Reduction of Top2/Top2b extends the life span of yeast, worm, and mice

(A) Schematic of top2 DAmP approach in S.cerevisiae. DAmP, decreased abundance by mRNA perturbation; KanR, kanamycin resistance.

(B) Relative top2 mRNA levels in BY4741 and BY4742 wild type (WT) strains and the corresponding top2 DAmP mutants, as measured by RT-qPCR.

(C) Replicative life span (RLS) of BY4741 and BY4742 WT strains and the top2 DAmP mutants.

(D) Schematic of top-2 RNAi approach in C. elegans. RNAi, RNA interference; RISC, RNA-induced silencing complex.

(E) Relative top-2 mRNA levels in C. elegans for the control group and the top-2 RNAi groups, as measured by RT-qPCR.

(F) Life span of the C. elegans for the control and top-2 RNAi groups.

(G) Schematic of Top2b knockdown by the CRISPR/CasRx system in mice. AAV, adeno-associated virus.

(H) Relative Top2b mRNA levels in the kidneys, lungs, skeletal muscles, hearts, and cortices of mice in the gLacZ control and gTop2b groups, measured by RT-qPCR.

(I-J) Relative Top2b protein levels in the kidneys, lungs, skeletal muscles, hearts, and cortices of mice in the gLacZ control and gTop2b groups, as measured by western blot.

(K) The life span of C57BL/6 mice for the gLacZ control and gTop2b groups.

Statistical analysis was performed using GraphPad Prism v8.0 software (https://www.graphpad.com). Data were considered statistically significant at P < 0.05 calculated by using Student’s t-test (B, E, H, and J) or log-rank test (C, F, and K). n.s. indicates not significant. All values are means ± SEM. The corresponding n values (number of cells, worms, or mice) are shown within each sub-plot.

Top2b/Top2 reduction improves the health span of mice and worms

(A) Frailty index (FI) scores for C57BL/6 mice in the gLacZ and gTop2b groups were recorded at 10 to 26 months of age, with assessments conducted once every two months. The number of mice assessed at each age is listed at the bottom of the plot.

(B) Bright-field images of gLacZ and gTop2b mice.

(C) Number of kyphosis in C57BL/6 mice of the gLacZ and gTop2b groups at 20 months of age.

(D) Body weight of C57BL/6 mice in the gLacZ and gTop2b groups at 12, 16, and 20 months of age.

(E) Falling velocity in rotarod tests for C57BL/6 mice in the gLacZ and gTop2b groups.

(F-I) Representative images of open field tests and number of zone transitions, entries in the center, and distance in the center for C57BL/6 mice in the gLacZ and gTop2b groups. (J-L) Representative images of Y-maze spontaneous alternation tests and total arm entries and alternation triplet times for C57BL/6 mice in the gLacZ and gTop2b groups. (M-P) Representative images of Y-maze novel arm tests and entries in novel arm, distance in novel arm, and alternation triplet times for C57BL/6 mice in the gLacZ and gTop2b groups.

(Q-S) Representative images of elevated zero maze tests and distance in zone-open and time in zone-open for C57BL/6 mice in the gLacZ and gTop2b groups.

(T-U) Representative images of novel object recognition tests and time in zone-new for C57BL/6 mice in the gLacZ and gTop2b groups.

(V-W) Representative images of tail suspension tests and mobility for C57BL/6 mice in the gLacZ and gTop2b groups.

(X-Z) Representative images of body bend and bending frequency and pharyngeal pumping rate in C. elegans fed with control RNAi and top-2 RNAi after 5 and 15 days.

Statistical analysis was performed using GraphPad Prism v8.0 software (https://www.graphpad.com). Data were considered statistically significant at P < 0.05 calculated by using Student’s t-test (A, D, E, G-I, K, L, N-P, R, S, U, W, Y, and Z) or Chi-squared test (C). n.s. indicates not significant. All values are means ± SEM or n (%). The corresponding n values (number of mice or worms) are shown in each sub-plot.

Top2b reduction mitigates the characteristics and pathologies associated with aging in multiple mouse tissues

Histology of various tissues of 23-month-old mice in the gLacZ control group and gTop2 group is analyzed (A-L).

(A) The longitudinal sections of the kidney tissues of were stained with HE. The right panels show a magnified view of the boxed area in the left panels. Scale bars represent 200 μm and 30 μm for the left and right panels.

(B) Average glomerular atrophy.

(C) The transverse sections of mouse skeletal muscle were stained with HE. The right panels show a magnified view of the boxed area in the left panels. Scale bars represent 200 μm for the left panels and 60 μm for the right panels.

(D) Average cross-sectional area of myofibers.

(E) Mouse liver tissues were stained with HE. The right panels show a magnified view of the boxed area in the left panels. Scale bars represent 60 μm for the left panels and 30 μm for the right panels. The yellow circles highlight the hepatic lobule. The red circles indicate anisokaryosis.

(F) Average anisokaryosis.

(G) Mouse lung tissues were stained with HE. The right panels show a magnified view of the boxed area in the left panels. Scale bars represent 200 μm for the left panels and 30 μm for the right panels.

(H) Average thickness of alveolar septa.

(I) Mouse skin tissues were stained with HE. The right panels show a magnified view of the boxed area in the left panels. Scale bars represent 200 μm for the left panels and 60 μm for the right panels.

(J) Average hair follicle density, average dermis layer thickness, and average epidermis layer thickness in mouse skin.

(K) The short axes of cardiac tissues from mice were stained with HE in the left ventricular wall regions. The right panels show a magnified view of the squared area in the left panels. Scale bars represent 600 μm and 30 μm for the left and right panels.

(L) The number of nuclei in each region of the left ventricular wall.

(M) Representative images of SA-β-Gal staining of various tissues from young (2m old), middle age (10m old), old (23m old) gLacZ control, and old (23m old) gTop2b groups.

(N) quantitation of percent of SA-β-Gal positive cells in various tissues.

Statistical analysis was performed using GraphPad Prism v8.0 software (https://www.graphpad.com). Data were considered statistically significant at P < 0.05 calculated by using Student’s t-test (B, D, F, H, J, and L) or one-way ANOVA (N). All values are means ± SEM. The corresponding n values (number of mice) are shown within each sub-plot.

Top2b knock down reduces various cellular aging hallmarks in human IMR-90 cells and in various mouse tissues

(A-H) IMR-90 cells were induced to undergo replicative, stress-induced, or oncogene-induced senescence. The protein levels of p16, p21, and γH2AX were detected by western blotting (A, B). γH2AX foci in DAPI-stained nuclei of IMR-90 cells were measured by immunofluorescent staining (C, D). The protein level of PARP1 was detected by western blotting (E, F). SA-β-Gal staining (blue-stained cells) and quantification of percent of SA-β-Gal positive cells were shown in G, H.

(I-P) Confocal fluorescence images of IMR-90 cells (I-K) and C. elegans (N-P) stained by LysoTracker Red DND-99 (LTR) and LysoSensorTM Green DND-189 (LSG), and the fluorescence intensity ratio of LSG/LTR was measured as an indicator of lysosomal acidity. (L, M) IMR-90 cells were induced to undergo replicative, stress-induced, or oncogene-induced senescence. The protein level of TFEB was detected by western blotting. The relative intensity of LSG/LTR and average lysosome diameter in IMR-90 cells (J, K) and C. elegans (O, P) were quantified.

(Q-R) Western blot analysis of nutrient-sensing mTOR signaling proteins in IMR-90 cells. (S-Y) Western blot analysis of nutrient-sensing mTOR signaling proteins in mouse kidney, lung, and skeletal muscle.

Statistical analysis was performed using GraphPad Prism v8.0 software (https://www.graphpad.com). Data were considered statistically significant at P < 0.05 calculated by using Student’s t-test (T-Y) or one-way ANOVA (B, D, F, G, M, R) or Kruskal-Wallis test (J, K, O, P). All values are means ± SEM. The corresponding number of samples for IMR-90 cells, number of worms, and number of mice are shown within each sub-plot.

Top2b knockdown led to changes of the global transcriptional program targeting multiple aging hallmarks.

(A) The adjusted Top2 FPKM levels between the control and top-2 RNAi-treated C. elegans. FPKM, fragments per kilobase of exon per million mapped fragments.

(B) Transcriptome analysis of the upregulated (up) and downregulated (down) DEGs between control and top-2 RNAi-treated C. elegans.

(C, D) GO analysis identified up (C) and down (D) regulated functional categories in DEGs between control and top-2 RNAi-treated C. elegans. BP, biological process; KEGG, Kyoto Encyclopedia of Genes and Genomes.

(E) The adjusted Top2b FPKM levels in the kidney, lung, and skeletal muscle from gLacZ and gTop2b mice

(F) Transcriptome analysis of the up and down DEGs in the kidney tissues between gLacZ and gTop2b mice.

(G, H) GO analysis identified up (G) and down (H) regulated categories in the kidney tissues between gLacZ and gTop2b mice.

(I) Transcriptome analysis of the up and down DEGs in the lung tissues between gLacZ and gTop2b mice.

(J, K) GO analysis identified up (J) and down (K) regulated categories in the lung tissues between gLacZ and gTop2b mice.

(L) Transcriptome analysis of the up and down DEGs in the skeletal muscle tissues between gLacZ and gTop2b mice.

(M, N) GO analysis identified up (M) and down (N) regulated categories in the skeletal muscle tissues between gLacZ and gTop2b mice.

(O) Circos plot illustrating connections between DEGs due to TOP2b knockdown in different tissues and hallmarks of aging.

Statistical analysis was performed using GraphPad Prism v8.0 software (https://www.graphpad.com). Data were considered statistically significant at P < 0.05 calculated by using Student’s t-test (A and E) or Fisher’s exact test and adjusted by the Benjamini-Hochberg method (C, D, G, H, J, K, M, and N). Genes with |log2fold change (FC)| > 1 and adjusted P value (by the Benjamini–Hochberg method) < 0.05 were considered DEGs (B, F, I, and L). n.s. indicates not significant. All values are means ± SEM. The corresponding n values (number of mice) are shown within each sub-plot.

Top2b reduction reprograms the epigenetic landscape and differentially down-regulates genes with active promoters/high abundance.

(A-D) Western blot analysis of histone modification markers in mouse kidney, lung, and skeletal muscle.

(E-G) The transcriptional state of the promoters of DEGs from mouse kidney, lung, and skeletal muscle. The distribution of the log2(fold change) of active vs. inactive genes (left panel) and percent of active and inactive genes in the up and down regulated DEGs (right panel) were shown.

(H) The ratio of genes up-regulated/down-regulated by Top2b knockdown in skeletal muscle decreases with the abundance in gLacZ mice. Genes were grouped into low, middle, high, and extremely high abundance groups according to the total FPKM values in gLacZ mice as indicated.

(I) Examples of DEGs with extremely high abundance in muscle.

(J) Heatmap depicting the expression levels of genes related to histone methyltransferases (HMTs) and histone lysine demethylases (KDMs) that regulate the trimethylation of histone H3 at lysine residues 4 (H3K4me3), 9 (H3K9me3), and 27 (H3K27me3) in skeletal muscle.

Statistical analysis was performed using GraphPad Prism v8.0 software (https://www.graphpad.com). Data were considered statistically significant at P < 0.05 calculated by using the Student’s t-test (A and E) or Chi-squared test (E-G). All values are means ± SEM or n (%). The corresponding n values (number of mice) are shown within each sub-plot. *** P<0.001.