It has recently been reported that apoE4 enhances APP expression, leading to increased Aβ production, in a pure excitatory human neuron culture system43. However, we did not observe a similar effect of apoE4 on APP expression in a mixed human neuronal culture system. This could be due to the following differences between the two experimental systems. First, the induced excitatory neurons were generated by artificially expressing a transcription factor Ngn2 in hiPSCs, without inhibitory neurons in the culture43. As reported in previous in vitro and in vivo studies, Aβ production is regulated by neuronal activity—increasing neuronal activity stimulates Aβ production and secretion44,45. Thus, in this pure excitatory neuronal culture, in which there is no inhibitory neurons to control the activity, Aβ production pathway and its regulation could be altered in a way that might not be physiologically relevant. In contrast, in the mixed neuronal culture, the presence of inhibitory neurons balances neuronal activities, as seen under a physiological condition, making Aβ production more physiologically controlled. Second, in the pure excitatory neuron study, conditioned media containing 294 nM (10 µg/ml) of
