Abstract

Abstract:

Introduction
Alzheimer’s disease (AD) is a progressive neurodegenerative disorder and the leading cause of dementia in seniors. Hallmarked by memory loss, cognitive impairment, and synaptic dysfunction, AD presents a growing public health challenge, with no effective disease- modifying therapies. Genetic studies have identified the apolipoprotein E (APOE) gene as the strongest risk factor for late-onset AD. The three major APOE alleles—APOE2, APOE3, and APOE4—confer varying risk levels: APOE4 significantly increases AD susceptibility, APOE3 is considered neutral, and APOE2 appears protective. However, the molecular mechanisms by which these isoforms differentially influence brain function and AD risk remain incompletely defined. This study aimed to investigate the transcriptomic landscapes associated with each APOE isoform in human APOE-targeted replaced aging mouse brains to elucidate their isoform-specific roles in AD pathogenesis.

Materials and Methods
Transcriptomic data were analyzed from two publicly available RNA-seq datasets (GSE188267 and GSE234711), comprising brain samples from human APOE-targeted replacement (TR) mice expressing APOE2, APOE3, or APOE4 under the control of the endogenous murine promoter. These mice, developed on a C57Bl/6 background, allow physiologically relevant expression of human APOE. Brain tissues were dissected from these mice at the specified time points 12- and 18-months old, and total RNA was extracted and sequenced. The two public datasets were re-analyzed by using R packages, and the differentially expressed genes (DEGs) were identified by FDR (false discovery rate) <0.05 and |log₂ fold change| > 0.25.

Results
Transcriptomic profiling revealed clear isoform-dependent expression differences, particularly in genes regulating immune responses, lipid metabolism, and neuronal function. In APOE2-expressing mice, we identified upregulation of several genes associated with protective and homeostatic functions: Prg2 (promotes innate immune activity and eosinophil function), Masp2 (activates the lectin complement pathway), Pla2g4e (regulates phospholipid metabolism), Angptl7 (modulates vascular homeostasis), Cxcl13 (guides B-cell chemotaxis), Itgax (CD11c; microglial activation marker), Il7r (supports lymphocyte homeostasis), Alox5 (leukotriene synthesis), Fgf7 (epithelial repair), Tekt1 (cytoskeletal structure), Pafah2 (reduces oxidative stress), Tlr2 (innate immune receptor), Cav1 (lipid raft formation and signaling), and Cd38 (NAD⁺ metabolism and calcium signaling). These findings suggest that APOE2 brains are primed for enhanced immune surveillance, lipid homeostasis, and neuroprotection, aligning with its observed protective effects in AD.
 
Conversely, APOE3 and especially APOE4 mice displayed gene expression patterns indicative of increased inflammation and metabolic imbalance. Upregulated genes included Ccl28 (chemokine recruiting CCR10+ lymphocytes), Tnfsf8 (T-cell costimulation), Nr4a1 (stress-responsive nuclear receptor), Cd59a (complement inhibition), Hdac1 (epigenetic gene regulator), Ccl9 (pro-inflammatory myeloid chemokine), and Hspa8 (heat shock protein involved in protein folding). These patterns reflect a pro-inflammatory, stress-prone environment in APOE3 and APOE4 brains.

Notably, APOE4 mice demonstrated a distinct signature marked by upregulation of multiple Serpina3 family members (Serpina3f, Serpina3k, Serpina3m, Serpina3c, Serpina3g, Serpina3n), known to inhibit proteases during inflammation and linked to reactive gliosis.
Additional upregulated genes included Ms4a15 (immune cell signaling), Gh (growth hormone involved in glial and neuronal function), and Pdk4 (regulates glycolytic shift under energy stress). These genes indicate elevated glial activation, metabolic dysregulation, and proteostatic burden, suggesting a transcriptomic environment conducive to neurodegeneration in APOE4 brains.

Conclusion
This study reveals that distinct APOE isoforms shape divergent molecular environments in the aging brain. APOE2 is associated with a gene expression profile favoring immune balance, lipid metabolism, and neuroprotection, whereas APOE3 and APOE4 promote pro- inflammatory and metabolically stressed states, with APOE4 showing the most pathogenic signature. These findings offer mechanistic insight into isoform-specific modulation of AD risk and provide a transcriptomic framework for developing targeted therapeutic strategies.