2012 Volume 6 Issue 4 Pages 183-191
Membrane rafts, rich in sphingolipids and cholesterol, play an important role in neuronal membrane domain-specific signaling events, maintaining synapses and dendritic spines. The purpose of this study is to examine the neuronal response to membrane raft disruption. Membrane rafts of 8 DIV primary neuronal cultures were isolated based on the resistance to Triton X-100 and ability to float in sucrose gradients. Membrane rafts from primary cortical neurons were also imaged using the membrane raft marker, cholera toxin subunit-B (CTxB), and were co-immunolabelled with the dendritic microtubule associated protein marker, MAP-2, the dendritic and axonal microtubule protein, β-III-Tubulin, and the axonal microtubule protein, Tau. Exposure of cortical neurons to either the cholesterol depleting compound, methyl-beta-cyclodextrin (MBC), or to the glycosphingolipid metabolism inhibiting agent D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), resulted in neuritic retraction prior to the appearance of neuronal death. Further investigation into the effects of MBC revealed a pronounced perturbation of microtubule protein association with membrane rafts during neuritic retraction. Interestingly, stabilizing microtubules with Paclitaxel did not prevent MBC-induced neuritic retraction, suggesting that neuritic retraction occurred independently of microtubule disassembly and that microtubule association with membrane rafts is critical for maintaining neuritic stability. Overall, the data indicated that membrane rafts play an important role in neurite stability and neuronal viability.