Fig 2: Effects of the APOE allele vs APOE contents on Ca2+-based excitability. aAPOE mRNA expression relative to Gapdh and 18S mRNA contents in APOE3 and APOE4 immortalized astrocytes, quantified by real time qPCR (N = 3). b Representative ApoE western blot of APOE3 cells transfected with scramble or APOE siRNA. c Representative traces and quantification of the magnitude of ATP-induced Ca2+ responses in APOE3 astrocytes transfected with lipofectamine (lipo), plus scramble (sc) or APOE siRNA, and APOE4 astrocytes treated only with lipofectamine (N = 4). d Representative images of immunocytochemistry of APOE3 and APOE4 cells transfected with plasmids expressing GFP, GFP-APOE4, and GFP-APOE3, as indicated. Scale bar represents 15 µm. e Representative traces and quantification of the magnitude of 100 µM ATP-elicited Ca2+ responses in APOE3 and APOE4 cells transfected with different plasmids as indicated. Ca2+ peaks after ATP stimulation are relative to the responses of APOE3 cells transfected with GFP. Only GFP fluorescent cells were analyzed (at least 30 cells from 4 independent experiments). f Quantification of the magnitude of Ca2+ responses elicited by 100 µM ATP in APOE3 astrocytes transfected with GFP or GFP-APOE4 plasmids, as indicated. Ca2+ peaks after ATP stimulation are relative to the responses of APOE3 cells transfected with GFP. Only GFP fluorescent cells were analyzed (at least 30 cells from 4 independent experiments). g Representative images of Lamp1 (red) and ApoE (green) immunocytochemistry in APOE3 cells. Merged images and amplifications of the area in the white frame, are displayed. Scale bar represents 15 µm. h Representative images of Lamp1 immunocytochemistry (red) and GFP-ApoE fluorescence (green) in APOE4 cells. Merged images and the amplification of a cell of each image, indicated with a white frame, are displayed. Scale bar represents 15 µm. Unpaired parametric T-test was used in a, and one-way ANOVA in c and e. p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****)

Fig 3: Effects of U373 conditioned medium on oxidative stress in neurons. Representative images in bright field and quantitative assessment of neuronal morphology of N1E-115. Cells were previously treated (+) or not (−) with rotenone (0.1 µM) for 16 h, and then cultured in fresh DMEM (Ctrl), in conditioned medium derived from control U373 (U373-Ctrl), NGF-pre-treated U373 (U373-NGF), and NGF-pre-treated U373 silenced for apoE (U373-NGF ApoE siRNA) for 48 h. n = 5 different experiments. Data represent means ± SD. Statistical analysis was assessed by using one-way ANOVA, followed by Tukey’s post hoc test. ** p < 0.01, *** p < 0.001.

Fig 4: ApoE secretion by NGF-treated U373 cells provides neuroprotection from oxidative stress. (A) Representative scheme of astrocyte–neuron co-cultures set up as described in the Materials and Methods Section. Neurite-bearing cells, neurite length, and cell number were assessed 48 h after the establishment of co-culture. (B) Representative images in bright field and quantitative assessment of neuronal morphology of N1E-115 cells, previously treated (+) or not (-) with rotenone (0.1 µM) for 16 h. N1E-115 were then kept in fresh DMEM (Ctrl), or co-cultured with control U373 (U373-Ctrl), NGF-pre-treated U373 (U373-NGF), and NGF-pre-treated U373 silenced for ApoE (U373-NGF ApoE siRNA) for 48 h. n = 5 different experiments. Data represent means ± SD. Statistical analysis was carried out by using one-way ANOVA, followed by Tukey’s post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Fig 5: Effects of p75NTR modulation by LM11A-31 on cholesterol metabolism and secretion. (A–D) Representative Western blot and densitometric analysis of HMGCR, ABCA1, NPC1, and ApoE in U373 cells treated with vehicle (Ctrl) and LM11A-31 (0.1 µM) for 48 h. n = 4 different experiments. Vinculin served as a housekeeping protein to normalize protein loading. (E) Quantification of ApoE amount (µg/mL) by ELISA assay in culture medium from vehicle- and LM11A-31-treated U373 cells. n = 3 different experiments. (F) Cholesterol quantification (total cholesterol, free cholesterol, and cholesteryl esters) in the culture medium of U373 cells treated with vehicle (Ctrl) and LM11A-31 (0.1 µM) for 48 h. n = 3 different experiments. Data represent means ± SD. Statistical analysis was carried out by using unpaired Student’s t test. *** p < 0.001.