Fig 2: miR33 is up-regulated in aged neurons in vitro.(A) The plot compares the miR33 levels (normalized by the housekeeping RNA U6) between 13 DIV, 16–17 DIV and 21 DIV neuronal cultures (fold change versus DIV). The microRNA levels were determined by RT–PCR (n = 5 independent culture). The graph shows the mean ± SEM. Statistical significance was analyzed by one-way ANOVA (P-value: 0.0410). Dunnet’s multiple comparisons test was used to compare the 21 DIV and the 16–17 DIV to the 13 DIV group (ns: non-significant; *P < 0.05; P-value for the 21 DIV to 13 DIV comparison: 0.0258). (B) Western blot analysis of lysates of N2A cells transfected with 40 nM of a non-targeting control mimic or 40 nM miR-33 mimic (miRNA-33). The blots were analyzed with antibodies against NPC1, ABCA1 and actin. (C) Plot comparing the relative protein levels of NPC1 between N2A cells transfected with 40 nM of a non-targeting control mimic (n = 4) or 40 nM miR33 mimic (miR-33; n = 4). (B) Blots were quantified from Western blot experiments as the one shown in panel (B). The graph shows the mean ± SEM and individual data points. Statistical significance was analyzed by two-tailed unpaired t test (**P < 0.05). (C, D) The plot compares the ABCA1 protein levels in the same experimental conditions as in panel (C). The graph shows the mean ± SEM and individual data points (n = 3). Statistical significance was analyzed by two-tailed unpaired t test (**P < 0.05). (E) Western blot analysis of brain lysates from 2 mo old WT or miR33 KO mice. Blots were tested with antibodies against NPC1 and HSP90. (F) Plots comparing the relative protein levels for NPC1 in the brain of WT (n = 5) and miR33 KO (n = 7) mice. (C) Protein levels were quantified from Western blot experiments as the one shown in panel (C). The graph shows the mean ± SEM and individual data points. Statistical significance was analyzed by two-tailed unpaired t test (*P < 0.05).

Fig 3: Insulin rescues MCP-1-mediated mRNA, total and surface protein suppression of ABCA1, ABCG1, and SR-B1 via PI3K/Akt activation in differentiated 3T3-L1 adipocytes. Cells were serum-starved for 6 h, followed by pretreatment with (+) or without (-) the PI3K inhibitor wortmannin for 45 min. The cells were then incubated with (+) or without (-) insulin (100 nM) and MCP-1 (40 ng/ml). (a–c) Insulin rescues MCP-1 mediated mRNA suppression of ABCA1, ABCG1and SR-B1. (d, e) Insulin rescues the total protein expression of the three receptors. (f) Insulin rescues the surface protein expression of the three receptors. Cell surface receptor levels and Na+/K+ ATPases were directly extracted using cell surface biotinylation and then measured using western blotting, as described in “Materials and Methods.” (g) Insulin rescues the expression of the three receptors by confocal microscopy (×63). ABCA1, SR-BI, and ABCG1 labeled with Alexa 546 (red), Alexa 633 (pink), and Alexa 488 (green), respectively, were detected by confocal microscopy as describe in “Materials and Methods.” Note that insulin reversed the MCP-1-mediated decreases in ABCA1, ABCG1, and SR-BI mRNA and protein levels, and this correction was inhibited by wortmannin. ((a–g): n = 3). *P < 0.05 compared with untreated cells; #P < 0.05 compared with MCP-1- or insulin-treated cells; +P < 0.05 compared with MCP-1 or insulin-treated cells

Fig 4: Immunofluorescence detection of ABCA1 expression in the MDA-MB-231 cell line after 24 h incubation with 30 µM of oxysterols. The intensity of fluorescence was quantified with MetaXpress software. Representative images of immunofluorescence after incubations with A: 7-ketocholesterol (7KC); B: cholestan-3a-5ß-6a-triol (triol); C: 5a-cholestane-3ß,6ß-diol/5a-cholestane-3ß,6a-diol (diol); or D: cholesterol (control); E: quantification of ABCA1 expression. Data are presented as the mean ± SEM from three independent experiments performed in triplicate. *p < 0.005 compared to control.

Fig 5: BODIPY-cholesterol accumulates in endosomal compartments in rat primary cortical neurons aged in vitro.(A) Representative confocal microscopy images of 14 and 24 DIV rat primary cortical neurons treated with 1 µM BODIPY-cholesterol (green) for 24 h and stained with antibodies against an early endosomal marker (EEA1, red), which stains endosomal compartments where internalized material ends up first, or a late endosomal/lysosomal marker (LIMP-I, red), which stains a mature state of endosomal compartments. 24 DIV neurons accumulate BODIPY-cholesterol in early and late-compartments (white arrows). (B) The plot compares the relative amount (% versus 12–14 DIV) of BODIPY-cholesterol taken by 12–14 DIV and 23–28 DIV primary cortical neurons in a 24 h period, calculated by the intracellular fluorescent signal (Ext 495 nm/Em 507) of BODIPY-cholesterol (nmol of cholesterol where from a BODIPY-cholesterol standard curve) and normalized by the total protein in the sample (% versus 12–14 DIV; n = 9 independent cultures). The graph shows the mean ± SEM. Statistical significance was analyzed by two-tailed unpaired t test (**P < 0.01; P-value: 0.0046). (A, C) Plot showing the percentage of the total EEA1-positive (n = 1,536) or LIMP-I positive (n = 1,269) compartments that accumulate BODIPY-cholesterol, calculated from confocal images as the ones shown in panel (A) (N = 3 independent experiments). The graph shows the mean ± SEM. Statistical significance was analyzed by two-tailed Mann–Whiitney Test (****P < 0.0001). (D) Western blot analysis of total lysates of 12–14 DIV, 18–21 DIV, and 23–28 DIV rat cortical neurons shows a pronounced decrease in NPC1 protein levels in 18–21 DIV neurons that is maintained in 23–28 DIV neurons. An antibody against beta-actin was used to normalize the protein levels. Below, a plot with the quantification of the bands of Western blot experiments as the one shown. Protein levels are relative to 12–14 DIV. The graph shows the mean ± SEM. Statistical significance was analyzed by one-way ANOVA (P-value: 0.0001). Comparison of the age-groups 18–21 DIV and 23–28 DIV to 12–14 DIV was performed by Dunnet’s multiple comparisons tests (n = 6 for 12–14 DIV and 18–21 DIV; n = 5 for 23–28 DIV, ***P < 0.001; P-value for 18–21 DIV to 12–14 DIV comparison: 0.0002; P-value for 23–28 DIV to 12–14 DIV comparison: 0.0004). (E) Western blot analysis of lysates of neuronal cultures at different aging points. The blot was tested with an antibody against the cholesterol transporter ABCA1 and actin. Below, a plot compares the relative ABCA1 protein levels between 12–14 DIV, 18–21 DIV and 23–28 DIV neuronal cultures. The bands corresponding to ABCA1 were quantified from blots such as the one shown above. Statistical significance was analyzed by Kruskal–Wallis Test (P-value 0.0022). Comparison of the age-groups 18–21 DIV and 23–28 DIV to 12–14 DIV was performed by Dunn’s Test (n = 9 independent lysates per group; **P < 0.01; P-value for 23–28 DIV to 12–14 DIV comparison: 0.0036).