# An NMR-Based Protocol for Profiling the Endo- and Exo-Metabolomes in Aβ1-42 Treated Human Astrocytes from Healthy and Alzheimer’s Disease Donors

## TL;DR

This study uses NMR to compare metabolic changes in astrocytes from healthy and Alzheimer’s patients after exposure to Aβ1-42, revealing potential differences in energy and antioxidant metabolism.

## Contribution

An optimized NMR-based protocol is introduced for profiling endo- and exo-metabolomes in human astrocytes under Aβ1-42 treatment.

## Key findings

- Aβ1-42 treatment reduced phosphocreatine in both AD and healthy astrocytes, suggesting impaired energy buffering.
- AD astrocytes showed a decrease in β-alanine, indicating altered antioxidant defense.
- Extracellular metabolite responses differed between AD and healthy astrocytes, but results were not statistically significant after correction.

## Abstract

Background/Objectives: Astrocytes play a critical role in maintaining brain homeostasis and are increasingly recognized as active contributors to neurodegenerative processes. Metabolic dysfunction in astrocytes has been implicated in the onset and progression of Alzheimer’s disease (AD), yet the underlying metabolic alterations remain poorly characterized. Methods: We used an optimized protocol for untargeted metabolomic profiling of both intracellular and extracellular compartments of primary human astrocytes derived from AD patients and healthy subjects (HS) using 1H nuclear magnetic resonance (NMR) spectroscopy. Cells were treated with oligomeric Aβ1-42 to model pathological conditions. Results: Aβ1-42 treatment induced intracellular metabolic alterations in both AD and HS astrocytes, including a consistent reduction in phosphocreatine, potentially indicating impaired energy-buffering capacity. Notably, a decrease in β-alanine was observed only in AD astrocytes, suggesting alterations in carnosine-related antioxidant defence. Analysis of conditioned media revealed differential responses between groups: AD astrocytes showed increased extracellular levels of 2-oxoglutarate, citrate, and glycine, whereas HS astrocytes exhibited reduced extracellular levels of leucine and isoleucine, suggesting distinct adaptive metabolic responses to Aβ-induced stress. However, none of these differences remained statistically significant after correction for multiple testing. Conclusions: These findings suggest that NMR-based metabolomics can detect subtle metabolic shifts in human astrocyte models of AD and HS exposed to amiloidogenic challenge. Given the limited sample size and the exploratory design adopted, the results should be interpreted as preliminary and require validation in larger, better-matched cohorts. Nevertheless, this study provides a methodological framework and generates biologically plausible hypotheses regarding astrocyte metabolic responses relevant to AD pathophysiology.