Journal: Neuroreport

Article Title: Glutaredoxin-1 attenuates transactive response DNA-binding protein 43–induced neurotoxicity by suppressing oxidative stress and transactive response DNA-binding protein 43 aggregation

doi: 10.1097/WNR.0000000000002266

Figure Lengend Snippet: Endogenous Grx1 is upregulated in N2a-hTDP-43 cells. (a) Validation of TDP-43 overexpression in N2a-hTDP-43 cells; 48 h post-transfection as indicated, TDP-43 was visualized with a TDP-43–specific antibody. Yellow arrows indicate cytoplasmic TDP-43 aggregates. (b, c) Intracellular ROS levels are increased in N2a-hTDP-43 cells. Intracellular ROS levels were measured using CellROX Deep Red and normalized to corresponding cell numbers ( n = 3, unpaired t test). (d, e) Validation of Grx1 antibody specificity. N2a cells were transfected with 20 nM Grx1-specific siRNA (siGrx1) or control siRNA (siCon). Endogenous Grx1 levels were normalized to corresponding cell numbers ( n = 3, unpaired t test). (f, g) Endogenous Grx1 levels are increased in N2a-hTDP-43 cells. Each level was normalized to the corresponding cell number and presented as a relative fold change to control ( n = 3, unpaired t test). Nuclei were stained with DAPI (blue). Scale bars, 25 µm. Grx1, glutaredoxin-1; N2a-hTDP-43, neuro-2a cells expressing human wild-type TDP-43; ROS, reactive oxygen species; TDP-43, transactive response DNA-binding protein 43.

Article Snippet: The coding sequences of full-length human wild-type TDP-43 (hTDP-43 , NM_007375 ) and Grx1( NM_002064.3 ) were cloned into pAAV-MCS (VPK-410, Cell Biolabs, San Diego, California, USA) and pCMV6-AC-Myc-DDK vector ( PS100001 , Origene Technologies, Rockville, Maryland, USA), respectively.

Techniques: Biomarker Discovery, Over Expression, Transfection, Control, Staining, Expressing, Binding Assay

Journal: Neuroreport

Article Title: Glutaredoxin-1 attenuates transactive response DNA-binding protein 43–induced neurotoxicity by suppressing oxidative stress and transactive response DNA-binding protein 43 aggregation

doi: 10.1097/WNR.0000000000002266

Figure Lengend Snippet: Increasing Grx1 decreases oxidative stress in N2a-hTDP-43 cells. (a, b) Validation of Grx1 overexpression in N2a-hTDP-43 cells; 48 h post-transfection as indicated, cells were simultaneously stained for TDP-43 (red) and Myc-tagged Grx1 (green) ( n = 3, one-way ANOVA). (c, d) Grx1 overexpression suppresses decreases ROS levels in N2a-hTDP-43 cells. Scale bars correspond to 25 µm. Each level was normalized to the corresponding cell number and presented as a relative fold change to control ( n = 3, one-way ANOVA). ANOVA, analysis of variance; Grx1, glutaredoxin-1; N2a-hTDP-43, neuro-2a cells expressing human wild-type TDP-43; ROS, reactive oxygen species; TDP-43, transactive response DNA-binding protein 43.

Article Snippet: The coding sequences of full-length human wild-type TDP-43 (hTDP-43 , NM_007375 ) and Grx1( NM_002064.3 ) were cloned into pAAV-MCS (VPK-410, Cell Biolabs, San Diego, California, USA) and pCMV6-AC-Myc-DDK vector ( PS100001 , Origene Technologies, Rockville, Maryland, USA), respectively.

Techniques: Biomarker Discovery, Over Expression, Transfection, Staining, Control, Expressing, Binding Assay

Journal: Neuroreport

Article Title: Glutaredoxin-1 attenuates transactive response DNA-binding protein 43–induced neurotoxicity by suppressing oxidative stress and transactive response DNA-binding protein 43 aggregation

doi: 10.1097/WNR.0000000000002266

Figure Lengend Snippet: Increasing Grx1 prevents TDP-43 aggregation in N2a-hTDP-43 cells. (a, b) Overexpressing Grx1 significantly reduces cytoplasmic TDP-43 aggregates in N2a-hTDP-43 cells. N2a cells were stained for TDP-43 (red) and DAPI (blue). Cells with cytoplasmic TDP-43 aggregates (yellow arrows) were presented as a percentage of cells ( n = 3, one-way ANOVA). Scale bars correspond to 25 µm. (c, d) Overexpressing Grx1 decreases total TDP-43 levels in N2a-hTDP-43 cells; 48 h post-transfection as indicated, total proteins were extracted using SDS-containing RIPA lysis buffer. TDP-43 protein levels were normalized to corresponding GAPDH levels ( n = 3, one-way ANOVA). (e–g) Overexpressing Grx1 decreases both soluble and insoluble TDP-43 levels in N2a-hTDP-43 cells. TDP-43 protein levels were assessed by western blot in Triton X-100 soluble (f) and insoluble fractions (g) and normalized to corresponding GAPDH and Ponceau S levels, respectively ( n = 3, one-way ANOVA). ANOVA, analysis of variance; Grx1, glutaredoxin-1; N2a-hTDP-43, neuro-2a cells expressing human wild-type TDP-43; TDP-43, transactive response DNA-binding protein 43.

Article Snippet: The coding sequences of full-length human wild-type TDP-43 (hTDP-43 , NM_007375 ) and Grx1( NM_002064.3 ) were cloned into pAAV-MCS (VPK-410, Cell Biolabs, San Diego, California, USA) and pCMV6-AC-Myc-DDK vector ( PS100001 , Origene Technologies, Rockville, Maryland, USA), respectively.

Techniques: Staining, Transfection, Lysis, Western Blot, Expressing, Binding Assay

Journal: Neuroreport

Article Title: Glutaredoxin-1 attenuates transactive response DNA-binding protein 43–induced neurotoxicity by suppressing oxidative stress and transactive response DNA-binding protein 43 aggregation

doi: 10.1097/WNR.0000000000002266

Figure Lengend Snippet: Increasing Grx1 attenuates neurotoxicity in N2a cells overexpressing hTDP-43; 48 h post-transfection as indicated, N2a cells were stained with cleaved caspase-3–specific antibody and DAPI (blue) (a). Scale bars correspond to 25 µm. Each level was normalized to the corresponding cell number and presented as a percentage of cells with cleaved caspase-3 signal (b) ( n = 3, one-way ANOVA). ANOVA, analysis of variance; Grx1, glutaredoxin-1; N2a, neuro-2a; TDP-43, transactive response DNA-binding protein 43

Article Snippet: The coding sequences of full-length human wild-type TDP-43 (hTDP-43 , NM_007375 ) and Grx1( NM_002064.3 ) were cloned into pAAV-MCS (VPK-410, Cell Biolabs, San Diego, California, USA) and pCMV6-AC-Myc-DDK vector ( PS100001 , Origene Technologies, Rockville, Maryland, USA), respectively.

Techniques: Transfection, Staining, Binding Assay

Journal: Nature Neuroscience

Article Title: In vivo CRISPR screen reveals regulation of macrophage states in neuroinflammation

doi: 10.1038/s41593-025-02151-6

Figure Lengend Snippet: a , Left, expression of each transcriptional signature related to lesion-promoting properties in Hoxb8FL control cells; right, phenotype of the KOs in Hoxb8FL-derived cells for the respective signature across macrophage clusters (Ma_Cx_1 to Ma_8) and overall (All) compared to the control cells. The shading/lines represent the distribution of log 2 (fold change or FC) of the KO compared to the control for all the genes that make up the signature, with the thick line being the median (quantile 50) log 2 (fold change), and the thinner overlaid shadows representing the lines of the adjacent quantiles: 40th and 60th quantiles of the log 2 (fold change) delimit the darkest shadow flanking the median line, in steps of 10 until the 10th and 90th quantiles delimiting the lightest shadow at the edges. For statistical analysis, gene-set enrichment analysis (GSEA) was used . b , Scheme illustrating the detection of oxidized/reduced cellular states by excitation ratiometric imaging of the Grx1-roGFP2 sensor. c , Experimental design of the intravital imaging experiments based on the transfer of Hoxb8FL-derived myeloid cells expressing the Grx1-roGFP2 sensor. d , Representative images of the dorsal lumbar spinal cord along the midline vein, showing a neuroinflammatory lesion infiltrated by Hoxb8FL-derived myeloid cells expressing the Grx1-roGFP2 sensor. Filled white arrowheads indicate myeloid cells in an oxidized state, while outlined arrowheads indicate myeloid cells in a reduced state. Scale bars, 50 μm (overview image) and 10 μm (inset). e , f , Quantification of the redox state of Ifngr1 -KO ( e ) or Tnfrsf1a- KO ( f ) Hoxb8FL-derived myeloid cells expressing the Grx1-roGFP2 sensor. Left, proportion of control (gray) or KO (purple) cells per animal that fall in each oxidation/reduction Grx1-roGFP2 ratio bin, where lower bin numbers mean low ratio; the whole range is divided into ten bins and each bin is equally sized; middle, cumulative distribution of the oxidized/reduced Grx1-roGFP2 ratios of all cells; right, percentage of cells per animal that show an oxidized/reduced ratio above the 75th percentile ratio of their experiment. N = 5 control and 4 Ifngr1 -KO animals from two independent experiments; control, 883, 587, 95, 172 and 677, and Ifngr1- KO, 2,394, 1,585, 96 and 99 cells per animal analyzed ( e ); N = 4 control and 4 Tnfrsf1a -KO from two different independent experiments; control, 33, 128, 36 and 342, and Tnfrsf1a- KO, 145, 1612, 123 and 828 cells per animal analyzed ( f ). K–S, Kolmogorov–Smirnov statistic. In the box plots, the line shows median, the box extends from the first quartile (Q1) to the third quartile (Q3), and the whiskers extend to the smallest and largest values within 1.5 times the interquartile range from Q1 and Q3. In a , KO names marked by an asterisk at the top of the plots and black ‘All’ labels indicate that the phenotype of the signature for that KO versus control in the global Hoxb8FL-derived cells is significant. Blue indicates downregulated and red indicates upregulated in the KO compared to the control. Asterisks and black cluster name labels indicate the phenotype is significant for that cluster for the Hoxb8FL-derived KO versus the control. Significance was determined with the GSEA pathway analysis algorithm for a nominal (NOM) P value < 0.05 or an FDR q value < 0.25 and absolute normalized enrichment score (NES) > 1.5 ; e , f , (left), Ordinary two-way ANOVA with a single pooled variance, F = 2.009, P = 0.0509 ( e ) and F = 0.2027, P = 0.9929 ( f ) for the interaction of genotype × bin. e , f (middle) K–S statistic. e , f , (right) one-tailed unpaired t -test. NS, P value > 0.05, * P value < 0.05, ** P value < 0.01, *** P value < 0.001, **** P value < 0.0001; figures show the mean ± s.d.

Article Snippet: The pMSCV-v2-U6-(BbsI)-Pgk-Grx1-roGFP2 vector was generated as follows: the pLPCX cyto Grx1-roGFP2 (Addgene) and pMSCV-v2-U6-(BbsI)-Pgk-Puro-T2A-BFP vectors were digested with BglII + ClaI and the Grx1-roGFP2 and pMSCV-v2-U6-(BbsI)-Pgk constructs were ligated with Quick Ligase.

Techniques: Expressing, Control, Derivative Assay, Imaging, One-tailed Test

Journal: Journal of Pharmaceutical Analysis

Article Title: Naringenin boosts Parkin-mediated mitophagy via estrogen receptor alpha to maintain mitochondrial quality control and heal diabetic foot ulcer

doi: 10.1016/j.jpha.2025.101333

Figure Lengend Snippet: Naringenin (NAR) alleviates oxidative stress and inhibition of autophagy induced by high glucose (HG). (A) HaCaT cells were treated with NAR (0.12, 0.37, 1.1, and 3.3 μM) for 24 h after being induced with HG (50 mM) for 24 h. Then, HaCaT cells were incubated with dihydroethidium (DHE) probes for 30 min, and the reactive oxygen species (ROS) levels in each group were detected using inverted fluorescence microscope. (B) HaCaT cells were incubated with DHE probes for 30 min, and the fluorescence intensity of DHE at different excitation wavelengths was subsequently detected using a multifunctional microplate detection platform. The 535 nm/370 nm ratio was quantified to assess ROS levels. (C) HaCaT cells were incubated with 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide (JC-1) dye for 30 min, and the JC-1 fluorescence was then examined using inverted fluorescence microscope. JC-1 aggregates (red) represent mitochondria with normal membrane potential, while JC-1 monomers (green) represent mitochondria with membrane potential depolarization. (D) The levels of oxidative stress-related proteins, including nicotinamide adenine dinucleotide phosphate (NAD(P)H):quinone oxidoreductase 1 (NQO1), superoxide dismutase 2 (SOD2), SOD1, and glutaredoxin 1 (GRX1), in HaCaT cells were measured by Western blotting. (E) Representative immunohistochemistry (IHC) staining images of NQO1 in wounds of mice at day 9 post-puncture. (F) The levels of sequestosome-1 (P62) and microtubule-associated proteins 1A/1B light chain 3C (LC3) in HaCaT cells were detected by Western blotting. (G) HaCaT cells were immunostained with antibody against LC3 and visualized using confocal microscope. (H) The LC3 puncta in the cells were quantified by ImageJ. Data are presented as mean ± standard deviation (SD). ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001, compared with the control group. GAPDH: glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: After blocking with 5% fat-free milk in Tris-buffered saline containing 0.1% Tween 20, the PVDF membrane was incubated with primary antibodies against β-actin (Santa Cruz Biotechnology), β-tubulin (Biodragon, Suzhou, China), p-H2A histone family member X (p-γH2AX) (Bioss, Beijing, China), adenosine triphosphate (ATP) synthase F1 subunit alpha (ATP5F1A) (Sangon Biotech Co., Ltd.), Cav-1 (Sangon Biotech Co., Ltd.), cytochrome C oxidase subunit 4 (COX IV) (Proteintech), dynamin-1-like protein (DRP1) (Sangon Biotech Co., Ltd.), ERα (Proteintech), ERβ (Sangon Biotech Co., Ltd.), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Proteintech), glutaredoxin 1 (GRX1) (Proteintech), histone H3 (Proteintech), heat shock protein 60 (HSP60) (Sangon Biotech Co., Ltd.), LaminB1 (Sangon Biotech Co., Ltd.), LC3 (MBL), mitofusin 2 (MFN2) (Proteintech), cytochrome C oxidase subunit 2 (MT-CO2) (Sangon Biotech Co., Ltd.), nicotinamide adenine dinucleotide (NADH) dehydrogenase (ubiquinone) iron-sulfur protein 4 (Ndufs4) (Sangon Biotech Co., Ltd.), nuclear factor-κB (NF-κB) (Proteintech), NQO1 (Sangon Biotech Co., Ltd.), nuclear respiratory factor 1 (NRF1) (Proteintech), cyclin-dependent kinase inhibitor 1A (P21) (Proteintech), sequestosome-1 (P62) (Proteintech), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) (Proteintech), PTEN-induced putative kinase 1 (PINK1) (Novus Biologicals, Littleton, CO, USA), p-NF-κB (S536) (Cell Signaling Technology), p-PINK1 (S228) (Thermo Fisher Scientific Inc.), succinate dehydrogenase complex flavoprotein subunit A (SDHA) (Proteintech), superoxide dismutase 1 (SOD1) (Proteintech), SOD2 (Proteintech), transcription factor A mitochondrial (TFAM) (Proteintech), translocase of inner mitochondrial membrane 23 (Tim23) (BD Biosciences, San Jose, CA, USA), Tomm20 (Proteintech), and voltage-dependent anion-selective channel protein 1 (VDAC1) (Sangon Biotech Co., Ltd.) overnight at 4 °C.

Techniques: Inhibition, Incubation, Fluorescence, Microscopy, Membrane, Western Blot, Immunohistochemistry, Standard Deviation, Control