Beneficial effects of physical exercise and an orally active mGluR2/3 antagonist pro-drug on neurogenesis and behavior in an Alzheimer's amyloidosis model

Abstract

Background: Modulation of physical activity represents an important intervention that may delay, slow, or prevent mild cognitive impairment (MCI) or dementia due to Alzheimer's disease (AD). One mechanism proposed to underlie the beneficial effect of physical exercise (PE) involves the apparent stimulation of adult hippocampal neurogenesis (AHN). BCI-838 is a pro-drug whose active metabolite BCI-632 is a negative allosteric modulator at group II metabotropic glutamate receptors (mGluR2/3). We previously demonstrated that administration of BCI-838 to a mouse model of brain accumulation of oligomeric Aβ^E22Q (APP ^E693Q = "Dutch APP") reduced learning behavior impairment and anxiety, both of which are associated with the phenotype of Dutch APP mice.

Methods: 3-month-old mice were administered BCI-838 and/or physical exercise for 1 month and then tested in novel object recognition, neurogenesis, and RNAseq.

Results: Here we show that (i) administration of BCI-838 and a combination of BCI-838 and PE enhanced AHN in a 4-month old mouse model of AD amyloid pathology (APP ^KM670/671NL /PSEN1 ^Δexon9= APP/PS1), (ii) administration of BCI-838 alone or with PE led to stimulation of AHN and improvement in recognition memory, (iii) the hippocampal dentate gyrus transcriptome of APP/PS1 mice following BCI-838 treatment showed up-regulation of brain-derived neurotrophic factor (BDNF), PIK3C2A of the PI3K-mTOR pathway, and metabotropic glutamate receptors, and down-regulation of EIF5A involved in modulation of mTOR activity by ketamine, and (iv) validation by qPCR of an association between increased BDNF levels and BCI-838 treatment.

Conclusion: Our study points to BCI-838 as a safe and orally active compound capable of mimicking the beneficial effect of PE on AHN and recognition memory in a mouse model of AD amyloid pathology.

Keywords: AD amyloid pathology; APP/PS1 mice; BDNF; mGlu2/3 antagonist; neurogenesis; physical exercise.

Figure 1

Experimental design and spontaneous activity. (A) APP/PS1 mice were divided into four groups: Group 1 no PE/no drug; Group 2 PE/no drug; Group 3, no PE/drug; and Group 4, PE + drug. Group 5 consisted of WT mice (C57BL/6). (B) Groups receiving PE were given ad libitum access to a running wheel and treatment with drug or vehicle was continued for 30 days. All studies used male mice and included three cohorts. The first cohort was processed for neurogenesis studies, following perfusion-fixation with PFA 4% (five animals per group). The second cohort was processed for RNA and biochemical studies. We sacrificed animals by CO₂ narcosis and the DG was removed for RNAseq and determination of BDNF by qPCR. We processed five animals per group. The third cohort was used for behavior testing (10-12 mice per group).

Figure 2

BCI-838 decreased spontaneous activity after 2 weeks of treatment. (A) Number of running wheel turns (# counts) are shown for the two groups that received running wheel access. APP/PS1 mice treated with BCI-838 decreased running wheel use during the last 2 weeks of drug treatment and running wheel exposure. Repeated measures ANOVA over the entire 30 days of treatment revealed a significant difference in running wheel activity within groups (F_3.8,61.1 = 3.695, p = 0.011) but no day*condition interaction effect (p = 0.052). A test of between subject effects over the 30 days revealed no significant effect of condition (F_1,16 = 2.399, p = 0.14). Tests of within subject effects revealed significant effects of running wheel activity if analyzed over 1–14 days (F_3.5,59.5 = 3.579, p = 0.014 for activity, F_3.5,59.5 = 2.062, p = 0.105 for day*condition) and 14–28 days (F_2.9,47.0 = 4.414, p = 0.008 for activity, F_2.9,47.0 = 4.414, p = 0.366 for day*condition). There was no difference in running wheel activity between groups over days 1–14 (F_{1, 17} = 0.799) whereas activity was reduced in mice that received PE + drug between days 14–28 (F_1,16 = 6.052, p = 0.026). Values significantly different between groups at individual time points are indicated by asterisks (*p < 0.05, **p < 0.01, unpaired t-tests). Values are expressed as mean ± SEM. 12 mice per group for each cohort were used. (B) Weights of mice before and after treatment with PE and/or BCI-838. There were no differences between groups analyzed pre-treatment (one way ANOVA, F_4,42 = 0.9107, p = 0.4666), but differences between WT and BCI-838 and PE + BCI-838 group were found post-treatment (F_4,42 = 5.588, p = 0.0011). However, a repeated measures ANOVA comparing pre-treatment to post-treatment revealed that mice gained weight after treatment (F_2.575,21.17 = 8.547, p = 0.0010). Asterisks indicate significant between group differences when selected groups were directly compared using Sidak's test (*p < 0.05, **p < 0.01, ***p < 0.001). (C) Running wheel activity in APP/PS1mice treated with PE or PE + BCI-838 is shown pre-treatment, after 15 days of treatment and post-treatment. A one-way ANOVA revealed significant between group differences when all 6 groups were compared F_(5,57) = 3.589, p = 0.0069. Asterisks indicate significant between group differences when indicated groups were directly compared using Sidak's test (*p < 0.05, **p < 0.01, ns, not significant).

Figure 3

Effect of BCI-838 treatment and PE on novel object recognition (NOR). (A) Training. No differences were found among groups during the training when the mice explored the identical objects, Object 1 (Ob1) and Object 2 (Ob2). (B) Long-term memory session (LTM). During the LTM testing conducted at 24 h after training, wild type mice spent more time exploring the novel object (NO) as compared to the familiar object (FO). APP/PS1 mice treated with vehicle explored the FO and the NO similar amounts of time whereas APP/PS1 mice exposed to PE, treated with BCI-838 or the combination of BCI-838 + PE for 1 month showed a preference for the NO compared to the FO. Values significantly different are indicated by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ANOVA followed by Sidak's test, comparing selected pairs of columns). (C) Total time spent exploring the objects during training is shown. The combination of BCI-838 + PE mice explored the objects more compared to WT mice (one-way ANOVA: F_4,48 = 3.286, p = 0.0185; *p < 0.05, Tukey's multiple comparisons test). (D) During LTM testing, APP/PS1 mice treated with BCI-838 alone were more exploratory than APP/PS1 mice treated with vehicle (F_2,47 = 3.308, p = 0.0181; *p < 0.05, Tukey's multiple comparisons test). (E) A discrimination index calculated for the training session showed no difference in object preference between groups (F_{4, 47} = 1.226, p = 0.3124). (F) A discrimination index calculated for LTM (F_4,47 = 6.131, p = 0.0005) found differences between APP/PS1 treated with vehicle and all other groups but no differences between treatments (** p < 0.01, ***p < 0.0001, Tukey's multiple comparisons test). Values are expressed as mean ± SEM). Ten to 12 mice per group were used for each cohort.

Figure 4

Quantification of neurogenesis in WT, APP/PS1 treated with vehicle, PE, BCI-838 or the combination. (a–e) Images of DCX staining in WT, APP/PS1, APP/PS1 + PE, APP/PS1 + BCI-838, and APP/PS1 combination. (f) Representative image of BrdU staining in a WT mouse. The hilus and granule cell layer (GCL) are indicated. Arrows mark BrdU-labeled cells in the subgranular zone. Scale bar 50 μm. (g) Total number of doublecortin-labeled cells was increased in the APP/PS1 + BCI-838 and APP/PS1 combination as compared to APP/PS1 control. Values are expressed as mean ± SEM and differences among groups are indicated by asterisks (*p < 0.05, **p < 0.01, one-way ANOVA followed by Tukey's test). (h) Total number of BrdU-labeled cells was increased in APP/PS1 + BCI-838 and APP/PS1 combination compared to APP/PS1 control. Values significantly different among groups are indicated by asterisks as in panel (g). 4–5 mice per group for each cohort were used.

Figure 5

Differential gene expression and enrichment analysis summary in dentate gyrus (DG) of APP/PS1 mice treated with BCI-838, PE or a combination of both BCI-838 + PE. RNA sequencing was performed on dentate gyrus for five groups of four-month-old APP/PS1 and WT mice, comprising a total of 24 samples. (A) Schematic overview of mouse AD transcriptome analysis. (B) Top DEGs in DG of APP/PS1 mice treated with BCI-838 vs. APP/PS1 treated with vehicle. (C) Top DEGs in DG of APP/PS1 mice treated with PE vs. APP/PS1 treated with vehicle. (D) Top DEGs in DG of APP/PS1 mice treated with a combination of both BCI-838 and PE vs. APP/PS1 treated with vehicle. (E) Top DEGs in DG of APP/PS1 mice treated with a combination of both BCI-838 and PE vs. APP/PS1 treated with PE. (F) Venn Diagram of DEGs and selected pathway enrichments of known or suspected relevance to AD pathophysiology, shared across different comparisons of APP/PS1 mice groups (transcription factor enrichments shown in red).

Figure 6

BDNF analysis in APP/PS1 mice treated with BCI-838 or PE. qPCR for BDNF mRNA. Asterisk indicates p = 0.025 (Kuskall–Wallis, uncorrected Dunn test). Five mice per group were used for the analysis.

Figure 7

Top drugs similar to BCI-838 based on transcriptomic activity.

Figure 8

Drug targets and side effects enriched among compounds with transcriptomic similarity to BCI-838.