Exercise-Induced Activated Platelets Increase Adult Hippocampal Precursor Proliferation and Promote Neuronal Differentiation

Abstract

Physical activity is a strong positive physiological modulator of adult neurogenesis in the hippocampal dentate gyrus. Although the underlying regulatory mechanisms are still unknown, systemic processes must be involved. Here we show that platelets are activated after acute periods of running, and that activated platelets promote neurogenesis, an effect that is likely mediated by platelet factor 4. Ex vivo, the beneficial effects of activated platelets and platelet factor 4 on neural precursor cells were dentate gyrus specific and not observed in the subventricular zone. Moreover, the depletion of circulating platelets in mice abolished the running-induced increase in precursor cell proliferation in the dentate gyrus following exercise. These findings demonstrate that platelets and their released factors can modulate adult neural precursor cells under physiological conditions and provide an intriguing link between running-induced platelet activation and the modulation of neurogenesis after exercise.

Keywords: adult mouse; dentate gyrus; exercise; neural precursor cell; neurogenesis; platelet activation; platelets.

Graphical abstract

Figure 1

Acute Exercise-Induced Peripheral Changes Are Platelet-Related and Increase the Number of DG-Derived Neurospheres (A) Animals ran for 4 days followed by serum and plasma collection in the morning of day 5. (B) Representative image of a neurosphere cultured with 0.01% mouse serum. Scale bar, 50 μm. (C) More neurospheres formed after the addition of 0.01% 4d RUN serum. Dashed line represents control cultures normalized to 100%. n = 3 independent experiments, ^∗p < 0.05, one-way ANOVA with Tukey test. Data represent the means ± SEM. (D) Enrichment of biological pathways of proteins with significantly increased plasma levels in 4d RUN mice (see also Table S1). Graph represents result of Reactome pathway analysis. Dashed line indicates false discovery rate (FDR) of 0.05.

Figure 2

Platelets Are Activated after Acute Periods of Running (A) Representative flow cytometry plots of platelet count and activation. Left: platelets and reference beads were first gated based on forward and side scatter. Platelets and activated platelets were defined by the expression of CD61 (middle) and CD62P (right), respectively. (B) Platelet count in STD and RUN mice measured by flow cytometry. n = 4 mice per group, ^∗∗p < 0.01, one-way ANOVA with Dunnett test. (C and D) Platelet count (C) and normalized platelet count (D) in a different cohort of mice determined by the Sysmex system. n = 4 mice per group, Student's t test. (E and F) Platelet activation in (E) C57BL/6 and (F) DBA/2 mice after running. n = 4 mice per group, ^∗p < 0.05, ^∗∗p < 0.01, one-way ANOVA with Dunnett test. (G) Time course of platelet activation. n = 4 mice per group, ^∗∗p < 0.01, ^∗∗∗p < 0.001, one-way ANOVA with Dunnett test. All data represent the means ± SEM.

Figure 3

Platelets Change Their Proteomic Signature after Acute Periods of Running Quantitative proteomic analysis of platelets from 4d RUN and STD mice revealed distinct platelet protein levels after physical activity (see also Table S2). Log10 p values were plotted against effect size (difference of means). 14-3-3γ showed the most significant increase after 4 days of running. The insert shows the log2-transformed and scaled protein values for 14-3-3γ. n = 4 mice per group, ^∗∗∗p < 0.001, Student's t test. Data represent the means ± SEM.

Figure 4

Activated Platelets Increase Neurogenesis and Directly Act on Hippocampal NPCs Ex Vivo (A) Neurosphere number after the addition of freshly isolated PPP and PRP. n = 7 independent experiments. (B) Platelet activation in the plasma of STD mice through cold and with phorbol-12-myristate-13-acetate (PMA). n = 4 samples of four individual mice, ^∗∗∗p < 0.001, one-way ANOVA with Dunnett test. (C) Representative flow cytometry plots showing cell populations as determined by forward and side scatter in freshly isolated (left) and cold-activated samples (right). Co-isolated cells are gated in P1, whereas P2 yields platelets. (D) Neurosphere assays with activated platelets in PPP and PRP. n = 11 independent experiments, ^∗p < 0.05, one-way ANOVA with Dunnett test. (E) Representative image of a neurosphere in control conditions (top) and a large neurosphere in RUN rich conditions (bottom). Scale bars, 100 μm. (F) Neurospheres (≥130 μm) cultured with activated platelets. n = 11 independent experiments, ^∗p < 0.05, one-way ANOVA with Dunnett test. (G) Flow cytometry plots of gating strategy to isolate lysophosphatidic acid receptor 1 (LPA₁)⁺ cells. Left: viable DG cells from LPA₁-GFP mice were determined by forward and side scatter. Right: plot of sorted LPA₁-GFP⁺ and LPA₁-GFP^- cells. (H) Neurosphere assay using LPA₁-GFP⁺ cells with activated PPP and PRP from STD mice. n = 7 independent experiments, ^∗∗p < 0.01, one-way ANOVA with Dunnett test. (I) Representative image of differentiated neurospheres showing GFAP⁺ astrocytes (green) and β-tubulin⁺ neurons (red). Scale bar, 50 μm. (J and K) Quantification of GFAP⁺ astrocytes is shown in (J) and β-tubulin⁺ neurons in (K). n = 4 independent experiments, ^∗p < 0.05, one-way ANOVA with Dunnett test. Dashed lines represent control cultures normalized to 100%. All data represent the means ± SEM.

Figure 5

Activated Platelets Do Not Increase Neurogenesis in SVZ-Derived Cultures (A) Animals were running for 4 days and received a single BrdU injection 12 h prior to perfusion. Graphs show quantification of BrdU⁺ cells in the DG and SVZ after 4 days of running. n = 10–13 mice per group. ^∗p < 0.05, Student's t test. See also Figure S1. (B) Neurosphere assays with activated platelets in PPP and PRP. n = 10 independent experiments, ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, one-way ANOVA with Dunnett test. (C) Representative image of differentiated SVZ neurospheres with GFAP⁺ astrocytes (green) and β-tubulin⁺ neurons (red). Scale bar, 100 μm. (D and E) Quantification of β-tubulin⁺ neurons (D) and GFAP⁺ astrocytes (E). n = 4 independent experiments. All data represent the means ± SEM.

Figure 6

Platelet Factor 4 Increases Neurogenesis in the DG (A) Relative plasma PF4 levels in STD and 4d RUN mice. n = 5 mice per group, ^∗p < 0.05, Student's t test. (B) Representative image of proliferating NPCs in monolayer culture. Scale bar, 50 μm. (C) Viability assay in NPC cultures with PF4. n = 5 independent experiments. (D) Neurosphere assays with different concentrations of PF4. n = 12 independent experiments, ^∗p < 0.05, one-way ANOVA with Dunnett test. (E and F) Quantification of β-tubulin⁺ neurons (E) and GFAP⁺ astrocytes (F) in differentiated neurosphere cultures treated with PF4. n = 3 independent experiments, ^∗∗p < 0.01, Student's t test. (G) Experimental setup of in vivo PF4 administration directly into the hippocampus of C57BL/6 mice. (H) Representative image of BrdU⁺ cells in the DG. Scale bar, 50 μm. (I) Quantification of BrdU⁺ cells in the SGZ of PF4-treated mice. n = 8 mice per group. (J) Representative images of newborn immature neurons stained with DCX. Scale bars, 50 μm. (K) Quantification of DCX⁺ cells in the DG of PF4-treated mice. n = 8 mice per group, ^∗p < 0.05, Student's t test. All data represent the means ± SEM.

Figure 7

In Vivo Platelet Depletion Abolishes the Running-Induced Increase in Precursor Cell Proliferation in the DG (A) Animals received a single injection of anti-platelet serum for 13 days, every second day. From days 3 to 13 the mice had access to a running wheel. (B) Platelet count of mice treated with anti-platelet and control serum (see also Table S3). n = 14–15 mice per group pooled from three independent experiments, ^∗∗∗∗p < 0.0001, one-way ANOVA with Sidak test. (C) Representative image of Ki67⁺ cells in the SGZ. Scale bar, 100 μm. (D) Quantification of Ki67⁺ cells in the SGZ. n = 14–15 mice per group pooled from three independent experiments, ^∗∗p < 0.01, one-way ANOVA with Sidak test. All data represent the means ± SEM.