Single Domain Antibody Fragments as New Tools for the Detection of Neuronal Tau Protein in Cells and in Mice Studies

ACS Chem Neurosci. 2019 Sep 18;10(9):3997-4006. doi: 10.1021/acschemneuro.9b00217. Epub 2019 Aug 19.

Abstract

Tau is a neuronal protein linked to pathologies called tauopathies, including Alzheimer's disease. In Alzheimer's disease, tau aggregates into filaments, leading to the observation of intraneuronal fibrillary tangles. Molecular mechanisms resulting in tau aggregation and in tau pathology spreading through the brain regions are still not fully understood. New tools are thus needed to decipher tau pathways involved in the diseases. In this context, a family of novel single domain antibody fragments, or VHHs, directed against tau were generated and characterized. Among the selected VHHs obtained from screening of a synthetic library, a family of six VHHs shared the same CDR3 recognition loop and recognized the same epitope, located in the C-terminal domain of tau. Affinity parameters characterizing the tau/VHHs interaction were next evaluated using surface plasmon resonance spectroscopy. The equilibrium constants KD were in the micromolar range, but despite conservation of the CDR3 loop sequence, a range of affinities was observed for this VHH family. One of these VHHs, named F8-2, was additionally shown to bind tau upon expression in a neuronal cell line model. Optimization of VHH F8-2 by yeast two-hybrid allowed the generation of an optimized VHH family characterized by lower KD than that of the F8-2 wild-type counterpart, and recognizing the same epitope. The optimized VHHs can also be used as antibodies for detecting tau in transgenic mice brain tissues. These results validate the use of these VHHs for in vitro studies, but also their potential for in-cell expression and assays in mouse models, to explore the mechanisms underlying tau physiopathology.

Keywords: Alzheimer’s disease; Variable domain of the heavy-chain only antibodies; nanobodies; nuclear magnetic resonance spectroscopy; tauopathies.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alzheimer Disease / genetics
  • Alzheimer Disease / metabolism
  • Alzheimer Disease / pathology
  • Amino Acid Sequence
  • Animals
  • Cell Line, Tumor
  • Immunoglobulin Fragments / genetics
  • Immunoglobulin Fragments / metabolism
  • Mice
  • Mice, Transgenic
  • Neurons / metabolism*
  • Neurons / pathology
  • Single-Domain Antibodies / genetics*
  • Single-Domain Antibodies / metabolism*
  • tau Proteins / genetics*
  • tau Proteins / metabolism*

Substances

  • Immunoglobulin Fragments
  • Single-Domain Antibodies
  • tau Proteins