Background: The early diagnosis of Alzheimer's disease (AD) is critical for the evaluation of new therapies and for patient follow-up. Aggregation of Aβ 1-40, Aβ 1-42 peptides in amyloid plaques, one of the major hallmarks of the pathology, begins more than 15years before neuropsychological symptoms. Few methods have been developed to detect amyloid plaques by MRI but they always required an invasive process to open the blood brain barrier (BBB). Heavy chain only antibody fragments (VHH), because of their small size and their basic isoelectric point, have the potential to cross the BBB. Hence, in order to detect amyloid plaques by MRI, we developed VHHs with high specificity against Aβ 1-40, Aβ 1-42. First, we evaluated the BBB crossing by in vivo multi-photon imaging after conjugation of our VHHs to a fluorochrome. Then, gadolinium (Gd) was conjugated to VHHs via a DOTA cage and the resulting contrast agent (VHH-DOTA/Gd) was evaluated by MRI.
      Methods: Conjugates were synthesized by conjugation of Alexa-Fluor488 or a DOTA/Gd synthon on VHHs. Conjugates were monitored by HPLC/MS, and binding properties were confirmed by ELISA and IHC. PS2APP mice aged of 90-100weeks were used (n=2/experiment). In vivo multi-photon experiments were realized after intravenous injections of VHH-AlexaFluor488. MRI acquisition were recorded following two different protocols 1) in vitro incubation of one hemisphere with VHH-DOTA/Gd (0.1mg/ml equivalent to 0.1mM of Gd) overnight, 2) Gd-staining, a reference technique to reveal amyloid plaques by soaking the hemisphere in a Gd-solution (2.5mM - 48h).
      Results: Live multi-photon imaging following intravenous injection showed gradual extravasation of the fluorescent VHHs from blood vessels and penetration in brain parenchyma with an exquisite tropism for amyloid plaques, as confirmed by IHC (Figure 1). MR images obtained after in vitro incubation with VHH-DOTA/Gd showed numerous hypointense spots in the cortex (Figure 2A). Moreover, several hypointense spots were colocalised (red arrows) with amyloid plaques revealed by the reference technique of Gd-staining (Figure 2B).
      Conclusions: This study demonstrates that VHHs cross the BBB, label amyloid plaques in vivo and can be detected by MRI following conjugation with a contrast agent. VHHs thus appear as promising tools with translational value for in vivo detection of amyloid deposits.