Epigallocatechin-3-gallate (EGCG) consumption in the Ts65Dn model of Down syndrome fails to improve behavioral deficits and is detrimental to skeletal phenotypes

Abstract

Down syndrome (DS) is caused by three copies of human chromosome 21 (Hsa21) and results in phenotypes including intellectual disability and skeletal deficits. Ts65Dn mice have three copies of ~50% of the genes homologous to Hsa21 and display phenotypes associated with DS, including cognitive deficits and skeletal abnormalities. DYRK1A is found in three copies in humans with Trisomy 21 and in Ts65Dn mice, and is involved in a number of critical pathways including neurological development and osteoclastogenesis. Epigallocatechin-3-gallate (EGCG), the main polyphenol in green tea, inhibits Dyrk1a activity. We have previously shown that EGCG treatment (~10mg/kg/day) improves skeletal abnormalities in Ts65Dn mice, yet the same dose, as well as ~20mg/kg/day did not rescue deficits in the Morris water maze spatial learning task (MWM), novel object recognition (NOR) or balance beam task (BB). In contrast, a recent study reported that an EGCG-containing supplement with a dose of 2-3mg per day (~40-60mg/kg/day) improved hippocampal-dependent task deficits in Ts65Dn mice. The current study investigated if an EGCG dosage similar to that study would yield similar improvements in either cognitive or skeletal deficits. Ts65Dn mice and euploid littermates were given EGCG [0.4mg/mL] or a water control, with treatments yielding average daily intakes of ~50mg/kg/day EGCG, and tested on the multivariate concentric square field (MCSF)-which assesses activity, exploratory behavior, risk assessment, risk taking, and shelter seeking-and NOR, BB, and MWM. EGCG treatment failed to improve cognitive deficits; EGCG also produced several detrimental effects on skeleton in both genotypes. In a refined HPLC-based assay, its first application in Ts65Dn mice, EGCG treatment significantly reduced kinase activity in femora but not in the cerebral cortex, cerebellum, or hippocampus. Counter to expectation, 9-week-old Ts65Dn mice exhibited a decrease in Dyrk1a protein levels in Western blot analysis in the cerebellum. The lack of beneficial therapeutic behavioral effects and potentially detrimental skeletal effects of EGCG found in Ts65Dn mice emphasize the importance of identifying dosages of EGCG that reliably improve DS phenotypes and linking those effects to actions of EGCG (or EGCG-containing supplements) in specific targets in brain and bone.

Keywords: Bone; Cognition; Down syndrome; EGCG; Mouse model; Trisomy 21.

Figure 1. Behavioral Study Timeline

Timeline for the current study beginning shortly after weaning (PD22). Animals received EGCG or water treatment throughout the behavioral testing, even on rest days.

Figure 2

a. MCSF Total Entries. Both Euploid (Eu) and Trisomic (Ts) mice increased the number of entries from Day 1 to Day 2. As indicated by (*), Eu+Water, Ts+Water, and Ts+EGCG groups had a significantly higher number of total entries on Day 2. Data are represented by mean ± SEM. b. MCSF Risk Assessment Behavior. The Trisomic (Ts) mice exhibited higher risk assessment behavior than the Euploid (Eu) mice (data collapsed across both days). As indicated by the (*), the Ts+EGCG group displayed significantly higher risk assessment behavior compared to both euploid groups. Data are represented by mean ± SEM. c. MCSF Risk Taking Behavior. There was a significant genotype × treatment interaction. Data are collapsed across days since there were no effects of day. Note that EGCG treatment had opposing effects on euploid and trisomic mice. Data are represented by mean ± SEM

Figure 3

a. MWM Acquisition Path Length. Acquisition in the Morris water maze spatial learning task by the Euploid (Eu) and Trisomic (Ts) groups. Each line represents the average path length (meters) for each acquisition day. All groups showed a decrease in latency over training days. However, trisomic mice of both treatment groups displayed higher path lengths versus the euploid groups (main effect of genotype, indicated by (*). Insert graph represents the total path length summed across all 7 acquisition days. Both trisomic groups exhibited increased total latency versus euploid-water controls (as indicated by the *). LSD post-hoc analysis revealed a significant increase in Trisomic + Water and Trisomic +EGCG latencies as compared to Euploid + Water mice (as indicated by the ×). Data are represented as mean ± SEM. b. MWM Acquisition Latency. Seconds spent locating the hidden platform by the Euploid (Eu) and Trisomic (Ts) groups. Each line represents the average time (latency) to find the hidden platform for each training day. All groups showed significant reductions in latencies over days (main effect of day). However, trisomic mice were significantly impaired versus controls (day × genotype interaction, p=0.041). LSD post-hoc analysis revealed a significant increase in Trisomic + Water and Trisomic +EGCG latencies as compared to Euploid + Water (as indicated by the ×), or increases in Trisomic + EGCG latencies versus Euploid + Water mice (as indicated by the +). Data are represented as mean ± SEM. c. MWM Acquisition Thigmotaxic Behavior. Percent of time spent in thigmotaxis (within 25.4 cm of the wall) by the Euploid (Eu) and Trisomic (Ts) groups. Each line represents the average percent of time for each acquisition day. There was an overall decrease in thigmotaxic behavior over training days, most evident in the Euploid-water group; however, mice receiving EGCG displayed higher thigmotaxis versus the water groups (main effect of treatment, p=0.039), due mainly to the increased thigmotaxic behavior of the Euploid-EGCG group relative to the euploid-water group. Data are represented as mean ± SEM. d. MWM Acquisition Floating Behavior. Seconds spent floating by the Euploid (Eu) and Trisomic (Ts) groups. Each line represents the average time (seconds) floating for each acquisition day. Trisomic mice displayed significantly less reduction in floating behavior over days than the euploid mice (day × genotype interaction, p=0.017). Data are represented as mean ± SEM.

Figure 4. MWM Probe Trial

Probe trial performance of Euploid (Eu) and Trisomic (Ts) mice of the two treatment groups on the probe trial (Day 8). The (*) comparing the Target and Non-target times of the euploid groups indicates a significant spatial bias for the Target location for both euploid groups ; the similar trend for the trisomic groups did not reach statistical significance. Bars represent the average time spent in the target and non-target quadrant, with error bars represented as SEM.

Figure 5. Dyrk1a Protein Levels-Cerebellum

Dyrk1a protein levels in the cerebellum of ~9-week old mice given 6 weeks of treatment with water or EGCG (mean ± SEM). Ts65Dn mice receiving water had significantly less Dyrk1a protein that euploid-water controls (as indicated by the *, genotype × treatment interaction, p=0.043).

Figure 6. Kinase activity-Femur

Kinase activity in the femur of ~9-week old mice given 6 weeks of treatment with water or EGCG (mean ± SEM). Mice receiving EGCG treatment had significantly lower kinase activity than mice receiving water (main effect of treatment p<0.05).