Animals

All animal experimental procedures were conducted in accordance with the animal care regulations of the National Institute of Health and were approved by Stanford University’s Administrative Panel on Laboratory Animal Care. The mice were maintained on a 12 h light/dark cycle in stable conditions in terms of temperature, humidity, and ventilation. Water and food were offered ad libitum. Animals were randomly assigned numbers and evaluated there after by a researcher blinded to both experimental condition and genotype.

In vivo model for chronic alcohol injury

Male C57BL/6 N (Charles River) (ALDH2*1/*1 i.e. WT controls) mice and ALDH2*2/*2 knock-in mice of the same background were used in these experiments [60]. Note that the deletion of the mitochondrial enzyme, nicotinamide nucleotide transhydrogenase (NNT) has been observed in the C57BL/6 J (Jackson Lab) mice. We, therefore, used C57BL/6 N mice, which have a complete and functional NNT gene [40]. Alda-1, dissolved in vehicle (50% PEG-400, 50% DMSO), or vehicle alone was delivered using 4-week osmotic pumps (Alzet; # 1004) at 10 mg/kg/day (0.11 μl daily volume). Pumps were surgically implanted subcutaneously in the back of 8–10-week-old mice, between the shoulders, under general anesthesia [23]. The wound was closed with metal clips, which were removed at 10–14 days post-implantation. Pumps were replaced twice, at 4-week intervals, up to 12 weeks of treatment with Alda-1. For ethanol challenge, mice were treated with 1 g/kg/day ethanol (i.p. 20% v/v in normal saline; 130 μl/injection) or an equivalent volume of saline by intraperitoneal injection for 11 weeks, beginning 1 week after implantation of pumps. The predicted blood alcohol levels are 10–15 mM based on previously reported observations in [30]. No evidence of peritonitis was evidenced in any of the experimental groups.

Primary neuron culture

Primary neuron cultures were prepared from cerebral cortices of embryonic day E17 ALDH2*2/*2 mice or WT (C57BL/6 N). In brief, cortices were dissected and dissociated using papain dissociation system (Worthington Biochemical Corporation). Cells were cultured at 20,000/ well of a 96 well plate coated with poly-D-lysine (Sigma) for cell viability assays. For seahorse experiments, 1 × 105 cells/well were seeded in XF 24-well cell culture microplate and cultured in Neurobasal medium (Invitrogen) supplemented with B-27 (Invitrogen) containing 25 mM glucose, 4 mM glutamine, 1 mM sodium pyruvate, and 5% FBS. At 24 h after seeding, the medium was changed to Neurobasal medium supplemented with B-27 and 0.5 mM glutamine. Cells were cultured at 37 °C in a humidified chamber of 95% air and 5% CO 2 . Cultures were used for experiments from 7 to 10 days after seeding.

Primary astrocyte culture

Primary astrocyte cultures were prepared from cerebral cortices of 2-day-old ALDH2*2/*2 mice or WT (C57BL/6 N) mice. In brief, dissociated cortical cells were suspended in DMEM/F12 50/50 (Life Technology) containing 25 mM glucose, 4 mM glutamine, 1 mM sodium pyruvate, and 10% FBS and plated on uncoated 25 cm2 flasks at a density of 6 × 105 cells cm2. Monolayers of type 1 astrocytes were obtained 12–14 days after plating. Cultures were gently shaken, and floating cells (microglia) were collected, resulting in more than 95% pure culture of astrocytes.

Patient-derived fibroblasts

AD patient fibroblasts (NG07613; NG07768; NG08170; NG08527; NG09955; NG10039; NG10788; NG11757; NG00364; NG04159; NG05809; NG05810; NG06205; NG06265; NG06840; AG04402; AG11414; AG05810; AG21158; AG11369) and fibroblasts of control healthy individuals (AG07123; AG04146, AG02258; AG02261) purchased from Coriell Institute, USA screened for ALDH2*2 mutation. AD patient fibroblasts (1:AG04402; 2:AG11414; 3:AG05810; 4:AG21158; 2*2/1 AD: AG11369) and fibroblasts of control healthy individuals (1:AG07123; 2:AG04146, 3: AG02258; 4: AG02261) were maintained in MEM supplemented with 15% (v/v) fetal bovine serum and 1% (v/v) penicillin/streptomycin at 37 °C in 5% CO2–95% air. On the bases of our previous primary cell culture data and assuming a statistical significance of 0.05 and a power of 0.8, we anticipate requiring a minimum of 3 patients in each arm of this study for assessment of mitochondrial dysfunction. For transfection experiments, cells were seeded in 96 well plates at 10,000 cells per well or 50,000 cells per well in 6 well plates at for 18-24 h before transfection. Plates were transfected with 1 μg of plasmid DNA using 3 μl of Lipofectamine 2000 reagent (Life Technologies). After 12 h, the media was replaced with fresh media to reduce toxicity of the Lipofectamine reagent. After 48 h cells were analyzed for markers of cellular health. Detailed information on fibroblasts used in the study is provided in Table 1.

Table 1 Details of patient-derived fibroblasts Full size table

Cell and mitochondrial function assays

Mitochondrial membrane potential

Cells were incubated with tetramethylrhodamine, methyl ester (TMRM), a cell-permeant, cationic, red-orange fluorescent dye that is readily sequestered by active mitochondria (200 nM; Invitrogen) in HBSS (Hank’s balanced salt solution) for 30 min at 37 °C, as before [31], and the fluorescence was analyzed using SpectraMax M2e (Molecular devices, using excitation at 360 nm and emission at 460 nm).

ATP measurements

Relative intracellular ATP levels were determined using ATP-based CellTiter-Glo Luminescent Cell Viability kit (Promega), which causes cell lysis and generates a luminescent signal proportional to the amount of ATP present. In brief for intracellular ATP levels, opaque-walled 96-well plates with cell lysate (50 μl) were prepared. An equal volume of the single-one-step reagent provided by the kit was added to each well and incubated for 30 min at room temperature. ATP content was measured using a luminescent plate reader SpectraMax M2e (Molecular devices).

ROS production

For cellular ROS detection, cells were incubated with 2,7 dichloro- fluorescein diacetate (DCFDA) (Abcam) 100 μM for 30 min at 37 °C in the dark, and fluorescence was analyzed with excitation/emission at 495/529 nm, using SpectraMax M2e (Molecular devices). Fluorescence intensity was then normalized for cell number. To determine mitochondrial ROS production, cells were treated with 5 μM MitoSOX™ Red, a mitochondrial superoxide indicator (Invitrogen) for 10 min at 37 °C, according to the manufacturer’s protocol, and fluorescence was analyzed with excitation/emission at 510/580 nm, using SpectraMax M2e (Molecular devices).

Bioenergetic profiles

Cells were plated in a Seahorse XF24 Cell Culture Microplate (Agilent). All seahorse experiments in neurons were performed at 24 h after individual stimuli. At the end of the treatment, cells were washed twice with Agilent Seahorse XF Media (Agilent) supplemented with 1 mM pyruvate, 2 mM L-glutamine, and 2 mM D-glucose; a final volume of 525 μl was placed in each well. Cells were then incubated in a 0% CO 2 chamber at 37 °C for 1 h before being placed into a Seahorse XFe24 Analyzer (Agilent). For oxygen consumption rate (OCR) and (extracellular acidification rate) ECAR experiments, cells were treated with 1 μM oligomycin, 2 μM carbonyl cyanide p-trifluoromethoxy phenylhydrazone (FCCP), and 0.5 μM rotenone/antimycin. A total of three OCR and pH measurements were taken after each compound was administered. All Seahorse experiments were repeated at least three times.

Cell death

Cytotoxicity was determined using Cytotoxicity Detection Kit, as before [33]. In brief, media was collected at endpoints (in phenol red-free DMEM) to measure the percentage of released lactate dehydrogenase activity (LDH). To quantify total LDH, cells were lysed with Triton X (1% in serum-free cell culture media) overnight at 4 °C; 50 ul media or lysate was transferred with 50 ul of reaction mix in a 96-well plate and incubated at RT for 30 min in the dark. Absorbance was measured at 490 nm using SpectraMax M2e (Molecular devices), and cell death is presented as a percent of released LDH of total LDH.

Elisa

Mouse IL-6, mouse IL-1α, mouse TNFα, mouse IL-1β and mouse MCP-1 ELISA kits (eBiosciences) were used to quantify cytokine levels in mouse tissue and cell supernatant according to manufactures protocols.

Caspase activity assay

Caspase 3 activities were determined using a colorimetric caspase 3 assay kit (Abcam), according to the manufacturer’s protocol. Cell lysates containing 200 μg protein were used for each assay. Each assay was repeated with three independent cell cultures. Absorbance at 400 nm was recorded using SpectraMax M2e (Molecular devices). Caspase activity was calculated in arbitrary units and represented as fold change of WT control.

Lipid peroxidation assay

MDA content was determined using MDA lipid peroxidation assay (Abcam) according to the manufacturer’s directions. For 4-HNE, Lipid Peroxidation (4-HNE) Assay Kit (Abcam) was used. Samples were homogenized in RIPA buffer with inhibitors and analyzed according to the manufacturer’s directions and represented as fold change of WT control.

Hydrogen peroxide assay

The rate of hydrogen peroxide production by fresh isolated mitochondria was determined by the Amplex Red Hydrogen Peroxide/Peroxidase Assay kit (Invitrogen) following the manufacturer’s instructions.

Aβ ELISA

Aβ levels in mitochondrial fractions and brain cortex were measured by using mouse Aβ 42 ELISA kit (Invitrogen) following the manufacturer’s instructions.

Measurement of ALDH2 activity

ALDH2 activity in patient derived fibroblasts was measured as before [16]. Cell lysates (200 μg) were added into a cuvette containing activity assay buffer and substrate; 50 mM sodium pyrophosphate buffer at pH 9.0, 2.5 mM NAD+ (nicotinamide adenine dinucleotide), 10 mM acetaldehyde, and 460 μl of H2O; with a final volume of 2 ml. Optical density at 340 nm was measured at 25 °C for increase of NADH (reduced form of NAD+) for 5 min. Blank control was no acetaldehyde.

Western blot analysis

Protein concentrations were determined using the Bradford assay (Thermo Fisher Scientific). Proteins were resuspended in Laemmli buffer containing 2-mercaptoethanol, loaded on SDS–PAGE, and transferred on to nitrocellulose membrane, 0.45 μm (Bio-Rad), as before [32]. Cell supernatant was cleared of cellular debris by centrifugation at 1000 g for 10 min. The total lysate was resuspended in Laemmli buffer containing 2-mercaptoethanol, loaded on SDS–PAGE, and transferred on to nitrocellulose membrane, 0.45 μm (Bio-Rad), as before [32]. Membranes were cut at appropriate molecular weights and then probed with the indicated antibody and visualized by ECL (0.225 mM p-coumaric acid; Sigma), 1.25 mM 3-aminophthalhydrazide (Luminol; Fluka) in 1 M Tris pH 8.5. Scanned images of the exposed X-ray film or images acquired with Azure Biosystems C600 were analyzed with ImageJ to determine relative band intensity. Quantification was performed on samples from independent cultures for each condition. The antibody details are listed in Table 2.

Table 2 Antibody details Full size table

RNA isolation and gene expression analysis

RNA isolation was performed using GenElute™ Mammalian Total RNA Miniprep Kit (Sigma Aldrich) according to manufacturer’s protocols. RNA concentration was measured using a Nanodrop (ND − 1000; NanoDrop Technologies, Rockland, DE, USA) and RNA integrity was assessed using a Bioanalyzer (2100; Agilent Technologies, Palo Alto, CA, USA). cDNA synthesis was performed using the Quantitect reverse transcription kit (Qiagen) according to manufacturer’s instructions, with a minimal input of 200 ng total RNA. Quantitative PCR (qPCR) was performed using the 7300 Real Time PCR system (Applied Biosystems, Foster City, USA) using the equivalent cDNA amount of 1–2 ng total RNA used in cDNA synthesis. SYBRgreen master mix (Applied Biosystems) and a 2 pmol/ml mix of forward and reverse primer sequences were used for 40 cycles of target gene amplification. The primer sequences used are listed in Table 3.

Table 3 qPCR primers Full size table

Statistical analysis

Prism 8.0 (GraphPad Software) was used for the statistical analysis. Data shown are the mean ± s.d. with P < 0.05 considered statistically significant. Group differences were analyzed with one-way analysis of variance (ANOVA) followed by Holms-Sidak multiple comparisons test for multiple groups. Data distribution was assumed to be normal, but this was not formally tested. No statistical methods were used to predetermine sample sizes.