Blood-brain barrier penetrating neprilysin degrades monomeric amyloid-beta in a mouse model of Alzheimer’s disease

Abstract BackgroundAggregation of the amyloid-b (Ab) peptide in the brain is one of the key pathological events in Alzheimer’s disease (AD). Reducing Ab levels in the brain by enhancing its degradation is one of the important strategies to develop new therapies for AD. Neprilysin (NEP) is a membrane bound metallopeptidase and one of the major Ab-degrading enzymes. The secreted soluble form of NEP (sNEP) has been previously suggested as a potential protein-therapy degrading Ab in AD. However, similar to other large molecule, peripherally administered sNEP is unable to reach the brain due to the presence of the blood-brain barrier (BBB). Methods To provide transcytosis across BBB, we recombinantly fused the TfR binding moiety (scFv8D3) to either sNEP or to a previously described variant of NEP (muNEP) having higher degradation efficiency on Ab compared to other NEP substrates. To provide long blood half-life, an Fc-based antibody fragment (scFc) was added to the designs, forming sNEP-scFc-scFv8D3 and muNEP-scFc-scFv8D3. Ability of the mentioned recombinant proteins to degrade Ab was first evaluated in vitro using synthetic Ab peptides followed by sandwich ELISA. For the in vivo studies, a single injection of 125-iodine labelled sNEP-scFc-scFv8D3 and muNEP-scFc-scFv8D3 was intravenously administered to tg-ArcSwe mouse model of AD, using scFc-scFv8D3 protein that lacks NEP as a negative control. Different ELISA setups were applied to quantify Ab concentration of different conformations, both in brain tissues and blood samples. ResultsWhen tested in vitro, sNEP-scFc-scFv8D3 retained sNEP enzymatic activity in degrading Ab. When intravenously injected, sNEP-scFc-scFv8D3 demonstrated 20-times higher brain uptake compared to sNEP. Both scFv8D3 fused NEP-proteins significantly reduced Ab levels in the blood of tg-ArcSwe mice, a transgenic mouse model of AD, following a single intravenous injection. However, the degradation of brain Ab was only evident with sNEP-scFc-scFv8D3 treatment, and both scFv8D3 fused NEP-proteins displayed a fast clearance from the brain. Conclusion Our study highlights the significance of BBB-penetrant proteins to study in details the effects of NEP on AD brains, avoiding the use of invasive intracranial administration or generation of NEP transgenic mice.