Running-Induced Systemic Cathepsin B Secretion Is Associated with Memory Function

Abstract

Peripheral processes that mediate beneficial effects of exercise on the brain remain sparsely explored. Here, we show that a muscle secretory factor, cathepsin B (CTSB) protein, is important for the cognitive and neurogenic benefits of running. Proteomic analysis revealed elevated levels of CTSB in conditioned medium derived from skeletal muscle cell cultures treated with AMP-kinase agonist AICAR. Consistently, running increased CTSB levels in mouse gastrocnemius muscle and plasma. Furthermore, recombinant CTSB application enhanced expression of brain-derived neurotrophic factor (BDNF) and doublecortin (DCX) in adult hippocampal progenitor cells through a mechanism dependent on the multifunctional protein P11. In vivo, in CTSB knockout (KO) mice, running did not enhance adult hippocampal neurogenesis and spatial memory function. Interestingly, in Rhesus monkeys and humans, treadmill exercise elevated CTSB in plasma. In humans, changes in CTSB levels correlated with fitness and hippocampus-dependent memory function. Our findings suggest CTSB as a mediator of effects of exercise on cognition.

Keywords: cathepsin B; exercise; hippocampus; humans; memory; mice; muscle.

Figure 1

CTSB as a candidate myokine. (A) CTSB is present in the conditioned media (CM) of AICAR (AIC, 100µM) and Vehicle (Veh, 0.1% DMSO) treated differentiated L6 myoblast cultures as indicated by WB analysis. PONCEAU staining was used as a loading control. (B) Flowchart indicating how CTSB was identified. (C, D, E) Ctsb gene expression and CTSB protein levels in cells and CM from cultures treated with Vehicle (0.1 % DMSO) or AICAR (100 µM). (C) Time-course analysis showed Ctsb mRNA increased 3 hours after AICAR treatment. (D) WB analysis of intracellular CTSB levels at indicated time-points (hours) after treatment with AICAR (100µM). The graph shows the relative CTSB intensity normalized by β-actin. There was no difference in intracellular CTSB levels. E) CTSB protein levels in the CM of AICAR (100 µM) treated cultures is increased at 6 and 12 hours as compared to control. (F–H) In vivo analyses of Ctsb gene expression and CTSB protein levels in plasma and gastrocnemius muscle tissue of sedentary (Sed) and running (Run) mice. (F) Running increased CTSB plasma levels at 14 and 30 days. (G) Ctsb gene expression was elevated in the mouse gastrocnemius muscle after 30 days of running. (H) CTSB protein levels are elevated in gastrocnemius muscle after 30 days of running. Specifically, WB analysis of CTSB levels and a graph showing the relative intensity normalized by β-actin. Data represent means ± S.E.M. *p < 0.05

Figure 2

Behavioral analyses of CTSB KO mice and WT littermates housed under sedentary or running conditions. (A) Total distance travelled in the open-field test did not differ between the groups (WT sedentary, WT-S; WT runner, WT-R; KO sedentary, KO-S; KO runner, KO-R). (B) Forced swim test showed increased immobility time in KO compared to WT mice. (C) Water maze acquisition over 7 days did not differ between the groups. (D) Probe trials (60-sec) were performed 24 hours and 48 hours after the last training session. WT-R mice preferred the target quadrant as compared to all other quadrants in both probe trials. WT-S mice searched preferentially in the target quadrant at 24 hours but not at 48 hours. Neither KO-S nor KO-R mice showed retention of spatial memory. (E) DCX⁺ Type C cell number/section was higher in WT than KO groups. (F) DCX⁺ Type D cell number/section in the WT-R group was greater than in all the other groups. (G) Representative DG images of DCX staining. (H–L) Patch-clamp recordings from mature DG cells in acute slices derived from WT (n=3 mice, 12 cells) and KO (n=3 mice, 15 cells). (H) Averaged data from WT (n=12) and KO (n=15) cells show a significant decrease in mIPSC frequency. (I–K) There was no change in (I) amplitude (J) rise time or (K) decay tau. (L) Representative traces of whole cell patch-clamp recordings of mIPSCs from WT (green trace) and KO (blue trace) mice. Data represent mean ± SEM.*p<0.05

Figure 3

Running-induced hippocampal Ctsb gene expression and analysis of CTSB function (A) Thirty days of running (R) increased hippocampal Ctsb mRNA levels compared to sedentary (S) controls. (B) aNPC cultures were treated with rCTSB (100 ng/mL) for 24 h and analyzed by Q-PCR arrays. (C–D) Dcx and Bdnf gene expression were among the top ten most elevated genes. RT-PCR analysis shows a significant increase in Dcx mRNA level after 48 hours and Bdnf mRNA level after 24 hours of rCTSB (100 ng/ml) treatment as compared to control (0 hour). (E) WB analysis of DCX, TrkB and BDNF protein levels in aNPCs treated with rCTSB (10 and 100 ng/ml) or Vehicle (Veh, Distilled water) for 24 h. The graphs show the relative intensity normalized by β-actin. rCTSB treatment significantly increased DCX and BDNF levels, as compared to Mock or Vehicle treatment. (F) P11 mRNA levels in PC12 cells are elevated 6, 12 and 24 hours after treatment with rCTSB, 100 ng/ml. Hsp90 gene was used for normalization. (G) Hippocampal CTSB and P11 levels in CTSB KO and WT littermates measured by WB analysis, β-actin was used as a control. P11 was significantly reduced in the CTSB KO. (H) Knockdown of P11 with si-RNA in PC12 cells decreased DCX protein level elevation by rCTSB (100 ng/ml), (Scr, scramble siRNA transfected cells) measured by WB analysis, β-actin was used as a control. (I, J) Effect of rCTSB (0.2, 2, 20 or 200 ng/ml) on cell migration. (I) Percentage of migrated PC12 cells treated with 5% FBS or 2ng/ml of rCTSB showed increased migration. (J) Knockdown of P11 in PC12 cells suppressed cell migration induced by rCTSB (2ng/ml) treatment for 5 hours as compared to Scr control. (K) Hippocampal 24-hydroxy cholesterol level was reduced in the CTSB KO mice. Data represent mean ± SEM. *p < 0.05. Veh, vehicle (Distilled water).

Figure 4

Effects of four months of treadmill exercise on correlations between plasma CTSB, fitness and complex figure recall (CF) in humans. (A) There is a positive correlation in humans between aerobic fitness change as measured by VO2 ventilatory threshold (VT) and CTSB plasma protein level change after 4 months of treadmill training intervention (p<0.016). (B) There is a positive correlation between change in CF score and CTSB level change (p<0.01, one-tailed; p=0.063, two-tailed) after 4 months of treadmill training.