Huettemann P, Mahadevan P, Lempart J, Tse E, Dehury B, Edwards BF, Southworth DR, Sahoo BR, Jakob U. Amyloid accelerator polyphosphate fits as the mystery density in α-synuclein fibrils. PLoS Biol. 2024 Oct;22(10):e3002650. Epub 2024 Oct 31 PubMed.
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MRC Laboratory of Molecular Biology
MRC Laboratory of Molecular Biology
Cryo-EM reconstructions of amyloid filaments made of the proteins tau, TDP-43, or α-synuclein that were extracted from human brains revealed the presence of additional densities, not attributable to these proteins themselves, whose identity remains unknown (Scheres et a;., 2024). Huettemann et al. propose that polyphosphate “fits” these densities in filaments made of α-synuclein with the multiple system atrophy (MSA) or Lewy folds. This carefully worded title is adequate in light of the presented data, which do not contain conclusive proof that the additional densities in filaments extracted from individuals with MSA or dementia with Lewy bodies are indeed polyphosphate. In fact, the paper contains no data on brain-derived filaments. Instead, the authors hypothesize that polyphosphate might fit the additional density based on molecular dynamics simulations. In addition, they show that recombinant α-synuclein filaments bind polyphosphate in vitro and that this binding is abolished when lysines 43 and 45 (which coordinate the additional densities in the MSA and Lewy folds) are mutated to alanines.
However, neither these computer simulations, nor the observations with recombinant protein constitute proof that polyphosphate is the compound that gives rise to the additional densities in filaments with the MSA and Lewy folds. A wide range of negatively charged molecules could fit these densities and be stable in computer simulations; filaments from recombinant α-synuclein have thus far adopted different folds than those extracted from human brains; and multiple negatively charged compounds have been found to interact with amyloid filaments in vitro; and such interactions likely occur through positively charged lysines.
Identification of the compounds responsible for the “mystery densities” is important, as this might shed light on the intriguing question why the same protein may form specific filament folds in different diseases. What would constitute solid proof for their identification? Preferably, one would provide direct evidence of the presence of any hypothesised compound in filaments that are extracted from the brains of individuals with the relevant disease. Possible approaches include mass spectrometry and analytical chemistry assays. Because brain-derived filament preparations typically contain many impurities, and a variety of compounds (especially negatively charged ones) may stick to these filaments in unspecific manners, the appropriate use of controls will be paramount to demonstrate significance of such findings.
This will not be an easy feat. Huettemann et al. themselves were “unable to directly visualize amyloid-associated polyP because it is not recognized by our polyP-binding probe” and they “considered it impossible to quantify any potential polyP associated with patient-derived fibrils as this method requires acid hydrolysis followed by inorganic phosphate determination which requires extremely pure starting material.” Therefore, it is likely that hypotheses derived from the detection of compounds in brain-derived filament samples will need to be backed up by additional data. One possibility would be to confirm that filaments of recombinant protein that are assembled in the presence of the hypothesised compounds adopt the same fold as observed in filaments extracted from human brains. Cryo-EM is the preferred method to confirm that the correct structures form, as negative stain EM or atomic force microscopy lack the required resolution. Again, this sets a high bar, as there may be other factors, besides the presence of these compounds, that determine which amyloid fold forms in vitro.
Also, although perhaps providing stronger support than computer simulations, the observation that disease-specific filaments form in vitro with the presence of a certain compound does not necessarily prove that this compound is indeed responsible for the additional densities in filaments extracted from human brain. One example here is the formation of filaments made of recombinant tau297-391 in the presence of sodium chloride (Lövestam et al., 2022). These filaments adopt the same fold as observed for filaments extracted from the brains of individuals with chronic traumatic encephalopathy (CTE) (Falcon et al., 2019), and there is compelling evidence to suggest that sodium chloride is responsible for the additional density inside the recombinant filaments. Yet, direct evidence that sodium chloride is also responsible for the additional density in brain-derived CTE filaments remains elusive.
In summary, although this paper proposes an interesting hypothesis, convincing proof is not provided and, for now, the nature of the additional densities in cryo-EM reconstructions of brain-derived filaments thus remains a mystery.
References:
Scheres SH, Ryskeldi-Falcon B, Goedert M. Molecular pathology of neurodegenerative diseases by cryo-EM of amyloids. Nature. 2023 Sep;621(7980):701-710. Epub 2023 Sep 27 PubMed.
Lövestam S, Koh FA, van Knippenberg B, Kotecha A, Murzin AG, Goedert M, Scheres SH. Assembly of recombinant tau into filaments identical to those of Alzheimer's disease and chronic traumatic encephalopathy. Elife. 2022 Mar 4;11 PubMed.
Falcon B, Zivanov J, Zhang W, Murzin AG, Garringer HJ, Vidal R, Crowther RA, Newell KL, Ghetti B, Goedert M, Scheres SH. Novel tau filament fold in chronic traumatic encephalopathy encloses hydrophobic molecules. Nature. 2019 Apr;568(7752):420-423. Epub 2019 Mar 20 PubMed.
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