Inflammation-enhanced synapse-specific phagocytosis by adult APP microglia in a microfluidic neuron–microglia co-culture model

Microglia play a critical role in synapse remodeling and neuroinflammation, both of which are dysregulated in Alzheimer’s disease (AD). However, most in vitro models rely on neonatal or immortalized microglia, limiting their relevance to adult pathophysiological context. Here, we present a compartmentalized microfluidic co-culture platform that enables spatially controlled interactions between primary cortical neurons and adult microglia from wild-type (WT) and APP-transgenic mice. This system allows precise functional analysis of microglia–synapse interactions under defined inflammatory conditions. Upon lipopolysaccharide (LPS) stimulation, APP microglia exhibited exaggerated morphological activation, elevated IL-1β secretion, and selectively increased engulfment of synaptic material. In contrast, phagocytosis of non-specific substrates such as pHrodo™ Zymosan remained unchanged, suggesting a substrate-specific enhancement of microglial phagocytic activity. Blocking the complement receptor CD11b abolished the LPS-induced increase in synaptic uptake, confirming the role of complement-dependent pathways. Transcriptomic profiling revealed robust inflammatory responses in both genotypes, with selectively heightened expression of proinflammatory genes in APP microglia, consistent with a primed immune phenotype. Importantly, increased synaptic uptake occurred without measurable loss of global synaptic connectivity, highlighting the specificity and sensitivity of the system to detect microglial functional changes. This model captures genotype-dependent microglial reactivity (revealing phenotypes not fully captured by transcriptomic rofiling) and provides a physiologically relevant, tractable in vitro platform for dissecting microglial contributions to synaptic pathology in neurodegenerative disease.