Journal: Nature Communications

Article Title: The aldehyde dehydrogenase 2 rs671 variant enhances amyloid β pathology

doi: 10.1038/s41467-024-46899-0

Figure Lengend Snippet: a Association of risk factors with ALDH2 rs671 polymorphism and Alzheimer’s disease (AD)-related neuropathologic changes after adjustment for age in Chinese populations. Odds ratios and P values were calculated by ordinal logistic regression with adjustment of age using SPSS software. n = 469. b Representative image of the anatomy of the right human brain with formalin fixation. Eight brain regions were immunostained for amyloid-β (Aβ) plaque deposition to assess Aβ pathology according to National Institute on Aging/Alzheimer Association guidelines for the neuropathologic assessment of AD. c Representative images of Aβ deposits labeled with 6E10 antibody (anti-β-amyloid 1–16 antibody) in six subregions from postmortem brains with pathological AD with different rs671 genotypes. rs671 GG genotype ( n = 18, 82.89 ± 7.76 yr), GA ( n = 18, 84.78 ± 4.40 yr), AA ( n = 8, 84.62 ± 9.76 yr). Percentage of amyloid plaque area was determined. Scale bar, 500 μm. d–f Quantification of Aβ42 and Aβ40 peptides in frozen ( d ) MFG homogenates, ( e ) STG homogenates, and ( f ) Hipp homogenates from the above-mentioned 44 postmortem brains with pathological AD, determined by enzyme-linked immunosorbent assay (ELISA). rs671 GG genotype ( n = 18, 82.89 ± 7.76 yr), GA ( n = 18, 84.78 ± 4.40 yr), AA ( n = 8, 84.62 ± 9.76 y). One datapoint represents one sample per genotype. All box plots include the median line, the box indicates the interquartile range, and whiskers indicate minima and maxima. g Western blot (WB) with anti-ALDH2 in frozen MFG, STG, and Hipp subregions from postmortem brains with pathological AD with rs671 GG (81.50 ± 1.91 yr), GA (84.25 ± 7.85 yr), and AA (90.40 ± 2.79 yr) genotypes. n = 4 or 5. IPL inferior parietal lobule, MFG middle frontal gyrus, STG superior temporal gyrus, Hipp hippocampus, BG basal ganglia, VC visual cortex. Data are presented as mean values ± SD. Statistical analysis was performed using one-way analysis of variance (ANOVA) with least significant difference (LSD) post-hoc test for multiple groups. Source data are provided as a Source Data file.

Article Snippet: The levels of Aβ42 and Aβ40 peptides were measured using a specific human amyloid-β peptide 1−42 ELISA Kit (Cusabio, CSB-E10684h) and a human amyloid-β peptide 1−40 ELISA Kit (Cusabio, CSB-E08299h), respectively.

Techniques: Software, Labeling, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Nature Communications

Article Title: The aldehyde dehydrogenase 2 rs671 variant enhances amyloid β pathology

doi: 10.1038/s41467-024-46899-0

Figure Lengend Snippet: a Schematic image of the endogenous loci to generate Aldh2 -knockout mice in a C57BL/6 background. (Image was created using Photoshop CS6, with elements from ChemBioDraw Ultra software 14.0.) b WB confirmed the Aldh2 expression in Aldh2 −/− mouse brains. All mice with Aldh2 −/− showed similar results. c Levels of Aβ42 and Aβ40 peptides in cortex (COR) homogenates from 3-month-old Aldh2 −/− mice ( n = 5 mice) and age-matched wild-type mice ( n = 4 mice) by ELISA. d Scheme illustrating intragastric administration of daidzin (150 mg/kg/day) in 2-month-old APP/PS1 mice for 2 months. Aβ pathology was assessed in 7-month-old mice. (Image was created using Photoshop CS6 and ChemBioDraw Ultra software 14.0.) e Immunohistochemical staining of Aβ plaque deposits in COR and Hipp and f quantification of Aβ40 and Aβ42 levels in COR from APP/PS1 transgenic mice with phosphate-buffered saline (PBS, n = 4 mice) or daidzin administration ( n = 4 mice). g and h ALDH2 knockdown by siRNA or daidzin (30–100 μM) treatment for 48 h in SH-SY5Y cells. g Enzymatic activity of ALDH2 in cell lysates, measured over 120 min by using a mitochondrial aldehyde dehydrogenase (ALDH2) Activity Assay Kit (Abcam, ab115348). n = 3 biologically independent samples. h Levels of intracellular Aβ42 and Aβ40 peptides. n = 3 biologically independent samples. i – k ALDH2 functional deficiency caused by ALDH2 silencing or combined with daidzin co-treatment for 48 h in HEK293T cells. i RT-qPCR of relative ALDH2 mRNA levels ( n = 3 biologically independent experiments) and j WB detection of ALDH2, APP, and PS1 expression levels. k Levels of intracellular Aβ42 and Aβ40 peptides. n = 3 or 4 biologically independent samples. siNC negative control small interfering RNA. Data are presented as mean values ± SD. All box plots include the median line, the box indicates the interquartile range, and whiskers indicate minima and maxima. Statistical analysis was performed using a two-tailed Student’s t -test for two groups ( c , e , f , h , i , k ) and one-way ANOVA with LSD post-hoc test for multiple groups ( h ). Source data are provided as a Source Data file.

Article Snippet: The levels of Aβ42 and Aβ40 peptides were measured using a specific human amyloid-β peptide 1−42 ELISA Kit (Cusabio, CSB-E10684h) and a human amyloid-β peptide 1−40 ELISA Kit (Cusabio, CSB-E08299h), respectively.

Techniques: Knock-Out, Software, Expressing, Enzyme-linked Immunosorbent Assay, Immunohistochemical staining, Staining, Transgenic Assay, Saline, Knockdown, Activity Assay, Functional Assay, Quantitative RT-PCR, Negative Control, Small Interfering RNA, Two Tailed Test

Journal: Nature Communications

Article Title: The aldehyde dehydrogenase 2 rs671 variant enhances amyloid β pathology

doi: 10.1038/s41467-024-46899-0

Figure Lengend Snippet: a Proteomic profiling was carried out using N2a-APPswe cells pretreated with PBS or 60 μM daidzin for 48 h. KEGG pathways analysis of the differentially expressed proteins (DEPs). b Representative images of 4-HNE immunohistochemical staining of paraffin sections of the human hippocampus with pathological AD. n = 3 per group. rs671 GG (86.33 ± 7.02 yr), GA/AA (82.33 ± 1.15 yr). Scale bar, 50 μm. c Representative images of 4-HNE immunostaining of frozen brain sections of 3-month-old male Aldh2 −/− mice ( n = 5) and wild-type mice ( n = 4). Scale bar, 50 μm. d Representative images of immunocytochemical staining with 4-HNE antibody (green) in SH-SY5Y cells after ALDH2 silencing. Three independent biological replicates were performed. Scale bar, 25 μm. e Separation of ( R )−4-HNE and ( S )−4-HNE from (±)−4-HNE by high-performance liquid chromatography (Shimadzu) with a chiral column (4.6 × 150 mm, CHIRALPAK AS-H). f and g SH-SY5Y cells were pretreated with PBS, or (±)−4-HNE, ( R )−4-HNE, or ( S )−4-HNE at 2 μM for 72 h or 10 μM for 4 h. f Autophagy was assessed by WB for LC3A-I/LC3A-II, the typical marker of autophagy. g Apoptosis was assessed by flow cytometry. The flow-cytometry gating strategies were shown in Supplementary Fig. . h and i Levels of intracellular Aβ40 and Aβ42 were determined by quantitative ELISA. h SH-SY5Y cells were treated with 0, 5, or 10 μM (±)−4-HNE, ( R )−4-HNE, or ( S )−4-HNE for 4 h. i SH-SY5Y cells were treated with 2 μM (±)−4-HNE, ( R )−4-HNE, or ( S )−4-HNE for 24 h. n = 3 biologically independent samples. Data are presented as mean values ± SD. All box plots include the median line, the box indicates the interquartile range, whiskers indicate minima and maxima. Statistical analysis was performed using two-tailed Student’s t -test for two groups ( b and c ) and one-way ANOVA with LSD post-hoc test for multiple groups ( h and i ). Source data are provided as a Source Data file.

Article Snippet: The levels of Aβ42 and Aβ40 peptides were measured using a specific human amyloid-β peptide 1−42 ELISA Kit (Cusabio, CSB-E10684h) and a human amyloid-β peptide 1−40 ELISA Kit (Cusabio, CSB-E08299h), respectively.

Techniques: Immunohistochemical staining, Staining, Immunostaining, High Performance Liquid Chromatography, Marker, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Nature Communications

Article Title: The aldehyde dehydrogenase 2 rs671 variant enhances amyloid β pathology

doi: 10.1038/s41467-024-46899-0

Figure Lengend Snippet: a The Schiff-base formation reaction of Ac-Leu–Lys–Lys–Lys–Gln (residues 52-56 of C99) with ( R )−4-HNE. Matrix-assisted laser desorption/ionization-time of flight MS (MALDI-TOF MS) detection of natural peptide Ac-Leu–Lys–Lys–Lys–Gln (left, upper) and 4-HNE-modified peptides (right, upper). Representative MS/MS spectra of natural peptide Ac-Leu–Lys–Lys–Lys–Gln (left, lower) and modified peptide (right, lower) with 4-HNE adduction on the first Lys residue (Lys53 of C99) via Schiff-base formation, the main product of this reaction. b WB verification of recombinant C99, Lys53Arg mutant C99 (C99 Lys53Arg ), and 4-HNE-modified HNE-C99 and HNE-C99 Lys53Arg . C99 was detected by C1/6.1 antibody. 4-HNE-modified proteins were detected by 4-HNE antibody. c Levels of generated Aβ40 and Aβ42 in in vitro γ-secretase cleavage assays from recombinant C99, C99 Lys53Arg , and their 4-HNE-modified forms, determined by ELISA assay. n = 3 biologically independent samples. d The predicted docking structures of human γ-secretase with peptide Val46–Lys55 of C99 (purple) or with modified peptide Val46–Lys53(HNE)–Lys55 (green) by ZDOCK and sequence alignment was done in PyMOL. The structures of PS1 (the main γ-secretase component) and natural Val46–Lys55 peptide were derived from the Cryo-EM structure ( PDB: 6IYC ). The 4-HNE modification on Lys53 (after Schiff-base formation) was manually added in PyMOL based on the natural peptide structure from 6IYC. The dashed lines and the numbers represent the distance between Asp385 or Asp257 of PS1 and –NH or C=O groups of residue Leu49 of C99 [purple, natural Val46-Lys53; green, modified Val46-Lys53(HNE)-Lys55]. Data are presented as mean values ± SD. Statistical analysis was performed using one-way ANOVA with LSD post-hoc test for multiple groups. Source data are provided as a Source Data file.

Article Snippet: The levels of Aβ42 and Aβ40 peptides were measured using a specific human amyloid-β peptide 1−42 ELISA Kit (Cusabio, CSB-E10684h) and a human amyloid-β peptide 1−40 ELISA Kit (Cusabio, CSB-E08299h), respectively.

Techniques: Modification, Tandem Mass Spectroscopy, Residue, Recombinant, Mutagenesis, Generated, In Vitro, Enzyme-linked Immunosorbent Assay, Sequencing, Derivative Assay, Cryo-EM Sample Prep