Di Maio R, Barrett PJ, Hoffman EK, Barrett CW, Zharikov A, Borah A, Hu X, McCoy J, Chu CT, Burton EA, Hastings TG, Greenamyre JT. α-Synuclein binds to TOM20 and inhibits mitochondrial protein import in Parkinson's disease. Sci Transl Med. 2016 Jun 8;8(342):342ra78. PubMed.

Recommends

Please login to recommend the paper.

Comments

  1. The study by Di Maio et al. is an important contribution to our understanding of the role of mitochondria and α-synuclein (α-syn) in the pathogenesis of Parkinson’s disease. The authors report here an aberrant interaction of certain modified or wild-type forms of α-syn with impaired mitochondrial protein import, giving us potential insight into two converging pathways that have been associated with early PD pathogenesis: α-syn modifications/misfolding and mitochondrial dysfunction. In a series of elegant experiments, mainly using a technique that allows probing for protein-protein interaction in ex vivo samples, the proximity ligation assay (Söderberg et al., 2006), the authors establish a robust interaction between α-syn and TOM20 that seems to correlate with pathology and lead to toxicity. Furthermore the authors are able to give us pointers toward one of the oldest questions in PD: What is it about dopaminergic neurons in the substantia nigra (SN), among all α-syn-expressing cells, that makes them particularly susceptible to α-syn mediated toxicity? Earlier work by the Surmeier group (Guzman et al., 2010) pointed toward the particular pace-making activity of SN neurons that leads to increased calcium flux and mitochondrial oxidative stress, something that, interestingly, recently has been linked to α-syn-mediated toxicity (Luth et al., 2014). The second, entirely novel connection presented here again converges at the mitochondria: Low baseline interaction between TOM20 and its co-receptor TOM22, which makes mitochondria especially susceptible to the disruptive effect of pathological α-syn species, seems to be a hallmark of dopaminergic neurons. In the future, strengthening the interaction of both TOM transporters or lowering the concentration of pathological α-syn could be promising therapeutic routes that need to be explored.

    An important question remains, though: What forms of α-syn mediate this toxic interaction? It has been a challenge for the field to address the question of what exactly the different pathology-associated α-syn species are. While the authors established the aberrant interaction of oligomeric, dopamine-modified, and S129E (phosphomimetic) α-syn (but not nitrosylated or fibrillar α-syn) with mitochondria in patient tissue, what these variants have in common remains elusive. The authors speculate that trimeric or tetrameric structures may be important (at least for dopamine-modified and S129E, which are found as defined species and should be part of the “smear of different species” in the oligomeric preparation). It seems unlikely that these species are the same physiological tetramer described by us (Bartels et al., 2011) and others (Burré et al., 2014), given that the former have significantly less secondary structure than the physiological tetramer. Oligomers, dopamine-modified, and S129E variants are characterized as unfolded by the authors, which leaves open the possibility that these interact with the mitochondrial membrane and form helical species just as the monomer does, pointing toward a normal physiological membrane interaction of α-syn that is modified in the disease condition, as described before (Kamp et al., 2010). Given that the authors describe a relatively strong and specific interaction of α-syn with TOM20, it should be possible to identify a specific structural determinant in the α-syn species that mediates the interaction, and it will be interesting to see if the authors (or another group) can provide that in subsequent studies. 

    A caveat is the mode of delivering purified recombinant α-syn to cultured cells. Given α-syn’s conformational flexibility and strong context-dependent structure, this approach is fraught with peril. While the synthetic forms of α-syn in model systems do recapitulate the toxicity seen in vivo in α-syn-overexpression systems, it is not clear that this extends to the α-syn-TOM20 interaction seen in patient brain. Further characterization of the synthetic species that mediate toxicity in vitro and finding common properties with TOM-20 associated α-syn species in patient samples would be necessary to specifically target these species in disease. But maybe that is not even necessary, as the authors correctly state that  ”it appears that strategies aimed at a general reduction of α-synuclein, rather than targeting specific modifications or aggregation states, may be most efficacious.” Thus, the most important new insight and potential new avenue toward PD disease-modifying drugs might be that stabilizing TOM20-TOM22 interactions and/or upregulating TOM20 expression could counter the detrimental effects of toxic α-syn species on mitochondria. 

    References:

    Söderberg O, Gullberg M, Jarvius M, Ridderstråle K, Leuchowius KJ, Jarvius J, Wester K, Hydbring P, Bahram F, Larsson LG, Landegren U. Direct observation of individual endogenous protein complexes in situ by proximity ligation. Nat Methods. 2006 Dec;3(12):995-1000. PubMed.

    Guzman JN, Sanchez-Padilla J, Wokosin D, Kondapalli J, Ilijic E, Schumacker PT, Surmeier DJ. Oxidant stress evoked by pacemaking in dopaminergic neurons is attenuated by DJ-1. Nature. 2010 Dec 2;468(7324):696-700. PubMed.

    Luth ES, Stavrovskaya IG, Bartels T, Kristal BS, Selkoe DJ. Soluble, prefibrillar α-synuclein oligomers promote complex I-dependent, Ca2+-induced mitochondrial dysfunction. J Biol Chem. 2014 Aug 1;289(31):21490-507. Epub 2014 Jun 18 PubMed.

    Bartels T, Choi JG, Selkoe DJ. α-Synuclein occurs physiologically as a helically folded tetramer that resists aggregation. Nature. 2011 Aug 14;477(7362):107-10. PubMed.

    Burré J, Sharma M, Südhof TC. α-Synuclein assembles into higher-order multimers upon membrane binding to promote SNARE complex formation. Proc Natl Acad Sci U S A. 2014 Oct 7;111(40):E4274-83. Epub 2014 Sep 22 PubMed.

    Kamp F, Exner N, Lutz AK, Wender N, Hegermann J, Brunner B, Nuscher B, Bartels T, Giese A, Beyer K, Eimer S, Winklhofer KF, Haass C. Inhibition of mitochondrial fusion by α-synuclein is rescued by PINK1, Parkin and DJ-1. EMBO J. 2010 Oct 20;29(20):3571-89. PubMed.

    View all comments by Ulf Dettmer

  2. We appreciate the comments by Tim Bartels and Ulf Dettmer, and we agree with everything they have said. As noted in our paper, and as Bartels and Dettmer reiterate, it will be very important to define the structural characteristics and commonalities of the species of α-synuclein that exert mitochondrial toxicity. Studies attempting to do so are underway.

    Bartels and Dettmer also point out that there are caveats associated with delivering purified recombinant proteins to cells, a point with which we agree. However, one major advantage of our approach is that it does not rely on overexpression, which may introduce artifactual protein interactions. In fact, the use of extrinsic α-synuclein did not measurably alter the levels of α-synuclein in cells. Additionally, our protocol allowed us to control, to a large extent, the specific species of post-translationally modified α-synuclein under study.

    A key outstanding question is: What species of α-synuclein are interacting with TOM20 in neurons in the parkinsonian brain? Given that only a tiny fraction of α-synuclein in a cell interacts with TOM20, and α-synuclein is an abundant protein, and TOM20 is an intrinsic membrane protein, conventional co-immunoprecipitation approaches are unlikely to be enlightening. On the other hand, as new α-synuclein antibodies are developed and validated, it may be possible to identify the toxic species in situ. In this regard, we have been able to demonstrate strong proximity ligation signal between endogenous S129-phospho-α-synuclein and TOM20 in ex vivo brain sections of substantia nigra, thereby confirming what we found in vitro with recombinant S129E-α-synuclein (Di Maio, Barrett et al., unpublished). It is hoped that other validated, conformer-specific antibodies will be similarly useful. Finally, as we pointed out, and as reiterated by Bartels and Dettmer, strategies aimed at reducing overall levels of α-synuclein may be useful, regardless of which specific forms of α-synuclein are the culprits. Similarly, strategies designed to mitigate α-synuclein toxicity to mitochondria, such as increasing levels of TOM20 or expressing a mitochondrial targeting signal peptide, may be useful therapeutically.

    Roberto Di Maio and Paul Barrett contributed to this comment.

    View all comments by Tim Greenamyre

Make a Comment

To make a comment you must login or register.