Citrulline prevents age-related LTP decline in old rats

Abstract

The prevalence of cognitive decline is increasing as the ageing population is considerably growing. Restricting this age-associated process has become a challenging public health issue. The age-related increase in oxidative stress plays a major role in cognitive decline, because of its harmful effect on functional plasticity of the brain, such as long-term potentiation (LTP). Here, we show that citrulline (Cit) has powerful antioxidant properties that can limit ex vivo oxidative stress-induced LTP impairment in the hippocampus. We also illustrate that a three-month Cit supplementation has a protective effect on LTP in aged rats in vivo. The identification of a Cit oxidation byproduct in vitro suggests that the antioxidant properties of Cit could result from its own oxidation. Cit supplementation may be a promising preventive nutritional approach to limit age-related cognitive decline.

Figure 1

Protective effect of Cit against H2O2-induced oxidative stress in SH-SY5Y cell line. (A) Cit effect on intracellular oxidative stress (IOS). IOS was assessed by GSH/Total Glutathione ratio and expressed as % of control. GSH/Total glutathione ratio has been assessed in control conditions (CTRL), after oxidative stress induced by H2O2 500 µM (H2O2), after H2O2 500 µM in the presence of Cit 5 mM (Cit 5 + H2O2) or Cit 10 mM (Cit 10 + H2O2). The significant decrease in GSH/Total glutathione ratio is shown by* (*p < 0.03). (B) Cit effect on H2O2-induced cell death. Cell death has been assessed in control condition (CTRL), after treatment with H2O2 500 µM (H2O2) alone and in the presence of Cit 5 mM (Cit 5 + H2O2) or Cit 10 mM (Cit 10 + H2O2). The results are expressed as % of control. The significances are designed as followed: *: H2O2 vs CTRL; #: Cit + H2O2 vs H2O2; +: Cit10 + H2O2 vs Cit5 + H2O2. (^****p < 0.0001; ^####p < 0.0001; ⁺⁺⁺p = 0.0001). Data in A and B are presented as the mean ± SEM of 4 independent experiments.

Figure 2

Protective effect of Cit on hippocampal LTP in ex-vivo slice preparation of young adult mice. All experiments in Fig. 2 are performed in mice. (A) Mean fEPSP/PFV slope ratio of AMPA receptor-mediated synaptic responses plotted against current intensity in control conditions (CTRL, n = 25 slices from 6 mice, white dots) or after application of H2O2 (150 µM, n = 11 slices from 8 mice, grey triangles) or H2O2 (150 µM) in the presence of Cit (5 mM) (n = 12 slices from 4 mice, black squares). (Repeated measures ANOVA, p = 0.91, NS). (B) Paired-pulse facilitation (PPF) of synaptic transmission was comparable in the three groups indicating that drug treatments did not alter presynaptic mechanisms underlying glutamate release (p =  0.66, ns, t-test). CTRL, n = 27 slices from 9 mice; H2O2, n = 12 slices from 7 mice; Cit + H2O2, n = 7 slices from 5 mice. (C) 3 × 100 Hz conditioning stimulus (black arrow) induces a long lasting potentiation of synaptic transmission in the 3 conditions. However, LTP stabilizes to a lower level in the presence of H2O2 (grey triangles) as compared to control conditions (white dots) (F(1,24) = 16.6, ***p < 0.001). This decrease in LTP expression is partially restored in the presence of Cit (black squares) (F(1.25) = 6.9, *p < 0.05). Drugs were applied in the superfusing medium at t = −20 min (white arrowhead) and maintained throughout the experiment. ndividual traces of fEPSPs are shown in the three conditions before (a) and 60 min after stimulation (b). (D) Mean fEPSP/PFV slope ratio of NMDA receptor-mediated synaptic responses plotted against current intensity in control conditions (CTRL, n = 10, white dots) and after application of H2O2 (150 µM) (n = 10, grey triangles) (p = 0.9, ns). (E) Mean fEPSP/PFV slope ratio of NMDA receptor-mediated synaptic responses plotted against current intensity in control conditions (CTRL, n = 10, white dots) and after application of H2O2 (150 µM) in presence of Cit (5mM) (n = 10, black squares) (p = 0.74, ns).

Figure 3

Theta Burst Stimulation-induced LTP is rescued in aged rats fed with Cit. Time course of changes in synaptic efficacy induced by a theta-burst conditioning stimulus (TBS, arrow) applied in the stratum radiatum of hippocampal CA1 area in aged control rats and aged rats fed with Cit for 12 weeks. While LTP is not promoted in control animal (white dots, 104.9 ± 4.2%, n = 12), a robust and significant long lasting potentiation is induced in Cit-supplemented animals (133.6 ± 4.7%, n = 12, grey diamonds). The difference in LTP expression between the two groups is statistically significant (F(1,22) = 22; ***p < 0.001).

Figure 4

UPLC-HRMS analysis of the products formed in vitro by the reaction between Cit and hydroxyl radicals. (A) Total ion current chromatograms of Cit 160 mM in H2O. (B) Total ion current chromatograms of Cit 160 mM in H2O containing H2O2 90 mM 6 h. Rt: retention time, A: area under curve. (C) Mass spectra displays at retention time 0.70 min for Cit 160 mM in H2O. (D) Mass spectra displays at retention time 1.46 min for Cit 160 mM in H2O containing H2O2 90 mM 6 h. Annotations were performed following fragmentation experiments on ions m/z 265.1279, 377.1915, 489.2556, 507.2264 and exact mass measurements (<5 ppm). (E) M_n correspond to condensation products of M1 formed in ion source of the mass spectrometer.