Human neurons lacking amyloid precursor protein exhibit cholesterol-associated developmental and presynaptic deficits

Abstract

Amyloid precursor protein (APP) produces aggregable {beta}-amyloid peptides and its mutations are associated with familial Alzheimers disease, which makes it one of the most studied proteins. However, APPs role in the human brain remains unclear despite years of investigation. One problem is that most studies on APP have been carried out in cell lines or model organisms, which are physiologically different from human neurons in the brain. Recently, human induced neurons (hiNs) derived from induced pluripotent stem cells provide a practical platform for studying the human brain in vitro. Here, we generated APP-null iPSCs using CRISPR/Cas9 genome editing technology and differentiate them to matured human neurons with functional synapses using a two-step procedure. During hiN differentiation and maturation, APP-null cells exhibited less neurite growth and reduced synaptogenesis in serum-free but not serum-containing media. We have found that cholesterol (Chol) remedies those developmental defects in APP-null cells, consistent with Chols role in neurodevelopment and synaptogenesis. Phenotypic rescue was also achieved by co-culturing those cells with wildtype mouse astrocytes, suggesting that APPs developmental role is likely astrocytic. Next, we examined matured hiNs using patch-clamp recording and detected reduced synaptic transmission in APP-null cells. This change was largely due to decreased synaptic vesicle (SV) release and retrieval, which was confirmed by live-cell imaging using two SV-specific fluorescent reporters. Adding Chol shortly before stimulation mitigated the SV deficits in APP-null iNs, indicating that APP facilitates presynaptic membrane Chol turnover during SV exo-/endocytosis cycle. Taken together, our study in hiNs supports the notion that APP contributes to neurodevelopment, synaptogenesis and neurotransmission via maintaining brain Chol homeostasis. Given the vital role of Chol in the central nervous system, the functional connection between APP and Chol bears important implication in the pathogenesis of Alzheimers disease.