y, MBP treatment was not toxic to primary astrocytes, microglia, oligodendrocytes or an endothelial cell line. Taken together, these data suggest that MBP induces neuron-specific toxicity. MBP binds to the extracellular surface of the neuronal plasma membrane To further explore the mechanism underlying the neuronspecific toxicity of MBP, we performed surface staining for MBP on neuronal cultures after incubation. To technically minimize background from non-specific binding, MBP with low centration was first used to incubate neurons. To our surprise, we found that 5-min incubation of mature neurons with 10 mg/mL MBP resulted in an extensive MBP binding to the surface, not only on the soma but also on all neurites. Incubation with higher MBP concentration gave the same binding pattern. MBP also bound to the surface of immature neurons and induced neurotoxicity. Furthermore, consistent with its specific neurotoxicity, MBP only bound to the extracellular surface of neurons, but not other cell types in DHMEQ web pubmed ID:http://www.ncbi.nlm.nih.gov/pubmed/19675955 the CNS, even if they were incubated for a longer time at the highest concentration. These results show that MBP selectively binds to the neuronal surface. Besides surface binding, we also asked if MBP can enter PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19674121 neurons by endocytosis. Our data showed no MBP entry into the neuronal cytoplasm. This was different from microglia, which took up MBP through endocytosis. Results MBP induces neuron-specific toxicity in vitro To investigate the effects of MBP on neurons, we used primary hippocampal neurons because of their physiological importance and the fact that there is extensive demyelination in the hippocampus during injury and neurodegenerative disease. According to the estimated concentration of released MBP after demyelination and previous MBP studies, MBP with concentrations ranging from 10 to 50 mg/mL was used in our in vitro study. We found that MBP was neurotoxic. First, MBP induced neurotoxicity, featured as typical somatic necrosis and the beaded degradation of neurites. Furthermore, MBP induced neurotoxicity in a dose-dependent manner, where no neurotoxicity was found after MBP treatment at a low MBP Induces Neuron-Specific Cell Death Surface binding and toxic effect of MBP is basicity-dependent It is well-known that in the intact myelin sheath, MBP interacts with acidic lipids located on the inner surface of the plasma membrane through MBP’s basic residues. We then hypothesized that MBP binds to neurons through electrostatic interaction. Since MBP is a basic protein with an isoelectric point of 10.8, according to our hypothesis, at a pH value of 10.8 MBP is neutral and would lose the ability to bind to acidic lipids on the neuronal surface. To test our hypothesis, we performed MBP surface staining at pH 10.8 and found that it did not bind to the neuronal surface. It is notable that MBP undergoes self-assembly in a basic solution. Moreover, we used another typical basic protein, protamine, whose isoelectric point is,1213, and found that pre-incubation of neurons with 5 mg/mL PRM for 30 min blocked all MBP surface binding. PRM also induced neurotoxicity on neurons. However, pre-incubation with 50 mg/mL BSA did not block any subsequent MBP surface binding. Next, we used insoluble liposomes to pre-treat MBP before its incubation with neurons, and accordingly found that MBP neutralized by acidic liposomes failed to bind to neurons, but pre-treatment of MBP with neutral liposomes had no such effect. Among the acidic lipids we tested, phosph