Figure 2. Ceramide is required for the p75^NTR-mediated regulation of Aβ generation.

(A–C) Primary neurons from WT and p75^NTR−/− animals were prepared as described in the Materials and methods section, and were then incubated in the absence or presence of the nSMase inhibitor manumycin A (100 μM). (A) Endogenous ceramide was analysed by TLC following prelabelling with [9,10-³H(N)]palmitic acid, as described in the Materials and methods section. (B) Aβ levels in the conditioned media were determined by standard sandwich ELISA using an anti-rodent Aβ antibody, as described in the Materials and methods section. The average concentration of Aβ_total found in the conditioned media of WT neurons was 11984±995 pmol/mg of protein. No difference in the Aβ₄₂/Aβ_total ratio was observed when comparing WT and p75^NTR−/− neurons (results not shown). *Significant difference from WT neurons (no treatment). Error bars represent the S.D. for six different determinations. (C) Western blot analysis of WT and p75^NTR−/− neurons maintained in the absence or presence of manumycin A. Total cell lysates were separated on 4–12% Bis-Tris SDS/PAGE gels, blotted on to a PVDF membrane, and probed with the appropriate antibodies (indicated in the Materials and methods section). (D) Schematic view to summarize the results observed with SK-N-BE (Figure 1) and primary neurons (Figure 2) showing that p75^NTR acts upstream of ceramide in the regulation of BACE1 steady-state levels and Aβ generation.