Hsiu-Chiung Yang

Biochemical and kinetic characterization of BACE1: investigation into the putative species-specificity for β- and β′-cleavage sites by human and murine BACE1

β‐amyloid peptides (Aβ) are produced by a sequential cleavage of amyloid precursor protein (APP) by β‐ and γ‐secretases. The lack of Aβ production in beta‐APP cleaving enzyme (BACE1)–/– mice suggests that BACE1 is the principal β‐secretase in mammalian neurons. Transfection of human APP and BACE1 into neurons derived from wild‐type and BACE1–/– mice supports cleavage of APP at the canonical β‐secretase site. However, these studies also revealed an alternative BACE1 cleavage site in APP, designated as β′, resulting in Aβ peptides starting at Glu11. The apparent inability of human BACE1 to make this β′‐cleavage in murine APP, and vice versa, led to the hypothesis that this alternative cleavage was species‐specific. In contrast, the results from human BACE1 transgenic mice demonstrated that the human BACE1 is able to cleave the endogenous murine APP at the β′‐cleavage site. To address this discrepancy, we designed fluorescent resonance energy transfer peptide substrates containing the β‐ and β′‐cleavage sites within human and murine APP to compare: (i) the enzymatic efficiency; (ii) binding kinetics of a BACE1 active site inhibitor LY2039911; and (iii) the pharmacological profiles for human and murine recombinant BACE1. Both BACE1 orthologs were able to cleave APP at the β‐ and β′‐sites, although with different efficiencies. Moreover, the inhibitory potency of LY2039911 toward recombinant human and native BACE1 from mouse or guinea pig was indistinguishable. In summary, we have demonstrated, for the first time, that recombinant BACE1 can recognize and cleave APP peptide substrates at the postulated β′‐cleavage site. It does not appear to be a significant species specificity to this cleavage.

Deriving functional human enteroendocrine cells from pluripotent stem cells

ABSTRACT Enteroendocrine cells (EECs) are a minor cell population in the intestine yet they play a major role in digestion, satiety and nutrient homeostasis. Recently developed human intestinal organoid models include EECs, but their rarity makes it difficult to study their formation and function. Here, we used the EEC-inducing property of the transcription factor NEUROG3 in human pluripotent stem cell-derived human intestinal organoids and colonic organoids to promote EEC development in vitro. An 8-h pulse of NEUROG3 expression induced expression of known target transcription factors and after 7 days organoids contained up to 25% EECs in the epithelium. EECs expressed a broad array of human hormones at the mRNA and/or protein level, including motilin, somatostatin, neurotensin, secretin, substance P, serotonin, vasoactive intestinal peptide, oxyntomodulin, GLP-1 and INSL5. EECs secreted several hormones including gastric inhibitory polypeptide (GIP), ghrelin, GLP-1 and oxyntomodulin. Injection of glucose into the lumen of organoids caused an increase in both GIP secretion and K-cell number. Lastly, we observed formation of all known small intestinal EEC subtypes following transplantation and growth of human intestinal organoids in mice. Summary: Manipulation of developmental signaling pathways and transcription factor expression allows differentiation of human pluripotent stem cells into nutrient-responsive, intestinal enteroendocrine cells.