Lipid rafts act as specialized domains for tetanus toxin binding and internalization into neurons

Tetanus (TeNT) is a zinc protease that blocks neurotransmission by cleaving the synaptic protein vesicle-associated membrane protein/synaptobrevin. Al though its intracellular catalytic activity is well established, the mechan ism by which this neurotoxin interacts with the neuronal surface is not kno wn. In this study, we characterize p15s, the first plasma membrane TeNT bin ding proteins and we show that they are glycosylphosphatidylinositol-anchor ed glycoproteins in nerve growth factor (NGF)-differentiated PC12 cells, sp inal cord cells, and purified motor neurons. We identify p15 as neuronal Th y-1 in NGF-differentiated PC12 cells. Fluorescence lifetime imaging microsc opy measurements confirm the close association of the binding domain of TeN T and Thy-1 at the plasma membrane. We find that TeNT is recruited to deter gent-insoluble lipid microdomains on the surface of neuronal cells. Finally , we show that cholesterol depletion affects a raft subpool and blocks the internalization and intracellular activity of the toxin. Our results indica te that TeNT interacts with target cells by binding to lipid rafts and that cholesterol is required for TeNT internalization and/or trafficking in neu rons.