Journal: Nature Communications

Article Title: PRDX1 promotes testosterone synthesis and attenuates aging via redox regulation of ATG4B to modulate lipophagy

doi: 10.1038/s41467-025-65328-4

Figure Lengend Snippet: a Representative H&E images of testicular biopsies of OA and NOA patients. n = 3 independent experiments with similar results. b Proteomic analyses by iTRAQ showing the relative protein level of PRDX1 in the testicular biopsies of OA versus NOA patients. Test-1, −2, −3 indicate the tests with three randomly paired samples. AVG, average; CV, coefficient of variation. c Representative IHC images showing the ranking of PRDX1 expression in testicular biopsies of OA and NOA groups. d PRDX1 expression ranking in a tissue microarray composed of testicular biopsies from NOA ( n = 30) and OA ( n = 25) patients. The differences in the PRDX1 expression between groups were tested using the Wilcoxon signed-rank test. e Venn diagram analysis integrating our proteomic data with three independent single-cell RNA sequencing datasets for human NOA, human LOH and mouse testis aging. Note that downregulation of PRDX1 was consistently involved in these pathologies. f Serum testosterone concentration of NOA ( n = 30) and OA ( n = 25) patients. g , h Co-immunofluorescence of PRDX1 (red) and DDX4 (green), a germ cell marker, in the testes of OA and NOA patients. Quantification data is presented in ( h ). n = 5 for each group. i , j Co-immunofluorescence of PRDX1 (red) and 3β-HSD1 (green), a LC marker, in the testes of OA and NOA cases. Quantification data is presented in ( j ). n = 5 for each group. k BODIPY staining showing lipid deposition in testes of OA and NOA patients. Quantification data for LD number in interstitial area are presented in ( l ). n = 5 for each group. Values in ( f ) represent Mean ± SD, others represent Mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001. Data in ( f ) ( h ), ( j ) and ( l ) were analyzed by t -test. Nuclei were stained with DAPI (blue). Scale bars = 100 μm in ( a ) and ( c ), 50 μm in ( g ), ( i ) and ( k ).

Article Snippet: The aged Ctrl and Prdx1 -cKO mice (19 months old) were intraperitoneally injected with ebselen (20 mg/kg/day; HY-13750, MedChemExpress, Elizabeth, NJ, USA.) once daily for 1 month.

Techniques: Multiplex sample analysis, Expressing, Microarray, RNA Sequencing, Concentration Assay, Immunofluorescence, Marker, Staining

Journal: Nature Communications

Article Title: PRDX1 promotes testosterone synthesis and attenuates aging via redox regulation of ATG4B to modulate lipophagy

doi: 10.1038/s41467-025-65328-4

Figure Lengend Snippet: a , b Co-immunofluorescence of PRDX1 (green) and SF1 (red) in testes from 1-month-old Ctrl and cKO mice. Quantification of PRDX1 intensity was showed in ( b ). c WB analyses of PRDX1 level in testes from 3-month-old Ctrl and cKO mice. d , e Representative H&E images showing testis morphology of 3- and 6-month-old Ctrl and cKO mice. d in box indicates the diameter of tubules and r indicates the radius. Quantification data were showed in ( e ). f , g Immunofluorescence of DDX4 (green), a germ cell marker in testes from 3- and 6-month-old Ctrl and cKO mice. Quantification of intensity was showed in ( g ). h Curves for the body (B) and testis (T) weight of Ctrl and cKO mice from 2 to 12 months old. i Curves for the cumulative litter size of Ctrl and cKO mice from 2 to 12 months old. Note that cKO mice became infertile at 6 months old, compared with 11 months old in Ctrl mice. j Sperm count of Ctrl and cKO mice from 2 to 12 months old. k Sperm motility of Ctrl and cKO mice from 2 to 12 months old. Note that the sperm motility of cKO mice at 2 months old equivalent to that of Ctrl mice at 8 months old. Type a, rapid progression; type b, low or sluggish progression; type c, nonprogressive. l Testicular testosterone levels of Ctrl and cKO mice from 2 to 12 months old. Testosterone levels were measured with total testis homogenates and values are presented as ng/mg protein. Five mice from each group were analyzed ( n = 5). * P < 0.05, ** P < 0.01, *** P < 0.001. n.s. not significant. All values represent Mean ± SEM. Data in ( b ) were analyzed by t -test, others by two-way ANOVA and sometimes followed by the Dunnett test for between-group differences. Nuclei were stained with DAPI (blue). Scale bars = 50 μm in ( a ), ( d ) and ( f ).

Article Snippet: The aged Ctrl and Prdx1 -cKO mice (19 months old) were intraperitoneally injected with ebselen (20 mg/kg/day; HY-13750, MedChemExpress, Elizabeth, NJ, USA.) once daily for 1 month.

Techniques: Immunofluorescence, Marker, Staining

Journal: Nature Communications

Article Title: PRDX1 promotes testosterone synthesis and attenuates aging via redox regulation of ATG4B to modulate lipophagy

doi: 10.1038/s41467-025-65328-4

Figure Lengend Snippet: a WB analyses of autophagy-related proteins in negative control (NC) and PRDX1 knockdown (KD) TM3 cells. The relative levels of LC3 and p62 were showed. b WB analyses of LC3 and p62 levels in cells after indicated treatments for 8 h. The relative levels of LC3 and p62 were showed. 3-MA (3-methyladenine, 10 mM), CQ (chloroquine, 20 μM), PA (pepstatin A,10 μg/mL), LP (leupeptin, 100 μM), Rapa (rapamycin, 10 nM). c WB analyses of LC3 and p62 levels in cells after treatment with gradient H O for 1 h. The relative levels of LC3 and p62 were showed. d – f Co-immunofluorescence of LC3 (green) and LAMP1 (red), a lysosome marker, in cells with or without H O treatment (100 μM, 1 h). Nuclei were stained with DAPI (blue). Relative intensity within the indicated area outlined in ( d ) was measured and the values indicate percentages of colocalizing signals ( e ). A.U., arbitrary units. Pearson’s colocalization coefficients were determined ( f ). n = 5 for each group. g , h LC3-GFP-RFP dual-fluorescence assay showing autophagic flux. Cells were transfected with lentivirus harboring LC3-GFP-RFP. The ratio of RFP + GFP + (presents autophagysome) to RFP + GFP - (presents autolysosome) was calculated and showed in ( h ). n = 5 for each group. i , j Proteinase K and Triton X-100 assay to examine the integrity of autophagosome membrane. Quantification of p62 level was showed in ( j ). n = 5 for each group. k – m Co-fluorescence of BODIPY 493/503 (green) and LC3 (red). Relative intensity within the indicated area outlined in ( k ) was measured and the values indicate percentages of overlapping signals ( l ). A.U., arbitrary units. Pearson’s colocalization coefficients were determined ( m ). n = 5 for each group. n – p Co-fluorescence of BODIPY 493/503 (green) and LysoTracker (red). Relative intensity within the indicated area outlined in ( n ) was measured and the values indicate percentages of colocalizing signals ( o ). A.U. arbitrary units. Pearson’s colocalization coefficients were determined ( p ). n = 5 for each group. q , r Lipoprotein uptake and degradation assay. Dil-HDL was added into media for 2 h and then withdrew for 8 h. Live-cell images were captured at indicated time-points. Average Dil-HDL intensity (red) per cell was measured and the curves were showed in ( r ). n = 5 for each group. s Time-lapse imaging of contacts between lipoproteins (Dil-HDL, red) and autophagic vacuoles (LC3, green). In NC group, lipoproteins were quickly degraded (white arrowheads), while in KD group, they kept steady (blue arrowheads). See Supplementary Movies and . t Time-lapse imaging of contacts between autophagic vacuoles (LC3, green) and lysosome (LysoTracker, red). In NC cells, LC3 puncta frequently contacted with lysosomes and then disappeared (gray arrowheads), with new LC3 puncta approaching lysosomes (cyan arrowheads). While in KD cells, a majority of LC3 puncta did not associated with lysosomes and remained visible (pink arrowheads), and even those having contacts with lysosomes kept visible (yellow arrowheads). See Supplementary Movies and . n = 50 cells examined over 5 independent experiments. *** P < 0.001. n.s. not significant. All values represent Mean ± SEM. Data in ( h ) were analyzed by chi-square test, those in ( m ) and ( p ) were analyzed by t -test, and others by two-way ANOVA followed by the Dunnett test for between-group differences. Scale bars = 5 μm in ( d ), ( k ) and ( n ), 100 μm in ( g ), 10 μm in ( q ), 1 μm in ( s ) and ( t ).

Article Snippet: The aged Ctrl and Prdx1 -cKO mice (19 months old) were intraperitoneally injected with ebselen (20 mg/kg/day; HY-13750, MedChemExpress, Elizabeth, NJ, USA.) once daily for 1 month.

Techniques: Negative Control, Knockdown, Immunofluorescence, Marker, Staining, Fluorescence, Transfection, Membrane, Degradation Assay, Imaging

Journal: Nature Communications

Article Title: PRDX1 promotes testosterone synthesis and attenuates aging via redox regulation of ATG4B to modulate lipophagy

doi: 10.1038/s41467-025-65328-4

Figure Lengend Snippet: a Co-IP/WB analyses of autophagy-related proteins in PRDX1 precipitates. Prdx1 -KD TM3 cells were transfected with an adenovirus carrying FLAG-PRDX1. Co-IP was performed using an anti-FLAG antibody. b Co-IP/WB analyses of PRDX1-ATG4B interaction with or without dithiothreitol (DTT, 1 mM), a disulfide bond reducing agent. Treatments were the same as in ( a ). c – e Co-immunofluorescence of ATG4B (red) and LC3 (green) in NC and KD cells, or NC cells treated with CQ (20 μM, 8 h) or H 2 O 2 (100 μM, 1 h). Relative intensity within the indicated area outlined in ( c ) was measured and the values indicate percentages of colocalizing signals ( d ). A.U., arbitrary units. Pearson’s colocalization coefficients were determined ( e ). n = 5 for each group. f Co-IP/WB analyses of ATG4B-LC3 interaction after PRDX1 knockdown or overexpression. NC and KD TM3 cells were transfected with GFP-LC3, and with or without FLAG-PRDX1 overexpression, finally treated with H 2 O 2 . Levels of PRDX1 and ATG4B in the GFP precipitates were detected by WB and quantified. g Roles of Cys52 and Cys173 in PRDX1 in association with ATG4B. HEK293 cells were transfected with plasmids of FLAG empty or FLAG-tagged WT PRDX1, or PRDX1 with single-point mutant C52A, C173A or C83A, or double-point mutant C52A/C173A. ATG4B level in the FLAG precipitates was detected by WB and quantified. h Effect of reconstitution of WT PRDX1 or mutants on the autophagic activity in KD cells. PRDX1-KD HEK293 cells were transfected with plasmids of FLAG-tagged WT PRDX1, or PRDX1 with C52A, C173A, C52A/C173A, or C83A mutation. Levels of LC3-II/I and p62 were detected by WB and quantified. i Roles of Cys74 and Cys78 in ATG4B in association with PRDX1 and LC3. HEK293 cells were transfected with plasmids of GFP tagged WT ATG4B, or ATG4B with Cys74S or Cys78S mutation, followed by treatment with or without H 2 O 2 . Levels of PRDX1 and LC3 were detected by WB and quantified. j GST pulldown assays of the molecular interaction between PRDX1 and ATG4B. 20 μg GST tag or GST-tagged PRDX1 proteins and 20 μg His-tagged ATG4B proteins were mixed, and pull downed by GSH magarose beads. k , l Measurement of the delipidation activity of ATG4B. The amount of LC3-I and LC3-II were quantified by gel densitometry, and the ratio of LC3I/LC3II was calculated as the readout for ATG4B delipidation activity. The quantification data were showed in ( l ). n = 5 for each group. m – o Co-immunofluorescence of ATG4B (GFP, green) and LC3 (red). HEK293 cells were transfected with plasmids of GFP-tagged WT ATG4B, or ATG4B with Cys74S or Cys78S mutation, followed by treatment with H 2 O 2 , with or without PRDX1 overexpression. Nuclei were stained with DAPI (blue). GFP/LC3 colocalization was analyzed ( n ). Quantification of LC3 puncta was showed in ( o ). n = 50 cells examined over 5 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001. n.s. not significant. All values represent Mean ± SEM. Data were analyzed by one-way ANOVA followed by the Dunnett test for between-group differences. Scale bars = 5 μm in ( c ) and ( m ).

Article Snippet: The aged Ctrl and Prdx1 -cKO mice (19 months old) were intraperitoneally injected with ebselen (20 mg/kg/day; HY-13750, MedChemExpress, Elizabeth, NJ, USA.) once daily for 1 month.

Techniques: Co-Immunoprecipitation Assay, Transfection, Immunofluorescence, Knockdown, Over Expression, Mutagenesis, Activity Assay, Staining

Journal: Nature Communications

Article Title: PRDX1 promotes testosterone synthesis and attenuates aging via redox regulation of ATG4B to modulate lipophagy

doi: 10.1038/s41467-025-65328-4

Figure Lengend Snippet: a , b Representative H&E images showing testis morphology of aged Ctrl and cKO mice, Ctrl and cKO mice with ebselen treatment, and cOE mice (conditional overexpression of PRDX1 in LCs). d in box indicates the diameter and r indicates the radius of tubules. Quantification data were showed in ( b ). Sperm count ( c ) and motility ( d ) of aged mice. e , f Testicular and serum testosterone levels of aged mice. g WB analyses of LC3 levels in testes of adult (4 months old) and aged mice (20 months old). The relative level of total LC3 to tubulin was showed. h – j Co-fluorescence of BODIPY 493/503 (green) and LC3 (red) in testes. Nuclei were stained with DAPI (blue). Relative intensity within the indicated area outlined in ( h ) was measured and the values indicate percentages of overlapping signals ( i ). A.U., arbitrary units. Pearson’s colocalization coefficients were determined ( j ). k , l TEM images displaying LDs and LD-autophagic vacuole (AV) contacts in the interstitial area of testes. Yellow asterisks (*) indicate LDs, blue triangles indicate AV-associated LDs. Quantification was showed in ( l ). m – p Memory, behavior and maximal exercise capacity in aged mice, revealed by Y-maze test ( m ), open field trail ( n ), and run-to-exhaustion test ( o ). Representative track images for the open field test were showed in ( p ). Five mice from each group were analyzed ( n = 5). * P < 0.05, ** P < 0.01, *** P < 0.001. n.s. not significant. Data were analyzed by one-way ANOVA followed by the Dunnett test for between-group differences. Scale bars = 20 μm in ( a ), 50 μm in ( h ), 1 μm in ( k ).

Article Snippet: The aged Ctrl and Prdx1 -cKO mice (19 months old) were intraperitoneally injected with ebselen (20 mg/kg/day; HY-13750, MedChemExpress, Elizabeth, NJ, USA.) once daily for 1 month.

Techniques: Over Expression, Fluorescence, Staining

Journal: Nature Communications

Article Title: PRDX1 promotes testosterone synthesis and attenuates aging via redox regulation of ATG4B to modulate lipophagy

doi: 10.1038/s41467-025-65328-4

Figure Lengend Snippet: A ATG4B serves as a “scissor” enzyme responsible for the delipidation of LC3II. Dimeric PRDX1 functions as a molecular shield by specifically targeting Cys78 of ATG4B via its Cys52 and Cys173 residues, thereby safeguarding the enzymatic activity of Cys74. This ensures the appropriate delipidation of LC3II on autophagic vacuoles and maintains the equilibrium between LC3II and LC3I, thus facilitating autophagosome maturation and the lipophagic process. Lipid metabolism proceeds seamlessly, ultimately leading to the production of testosterone, which is crucial for spermatogenesis and systemic metabolism. B When PRDX1 is inactivated or when oxidative stress disrupts the PRDX1-ATG4B interaction, Cys78 becomes oxidized, impairing the delipidating activity of Cys74. Consequently, autophagosome maturation is compromised, and lipophagy and lipid metabolism are disrupted, resulting in testosterone deficiency. This deficiency ultimately leads to male infertility and accelerates the processes of LOH and aging. Notably, all these disorders can be effectively mitigated by the administration of ebselen. Created in BioRender. Keke, M. (2025) https://BioRender.com/hb6bsv1 .

Article Snippet: The aged Ctrl and Prdx1 -cKO mice (19 months old) were intraperitoneally injected with ebselen (20 mg/kg/day; HY-13750, MedChemExpress, Elizabeth, NJ, USA.) once daily for 1 month.

Techniques: Activity Assay