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The interactome of stabilized α-synuclein oligomers and neuronal proteins

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  • Femke van Diggelen, Crossbeta Biosciences BV, Utrecht, The Netherlands.
  • ,
  • Signe Andrea Frank
  • ,
  • Arun Kumar Somavarapu
  • ,
  • Carsten Scavenius
  • Mihaela M Apetri, Crossbeta Biosciences BV, Utrecht, The Netherlands.
  • ,
  • Janni Nielsen
  • Armand W J W Tepper, Crossbeta Biosciences BV, Utrecht, The Netherlands.
  • ,
  • Jan J Enghild
  • Daniel E Otzen

While it is generally accepted that α-synuclein oligomers (αSOs) play an important role in neurodegeneration in Parkinson's disease, the basis for their cytotoxicity remains unclear. We have previously shown that docosahexaenoic acid (DHA) stabilizes αSOs against dissociation without compromising their ability to colocalize with glutamatergic synapses of primary hippocampal neurons, suggesting that they bind to synaptic proteins. Here, we develop a proteomic screen for putative αSO binding partners in rat primary neurons using DHA-stabilized human αSOs as a bait protein. The protocol involved co-immunoprecipitation in combination with a photoactivatable heterobifunctional sulfo-LC-SDA crosslinker which did not compromise neuronal binding and preserved the interaction between the αSOs-binding partners. We identify in total 29 proteins associated with DHA-αSO of which eleven are membrane proteins, including synaptobrevin-2B (VAMP-2B), the sodium–potassium pump (Na +/K + ATPase), the V-type ATPase, the voltage-dependent anion channel and calcium-/calmodulin-dependent protein kinase type II subunit gamma; only these five hits were also found in previous studies which used unmodified αSOs as bait. We also identified Rab-3A as a target with likely disease relevance. Three out of four selected hits were subsequently validated with dot-blot binding assays. In addition, likely binding sites on these ligands were identified by computational analysis, highlighting a diversity of possible interactions between αSOs and target proteins. These results constitute an important step in the search for disease-modifying treatments targeting toxic αSOs.

Original languageEnglish
JournalThe FEBS Journal
Pages (from-to)2037-2054
Number of pages18
Publication statusPublished - 2020

    Research areas

  • DHA stabilization, Rab-3A, co-immunoprecipitation, computational binding site prediction, proteomics, DEPENDENT ANION CHANNEL, RAB3A, ALZHEIMERS-DISEASE, PARKINSON DISEASE, TOXICITY, SYNAPTIC PLASTICITY, IN-VIVO, ELECTROSTATICS, BINDING, SUBUNIT

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