That Mystery “Density” in α-Syn Fibrils: Polyphosphate, Anyone?

In recent years, CryoEM has enabled scientists to decipher the structures of the fibrils that form various deposits in neurodegenerative disease. They have not only solved disease-specific folding structures for fibrils of Aβ, tau, TDP-43, TMEM106b, and α-synuclein, but also revealed mysterious components. Visible as electron-dense patches, these cluster within some of these fibrils from patients’ brains. What might they be? A paper published October 31 in PLoS Biology proposes that, for α-synuclein, these “mystery densities” comprise polyphosphate.

  • Polyphosphate would fit a pocket in α-synuclein that houses mysterious densities.
  • Removing positively charged residues from the pocket eliminates polyphosphate binding.
  • Proving that fibrils from patient brains contain polyphosphate may be difficult.

Led by Ursula Jakob and Bikash Sahoo, University of Michigan, Ann Arbor, the authors based their conclusions on computer modeling of how polyphosphate binds to α-synuclein, as well as on experiments with recombinant α-synuclein.

Forming chains of up to 1,000 molecules long, polyphosphate is found in the brain both inside and outside of neurons. Jakob and colleagues had previously shown that polymer accelerates the formation of α-synuclein fibrils. Curiously, they found it also protects neurons from the fibrils’ toxic effects (Cremers et al., 2016; Lempart et al., 2019). Jakob believes that polyphosphate supports and stabilizes α-synuclein fibrils, preventing them from fragmenting into smaller, more dangerous oligomers. “If this is really true, then as the brain polyphosphate levels decrease, people might get more predisposed to the toxic effects of the amyloids,” said Jakob. Levels of polyphosphate in the brains of rats decline with age, though it is not known if this is true for people (Lorenz et al., 1997). 

How would polyphosphate fit into α-synuclein fibrils? When researchers led by Sjors Scheres and Michel Goedert at the MRC Laboratory of Molecular Biology in Cambridge, U.K., determined the shape of the fibrils, Jakob’s team realized the structure might explain the effects of polyphosphate on synuclein fibrillogenesis (Schweighauser et al., 2020; Yang et al., 2022). The MRC scientists had found “mystery densities” that lay in positively charged pockets in the fibrils. Polyphosphate has a strong negative charge, which could allow it to bind to and neutralize those pockets, Jakob posited. The pockets are long and skinny, just the right size and shape to hold polyphosphate, she said.

“There are not a lot of physiologically known molecules that could fit all the criteria [for binding these pockets],” she said.

Just the Ticket? This artist’s rendition shows how polyphosphate (red) might punch its way through a long fibril comprising stacks of α-synuclein monomers (blue). The polyphosphate would fill a long, positively charged cavity that runs through the fibril. [Courtesy of Pavithra Mahadevan and Bikash Sahoo.]

To investigate, co-first authors Philipp Huettemann and Pavithra Mahadevan used the structures of α-synuclein fibrils and polyphosphate to simulate their binding. Molecular docking and molecular dynamics simulations showed that polyphosphate could bind to the fibrils in the same places as the mystery densities, forming hydrogen bonds with amino side chains on lysines 43 and 45.

Fitting In. Molecular modelling shows how polyphosphate (red) could fit into an α-synuclein fibril from a patient with multiple system atrophy. [Courtesy of Huettemann et al., 2024.]

Next, the scientists engineered α-synuclein to carry neutral instead of positively charged residues in the binding pocket. Polyphosphate neither bound this recombinant α-synuclein nor accelerated fibril formation nor protected neurons from fibril toxicity.

“Although this paper proposes an interesting hypothesis, convincing proof for this is not provided,” Scheres and Goedert wrote to Alzforum. For now, the nature of the additional densities in cryo-EM reconstructions of brain-derived filaments therefor remains a mystery.

To demonstrate what the densities are made of, scientists would ideally show direct evidence in filaments that are extracted from the brains of individuals with the relevant disease, Scheres and Goedert added. Jakob said her team wasn’t able to use patient-derived proteins because their techniques required extremely pure fibrils. Samples from patients’ brains contain many impurities, including inorganic phosphates that are difficult to distinguish from polyphosphate once it has been hydrolyzed from the protein and quantified.

Fibrils that form in vitro are known to adopt different shapes than fibrils found in people’s brains. If the scientists had shown that wild-type synuclein adopts the same structures as patient-derived fibrils when exposed to polyphosphate in vitro, that would have been stronger evidence for the polyphosphate ID, wrote Scheres and Goedert. Huettemann and colleagues tried this, but were unable to solve the structure by CryoEM.

Jakob acknowledged the study’s limitations. “We are not saying definitively that this is polyphosphate in those patient-derived fibrils,” she said. She thinks it is a strong candidate and hopes more scientists will tackle the question. Scheres and Goedert acknowledged that identifying the mystery molecule(s) will not be an easy feat.—Nala Rogers

Nala Rogers is a freelance science writer based in Silver Spring, Maryland.

Comments

  1. 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.

    View all comments by Sjors Scheres View all comments by Michel Goedert

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References

Paper Citations

  1. Cremers CM, Knoefler D, Gates S, Martin N, Dahl JU, Lempart J, Xie L, Chapman MR, Galvan V, Southworth DR, Jakob U. Polyphosphate: A Conserved Modifier of Amyloidogenic Processes. Mol Cell. 2016 Sep 1;63(5):768-80. Epub 2016 Aug 25 PubMed.
  2. Lempart J, Tse E, Lauer JA, Ivanova MI, Sutter A, Yoo N, Huettemann P, Southworth D, Jakob U. Mechanistic insights into the protective roles of polyphosphate against amyloid cytotoxicity. Life Sci Alliance. 2019 Oct;2(5) Print 2019 Oct PubMed.
  3. Lorenz B, Münkner J, Oliveira MP, Kuusksalu A, Leitão JM, Müller WE, Schröder HC. Changes in metabolism of inorganic polyphosphate in rat tissues and human cells during development and apoptosis. Biochim Biophys Acta. 1997 Apr 17;1335(1-2):51-60. PubMed.
  4. Schweighauser M, Shi Y, Tarutani A, Kametani F, Murzin AG, Ghetti B, Matsubara T, Tomita T, Ando T, Hasegawa K, Murayama S, Yoshida M, Hasegawa M, Scheres SH, Goedert M. Structures of α-synuclein filaments from multiple system atrophy. Nature. 2020 Sep;585(7825):464-469. Epub 2020 May 27 PubMed.
  5. Yang Y, Shi Y, Schweighauser M, Zhang X, Kotecha A, Murzin AG, Garringer HJ, Cullinane PW, Saito Y, Foroud T, Warner TT, Hasegawa K, Vidal R, Murayama S, Revesz T, Ghetti B, Hasegawa M, Lashley T, Scheres SH, Goedert M. Structures of α-synuclein filaments from human brains with Lewy pathology. Nature. 2022 Oct;610(7933):791-795. Epub 2022 Sep 15 PubMed. bioRxiv. PubMed

Further Reading

Primary Papers

  1. 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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