(A) Representative immunostaining images for p16INK4A, HMGB1 and MAP2 in DIV14 neurons treated with insulin for different time courses. The appearance of nuclear p16INK4A signals and the loss of nuclear HMGB signals are well-established markers of senescence. Quantification of the p16INK4A+ and HMGB+ neurons is shown on the right (n=15, ***P=0.0001; **P=0.001, P=0.01, ns=non-significant, one-way ANOVA). (B) qPCR for Cdkn2a and Cdkn1a gene expression in neuronal cultures subjected to different period of chronic insulin treatment (n=10, ***P=0.001, **P=0.009, *P=0.032, one-way ANOVA). (C) Representative image of SA-β-gal in cerebellum of young insulin resistant mice (n=10). (D) Representative immunostaining images for p16INK4A, MAP2 and SA-β-gal in frontal cortex and hippocampal regions of young insulin resistant mice. Region with extensive neurite loss is highlighted in the dash boxes (n=10). (E) qPCR for Cdkn2a and Cdkn1a gene expression levels in frontal cortex and hippocampus tissues of young and aged mice (repeated independently for four times; n=10 each group, *** P=0.0005; **P=0.001, *P=0.02, #P=0.03; one-way ANOVA). (F-G) Representative images of (F) EGFP fluorescence signals and SA-β-gal histochemistry signals in sections harvested from mice stereotaxically injected AAV9 carrying EGFP scrambled or HK2 shRNA shuttles into hippocampal CA2 region. Quantification of SA-β-gal- and GFP-double positive hippocampal neurons Fig. 5e (Scrambled vs HK2 shRNA: n=11 vs n=10, ***P=0.0001, two-tailed unpaired t-test). (G) qPCR for Cdkn2a and Cdkn1a gene expression in hippocampal is shown (n=10, **P=0.009; *P<0.034, two-tailed unpaired t-test). (H-J) Escape latency during the (H) training phase and (I) probe trial of Morris water maze (MWM) test (Scrambled vs HK2 shRNA: n=11 vs n=9, *P=0.037; two-tailed unpaired t-test). (J) Percentage time spent in target quadrant (TQ) during the probe trial of MWM (Scrambled vs HK2 shRNA: n=11 vs n=9, #P=0.045; two-tailed unpaired t-test). (K-L) Correlation between (K) GFP-only (n=10, r=-0.7852, P=0.0071) or (L) SA-β-gal-and-GFP-double positive (n=10, r=-0.8654, P=0.0012) neurons in hippocampus of individual animal received HK2-shRNA with corresponding percentage time in TQ by two-tailed Spearman’s rank analyses. (M-N) SA-β-gal staining in (M) frontal cortex and (N) hippocampus, followed by immunohistochemistry for p16INK4A, p21 and MAP2 in the same hippocampal brain sections. Region with extensive neurite loss is highlighted in white boxes. Age and insulin resistance status are indicated (n=10, repeated independently for three times). (O) Quantification the percent SA-β-gal and p16INK4A double-positive cells per section (n=10, ***P=0.0001, P=0.001; one-way ANOVA). (P-T) Correlations of the abundance of SA-β-gal-positive neurons in cortex with mouse behavioral performance in (P) Morris water maze (n=24, r=-0.8069, P=0.0001); (Q) Y-maze maze (n=24, r=-0.5717, P=0.0035); (R) rotarod (n=24, r=0.3477, ns=non-significant); (S) forced swim (n=24, r=0.094, ns=non-significant) and (T) open field test (n=24, r=0.189, ns=non-significant) paradigms by two-sided Spearman’s rank analysis. Unless otherwise specified, all in vitro insulin treatments were performed at 100nM. Values represent the mean ± SEM.