Serum Autophagy-Related Protein 5 and Clinically Defined Cognitive Status in Older Adults: A Plate-Stratified Cross-Sectional Study Across: Normal Cognition, Mild Cognitive Impairment, and Alzheimer's Disease

Background and Objectives: Impaired autophagy has been implicated in neurodegeneration, but circulating autophagy-related proteins have shown inconsistent associations with cognitive impairment. We compared serum autophagy-related protein 5 (ATG5) among older adults with normal cognition, mild cognitive impairment (MCI), and clinically diagnosed Alzheimer's disease (AD), and examined its relationship with global cognitive performance. Materials and Methods: This single-centre cross-sectional study enrolled 164 older adults (55 with normal cognition, 55 with MCI, and 54 with clinically diagnosed AD). Cognitive status was determined through integrated clinical assessment rather than a single test cut-off. An enzyme-linked immunosorbent assay was employed to quantify serum ATG5, yielding quantifiable values for 156 participants, right-censored observations above the highest calibrator for six, and unmeasurable results for two. Since the two 96-well plates, drawn from one ELISA kit lot and processed on the same day, exhibited a substantial discrepancy in measurement scale across runs, they were analysed as distinct strata, with the principal group contrast estimated via a right-censored Tobit regression of log-transformed ATG5 within each plate, controlling for cognitive group, age and sex; a pooled plate-adjusted model also provided support. Supportive analyses used within-plate z-standardised ATG5. Associations with clinical variables were assessed using age- and sex-adjusted partial Spearman correlations with false discovery rate correction. Results: Across both analytical strata (plate 1 omnibus p = 0.293; plate 2 p = 0.264) and within the supportive pooled model (p = 0.143; AD vs. normal geometric mean ratio 0.89, 95% CI 0.73-1.09), serum ATG5 concentrations did not differ significantly between cognitive groups. Concentrations on plate 2 were 47% lower (GMR 0.53, 95% CI 0.45-0.62), a gap exceeding the inter-assay imprecision declared by the manufacturer and unexplained by the calibrator values from plate 2; since relative dispersion matched across both plates (likelihood ratio p = 0.730), the pattern suggests a multiplicative scale shift of unknown origin. Serum ATG5 levels were weakly associated with global cognitive performance assessed via the S-MMSE (partial ρ = 0.283, p < 0.001, FDR p = 0.010), an association that persisted after adjusting for education, albumin and folate and retained a similar magnitude within a linear model (β = 0.044, p = 0.014). Incorporating ATG5 into a model that included age, sex and education failed to enhance discrimination (ΔAUC +0.012 and +0.000). Conclusions: Serum ATG5 did not discriminate normal cognition, MCI, and clinically diagnosed AD. A weak rank-based association with S-MMSE was observed, but the effect was small. These findings do not support serum ATG5 as a diagnostic discriminator for clinically defined cognitive status.