Figure 6.

BDNF causes an increase in presynaptic proteins that accumulate in lipid rafts. Cortical neurons were cultured in the presence or absence of BDNF (200 ng/ml) and in the presence or absence of 10 μm mevastatin or 100 μm zaragozic acid for 3 d. A, BDNF-increased the number of synaptophysin-positive puncta. Cortical neurons were stained by using antibodies against MAP-2 (green; neuron marker) and synaptophysin (red; presynaptic marker). Scale bar, 50 μm. B, BDNF increases the presynaptic protein content of lipid rafts. Lipid raft and nonraft fractions were prepared as in Figure 5. Presynaptic proteins were detected with the indicated antibodies. C, Effect of mevastatin on the BDNF-induced increase in presynaptic puncta. Immunostaining was performed as in A. The synaptophysin-positive puncta were determined by using Scion Image as described in Materials and Methods. Results (the means ± SEM) are presented relative to −BDNF samples. The asterisk indicates a statistically significant difference from −BDNF samples (Student's t test; *p < 0.01). D, E, Cholesterol synthesis inhibitors prevent the BDNF-dependent increase in lipid raft-associated presynaptic proteins. Total cell lysates were immunoblotted with the indicated antibodies. The BDNF-dependent increase in presynaptic proteins was inhibited by mevastatin (D) and zaragozic acid (E).