Macrophages share core phagocytic and lysosomal clearance programs with microglia and are widely used as human cellular models to study mechanisms relevant to amyloid-β (Aβ) handling in Alzheimer’s disease (AD). Genetic variation at the CD33 locus is a well-established risk factor for late-onset AD, encoding multiple isoforms with distinct functional properties. While the CD33 short isoform (CD33-S) has been associated with enhanced phagocytic uptake, whether this translates into effective intracellular degradation and true Aβ clearance remains unclear.

Here, we employed human macrophage models engineered to express either the long (CD33-L) or short (CD33-S) CD33 isoform to dissect isoform-specific regulation of Aβ processing. Using quantitative confocal imaging with time-resolved uptake and degradation analyses, together with transcriptomic profiling (RNA-seq), label-free mass spectrometry, and targeted protein validation, we examined how CD33 isoforms coordinate phagocytosis with downstream intracellular handling of fibrillar Aβ.

Consistent with previous observations, macrophages expressing CD33-S displayed significantly increased Aβ uptake compared with CD33-L–expressing cells. Importantly, this enhanced uptake was accompanied by more efficient intracellular degradation of internalised Aβ, rather than simple accumulation. Multi-omics analyses revealed coordinated up-regulation of phagocytosis-associated components, including cytoskeletal and adaptor proteins, alongside activation of autophagy–lysosome machinery, such as ATG5- and LAMP2-associated pathways. These molecular programmes linked early internalisation to effective lysosomal processing, resulting in a marked increase in net Aβ clearance capacity in CD33-S macrophages.

Together, these findings demonstrate that the functional impact of the CD33 short isoform extends beyond enhanced uptake to promote intracellular degradation pathways critical for efficient Aβ clearance. Our work highlights CD33 isoform balance as a key regulator of macrophage clearance efficiency and provides mechanistic insight relevant to microglia-mediated amyloid handling in the context of AD.