Microglial phagocytosis of amyloid plaques in an ex vivo model of Alzheimer's Disease

Alzheimer’s disease (AD) is one of the most severe neurodegenerative disorders defined by deposition of amyloid plaques and neurofibrillary tangles. An important role in AD is also exerted by neuroinflammation and microglial activation is one of the hallmarks of the disease pathology. Although microglia are known to be recruited and to cluster around amyloid plaques in the AD brain, their involvement in amyloid plaque clearance over the course of AD is still controversial. Impaired microglial function resulting in decreased Aβ clearance was reported in sporadic AD cases as well as in mouse models of AD. Moreover, several recently identified genetic risk alleles for AD have been linked to microglial function and phagocytosis. To study the contribution of microglial phagocytosis in amyloid plaque clearance, I established a novel ex vivo co-culture model where I combined organotypic brain slices from aged, amyloid plaque-bearing mice (APPPS1) together with slices from young, neonatal wild-type (WT) mice. In the ex vivo co-culture model, I observed changes in amyloid plaque morphology over 14 days in vitro manifested by clearance of the plaque halo bearing diffuse Aβ and increased number of core-only plaques. Additionally, I found a strong increase in immunoreactivity of CD68, a lysosomal marker of activated microglia/macrophages in the old APPPS1 tissue. Specific recruitment and clustering of CD68 positive cells around amyloid plaques was paralleled by a decrease in plaque size upon co-culturing of old and young brain slices. Pharmacological inhibition of phagocytosis by cytochalasin D prevented clearance of the plaque halo, suggesting that CD68 positive microglial cells detected at amyloid plaques are phagocytosing Aβ. Furthermore, specific removal of either old or young microglial cells by clodronate hindered amyloid plaque clearance, suggesting a synergistic contribution of both microglial populations in plaque phagocytosis. To discriminate between young and old microglial cells, I co-cultured either young slices from CX3CR1+/GFP reporter mice with old APPPS1 brain slices or old APPPS1/CX3CR1+/GFP brain slices together with young WT slices. Surprisingly, only the old APPPS1 microglial cells were found in the vicinity of amyloid plaques and thus identified as cells responsible for Aβ uptake in our ex vivo co-culture model. Intriguingly, culturing old APPPS1 brain slices in conditioned media collected from young WT brain slices or from cultured young primary microglia was sufficient to increase amyloid plaque clearance, in contrast to media obtained from microglia-depleted young slices. These data suggested that soluble factors released by young microglia promote Aβ uptake by the old APPPS1 microglial cells. Hence, I tested action of several pro- and anti-inflammatory cytokines on amyloid plaque clearance and found enhanced plaque phagocytosis upon direct addition of granulocyte-macrophage colony-stimulating factor (GM-CSF) to old APPPS1 brain slices, mimicking the co-culture condition. Moreover, the GM-CSF treatment increased numbers of CD68 positive cells confirming its mitogenic potential on myeloid cells. Increased numbers of proliferating microglial were also detected upon co-culturing of old and young brain slices. Nevertheless, co-culturing of young slices from GM-CSF-/- mice with old APPPS1 brain slices still resulted in increased numbers of core-only plaques and, accordingly, increased CD68 coverage. Therefore, GM-CSF is not the sole factor triggering microglial proliferation and Aβ uptake in the ex vivo co-culture model. However, proliferation of old microglial cells was necessary for amyloid plaque clearance, as exposure of the old APPPS1 tissue to the young WT conditioned media obtained after treatment with the proliferative inhibitor AraC prevented amyloid plaque clearance. This study suggests that impaired amyloid plaque clearance of aged microglia in AD may be reversed and microglial phagocytic capacity may be restored upon a proper stimulus. The novel ex vivo model system can be used as a platform to screen, test and identify factors or compounds directed to therapeutically modulate phagocytic competence of microglia.