Lack of N-glycosylation increases amyloidogenic processing of the amyloid precursor protein.

The amyloid precursor protein (APP) is a ubiquitously expressed type 1 transmembrane protein mostly known for serving as a precursor to the amyloid-ß peptide (Aß), a culprit in Alzheimer disease (AD). However, APP also has important physiological functions by being implicated in, for instance, adhesion, signaling, neuronal development, and synaptic function. Human APP contains 2 N-glycosylation sites, at asparagine (N) 467 (N467) and N496. Here, we studied the role of N-glycosylation on APP trafficking and processing by constructing APP-SNAP plasmid vectors for wildtype APP and N-glycosylation site mutants in which N467 or N496 was replaced by glutamine (Q) and expressed these in HEK293T cells. Lack of either of the 2 N-glycans resulted in a reduction in the size of intracellular APP-SNAP-positive vesicles and a reduction of APP-SNAP in the plasma membrane and lysosomes. Importantly, loss of either of the 2 N-glycans resulted in elevated levels of intracellular as well as secreted Aß42. These data suggest that N-glycans have a major impact on trafficking and processing of APP and could play an important role in the development of AD.

Engineered Human Induced Pluripotent Cells Enable Genetic Code Expansion in Brain Organoids.

Human induced pluripotent stem cell (hiPSC) technology has revolutionized studies on human biology. A wide range of cell types and tissue models can be derived from hiPSCs to study complex human diseases. Here, we use PiggyBac-mediated transgenesis to engineer hiPSCs with an expanded genetic code. We demonstrate that genomic integration of expression cassettes for a pyrrolysyl-tRNA synthetase (PylRS), pyrrolysyl-tRNA (PylT) and the target protein of interest enables site-specific incorporation of a non-canonical amino acid (ncAA) in response to an amber stop codon. Neural stem cells, neurons and brain organoids derived from the engineered hiPSCs continue to express the amber suppression machinery and produce ncAA-bearing reporter. The incorporated ncAA can serve as a minimal bioorthogonal handle for further modifications by labeling with fluorescent dyes. Site-directed ncAA mutagenesis will open a wide range of applications to probe and manipulate proteins in brain organoids and other hiPSC-derived cell types and complex tissue models.

Dual Bioorthogonal Labeling of the Amyloid-ß Protein Precursor Facilitates Simultaneous Visualization of the Protein and Its Cleavage Products.

The amyloid-ß protein precursor (AßPP) is critical in the pathophysiology of Alzheimer's disease (AD), since two-step proteolytic processing of AßPP generates the neurotoxic amyloid-ß peptide (Aß). We developed a dual fluorescence labeling system to study the exact subcellular location of γ-secretase cleavage of AßPP. The C-terminal tail of AßPP was fluorescently labeled using a SNAP-tag, while the Aß region of AßPP was fluorescently tagged with a dye at a genetically-encoded noncanonical amino acid (ncAA). The ncAA was introduced at specific positions in AßPP using a genetic code expansion strategy and afterwards, the reactive side-chain of the ncAA was coupled to the dye using a bioorthogonal labeling chemistry. In proof-of-concept experiments, HEK293T cells were transfected with plasmids containing engineered AßPP harboring an amber mutation and an amber codon suppression system with an evolved tRNA synthetase/tRNA pair and grown in the presence of a lysine-derived ncAA. Processing of the AßPP variants was validated with ELISA and immunoblotting, and seven AßPP mutants that showed similar cleavage pattern as wild-type AßPP were identified. The AßPP mutant was fluorescently labeled with 6-methyl-tetrazine-BDP-FL and TMR-Star at the ncAA and SNAP-tag, respectively. Using this approach, AßPP was fluorescently labeled at two sites in living cells with minimal background to allow monitoring of Aß and C-terminal cleavage products simultaneously. The method described provides a powerful tool to label Aß with minimal perturbations of its processing, thus enabling studies of the trafficking of the cleavage products of AßPP.