A nanobody-based fluorescent reporter reveals human α-synuclein in the cell cytosol

Nat Commun. 2020 Jun 1;11(1):2729. doi: 10.1038/s41467-020-16575-0.

Abstract

Aggregation and spreading of α-Synuclein (αSyn) are hallmarks of several neurodegenerative diseases, thus monitoring human αSyn (hαSyn) in animal models or cell cultures is vital for the field. However, the detection of native hαSyn in such systems is challenging. We show that the nanobody NbSyn87, previously-described to bind hαSyn, also shows cross-reactivity for the proteasomal subunit Rpn10. As such, when the NbSyn87 is expressed in the absence of hαSyn, it is continuously degraded by the proteasome, while it is stabilized when it binds to hαSyn. Here, we exploit this feature to design a new Fluorescent Reporter for hαSyn (FluoReSyn) by fusing NbSyn87 to fluorescent proteins, which results in fluorescence signal fluctuations depending on the presence and amounts of intracellular hαSyn. We characterize this biosensor in cells and tissues to finally reveal the presence of transmittable αSyn in human cerebrospinal fluid, demonstrating the potential of FluoReSyn for clinical research and diagnostics.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Aged
  • Aged, 80 and over
  • Animals
  • Cells, Cultured
  • Cytosol / chemistry
  • Cytosol / metabolism*
  • Female
  • Fluorescence
  • HEK293 Cells
  • Humans
  • Luminescent Proteins / chemistry
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism*
  • Male
  • Microscopy, Fluorescence, Multiphoton
  • Middle Aged
  • Neurons / cytology
  • Neurons / metabolism
  • Rats, Wistar
  • Red Fluorescent Protein
  • Single-Domain Antibodies / genetics
  • Single-Domain Antibodies / metabolism*
  • alpha-Synuclein / cerebrospinal fluid
  • alpha-Synuclein / genetics
  • alpha-Synuclein / metabolism*

Substances

  • Luminescent Proteins
  • Single-Domain Antibodies
  • alpha-Synuclein