Figure 2.
ApoE-containing lipoproteins, but not apoE alone, cholesterol alone, or apoA1/apoJ-containing lipoproteins, prevent apoptosis of RGCs.
A
, Recombinant human apoE3 and/or cholesterol (5 and 1 μg/ml, respectively) were added to BM(−) and incubated with RGCs for 24 h. Control cultures were given BM(−) or BM(+). The number of apoptotic neurons as a percentage of all neurons was determined by Hoechst staining.
B
, Immunoblot of apoE, apoA1, and apoJ in lipoproteins isolated by ultracentrifugation from cultures of ApoE
+/+ and ApoE
−/− glia. ApoA1-containing lipoproteins (+A1) were generated by addition of human apoA1 to glia isolated from ApoE
−/− mice.
C
, Apoptosis was assessed in RGCs incubated in BM(−) supplemented with lipoproteins (120 μg of protein/ml) isolated from glial cultures from ApoE
+/+ and ApoE
−/− mice.
D
, BM(−) was supplemented with apoE-containing lipoproteins (ApoE
+/+) or apoA1-containing lipoproteins (ApoE
−/− + A1) so that the concentration of cholesterol in the medium was 1 μg/ml.
E
, rHDLs that contained apoE, phosphatidylcholine (PC), and cholesterol (molar ratio, 1:100:10) were prepared. BM(−) was supplemented with rHDLs so that the amount of apoE was the same as (1.0), 50% (0.5), or 20% (0.2) of that in glial LPs. Liposomes lacking apoE, but containing the same amount of phosphatidylcholine and cholesterol as in rHDLs (PC + chol), did not prevent apoptosis.
F
, RGCs were incubated in BM(−) for 24 h with rHDLs that contained the same amount of apoE as in LPs and either contained (chol/PC) or lacked (PC) cholesterol. For all experiments, fragmented/shrunken nuclei were detected by Hoechst staining and designated as apoptotic cells. Data are means ± SE from three or four independent experiments. chol, Cholesterol.