Single-cell analysis of sterol-induced Ca2+ signaling in human astrocytes by dynamic mode decomposition

Ca2+ signaling in astrocytes is a central mechanism of intercellular communication in the brain and plays a key role in regulating neuronal excitability, synaptic plasticity, and energy metabolism. Disruption of astrocytic Ca2+ dynamics is a characteristic of neurodegenerative diseases, as are deviations in cholesterol trafficking and metabolism, which are essential for maintaining membrane structure and function. Although recent studies have begun to explore links between Ca2+ signaling and sterol homeostasis in astrocytes, unbiased analytical workflows and mechanistic insight into how cholesterol and related sterols regulate astrocytic Ca2+ dynamics remain limited. Here, we apply dynamic mode decomposition to dissect and classify Ca2+ signals obtained from time-lapse imaging of human astrocytes. Using both synthetic and experimental datasets, we show that delay-embedded dynamic mode decomposition combined with clustering separates heterogeneous Ca2+ activity into distinct dynamical states. This analysis reveals that increasing cholesterol levels shift astrocytes toward more active oscillatory states, whereas acute cholesterol depletion suppresses Ca2+ activity. In addition, pretreatment with the oxysterols 24-, 25-, and 27-hydroxycholesterol impaired cholesterolinduced Ca2+ oscillations. Together, this work presents a general computational framework for decomposing and analyzing complex spatiotemporal Ca2+ signals, with broad applicability to quantitative imaging in cell biology.