Journal: Cell Death & Disease

Article Title: Homocysteine interferes with Ndufa1 leading to mitochondrial dysfunction through repression of the NAD + /Sirt1 pathway in the brain: a possible link between hyperhomocysteinemia and neurodegeneration

doi: 10.1038/s41419-025-07834-3

Figure Lengend Snippet: A , B Representative TEM images of mitochondria during Hcy treatment. The red “m” in the images represented mitochondria ( n = 5 for each group, 6 serial sections per rat). C Relative mtDNA/nDNA ratio in the hippocampus of Con and HHcy rats. D , E Western blotting of p-AMPKα1/AMPKα, m-PGC1α/PGC1α, NRF1, and TFAM protein abundance in the hippocampus of Con and HHcy rats. F , G Red arrows indicated damaged mitochondria. All mitochondria were divided into 4 levels according to the degree of damage from mild to severe. The scoring criteria referred to . H , I Western blotting measured the expression of LC3B, P62, PINK1, PARKIN, and TOMM40 in the hippocampus of Con and Hcy groups. J Representative confocal fluorescence micrographs from the CA1 region of the hippocampus stimulated with or without Hcy and subjected to immunofluorescence labeling of TOMM40 (green) and LC3B (red). K The representative line scans were from the LC3B puncta colocalizing with TOMM40. L Relative PINK1 activity in the hippocampus of Con and HHcy rats. M–Q Relative ATP, MDA, MitoSOX level, and SOD activity in the hippocampus of Con and HHcy rats. R , S Immunoblot of COX5A, SDHB, UQCRC2, and ATP5A in the hippocampus of Con and HHcy rats. T The relative activity of Sirt1 in the hippocampus between Con and Hcy groups was measured using the Sirt1 activity assay. U RT-qPCR verification of Sirt1. Gapdh normalized the data. V , W Immunoblot analysis of p-Sirt1/Sirt1 in the hippocampus of Con and HHcy rats. X–Z NAD + , NADH, and NAD + /NADH levels of the hippocampus in rats between Con and Hcy groups were measured by a related kit ( n = 5 for each group). Data were presented as mean ± SEM. Unpaired t -test was used to analyze the data (* P < 0.05, ** P < 0.01, *** P < 0.001; n = 6 for each group).

Article Snippet: p-Sirt1 , Phosoho-Sirt1 (Ser47) , Poly- , 1:1000 , Bioss , Bs-3393R.

Techniques: Western Blot, Quantitative Proteomics, Expressing, Fluorescence, Immunofluorescence, Labeling, Activity Assay, Quantitative RT-PCR

Journal: Cell Death & Disease

Article Title: Homocysteine interferes with Ndufa1 leading to mitochondrial dysfunction through repression of the NAD + /Sirt1 pathway in the brain: a possible link between hyperhomocysteinemia and neurodegeneration

doi: 10.1038/s41419-025-07834-3

Figure Lengend Snippet: A Relative complex I activity of hippocampus in rats between Con and Hcy groups. B Volcano plot of mitochondrial function-related DEGs. The DEGs in the volcano plot were derived from the mitochondrial gene dataset in Fig. . Blue indicated downregulated genes and red indicated upregulated genes. The location of the arrow indicated Ndufa1. C Diagram of the role of Ndufa1 as an assembly and active factor in complex I of the mitochondrial ETC. D The mRNA expression of Ndufa1 in the hippocampus of Con and HHcy rats. The data were normalized by Gapdh. E , F Images and quantification of immunoblotted Ndufa1 signal in the hippocampus from Con and HHcy rats. G The mRNA level of Ndufa1 was negatively correlated with Hcy content in humans and mice, and the brain was defined as “high-Hcy responsive” tissue. The conclusion was from . H The mRNA level of Ndufa1 in N2a cells decreased significantly after transfection with siNdufa1. I The relative ATP level was decreased in N2a cells treated with siNdufa1. J The expression level of Ndufa1 in brain tissue and blood from AD and MCI patients showed a statistically significant and decreasing trend. The conclusion was from . K–N Following siNdufa1 treatment, both mitochondrial membrane potential (MMP) and MitoSOX levels were elevated in N2a cells. O , P Complex I activity and NAD + level were detected by relevant kits. Q–S Western blotting and an ELISA kit were used to detect Sirt1 expression and activity. Data were presented as mean ± SEM. Unpaired t -test was used to analyze the data (* P < 0.05, ** P < 0.01, *** P < 0.001; n = 6 for each group).

Article Snippet: p-Sirt1 , Phosoho-Sirt1 (Ser47) , Poly- , 1:1000 , Bioss , Bs-3393R.

Techniques: Activity Assay, Derivative Assay, Expressing, Transfection, Membrane, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Cell Death & Disease

Article Title: Homocysteine interferes with Ndufa1 leading to mitochondrial dysfunction through repression of the NAD + /Sirt1 pathway in the brain: a possible link between hyperhomocysteinemia and neurodegeneration

doi: 10.1038/s41419-025-07834-3

Figure Lengend Snippet: A–C The NAD + and Sirt1 levels were detected and quantified by RT-qPCR, western blotting, and a related kit. D Meanwhile, Sirt1 activity was measured using an ELISA kit for Sirt1. E PGC1α acetylation levels in the hippocampus of rats in 3 groups. First, PGC1α immunoprecipitation (IP) was performed. Total PGC1α and acetylated PGC1α were then detected by western blotting (IB) using anti-PGC1α and anti-acetyl-lysine antibodies, respectively ( n = 3 for each group). F , G Western blotting measured the expression of p-AMPKα/AMPKα1, m-PGC1α/PGC1α, NRF1, and TFAM in the hippocampus of rats. H , I In parallel, western blotting quantified mitophagy-related protein content in the hippocampus. β-actin was used as an internal reference protein for correction. J The related kits tested relative NAD + levels after transfecting in N2a cells. K–M The protein and activity levels of Sirt1 were detected and quantified by western blotting and an ELISA kit. N , O SDS-PAGE and IB analysis of p-AMPKα1/AMPKα, m-PGC1α/PGC1α, NRF1, and TFAM in N2a cells. P , Q Representative confocal live images of mito-tracker (red) and lyso-tracker (green) targeting mitochondria in N2a cells. Cells were kept untreated or treated with Hcy and/or plasmid. Qualification of the Pearson’s correlation coefficient was calculated by Image J ( n = 5 for each group). R , S SDS-PAGE and IB analysis of LC3B, P62, PINK1, and PARKIN in N2a cells. Data were presented as mean ± SEM. Unpaired t -test for ( C and M ). One-way ANOVA followed by Bonferroni’s post hoc was used for others (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns no significance; n = 6 for each group).

Article Snippet: p-Sirt1 , Phosoho-Sirt1 (Ser47) , Poly- , 1:1000 , Bioss , Bs-3393R.

Techniques: Quantitative RT-PCR, Western Blot, Activity Assay, Enzyme-linked Immunosorbent Assay, Immunoprecipitation, Expressing, SDS Page, Plasmid Preparation