Article Figures & Data
Figures
- Figure 1.
Characterization of hypothalamic and microglial exosomes; effects of alcohol. Nanoparticle tracking analysis, TEM, and Western blot verification of exosomes prepared from the tissue of MBH obtained from postnatal day 6 rats, which were fed daily with ethanol-containing milk formula (AF), pair-fed isocaloric milk formula (PF), or left undisturbed in the litter with mother (AD) (A, D, G, I); or from primary cultures of microglial cells treated with 50 mm ethanol for 24 h (B, C, E, F, H, J). For nanoparticle tracking analysis, the calculated size distribution is depicted as a mean (black line) with SE (red shaded area). Mean particle size, mode particle size, and concentration of particles in exosomes collected from MBH from AD rats (A) or microglia treated with vehicle control (B) or 50 mm ethanol (C). Representative images of exosomes from MBH of AD rats (D) and from control (E) or ethanol-treated microglia (F) under TEM. The width and length of each exosome are represented on the individual exosome. Representative bands for the protein levels of cellular proteins, mitochondria marker cytochrome c, Golgi apparatus marker GM130, and endoplasmic reticulum marker calnexin in MBH exosomes or in liver lysates (G) as determined by Western blot analysis. No BSA band was found in microglial-derived exosomes (H). Exosome marker proteins CD9, HSP70, and CD63 were detectable in Western blot analysis of exosomes prepared from MBH tissues of AD, PF, and AD rats (I) or from control (EC1, EC2) or ethanol (EE1, EE2)-treated microglial cultures (J). An equal amount of protein lysate was used from each group. Data represent three or more independent experiments.
- Figure 2.
Alcohol effects in the hypothalamic and microglial exosomes. In in vivo studies, exosomal activity was determined by measuring CD13 protein levels in the MBH of AD, PF, and AF offspring (A), and CD13 activity levels (at 60, 120, 240, and 360 min) in the MBH for AD, PF, and AF without (B) and with minocycline (C). Data are mean ± SEM (n = 4-7/group). *p <0.05, AF versus AD and PF. **p < 0.01, AF versus AD. #p < 0.01, AF versus PF. In in vitro studies, the activity of exosomes was determined by measuring the level of CD13 proteins 24 h after 0 or 50 mm ethanol treatment (D), CD13 activity levels at various time points (0-240 min) with various doses of ethanol (0-100 mm; E), and changes of MMP2 protein levels 24 h after treatment with various doses of ethanol (F) in microglial cells in primary cultures. Data are mean ± SEM (n = 4-6/group). **p < 0.01, ethanol versus control. *p < 0.05, 25 mm ethanol versus control. ***p < 0.001, 50 mm and 100 mm ethanol versus control.
- Figure 3.
Involvement of exosomes in ethanol-induced β-endorphin neuronal apoptosis. Effect of PAE with or without GW4869 on β-endorphin neuronal number in the arcuate nucleus (A, B), exosome release from microglia in primary cultures (C–F), exosome deposition on β-endorphin neurons in primary cultures (G), and β-endorphin neuronal apoptosis following addition of ethanol-activated exosomes in primary cultures (H). Representative images of (A) β-endorphin-positive staining (green) along with a histogram (B) representing the mean ± SEM values in AD, PF, AF, and AF+GW4869 treated rat pups (n = 5 or 6). Scale bars, 200 µm. *p< 0.05; **p< 0.01; compared with AF. Showing data of nanoparticle tracking analysis, the calculated size distribution is depicted as a mean (black line) with SE (red shaded area). Mean particle size, mode particle size, and concentration of particles collected from microglial exosomes treated with vehicle control (C), ethanol (D), or ethanol and GW4869 (E). F, Histogram represents the mean ± SEM values (n = 3) of exosome release as determined by nanoparticle tracking analysis in microglial cultures following treatment with ethanol with or without GW4869. Shown is a significant number of fluorescently labeled exosomes (green) on β-endorphin neurons (red) in primary cultures (G). Nucleus was stained with DAPI (blue). Arrows indicate uptake of exosomes in these neurons. Scale bars, 50 µm. β-endorphin neuronal apoptosis as determined by nucleosome unit following incubation with microglial exosomes treated with various doses of ethanol for a period of 24 h in primary cultures (H). Data are mean ± SEM (n = 4-7/group). *p< 0.05, ***p< 0.001.
- Figure 4.
Proteomics characterization of exosome cargo from microglial cells treated with and without ethanol. Exosomes were prepared from primary microglia cells treated with 50 mm ethanol (E) or vehicle control (C) for 24 h and used for LC-MS/MS analysis. Comparative proteomic data were presented in a volcano plot, which was constructed by plotting the negative log of the p value on the y axis. Each dot represents the proteomes from three biological replicates per group. A, Volcano plot showed significant downregulated and upregulated proteins as determined by proteomic analysis in both ethanol and vehicle control. B, IPA identified the significantly affected canonical pathways, including complement pathways (highlighted in red), in ethanol-treated exosomes. C, The complement proteins, which are affected by ethanol (increase highlighted in pink; hub gene highlighted in red) in the complement pathway.
- Figure 5.
Regulation of ethanol-induced changes in complement system in the hypothalamus. The effects of PAE with or without minocycline administration on C1q expression (A, B)and colocalization on β-endorphin neurons in the MBH (C–F), ethanol-induced changes in the levels of complements in exosomes derived from microglial cells in cultures (G–J), and ethanol-induced changes in deposition of microglia derived exosomal C1q on β-endorphin neurons in cultures (K, L). A, B, Bar graphs represent C1q protein levels along with representative bands for C1q and CD63 (used for normalizing C1q protein levels) (Kavanagh et al., 2017) measured by Western blot analysis. Histograms represent the mean ± SEM in AD, PF, AF, AD+Mino, PF+Mino, and AF+Mino rat pups on PND 6 (n = 5-8). **p< 0.01, AF versus AD, PF. C–F, Representative images of C1q-positive staining (red) and β-endorphin-positive staining (green), along with merged images and a zoomed-in picture to demonstrate colocalization. Scale bars, 200 µm. Histograms represent the mean ± SEM values of pixel density (D) or Pearson's correlation of C1q colocalization with β-endorphin (F) in AD, PF, AF, and AD+Mino, PF+Mino, and AF+Mino rat pups on PND 6 (n = 5-7). **p < 0.01 versus AF. *p < 0.01 versus AF. G–J, Histograms represent complement protein levels measured by ELISA in exosomes derived from microglial cells treated with vehicle (control) or 50 mm ethanol for 24 h. N = 4-6. *p < 0.05. **p < 0.01. Representative images of C1q-positive staining C1q-positive staining (Green) and β-endorphin-positive staining (Red) in β-endorphin neuronal culture after 24 h of incubation with exosomes harvested from control or ethanol-treated (50 mm) microglia cultures. Nucleus was stained with DAPI (blue). Arrows indicate C1q proteins inside the cells. Scale bars, 50 µm. Histograms represent the mean ± SEM values of Pearson's correlation of C1q colocalization with β-endorphin in cells of control and alcohol-treated groups. N = 6. **p < 0.01.
- Figure 6.
Role of the C1q-activated complement pathway in ethanol-induced cell death of β-endorphin neurons in the hypothalamus. A, B, Representative images of β-endorphin-positive staining (red) and C5b9-positive staining (green), along with merged images and zoomed pictures to demonstrate colocalization (A). Scale bars, 50 μm. Histograms represent the mean ± SEM values of Pearson's correlation of C5b9 colocalization with β-endorphin (B) in AD-, PF-, AF-, and AD+C1NH (100 U/kg)-treated rat pups on PND 6 (n = 7-9). C, D, Representative images of β-endorphin-positive staining (red) and caspase 3-positive staining (green), along with merged images and zoomed pictures to demonstrate colocalization (C). Scale bars, 50 μm. Bar graphs representing mean ± SEM values of Pearson's correlation of caspase 3 colocalization with β-endorphin (D) in AD-, PF-, AF-, and AD+C1NH-treated rat pups on PND6 (n = 7). E–G, Flowcytometric analysis of C3a/b (E), C4 (F), and C5b-9 expression (G) on β-endorphin neurons in primary cultures treated with ethanol-activated (50 mm) microglial exosomes or recombinant C1q protein (500 ng) with or without anti-C1q (C1q neutralizing antibody; 1:25 dilution) for 2 h. H, I, Flowcytometric analysis of cellular ROS by H2DCFDA (H) or mitochondrial ROS by MITO-SOX RED (I) on β-endorphin neurons treated with H2O2 (40 μm) for 30 min, ethanol-activated (50 mm) microglial exosomes, or recombinant C1q protein (500 ng) with or without anti-C1q (C1q neutralizing antibody; 1:25 dilution) for 2 h. J, K, Effects of anti-C1q (C1q neutralizing antibody; 1;25 dilution; J) or anti-ROS NAC (10 μm; K) in control-treated microglial exosomes (C-Ex) or ethanol-treated (50 mm) microglial exosomes (E-Ex)-induced changes in β-endorphin neuronal apoptosis as determined by nucleosome assay in primary cultures of β-endorphin neuronal cells. Data are mean ± SEM (N = 4-6). *p< 0.05, **p< 0.01, ***p< 0.001.
- Figure 7.
A schematic diagram showing a proposed mechanism by which ethanol-activated microglial exosomes uses complement C1q system to induce β-endorphin-producing POMC neuronal death. C1q deposition on POMC neurons may increase expression of C3a/b and C4 and MAC/C5b9 proteins in POMC neurons and may induce cell death in these cells. C1q deposition may also drive ROS production and induce oxidative stress to cause apoptotic-type cell death of POMC neurons. A complement blocker (anti-C1q) or an antioxidant NAC protects POMC neurons from ethanol-induced cellular death.
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