Figure 7.
Differentiation-occurring increase in cholesterol-24-hydorxylase is sufficient and necessary for TrkB activation. (A) Compared with 10 DIV neurons, and after normalization for actin mRNA, mRNA expression levels of cholesterol-24-hydroxylase (CYP46) increase at 26 DIV. Variations related to 10 DIV (value of 1) are indicated. (B) Immunofluorescence analysis of the expression levels of cholesterol-24-hydroxylase in hippocampal neurons maintained for 10 and 26 DIV. Antibody concentration and image exposure were identical for all three time points. Higher reactivity occurs in the 26 DIV neurons, consistent with the higher expression of the messenger (see A). (C) Hippocampal neurons in suspension were transfected either with the plasmid expressing cyp46a1 (p46+) or scrambled vector (pLL3.7) or with the plasmid expressing the cyp46a1-siRNA (pSi46). TrkB activity was measured at 7 DIV or 10 DIV. Note how the increased expression of the cholesterol-24-hydroxylase (CYP46) resulted in high levels of active TrKB in these cells, and reduction of the cholesterol 24-hydroxylase by siRNA resulted in lower levels of Trk phosphorylation. The amount of pTrk/tubulin either in pSi46 or p46+ transfected cells respect to the control is shown. Quantification of the Western blottings performed using anti-cholesterol-24-hydroxylase antibody reveals the efficacy of the knockdown strategy. (D) Immunofluorescence microscopy with anti phospho-Trk antibody (p-Trk) reveals that reduction in cholesterol-24-hydroxylase results in reduced levels of active Trk (IF:pTrk). The efficacy of the knockdown strategy was analyzed with anti-cholesterol-24-hydroxylase antibody reveals (see IF: CYP46).