Combinatorial targeting of NMDARs and 5-HT4Rs exerts beneficial effects in a mouse model of Alzheimer's disease

Abstract

Background: Alzheimer's disease (AD) is the leading cause of dementia. There are limited approved medications that delay cognitive decline or lessen neuropsychiatric symptoms. Numerous clinical trials for AD using a single drug administration have failed to meet therapeutic endpoints, which is most likely due to the complexity of AD. A multimodal therapeutic intervention is more likely to improve symptoms by targeting multiple targets implicated in AD. Here, we investigated if targeting both N-Methyl-D-aspartic acid receptors (NMDARs) and serotonin type 4 receptors (5-HT₄R) may have beneficial effects in a mouse model of AD, as they have separately been shown to improve cognition and/or mood.

Methods: Male and female control (Ctrl) or APP/PS1 mice were administered single, intermittent, or chronic administration of 1) saline; 2) (R,S)-ketamine, an NMDAR antagonist; 3) prucalopride, a 5-HT₄R agonist; or 4) (R,S)-ketamine + prucalopride to simultaneously target co-morbid neuropsychiatric and cognitive deficits. Behavioral assays were then administered to measure cognition, perseverative behavior, hyponeophagia, and/or sleep. Brains were processed for glial fibrillary acidic protein (GFAP) immunohistochemistry.

Results: Single and chronic administration of (R,S)-ketamine + prucalopride administration improved cognitive decline by increasing memory retrieval in a contextual fear conditioning (CFC) paradigm in APP/PS1 mice. Drug efficacy was less effective in females than in males and was age dependent. Hippocampal GFAP immunoreactivity was decreased by chronic (R,S)-ketamine + prucalopride treatment in females.

Conclusions: Our results indicate that combined administration of (R,S)-ketamine + prucalopride is a novel multimodal therapeutic strategy to treat cognitive decline in AD. Future work will further characterize these interactions with the goal of clinical development.

Keywords: Adjunctive treatment; Ketamine; Neurodegeneration; Neuroinflammation; Prucalopride.

Fig. 1

A single administration of combined (R,S)-ketamine + prucalopride improves memory retrieval in male APP/PS1 mice. A Experimental design. B-C Mice generally exhibited increased activity at night. In APP/PS1 mice, at ZT 12–14, K + P (10 + 3 mg/kg)-administered mice exhibited reduced activity in relation to other experimental groups. D-E However, overall sleep and sleep amplitude were comparable across all groups. F During the light phase, K (10 mg/kg)-administered APP/PS1 mice unexpectedly exhibited less overall sleep compared to saline-administered APP/PS1 and K (10 mg/kg)-administered Ctrl mice. K + P-administered APP/PS1 mice also exhibited increased sleep relative to their respective Ctrl group. G There was no effect of Drug or Genotype in the dark phase. H–L Behavior in the NSF assay was similar in all groups. M Sal-administered APP/PS1 mice buried significantly less marbles in relation to Sal-administered Ctrl mice. N-P K (30 mg/kg) and combined K + P (10 + 3 mg/kg)-administered APP/PS1 mice exhibited significantly increased freezing in comparison to Sal-administered APP/PS1 mice. (n = 4–12 male mice per group). Error bars represent ± SEM. * p < 0.05. ** p < 0.01. *** p < 0.001. **** p < 0.0001. Sal, saline; K, (R,S)-ketamine; P, prucalopride; NSF, novelty suppressed feeding; MB, marble burying; CFC, contextual fear conditioning; Ctrl, control; AD, Alzheimer’s disease; ZT, zeitgeiber; mg, milligram; kg, kilogram; OF, open field; sec, seconds; g, grams

Fig. 2

Single administration of (R,S)-ketamine + prucalopride does not significantly impact behavior in female APP/PS1 mice. A Behavioral paradigm. B-D Activity and overall sleep were not significantly impacted by drug treatment in Ctrl and APP/PS1 mice. E K (10 mg/kg) and P (1.5 mg/kg), but not K + P administration, reduced sleep amplitude in Ctrl mice relative to Sal. F-G However, sleep in the light and dark phase were not altered across drug and genotype groups. H-I There was a trending (p = 0.0500), but not significant, effect of Drug on latency to feed in the novel arena in Ctrl, but not APP/PS1 mice during the NSF. All other behaviors in the (J-L) NSF, (M) MB, and (N-P) CFC assays were comparable across all groups. (n = 4–13 female mice per group). Error bars represent ± SEM. * p < 0.05. Sal, saline; K, (R,S)-ketamine; P, prucalopride; Ctrl, control; AD, Alzheimer’s disease; NSF, novelty suppressed feeding; MB, marble burying; CFC, contextual fear conditioning; ZT, zeitgeiber; mg, milligram; kg, kilogram; OF, open field; sec, seconds; g, grams

Fig. 3

Chronic, combined (R,S)-ketamine + prucalopride enhances memory retrieval in 2-month-old male APP/PS1 mice. A Behavioral paradigm. B Chronic drug administration did not significantly affect body weight. C Freezing during CFC training was comparable across all groups. D However, during CFC re-exposure, combined K + P (10 mg/kg, 1X + 3 mg/kg, 7X) significantly increased freezing in APP/PS1 mice in relation to K + P (10 mg/kg, 1X + 3 mg/kg, 7X)-administered Ctrl and Sal-administered APP/PS1 mice. In addition, P (3 mg/kg, 7X) also significantly increased freezing relative to Sal-administered APP/PS1 mice. E K (10 mg/kg, 2X) reduced immobility time in APP/PS1, but not Ctrl mice during FST day 1. F Behavior during FST day 2 was not significantly impacted by Drug treatment. G K (10 mg/kg, 2X), K (30 mg/kg, 1X), K (30 mg/kg, 2X), P (3 mg/kg, 7X), and K + P (10 mg/kg, 1X + 1.5 mg/kg, 7X) significantly reduced marbles buried in APP/PS1 mice relative to Ctrl mice. (n = 5–13 2-month-old male mice per group). Error bars represent ± SEM. * p < 0.05. ** p < 0.01. *** p < 0.001. i.p., intraperitoneal; CFC, contextual fear conditioning; RE, re-exposure; FST, forced swim test; MB, marble burying; Sal, saline; K, (R,S)-ketamine; K + P, (R,S)-ketamine + prucalopride; X, times; Ctrl, control; AD, Alzheimer’s disease; sec, seconds

Fig. 4

Chronic, combined (R,S)-ketamine + prucalopride increases memory retrieval in 6-month-old male APP/PS1 mice. A Behavioral paradigm. B Body weight was comparable across all groups. C In Ctrl mice, K (30 mg/kg, 2X) increased freezing relative to saline-administered Ctrl and K (30 mg/kg, 2X)-administered APP/PS1 mice. K + P (10 mg/kg, 1X + 3 mg/kg, 7X) enhanced freezing in APP/PS1 mice compared to its respective Ctrl group. D Sal-administered APP/PS1 male mice exhibited reduced freezing relative to saline-administered Ctrl mice. This deficit in freezing was rescued by administration of K + P (10 mg/kg, 1X + 3 mg/kg, 7X). K (30 mg/kg, 2X), P (3 mg/kg, 7X), and K + P (10 mg/kg, 1X + 3 mg/kg, 7X) increased freezing in APP/PS1 mice relative to respective Ctrl groups. E On FST day 1, Sal-administered APP/PS1 mice had less immobility than Sal-administered Ctrl mice. K (10 mg/kg, 2X) and K + P (10 mg/kg, 1X + 3 mg/kg, 7X) increased immobility in APP/PS1 mice in comparison to respective Ctrl groups. F On FST day 2, in Ctrl mice, K (10 mg/kg, 2X), P (1.5 mg/kg, 7X), and K + P (10 mg/kg, 1X + 1.5 mg/kg, 7X) reduced immobility time compared to saline. K (10 mg/kg, 2X) and K (30 mg/kg, 1X) increased immobility in APP/PS1 mice compared to respective Ctrl groups, while K + P (10 mg/kg, 1X + 3 mg/kg, 7X) reduced immobility time in APP/PS1 mice compared to K + P-administered Ctrl mice. G K + P (10 mg/kg, 1X + 3 mg/kg, 7X) reduced marble burying in APP/PS1 mice relative to K + P-administered Ctrl mice. (n = 5–11 6-month-old male mice per group). Error bars represent ± SEM. * p < 0.05. ** p < 0.01. *** p < 0.001. **** p < 0.0001. i.p., intraperitoneal; CFC, contextual fear conditioning; RE, re-exposure, FST, forced swim test; MB, marble burying; X, times; Sal, saline; K, (R,S)-ketamine; P, prucalopride; K + P, (R,S)-ketamine + prucalopride; AD, Alzheimer’s disease; sec, seconds

Fig. 5

Chronic, combined (R,S)-ketamine + prucalopride administration rescues hippocampal GFAP expression in 6-month-old female APP/PS1 mice. A-B Representative image of GFAP immunostaining (green) with Hoechst (blue) in 6-month-old mice. Inset reveals close-up of hippocampal GFAP expression. C In male HPC, P increased GFAP in Ctrl mice relative to Sal-administered Ctrl and P-administered APP/PS1 mice. K increased GFAP relative to Sal-administered mice. D In female HPC, GFAP was increased in Sal-administered APP/PS1 mice relative to Sal-administered Ctrl mice; this increase in GFAP was rescued by chronic K + P. P-administered APP/PS1 mice exhibited increased GFAP relative to its respective Ctrl group. E In male Ctrl mice, P administration increased GFAP expression in the DG-sg. F In female mice, there was a significant effect of Drug, but not Genotype or a Genotype x Drug in the DG-sg. G In DG-mo of male APP/PS1 mice, K increased GFAP expression relative to Sal-administered APP/PS1 mice and K-administered Ctrl mice. H In female DG-mo, APP/PS1 Sal-administered mice exhibited increased GFAP relative to Sal-administered Ctrl mice, which was rescued in K + P-administered APP/PS1 mice. P-administered APP/PS1 mice ad increased GFAP relative to its respective Ctrl mice. I In SLM, P-administered Ctrl male mice had increased GFAP expression in comparison to Sal-administered Ctrl mice and P-administered APP/PS1 mice. J In female SLM, there was a significant effect of Drug, but not Genotype or Genotype x Drug. K In male CA1, P-administered Ctrl male mice had increased GFAP expression in comparison to Sal-administered Ctrl mice and P-administered APP/PS1 mice. In APP/PS1 mice, K increased GFAP relative to Sal. L In female CA1, K-administered APP/PS1 mice had increased GFAP relative to K-administered Ctrl mice and Sal-administered APP/PS1 mice. (n = 3–6 mice per group). Error bars represent ± SEM. ** p < 0.01. *** p < 0.001. **** p < 0.0001. HPC, hippocampus; DG-mo, dentate gyrus molecular layer; DG-sg, dentate gyrus granule cell layer; SLM, stratum lacunosum-moleculare; CA1, field CA1 stratum radiatum; μm, microns; GFAP, glial fibrillary acidic protein; Ctrl; control; AD, Alzheimer’s disease; Sal, saline; K, (R,S)-ketamine; P, prucalopride; K + P, (R,S)-ketamine + prucalopride