Low-Affinity/High-Selectivity Dopamine Transport Inhibition Sufficient to Rescue Cognitive Functions in the Aging Rat

Abstract

The worldwide increase in cognitive decline, both in aging and with psychiatric disorders, warrants a search for pharmacological treatment. Although dopaminergic treatment approaches represent a major step forward, current dopamine transporter (DAT) inhibitors are not sufficiently specific as they also target other transporters and receptors, thus showing unwanted side effects. Herein, we describe an enantiomerically pure, highly specific DAT inhibitor, S-CE-123, synthetized in our laboratory. Following binding studies to DAT, NET and SERT, GPCR and kinome screening, pharmacokinetics and a basic neurotoxic screen, S-CE-123 was tested for its potential to enhance and/or rescue cognitive functions in young and in aged rats in the non-invasive reward-motivated paradigm of a hole-board test for spatial learning. In addition, an open field study with young rats was carried out. We demonstrated that S-CE-123 is a low-affinity but highly selective dopamine reuptake inhibitor with good bioavailability. S-CE-123 did not induce hyperlocomotion or anxiogenic or stereotypic behaviour in young rats. Our compound improved the performance of aged but not young rats in a reward-motivated task. The well-described impairment of the dopaminergic system in aging may underlie the age-specific effect. We propose S-CE-123 as a possible candidate for developing a tentative therapeutic strategy for age-related cognitive decline and cognitive dysfunction in psychiatric disorders.

Keywords: DAT; aging; dopamine; learning and memory; reward.

Figure 1

(A) S-CE-123 competes for the binding of radioligand specific to the hDAT with IC50 = 1.1E-06 M and K_i = 6.1E-07 M. (B) The plasma concentrations of S-CE-123 after a single i.p. administration at 10 mg/kg. (C) The brain concentrations of S-CE-123 after a single i.p. administration at 10 mg/kg. (D,E) S-CE-123 did not show neurotoxic effect on cell viability or neurite outgrowth of cortical neurons in vitro. Data are presented as a mean ± SEM, n = 3.

Figure 2

Effect of S-CE-123 on locomotor activity in an open field apparatus. (A) A novelty test did not show significant effects on distance travelled at 10 mg/kg (p > 0.05, unpaired t-test, n = 7–8). (B–E) Familiar OF. (B) Schematic demonstration of experimental design and representative traces of locomotor activity recorded during test sessions with 0, 1, 10 and 20 mg/kg S-CE-123. (C) Number of entries to the center zone. (D₁) Total distance travelled over 10 min and (D₂) 1 min intervals. (E) Baseline activity measured during habituation sessions (all rats received vehicle at 1 mL/kg; H1–novelty session, H2-4–familiar sessions). One-way RM-ANOVA followed by Dunnett post hoc test for multiple comparisons; * p < 0.05, *** p < 0.001, **** p < 0.0001. Values are presented as mean ± SEM, n = 10.

Figure 3

(A) Schematic of the hole-board maze, a non-sophisticated paradigm for the evaluation of reward-motivated spatial learning and memory. (B) A cartoon depicting the timeline of experiments. (C,D) Performance of young rats (5 months) treated with S-CE-123 at 1, 5 and 10 mg/kg body weight or vehicle in hole-board task. There were no significant differences in (C) RMIs (F_3,36 = 0.4642, p = 0.7090, two-way RM-ANOVA) or (D) latency (F_3,36 = 0.1431, p = 0.9335, two-way RM-ANOVA) between four groups (n = 10/group). (E) A large cohort of 22–24 months old rats (n = 160) [34] was behaviorally characterized using a hole-board test and based on their mean RMIs derived from trial 6 and 10, rats were characterized as impaired, unimpaired or “intermediate”. Thirty-eight “intermediate” rats of age 22–23 months were selected, randomly grouped and used for treatment. (F) Four groups of aged rats were equal in terms of RMIs in the first hole-board test. (G–I) Performance of old (24–25 months) rats treated with S-CE-123 at 1, 5 and 10 mg/kg body weight or vehicle in hole-board task (n = 9–10/group). There was a significant treatment effect with improved (G) RMIs (day1 (F_3,34 = 4.050, p = 0.0145, two-way RM-ANOVA), day2 (F_3,34 = 8.021, p = 0.0004, two-way RM-ANOVA); day3 (F_3,34 = 3.273, p = 0.0328, one-way ANOVA) and decreased (H) latency (day1 (F_3,34 = 5.405, p = 0.0038, two-way RM-ANOVA), day2 (F_3,34 = 3.698, p = 0.0209, two-way RM-ANOVA); day3 (F_3,34 = 2.281, p = 0.0969, one-way ANOVA) in S-CE-123 groups. * ANOVA general treatment effect, multiple comparison, & vehicle vs. 1 mg/kg and # vehicle vs. 10 mg/kg. (I) Heat map of hole visits of aged rats. (J) In the first trial on day1, aged S-CE-123-treated animals spent significantly less time immobile compared to vehicle treated rats (p = 0.0038, Mann–Whitney test), whereas, in young animals, treatment had no effect on immobility (p = 0.4988, Mann–Whitney test). Values are expressed as mean ± SEM, (*/&/# p < 0.05; **/&& p < 0.01; ***/### p < 0.001).

Figure 4

Analyses of hole-board RMIs in the first test (no treatment) compared to the second test (treatment) for (A) vehicle group (day1 (F_1,9 = 0.1125, p = 0.7450, 2-way RM-ANOVA); day2 (F_1,9 = 1.075, p = 0.3270, 2-way RM-ANOVA); day3 (p = 0.3925, paired t-test)) and for S-CE-123 groups at (B) 1 mg/kg (day1 (F_1,8 = 12.38, p = 0.0079, 2-way RM-ANOVA); day2 (F_1,8 = 11.93, p = 0.0086, 2-way RM-ANOVA); day3 (p = 0.0322, paired t-test)), (C) 5 mg/kg (day1 (F_1,8 = 3.386, p = 0.1030, 2-way RM-ANOVA); day2 (F_1,8 = 2.448, p = 0.1563, 2-way RM-ANOVA); day3 (p = 0.2071, paired t-test)) and (D) 10 mg/kg (day1 (F_1,9 = 30.62, p = 0.0004, 2-way RM-ANOVA); day2 (F_1,9 = 9.210, p = 0.0141, 2-way RM-ANOVA); day3 (p = 0.0024, paired t-test)). (E–I) Different sample of old rats (21–22 months) were tested in a MWM spatial memory task. (E) Schematic demonstration of experimental design and treatment. There was no significant difference between vehicle and 10 mg/kg S-CE-123 in (F) escape latency during four days of training, in (G) time spent in target quadrant during probe trial, (H) in escape latency during four days of training in reversal learning and in (I) time in target quadrant during probe trial of reversal test. Values are expressed as mean ± SEM (n = 10; * p < 0.05; ** p < 0.01).

Figure 5

(A) Representative images of CD68 immunostaining in DG hippocampal subregion (scale bars represent 250 μm). (B) Quantification of CD68 immunostaining in the DG of young and aged rats treated with 10 mg/kg S-CE-123 or vehicle. Each dot represents the mean of values across the three sections in an individual animal. Values are expressed as mean ± SEM.