Figure 4.
Moderate still continuous loss of cholesterol from the plasma membrane of differentiated hippocampal neurons in vitro determines TrkB DRM partitioning. (A) Colorimetric measurement of plasma membrane cholesterol from hippocampal neurons kept for 10, 15, and 26 DIV. Mean ± SD values (nmol/mg protein, n = 4) were 0.61 ± 0.029, 0.55 ± 0.047, and 0.43 ± 0.023 in 10, 15, and 26 DIV neurons, respectively. (B) TLC analysis of the membrane cholesterol content in DRMs of 10, 15, and 26 DIV neurons; cholesterol is clearly and progressively reduced in DRMs from 15 and 26 DIV neurons. Percentages of cholesterol reduction in DRMs (fractions 4 and 5) and non-DRMs (fractions 6–8) were measured by densitometry and normalized for sphingomyelin values of different sucrose fractions (n = 2). In DRMs, cholesterol was decreased to 43.29% in 15 DIV cells, and to 25.92% in 26 DIV cells. The migration of the cholesterol standard is indicated (cholesterol). (C) Western blotting with anti-total Trk, anti-phospho Trk, and anti-prion protein (PrPc) antibodies, of 4°C detergent-extracted, sucrose-gradient centrifuged membranes from 10 and 26 DIV hippocampal neurons in vitro. Note that receptor activity is exclusively present in the DRM domains (fractions 4 and 5) of 26 DIV neurons. The PrPc lane demonstrates that the presence in fractions 4 and 5 truly reflect DRM partitioning. (D) Immunoprecipitations of TrkB from the DRMs and non-DRMs of 10 and 26 DIV neurons, probed with antibodies against phospho Trk and the anti-p85 subunit of PI3K. Note that the downstream survival effector of TrkB is precipitated exclusively from the DRM domains of 26 DIV neurons. (E) Control for correct separation between DRMs and non-DRMs fractions. Lysates utilized in C were hybridized with antibodies against the non-DRM protein Transferrin receptor (IB: TfR) or the DRM protein PrPc (IB: PrPc).