CREB5 Upregulation Is Reproducible Across Human Astrocyte Datasets in Alzheimer’s Disease

Human Alzheimer’s disease (AD) astrocytes are transcriptionally heterogeneous across brain regions, pathological stages and cohorts, complicating the distinction between reproducible disease signals and dataset-specific effects. We asked whether CREB5, identified by donor-level pseudobulk analysis in SEA-AD middle temporal gyrus (MTG), retained a positive astrocyte association across sequential regional, independent-cohort, confirmatory and prespecified external-validation analyses, and then examined the neuropathological context of this replicated signal. CREB5 was increased in SEA-AD MTG (log2FC 1.203, FDR 0.019), remained positive in SEA-AD dorsolateral prefrontal cortex and both regions of GSE160936, and was independently confirmed in a diagnosis- and target-insulated reanalysis of GSE157827 (log2FC 0.857, P = 3.78×10−5). Three additional accessions were then analyzed under a fixed internal validation plan. GSE188545 supported the association after Holm correction, whereas GSE138852 and GSE174367 yielded smaller, nonsignificant positive estimates. Their prespecified fixed-effect meta-analysis gave a pooled log2FC of 0.678 (95% CI 0.294–1.062; P = 5.36×10−4), with moderate heterogeneity (I² = 58.6%); random-effects sensitivity remained positive. Across six cohorts and eight primary regional or cohort analyses, all eight estimates were positive. After the primary validation sequence was frozen, author-provided sample-level astrocyte data from the five-region AD Progression Atlas (GSE268599; 32 donors) were analyzed with donor as the biological unit. CREB5 was higher in Pathology Groups 3–4 than in Group 1 (donor-level Welch P = 6.19×10−5), and a region-adjusted analysis with donor-clustered standard errors supported the same association (β = 0.379, 95% CI 0.209–0.548; P = 1.16×10−5). Portal-reported correlations with local Aβ plaque or pTau/total-tau burden remained nonsignificant after correction. A separate atlas-scale Malva audit showed heterogeneous CREB5 behavior across additional AD astrocyte studies and was retained as contextual rather than inferential evidence. Together, these findings support CREB5 as a reproducible human AD astrocyte-associated signal with broader neuropathological-stage context, while arguing against a universal effect or a simple linear relationship with local plaque or tau burden.