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<p>Parkinson’s disease involves the accumulation of aggregates of ɑ-synuclein (ɑ-Syn), both as intracellular fibrils and as cytotoxic soluble oligomeric species (ɑSOs). No available nanobodies show exclusive preference for the oligomeric state of ɑ-Syn. Here, we describe two nanobodies NB1 and NB2, obtained by immunizing a llama with αSOs, which bind ɑSOs with nM affinity and do not show any measurable affinity for monomeric ɑ-Syn or ɑ-Syn fibrils. While the nanobodies were not useful for high-throughput screening for therapeutic compounds or high-resolution cryoEM, they retained their ability to discriminate against ɑ-Syn monomers in brain tissue and were able to detect ɑ-Syn aggregates in diseased tissue. In addition, αSO binding affinity was improved by DNA-scaffold-mediated NB1 dimerization compared to scaffolded monomeric NB1. The nanobodies promote the uptake of ɑSOs into HEK93 cells via the Sortilin receptor pathway. Their absolute specificity for oligomeric ɑ-Syn makes them promising reagents to detect oligomeric ɑ-Syn in patient samples.</p>

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Nanobodies raised against the cytotoxic α-synuclein oligomer are oligomer-specific and promote its cellular uptake

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年6月2日

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  • Janni Nielsen,
  • Jannik N. Pedersen,
  • Giulia Kleijwegt,
  • Jan S. Nowak,
  • Faranak Nami,
  • Charlie Johansen,
  • Elisa Sassetti,
  • Bjørn Bjerre Berg,
  • Nanna Miang Lyngsø,
  • Bo Volf Brøchner,
  • Johanne Holm Carlson,
  • Amanda Simonsen,
  • William Pallisgaard Olsen,
  • Bine Wissendorf Simonsen,
  • Jakob Hauge Mikkelsen,
  • Vita Sereika-Bejder,
  • Johanne Aarup Lauritsen,
  • Kirstine Friis Merrild,
  • Mette Galsgaard Malle,
  • Julián Valero,
  • Jørgen Kjems,
  • Andreas Bøggild,
  • Thomas Boesen,
  • Svend Birkelund,
  • Gunna Christiansen,
  • Simon Boje Hansen,
  • Marianne Kristensen,
  • Peder Søndergaard Madsen,
  • Kristian Strømgaard,
  • Marina Romero-Ramos,
  • Camilla Gustafsen,
  • Simon Glerup,
  • Kasper Røjkjær Andersen,
  • Mads Hartvig Clausen,
  • Daniel E. Otzen

摘要

Parkinson’s disease involves the accumulation of aggregates of ɑ-synuclein (ɑ-Syn), both as intracellular fibrils and as cytotoxic soluble oligomeric species (ɑSOs). No available nanobodies show exclusive preference for the oligomeric state of ɑ-Syn. Here, we describe two nanobodies NB1 and NB2, obtained by immunizing a llama with αSOs, which bind ɑSOs with nM affinity and do not show any measurable affinity for monomeric ɑ-Syn or ɑ-Syn fibrils. While the nanobodies were not useful for high-throughput screening for therapeutic compounds or high-resolution cryoEM, they retained their ability to discriminate against ɑ-Syn monomers in brain tissue and were able to detect ɑ-Syn aggregates in diseased tissue. In addition, αSO binding affinity was improved by DNA-scaffold-mediated NB1 dimerization compared to scaffolded monomeric NB1. The nanobodies promote the uptake of ɑSOs into HEK93 cells via the Sortilin receptor pathway. Their absolute specificity for oligomeric ɑ-Syn makes them promising reagents to detect oligomeric ɑ-Syn in patient samples.