Genetic code expansion in neurons for studying pathogenic mechanisms of Alzheimer disease

Abstract

Every three seconds a new patient is diagnosed with Alzheimer disease (AD). The primary risk factor for this irreversible, progressive brain disorder is old age. It is a growing health concern, not only for the patients, but also for their relatives and society, because the number of affected people is expected to double every 20 years. New tools are necessary to expand the knowledge about the molecular mechanisms behind AD. The small neurotoxic amyloid β-peptide (Aβ) is produced by processing of the Amyloid Precursor Protein (APP) through a cascade of secretase cleavages. However, it is not fully understood in which subcellular location the processing takes place and how this is changed during the course of the disease. A major problem is the lack of methods labeling small peptides like Aβ in living cells. To investigate where the processing takes place, we developed a dual fluorescence labeling system. The C-terminal tail of APP is fluorescently labeled using a SNAP-tag, while the Aβ region of APP is fluorescently tagged with a turn-on dye at a non-canonical amino acid (ncAA). The ncAA is introduced at specific positions in APP using a genetic code expansion (GCE) strategy. Using this approach, APP can be fluorescently labeled at two sites in living HEK293T cells with minimal background. However, a key to understand the underlying molecular mechanisms in AD are appropriate model systems. Therefore, we produced Baculoviruses to transduce primary cortical neurons from mice with the GCE machinery and APP with a SNAPtag and an amber codon. We reduced background labeling caused by tetrazine dyes in the neurons by developing a two-step labeling protocol to block excessive ncAA. In parallel, we also established GCE in human induced pluripotent stem cells (hiPSCs) and differentiated them to neurons and brain organoids. The stable hiPSCs expressing the GCE machinery provide a unique platform to study and manipulate proteins in different cell types and development stages.