Fit-Derived Phasor Analysis of Metabolic Signatures of Cerebral Endothelial Dysfunction in Alzheimer’s Disease: A Proof-of-Concept Study

Vascular dysfunction at the level of cerebral endothelial cells is an early component of Alzheimer’s disease pathogenesis. Fluorescence lifetime imaging microscopy (FLIM) with phasor analysis enables label-free assessment of cellular metabolism based on NAD(P)H autofluorescence. This proof-of-concept study aimed to evaluate the potential of FLIM with fit-derived phasor analysis to compare cerebral endothelial cells (CECs) derived from a healthy donor and a patient with Alzheimer’s disease (AD) and to characterize their responses to metabolic modulators. CECs were differentiated from induced pluripotent stem cells obtained from a healthy donor (PO2) and a patient with AD (Alz9L, APP V717I mutation). Two-photon NAD(P)H FLIM was performed under basal conditions and following treatment with 3-chloro-5-hydroxybenzoic acid (3CLBA) and sodium lactate. The analysis included mean lifetime (τmean), phase lifetime (τφ), biexponential fitting parameters, and phasor coordinates reconstructed from these parameters. Under basal conditions, the phasor distributions of PO2 and Alz9L cells differed significantly despite similar mean NAD(P)H lifetimes. Alz9L cells exhibited a lower τφ and a higher lifetime of the short-lived component, τ1. Following sodium lactate treatment, Alz9L cells exhibited a higher a1/a2 ratio and lower τmean and τ1 values than PO2 cells. Comparison of the basal and post-lactate states revealed pronounced decreases in τmean and τ1 in Alz9L cells. Fit-derived phasor analysis jointly visualizes fitted NAD(P)H decay parameters and illustrates differences between the two studied CEC lines despite similar basal mean lifetimes. These proof-of-concept observations require validation in additional cell lines from independent donors.