Fanlo-Ucar, H. | bioRxiv Science

bioRxiv2025

Network-Based Analysis of Human Astrocytes Links Aging to Neurodegenerative and Cardiovascular Diseases

Astrocytes are central to brain homeostasis, supporting neuronal metabolism, synaptic activity, and the blood-brain barrier. With aging, these glial cells undergo molecular and functional changes that weaken support functions and promote neuroinflammation, contributing to neurodegeneration. Yet the systems-level mechanisms of astrocytic aging remain poorly defined in human models. Because aging also heightens risk for cardiovascular disease, cognitive impairment, type 2 diabetes, and systemic inflammation, clarifying shared astrocytic pathways is critical for understanding brain-body crosstalk. Using an in vitro human astrocyte model exposed to sublethal oxidative stress (10 {micro}M H2O2), we profiled transcriptomic changes and identified differentially expressed genes across antioxidant defences, proteostasis, transcriptional regulation, vesicular trafficking, and inflammatory signalling. We then performed seven network-prioritization analyses on a curated human protein-protein interactome: one seeded with the astrocyte H2O2-responsive genes and six with phenotype-associated gene sets (Alzheimers disease, cardiovascular disease, cognitive impairment, type 2 diabetes, oxidative stress, and inflammation). Intersecting the top 5% scoring genes from each run yielded a 127-gene core shared across all seven, enriched for proteostasis, DNA repair, mitochondrial regulation, and telomere and nuclear envelope maintenance. Structure-guided analyses highlighted vulnerable interfaces, including lamin A/C-lamin B1, -actinin-filamins, 14-3-3 dimers, and aminoacyl-tRNA synthetase assemblies, where pathogenic variants are predicted to destabilize or aberrantly stabilize protein interactions. Structure-based interface predictions also highlight potential interactions between APP-VCP/p97 and p53-14-3-3{zeta} that link proteostasis and stress signalling. Together, these findings define a conserved astrocytic vulnerability network that may couple neurodegeneration with cardiovascular disease and nominate structurally testable targets for biomarkers and interventions.

bioinformatics↗

bioRxiv2025

Phospho-eIF2B-epsilon links stress response-driven translation to amyloidogenesis in Alzheimer's disease

A central etiopathogenic event in Alzheimers disease (AD) is the accumulation of amyloid {beta}-peptide (A{beta}) derived from the amyloidogenic processing of the amyloid precursor protein, a pathway initiated by BACE1. Chronic activation of the Integrated Stress Response is linked to AD, typically through eIF2 phosphorylation, which selectively enhances translation of key stress-responsive mRNAs like ATF4 and BACE1. We investigated a novel regulatory mechanism mediated by Glycogen Synthase Kinase-3{beta} (GSK-3{beta}) a hyperactive kinase in AD, on the translational factor eIF2B, the guanine nucleotide exchange factor (GEF) for eIF2. The methodology combined cellular and molecular biology approaches (western blot, ELISA, immunofluorescence, and luciferase assays) using pharmacological inhibitors and plasmid transfection in established cell models, along with computational structural modeling (AlphaFold3). Findings were validated in post-mortem human brain tissue, with statistical analyses (t-tests and ANOVA) applied throughout. We treated cells (SH-SY5Y) with a GSK-3{beta} inhibitor (NP031112) or overexpressed a constitutively active GSK-3{beta} mutant (GSK-3{beta}-S9A) to modulate GSK-3{beta} activity. GSK-3{beta} inhibition significantly reduced ATF4 and BACE1 protein levels in SH-SY5Y cells in a dose-dependent manner as we tested by western blot (P<0.001), independent of eIF2 phosphorylation. This effect was translational, as the inhibitor still reduced BACE1 levels when transcription was blocked. Crucially, NP031112 reversed stress-induced eIF2B{varepsilon} phosphorylation at Ser540 (P<0.001). Furthermore, H2O2-induced A{beta}1-42 secretion by SH-SY5Y cells was significantly reversed by GSK-3{beta} inhibition (P<0.001). Luciferase reporter assays using the BACE1 5 untranslated region (5 UTR) confirmed that both eIF2B{varepsilon} silencing and a phosphomimetic mutant eIF2B{varepsilon}-S540E increased BACE1 5 UTR-driven translation (P<0.001), demonstrating that reduced functional eIF2B enhances BACE1 translation. Finally, immunohistofluorescence and western blot analysis of human AD hippocampi showed a significant increase in pS540-eIF2B{varepsilon}, ATF4, and BACE1 levels in AD patients compared to non-demented controls (P<0.001 for all three). Our findings establish a novel GSK-3{beta}-eIF2B{varepsilon}-BACE1 axis that links stress response dysregulation to amyloidogenesis, independent of canonical eIF2 phosphorylation. The phosphorylation of eIF2B{varepsilon} functionally mimics the effects of eIF2 phosphorylation by reducing the available functional eIF2B pool, thereby reprogramming translation to favour the production of stress proteins like ATF4 and BACE1. We proposed eIF2B{varepsilon} phosphorylation is a key etiological mechanism in AD.

neuroscience↗