Characterization of Intra- and Extracellular Metabolic Profiles by ¹H NMR in Aβ1-42-Treated Human Astrocytes from Healthy and Alzheimer's Disease Subjects

Alzheimer’s disease (AD) is the leading cause of dementia and is characterized by progressive neurodegeneration, synaptic dysfunction, and metabolic impairment. Astrocytes play a critical role in maintaining brain homeostasis and are increasingly recognized as active contributors to AD pathophysiology, yet the metabolic changes underlying their dysfunction remain incompletely understood. In this study, we established an optimized Nuclear Magnetic Resonance (NMR)-based metabolomics workflow to profile both intracellular (endo-) and extracellular (exo-) metabolomes of primary human astrocytes derived from healthy subjects (HS) and AD patients. Cells were exposed to oligomeric Aβ_1–42 to model amyloid-induced stress. Using ^1H NMR spectroscopy at 600 MHz, we quantified 40 intracellular and 33 extracellular metabolites, enabling a comprehensive assessment of astrocyte metabolic responses. Aβ_1–42 treatment induced metabolic alterations in both HS and AD astrocytes, particularly affecting energy metabolism and amino acid pathways. Notably, intracellular phosphocreatine levels decreased consistently, suggesting impaired energy-buffering capacity under stress. AD-derived astrocytes also showed reduced β-alanine levels, potentially reflecting altered antioxidant defense mechanisms. Analysis of conditioned media revealed distinct extracellular responses, with AD astrocytes exhibiting increased levels of metabolites linked to tricarboxylic acid cycle activity and amino acid metabolism, whereas HS astrocytes displayed changes consistent with altered substrate utilization.Although none of these differences remained statistically significant after correction for multiple testing, likely due to the limited sample size, the observed trends are biologically plausible and provide preliminary insights into astrocyte metabolic adaptations to amyloid stress.Overall, this work establishes a robust and reproducible NMR-based workflow for integrated endo- and exo-metabolomic analysis of human astrocytes. The findings highlight the potential of metabolomics to uncover subtle metabolic alterations relevant to AD and provide a foundation for future studies in larger, well-characterized cohorts.