PROTEOMIC SIGNATURES OF AMYLOID BETA–INDUCED ASTROCYTE SENESCENCE DIFFER BETWEEN 2D CULTURES AND 3D SPHEROIDS

Astrocyte senescence contributes to brain aging and neurodegeneration, yet how cellular organization modulates stress-induced proteomic remodelling remains insufficiently understood. The aim of this study was to investigate amyloid beta (Aβ)–induced astrocyte senescence with a focus on proteomic and metabolic alterations in two-dimensional (2D) cultures and three-dimensional (3D) spheroid models. Human astrocyte cell lines CCF-STTG1 and IM-HA were exposed to Aβ₄₂, and senescence was evaluated using growth arrest, senescence-associated beta-galactosidase activity, DNA damage marker gammaH2AX, and expression of p16 and p21. Oxidative stress pathways were examined through analysis of NADPH oxidase 4 (NOX4) expression and hydrogen peroxide release. Proteomic profiling of astrocytes grown in 2D and 3D conditions was performed to identify culture-dependent differences in protein synthesis, mitochondrial function, and metabolic regulation. Aβ exposure induced a robust senescent phenotype in both cell lines, accompanied by DNA damage and activation of oxidative stress signalling. Proteomic analysis revealed pronounced differences between 2D and 3D astrocytes, with 3D spheroids displaying altered expression of cytosolic and mitochondrial ribosomal subunits, proteins involved in mitochondrial translation, and enzymes associated with glycolysis. These changes were more pronounced in the tumour-derived CCF-STTG1 cells compared to IM-HA astrocytes. Consistently, metabolic analyses demonstrated a shift toward glycolytic ATP production following Aβ treatment, particularly in 2D cultures. In contrast, 3D spheroids exhibited distinct proteomic and metabolic adaptations suggestive of increased cellular resilience to stress. In conclusion, amyloid beta–induced astrocyte senescence is associated with profound proteomic remodelling that is strongly influenced by culture dimensionality.