A Nanobody-Based Toolbox to Probe ApoE4 in the Secretory Pathway and Cytosol

Apolipoprotein E4 (apoE4) is the strongest genetic risk factor for late-onset Alzheimer's disease (AD). Yet the molecular mechanisms underlying its contribution to AD remain to be fully elucidated. Here, we developed and characterized a set of apoE-specific single-domain antibodies (nanobodies) as a molecular toolbox to investigate intracellular apoE4. The nanobodies bind human apoE with nanomolar to sub-nanomolar affinity and recognize both apoE3 and apoE4. Domain-level epitope mapping revealed nanobodies that selectively bind either an N-terminal (residues 1-173) or C-terminal (residues 170-299) apoE4 fragment. Several nanobodies were validated as endoplasmic reticulum-targeted intrabodies that bind apoE4 intracellularly and promote its intracellular retention. These nanobodies constitute a versatile toolbox for probing and manipulating apoE4 in cellular models. As an exploratory application of this nanobody toolbox, we examined cytosolic apoE4, motivated by previous studies suggesting that cytosolic apoE4 fragments may influence AD-related cellular processes. Cytosolic expression of apoE4 resulted in perinuclear protein assemblies and the appearance of a ~25 kDa apoE4 fragment. Using a nanobody-based nuclear relocalization assay, we showed that cytosolic apoE4 remains accessible for nanobody binding but was not relocated to the nucleus by a nuclear localization signal-equipped nanobody. Altogether, this study introduces a nanobody-based toolbox to investigate apoE4 in distinct intracellular contexts, which can be relevant to AD.