Background: functional network segregation, defined as the normalized difference between mean within-system connectivity and mean between-system connectivity, has been proposed as a potential neural substrate of cognitive reserve (CR). But its moderating role on the relationship between amyloid-β (Aβ) pathology and cognition remains unknown.

Methods: We analyzed 375 participants from the Alzheimer’s Disease Neuroimaging Initiative (ADNI). Global segregation was computed from resting-state fMRI using a 502-node parcellation. Aβ burden was measured by [¹⁸F]-florbetapir positron emission tomography (PET) composite standardized uptake value ratio (SUVR). Cognition was assessed by modified Preclinical Alzheimer Cognitive Composite (PACC). Multiple regression tested whether system segregation moderated the cross-sectional and longitudinal associations between Aβ burden and cognition, adjusting for age, sex, education, and apolipoprotein E (APOE) ε4 status. Restricted cubic spline (RCS) analyses were used to find potential nonlinear patterns in the moderation.

Results: Higher global system segregation was associated with better baseline cognition and slower subsequent cognitive decline (all p<0.001). System segregation significantly attenuated the adverse association between Aβ burden and PACC both cross-sectionally (B=66.9, p<0.001) and longitudinally (B=9.44, p<0.001). RCS analyses revealed a rightward shift of the change point in the high-segregation group, without evidence of steeper decline beyond this point.

Conclusions: These findings suggest that network segregation may confer resilience to amyloid pathology and highlight the need for longitudinal studies to guide early intervention and identify the protective window of CR.