In development and neurodegeneration, synapses are eliminated partly through engulfment of neurons and their synapses. As a member of the tripartite synapse, astrocytes are well-placed to monitor synaptic function and engulf stressed neurons or undesired synapses. Milk fat globule epidermal growth factor-like factor 8 (MFG-E8), a phosphatidylserine (PS)-binding opsonin secreted by astrocytes, has been reported to participate to the phenotypic engulfment of synaptosomes by astrocytes. However, the molecular mechanisms of this phenomenon have not been fully elucidated. In this thesis, a phagocytosis assay was developed using confocal microscopy to investigate the engulfment capacity of the astrocytoma cell line U-138 MG when challenged with apoptotic neuronal SH-SY5Y cells. In the presence of fetal bovine serum (FBS), U-138 MG cells challenged with apoptotic SH-SY5Y cells were found to increase their relative engulfment ability and ratio of phagocytosing cells significantly, suggesting that U-138 MG cells are capable of engulfment. In the presence of MFG-E8 alone, a trend of increased relative engulfment ability and ratio of phagocytosing cells was observed, but the statistical significance of the increase was inconsistent across analyses. Inhibition of MFG-E8’s receptors integrin αVβ3/5 was found to significantly attenuate increased MFG-E8-induced uptake. Inhibition of Mer receptor tyrosine kinase (MERTK), a phagocytic receptor known to participate in astrocytic phagocytosis of synapses in development and to cross talk with MFG-E8 in other cell types, did not significantly attenuate the effect, despite slightly decreasing the relative engulfment ability and ratio of phagocytosing U-138 MG cells. Taken together, the data suggest that the U-138 MG model cells used herein may be appropriate to study astrocytic engulfment but may be insufficient to reveal the exact role of MFG-E8 in astrocyte-mediated phagocytosis in the brain. Further investigation is needed to determine whether MFG-E8 plays a significant role in U-138 MG engulfment and whether MFG-E8-MERTK cross talk is a relevant phenomenon in astrocytes. Continued study of the molecular details of astrocytic engulfment mechanisms may reveal potential therapeutic avenues for the treatment of pathologies with aberrant synapse elimination.
Marisa G. Aviani (Fri,) studied this question.