*4.5. Generation and Characterization of Human iPS-Derived Neurons*

Human iPS cells of Alzheimer's patients were generated as described from skin fibroblasts [43,57] and maintained using StemFit AK02N medium (Ajinomoto, Tokyo, Japan) [64] and expanded for neural differentiation. To establish a robust and rapid differentiation method, we utilized direct conversion technology. We differentiated iPS cells into neurons by using a direct conversion method, as previously described [43]. Briefly, we transduced human NGN2 cDNA by using the piggyBac transposon system, transiently under tetracycline-inducible promoter (tetO), and converted iPS cells into neuronal cells with more than 96% purity.

#### *4.6. Electrochemiluminescence Assays*

Aβspecies in culture media after 24 h cultivation with PKC ligands were measured by human (6E10) Aβ 3-VPlex Kit (Meso Scale Discovery; Rockville, MD, USA). This assay uses the 6E10 anti-β-amyloid antibody to capture Aβ peptides and SULFO-TAG-labeled C-terminus specific anti-Aβ antibodies for detection by electrochemiluminescence with Sector Imager 2400 (Meso Scale Discovery). Quantified Aβ values were adjusted using total protein in neurons and compared among conditions.
