufs3, as assessed by immunocytochemistry, in the PD brains. Greenamyre is currently characterizing which species of α-synuclein bind TOM20 in these brain samples.
The identification of this mitochondrial mechanism suggests new therapeutic strategies, Greenamyre believes. In dopaminergic cell cultures treated with toxic forms of α-synuclein, a fourfold overexpression of TOM20 prevented mitochondrial import problems and maintained cellular respiration. Surprisingly, expressing just an MTS sequence also protected cells from α-synuclein. Because MTS binds TOM20, the authors expected it might also poison mitochondria. Instead, the peptide appeared to block α-synuclein binding to TOM20, while not interfering with the TOM20-TOM22 interaction. The reason is not clear, but possibly MTS jumps quickly on and off TOM20, in contrast to the more stable binding of α-synuclein, Greenamyre speculated. He is currently testing these treatment strategies in animal models and looking for improvements in pathology and behavior.
The interaction between α-synuclein and mitochondria may provide clues as to why dopaminergic neurons in particular fail in PD, Greenamyre noted. In control rats, these neurons gave off only a weak TOM20-TOM22 interaction signal compared to other neurons, suggesting their mitochondrial import machinery might be feeble and easily perturbed. Synuclein toxicity, such as that caused by the abundant dopamine in these cells, might be the culprit, Greenamyre hypothesized (see May 2002 news). To test this, the authors knocked down α-synuclein in the substantia nigra of wild-type rats. The TOM20-TOM22 interaction picked up, and protein oxidation dropped by about half, suggesting rescue of mitochondrial function. Wolozin found the data intriguing. “This may answer the age-old question: What is it about dopaminergic neurons that renders them most vulnerable to Parkinson’s disease?” he said.—Madolyn Bowman Rogers
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