Fig 2: Recovery of FAM134A suppresses ethanol-induced synaptic dysfunction and neuronal cell death. (A) SH-SY5Ys were transfected with pcDNA3.1-GFP or pcDNA3.1-FAM134A-GFP before exposure to EtOH for 24 h. ER stress marker proteins were detected by western blotting. n = 5. (B) iPSC-neurons were pretreated with JQ1 (25 nM) for 30 min prior to treatment of EtOH for 24 h. Protein levels of ER stress markers were determined by western blotting. n = 5. (C-D) iPSC-neurons and hippocampal neurons were pretreated with JQ1 (25 nM) for 30 min prior to treatment of EtOH for 48 h, respectively. (C) Protein expression of PSD95 and Synaptophysin was analyzed by western blotting. n = 5. (D) Immunofluorescence staining of PSD95 and Synaptophysin was visualized with confocal microscope system. Scale bars, 20 μm. n = 5. (E) SH-SY5Ys were transfected with pcDNA3.1-GFP or pcDNA3.1-FAM134A-GFP before exposure to EtOH for 48 h. Western blot was performed to analyze the protein expression of cleaved caspase-9 and cleaved caspase-3. n = 5. (F-H) iPSC-neurons were pretreated with JQ1 (25 nM) for 30 min prior to treatment of EtOH for 48 h. (F) The protein levels of cleaved caspase-9 and cleaved caspase-3 were determined by western blotting. n = 5. (G) Neuronal apoptosis was assessed using a TUNEL assay. Scale bars, 20 μm. n = 5. (H) Apoptotic cells were measured by annexin V/PI analysis assay. n = 5. All data are representative. Quantitative data are presented as a mean ± SD. * indicates p < 0.05 versus control, # indicates p < 0.05 versus EtOH.

Fig 3: Inhibition of BRD4 prevents synaptic dysfunction, neuronal apoptosis, and cognitive impairment in ethanol-fed mice. (A-I) The experimental mice were treated with vehicle, ethanol, ethanol with JQ1, and JQ1 alone for 4 weeks. Samples were obtained from mice hippocampus. (A) Hippocampal FAM134A, CANX, and CLIMP63 were detected by western blot. n = 5. (B) Immunohistochemistry images showing LAMP1 and CANX. Scale bars, 20 μm. n = 4. (C) Hippocampal PSD95 and Synaptophysin were detected by western blot. n = 5. (D) Hippocampal IHC slides were immunostained with PSD95 and Synaptophysin. Scale bars, 20 μm. n = 4. (E) Protein levels of the hippocampal cleaved caspase-9 and cleaved caspase-3 were investigated. n = 5. (F) Tissue slides for immunohistochemistry were immunostained with cleaved caspase-3 -specific antibody. Scale bars, 20 μm. n = 4. (G-I) The mice were subjected to open field test, NOR test, and Y-maze test, respectively. n = 5. (J) The schematic model for inhibition of BRD4-mediated FAM134A-dependent ER-phagy by ethanol was shown. All data are representative. Quantitative data are presented as a mean ± SD. * indicates p < 0.05 versus control, # indicates p < 0.05 versus EtOH.

Fig 4: BRD4 regulates FAM134A gene expression through G9a-mediated H3K9me2. (A) iPSC-neurons were incubated with EtOH for 12 h. BRD4 was co-immunoprecipitated with anti-G9a antibody (the left panel). Total protein expression of G9a and BRD4 is shown in right panel. n = 4. (B-C) DNA was immunoprecipitated with H3K9me2. The immunoprecipitation and input samples were amplified with primers of GAPDH and FAM134A. (B) iPSC-neurons were pretreated with JQ1 (25 nM) for 30 min prior to treatment of EtOH for 12 h. n = 4. (C) iPSC-neurons were pretreated with BIX01294 (1 μM) for 30 min prior to treatment of EtOH for 12 h. n = 4. (D) 1000 base pairs upstream of the first codon of the FAM134A was described and the putative XBP1s binding sequences were emphasized with yellow labeling. (E) iPSC-neurons were pretreated with JQ1 (25 nM) for 30 min prior to treatment of EtOH for 12 h. DNA was immunoprecipitated with XBP1s. The immunoprecipitation and input samples were amplified with primers of GAPDH and FAM134A. n = 4. (F-G) iPSC-neurons were pretreated with JQ1 (25 nM) for 30 min prior to treatment of EtOH for 24 h. (F) The protein levels of FAM134A were detected by western blotting. n = 5. (G) Immunofluorescence staining of FAM134A was visualized. Scale bars, 20 μm. n = 5. (H) iPSC-neurons were pretreated with BIX01294 (1 μM) for 30 min prior to treatment of EtOH for 24 h. FAM134A expression was analyzed by western blotting. n = 5. All data are representative. Quantitative data are presented as a mean ± SD. * indicates p < 0.05 versus control, # indicates p < 0.05 versus EtOH.

Fig 5: Ethanol inhibits ER-phagy in neuronal cells. (A) Schematic illustration of the mCherry-Cleaved ER-phagy Reporter (CCER) assay. When ER-phagy occurs, lysosomal cleavage of RAMP4-mCherry results in the formation of free mCherry product. (B) SH-SY5Ys were transfected with the pcDNA3.1-RAMP4-mCherry vector to establish the CCER system. The protein expression of mCherry and mCherry-RAMP4 were detected by western blotting. n = 4. (C) Schematic of the ER Autophagy Tandem Reporter (EATR) assay. When ER-phagy occurs, eGFP is quenched in autolysosome-induced low pH situations. (D) SH-SY5Ys were transfected with the Tet-On-mCherry-eGFP-RAMP4 vector to establish the EATR system and visualized after EtOH exposure (50 mM) for 24 h. Scale bars, 5 μm. n = 5. (E) Hippocampal neurons were exposed to EtOH for 24 h. Immunofluorescence staining of LAMP1 (lysosomal marker), CANX (ER marker), and DAPI was visualized. Scale bars, 5 μm. n = 4. (F) iPSC-neurons were exposed to EtOH for 12 h. The mRNA expression levels of ER-phagy receptors were analyzed by using real time PCR. Data were normalized by the ACTB mRNA expression level. n = 4. (G) iPSC-neurons were treated with EtOH for 24 h. The protein levels of ER-phagy receptors were detected by western blotting. n = 4. (H) Hippocampal neurons were treated with EtOH for 24 h. Immunofluorescence staining of FAM134A, LC3, and DAPI was visualized. Scale bars, 5 μm. n = 4. All data are representative. Quantitative data are presented as a mean ± SD. * indicates p < 0.05 versus control.