Ludtmann MH, Angelova PR, Horrocks MH, Choi ML, Rodrigues M, Baev AY, Berezhnov AV, Yao Z, Little D, Banushi B, Al-Menhali AS, Ranasinghe RT, Whiten DR, Yapom R, Dolt KS, Devine MJ, Gissen P, Kunath T, Jaganjac M, Pavlov EV, Klenerman D, Abramov AY, Gandhi S. α-synuclein oligomers interact with ATP synthase and open the permeability transition pore in Parkinson's disease. Nat Commun. 2018 Jun 12;9(1):2293. PubMed.
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Comments
Aarhus University
This paper by Ludtmann compares the effect of recombinant purified monomeric α-synuclein (AS) to a pool of monomers that have been allowed to aggregate by agitation at 37ºC. This pool thus contains a mixture of species including about 1 percent oligomers that are rich in β-sheets. Basically, the paper presents a comparison of the two preparations. First it is reported that the oligomers are toxic to primary co-cultures of rat neurons and astrocytes and induce the accumulation of NADH in mitochondria. Subsequently, a range of analyses are conducted that aims to demonstrate targeting of the mitochondrial ATP-synthase by aggregated AS. This interaction has previously been reported by our group (Betzer et al., 2015), but here it is reported that the interaction causes oxidation of amino acids in the ATP synthase. In fact it is demonstrated that the preparation of oligomers induces non-enzymatic production of reactive oxygen species in a cell free assay.
A problem with the paper is the oligomer preparation, because it is stated this preparation contains 0.01 percent sodium azide, which was added to prevent bacterial growth. This is apparently not present in the monomers—at least it is not stated. When applying the oligomers to the cells or mitochondria, the authors diluted them from 170 micromolar to 0.5 micromolar, or a 140-fold dilution. This means the sodium azide is diluted from 0.01 percent, equivalent to 1.5 mM, to about 10 micromolar. Barret reported that 10 micromolar sodium azide applied to N2A cells reduced the cytochrome C activity by 50 percent within 24 hours (Barret et al., 2004). This papers refers to other papers demonstrating sodium azide is a mitochondrial toxin. Moreover, sodium azide can directly oxidize amino acids when present during hydrolysis (Manneberg et al., 1995, and other papers by the same group) suggesting the non-enzymatic pro-oxidant activity of the oligomer preparation also could be caused by the bacteriostatic supplement.
The Ludtmann paper is, indeed, intriguing, but the reported findings may be caused by the supplemented mitochondrial toxin sodium azide rather than the oligomers.
References:
Betzer C, Movius AJ, Shi M, Gai WP, Zhang J, Jensen PH. Identification of synaptosomal proteins binding to monomeric and oligomeric α-synuclein. PLoS One. 2015;10(2):e0116473. Epub 2015 Feb 6 PubMed.
Barrett MJ, Alones V, Wang KX, Phan L, Swerdlow RH. Mitochondria-derived oxidative stress induces a heat shock protein response. J Neurosci Res. 2004 Nov 1;78(3):420-9. PubMed.
Manneberg M, Lahm HW, Fountoulakis M. Oxidation of cysteine and methionine residues during acid hydrolysis of proteins in the presence of sodium azide. Anal Biochem. 1995 Jan 1;224(1):122-7. PubMed.
In the study by Ludtmann et al., 2018, we reported the effect of the oligomeric conformation of α-synuclein on mitochondrial function, and its interaction with ATP synthase in two systems: exogenous application of recombinant protein aggregates to primary cultures, and the endogenous generated oligomers in human iPSC derived neurons.
The comment above has raised the possibility that the presence of azide in the buffer (used to inhibit bacterial growth during aggregation) of the recombinant aggregates may have confounded the results on mitochondrial function.
We draw the attention of the author and readers to the paper, which states: “As a paired control to the oligomer preparation, monomer sample from the starting time point of the aggregation was used.”
Utilizing the paired monomer in the same aggregation buffer allows us to (1) ensure that the monomeric protein, taken at time = zero hours, does not contain any aggregates by single molecule ThT imaging while the oligomeric sample taken at time = 8 hours does, and (2) standardize the buffer components (including the azide concentration) across all samples to prevent any confounding effects from the buffer. As all our experiments are compared to the monomer sample as the control, we are able to conclude with confidence that the effect of the oligomeric sample is solely due to the presence of the oligomers.
References:
Ludtmann MH, Angelova PR, Horrocks MH, Choi ML, Rodrigues M, Baev AY, Berezhnov AV, Yao Z, Little D, Banushi B, Al-Menhali AS, Ranasinghe RT, Whiten DR, Yapom R, Dolt KS, Devine MJ, Gissen P, Kunath T, Jaganjac M, Pavlov EV, Klenerman D, Abramov AY, Gandhi S. α-synuclein oligomers interact with ATP synthase and open the permeability transition pore in Parkinson's disease. Nat Commun. 2018 Jun 12;9(1):2293. PubMed.
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