Effect of Epigallocatechin-3-gallate on Skeletal and Cognitive Phenotypes in a Down Syndrome Mouse Model

Indiana University-Purdue University Indianapolis (IUPUI)Down syndrome (DS), a genetic disorder that affects ~1 in 700 live births, is caused by trisomy of human chromosome 21 (Hsa21). Individuals with DS are affected by a wide spectrum of phenotypes which vary in severity and penetrance. However, cognitive and skeletal impairments can be commonly observed in all individuals with DS. To study these phenotypes, we utilized the Ts65Dn mouse model that carries three copies of approximately half the gene orthologs found on Hsa21 and exhibit similar phenotypes as observed in humans with DS. Individuals with DS and Ts65Dn mice have deficits in bone mineral density (BMD), bone architecture, bone strength, learning and memory. Over-expression of DYRK1A, a serine-threonine kinase encoded on Hsa21, has been linked to deficiencies in DS bone homeostasis and cognition. Epigallocatechin-3-gallate (EGCG), an aromatic polyphenol found in high concentrations in green tea, is a selective inhibitor of DYRK1A activity. Normalization of DYRK1A activity by EGCG therefore may have the potential to ameliorate skeletal and cognitive deficits. We hypothesized that supplements containing EGCG obtained from health food stores/ online vendors will not be as effective as EGCG from a chemical company in correcting bone deficits associated with DS. Our results suggest that EGCG improves the bone mineral density of trisomic femurs significantly better than the supplements while the EGCgNOW supplement from NOW FOODS improves trabecular and cortical bone structure. The results from HPLC analysis of supplements showed the presence of other catechins in EGCgNOW and degradation analysis revealed the rapid degradation of supplements. Therefore we hypothesize that the presence of EGCG degradation products and other green tea catechins in supplements may play a role in the differential skeletal effects we observed. We further hypothesized that a three week treatment of adolescent mice with EGCG will lead to an improvement in the learning and memory deficits that are observed in trisomic animals in comparison to control mice. However, our results indicate that three weeks of low-dose EGCG treatment during adolescence is insufficient to improve hippocampal dependent learning and memory deficits of Ts65Dn mice. The possibility remains that a higher dose of EGCG that begins at three weeks but lasts throughout the behavioral test period may result in improvement in learning and memory deficit of Ts65Dn mice

Extended Treatment with a High Dosage of EGCG to Rescue Appendicular Bone Abnormalities in a Down Syndrome Mouse Model

poster abstractIndividuals with Down syndrome (DS) show significant abnormalities in cognitive abilities, muscle tone, and bone homeostasis. DS is caused by a triplication of the 21st human chromosome (Hsa21). Previous research conducted by our lab using mouse models indicates that three copies of Dyrk1a causes the appendicular skeletal deficits associated with DS. Ts65Dn mouse model carries 50% of the genes homologous to Hsa21, and exhibit excellent phenotypic model for the skeletal deficits seen in individuals with DS, such as low bone mineral density, altered bone structure, and decreased cortical bone. Epigallocatechin-3-gallate (EGCG) is a green tea polyphenol that inhibits Dyrk1a activity. In a previous study, we showed that a three-week, low dose (10mg/kg/day) treatment of EGCG rescued bone mineral density, and trabecular bone to that of euploid levels, but not cortical bone. We hypothesize that increasing the concentration and duration of the treatment will be sufficient enough to more fully restore bone abnormalities by rescuing femoral bone mineral density, bone volume, and improving overall bone strength. This project explores the effects of using a prolonged seven-week, high dosage (100mg/kg/day) treatment on specific bone phenotypes. Dual Energy X-ray absorptiometry (DXA), MicroCT, and mechanical testing will be used as our means of analysis of the treated and untreated bones

Effects of Epigallocatechin-3-gallate Treatment on Cognitive Deficits in a Down Syndrome Mouse Model

poster abstractDown syndrome (DS) is caused by three copies of human chromosome 21 (Hsa21) and results in a constellation of phenotypes that include intellectual disability (ID) and skeletal abnormalities. Ts65Dn mice, the most extensively studied model of DS, have three copies of approximately half the genes on Hsa21 and display many DS related phenotypes including skeletal and ID deficits. DYRK1A is found in three copies both in humans with DS and in Ts65Dn mice; DYRK1A has increased expression in humans with DS and is involved in a number of critical pathways including CNS development and osteoclastogenesis. Epigallcatechin-3-gallate (EGCG), the main polyphenolic compound found in green tea, inhibits Dyrk1a activity, and we have shown previously that a three-week treatment with EGCG during adolescence normalizes some skeletal abnormalities in Ts65Dn mice. The current study tested the hypothesis that a similar 3-week treatment with EGCG will also rescue cognitive deficits observed in Ts65Dn mice. Trisomic mice and euploid littermates were given EGCG or water (control) for three weeks during adolescence. Following termination of the treatment, the mice were tested sequentially (over 5 weeks) on locomotor activity (two daily 30-min sessions in an activity chamber), novel object recognition (NOR) memory, acquisition of delayed non-matching to place (DNMP) spatial working memory in a tmaze, or spatial learning and memory in the Morris water maze (MWM). Results to date indicate that Ts65Dn mice exhibit deficits in the learning and memory tasks compared to controls, but the 3-week EGCG treatment did not significantly improve their performance.We hypothesize that for EGCG to be effective for improving cognitive deficits of the Ts65Dn mice, it needs to be present in the brain during the behavioral testing period; our ongoing studies are testing this with continuous EGCG treatment throughout the behavioral testing process

Evaluation of osteoclastogenesis in the Ts65Dn Down Syndrome Mouse Model

poster abstractDown Syndrome (DS) affects ~1 in 700 live births and is caused by trisomy of human chromosome 21 (Hsa21). DS is characterized by a wide spectrum of phenotypes including cognitive and skeletal abnormalities that affect all individuals with DS. To study these phenotypes, we utilize the Ts65Dn mouse model, which contains three copies of approximately half the gene orthologous found on Hsa21 and exhibits similar phenotypes as found in humans with DS. Individuals with DS and Ts65Dn mice have deficits in bone mineral density (BMD), bone architecture, and bone strength. Three copies of DYRK1A, a serine-threonine kinase encoded on Hsa21, has been linked to deficiencies in bone homeostasis in DS mouse models and individuals with DS. DYRK1A is thought to act via NFATc1, a master regulator of osteoclastogenesis. Epigallocatechin-3-gallate (EGCG), a polyphenol found in high concentrations in green tea, is a known inhibitor of DYRK1A activity. We propose that the DS bone phenotype arises from an increase in osteoclastogenesis and/or maturation which results in increased bone resorption and disrupted bone homeostasis. We hypothesize that treatment of the mice during adolescence with 100 mg/kg/day EGCG would result in normalization of osteoclast numbers in trisomic mice to that of the controls. Osteoclast precursors from femur and spleen were isolated from 8-10 week old mice treated with 100 mg/kg/day EGCG or water from three weeks of age onwards. The cells were grown in the presence of M-CSF & RANK-L to promote osteoclast differentiation. Following 3 weeks in culture, the cells were fixed, TRAP stained, and multinucleated osteoclasts from control and Ts65Dn treated and untreated mice were counted. Mentor: Randall Roper, Department of Biology, IUPUI School of Science, Indianapolis, I

EGCG Treatment on Ts65Dn Mice Suggests a Possible Correlation in Cognitive Development Deficit Reduction

poster abstractDown syndrome (DS) is caused by trisomy of human chromosome 21 (Ts21), affecting 1 in 700 live births. Ts21 results in about 80 phenotypes of which intellectual disability (ID) is one of the most debilitating. DYRK1A, found in 3 copies in individuals with Ts21 has been linked to alterations in morphology and function of the brain resulting in ID. Epigallocatechin-3-gallate (EGCG), a specific inhibitor of Dyrk1a activity has been hypothesized as a possible treatment for the overexpression of this gene, reducing the deficits caused by Dryk1a. Using the Ts65Dn mouse model, we examined the effects on hippocampal dependent learning and memory in the novel object recognition task (NOR) using mice of 3-6 weeks of age (adolescent mice). They were given free access to EGCG (0.124 mg/mL) in their drinking water for 21 days. They were then tested for cognitive improvement through NOR. Ts65Dn and control mice (treated and untreated) were subjected to 3 days of testing with 15 minute sessions per day consisting of habituation, exposure, and test day. All procedures were recorded and analyzed to determine time spent exploring novel object in relation to familiar. Our current results suggest that s65Dn mice do not spend as much time exploring the novel object as euploid mice and there exists a genotype effect, but treatment is not correcting the learning and memory deficit. We hypothesize that continuous EGCG treatment may be needed in order to see cognitive deficit reduction in adolescent mice

Rescue of the abnormal skeletal phenotype in Ts65Dn Down syndrome mice using genetic and therapeutic modulation of trisomic Dyrk1a

Trisomy 21 causes skeletal alterations in individuals with Down syndrome (DS), but the causative trisomic gene and a therapeutic approach to rescue these abnormalities are unknown. Individuals with DS display skeletal alterations including reduced bone mineral density, modified bone structure and distinctive facial features. Due to peripheral skeletal anomalies and extended longevity, individuals with DS are increasingly more susceptible to bone fractures. Understanding the genetic and developmental origin of DS skeletal abnormalities would facilitate the development of therapies to rescue these and other deficiencies associated with DS. DYRK1A is found in three copies in individuals with DS and Ts65Dn DS mice and has been hypothesized to be involved in many Trisomy 21 phenotypes including skeletal abnormalities. Return of Dyrk1a copy number to normal levels in Ts65Dn mice rescued the appendicular bone abnormalities, suggesting that appropriate levels of DYRK1A expression are critical for the development and maintenance of the DS appendicular skeleton. Therapy using the DYRK1A inhibitor epigallocatechin-3-gallate improved Ts65Dn skeletal phenotypes. These outcomes suggest that the osteopenic phenotype associated with DS may be rescued postnatally by targeting trisomic Dyrk1a

Effects of Increased Dosage EGCG Treatment on Cognitive Deficits in the Ts65Dn Down Syndrome Mouse Model

poster abstractDown syndrome (DS), caused by trisomy of human chromosome 21 (Hsa21), is the leading genetic cause of cognitive impairment and results in a constellation of phenotypes. Although symptomatic and therapeutic treatments exist for some DS phenotypes, treatments generally do not address the genetic etiology. The Ts65Dn mouse model, which contains a triplication of approximately half the gene orthologs of Hsa21, exhibits hippocampal learning and memory deficits as well as cerebellar motor and spatial deficits similar to those present in individuals with DS. DYRK1A, one of the genes overexpressed in DS, has been identified as a potential cause of cognitive impairment; therefore normalization of DYRK1A activity may be a valid form of treatment. We have shown that Epigallocatechin-3-gallate (EGCG), a major polyphenol of green tea, can rescue skeletal deficits found in the Ts65Dn mouse model at a low dosage. When this same low dosage was used to rescue behavioral deficits, however, it was ineffective. We hypothesize that high dose EGCG treatment lasting throughout the behavioral testing period will rescue the cognitive deficits observed in Ts65Dn mice. Trisomic mice and euploid littermates were given EGCG or water (control) for 7 weeks while being tested sequentially on novel object recognition (NOR) and Morris water maze (MWM). Our current data set shows that Ts65Dn mice exhibit deficits in learning and memory; further data will be collected in order to identify the effect of EGCG. Data showing pure EGCG as being ineffective will suggest the importance adding a supplemental compound, while data showing pure EGCG as an effective form of treatment will strongly support use of EGCG in translational studies in individuals with Down syndrome

EGCG from different sources: differential stability and effects on treating bone phenotypes related to Down syndrome

poster abstractDown Syndrome (DS) is a genetic disorder caused by trisomy of human chromosome 21 (Hsa21). DS phenotypes include cognitive impairment, craniofacial abnormalities, low muscle tone, and skeletal deficiencies. The Ts65Dn mouse model exhibits similar phenotypes as found in humans with DS, including deficits in skeletal bone. Over-expression of DYRK1A, a serine-threonine kinase encoded on Hsa21, has been linked to deficiencies in DS bone homeostasis. Epigallocatechin-3-gallate (EGCG), an aromatic polyphenol found in green tea (GT), is a known inhibitor of Dyrk1a activity. Normalization of Dyrk1a activity by EGCG may have the potential to regulate bone homeostasis, by increasing bone mineral density (BMD) and bone strength. We hypothesized that EGCG obtained from different vendors would differ in stability as well as success in ameliorating skeletal deficiencies. EGCG from different sources was subjected to degradation analysis because of its low bioavailability due to strong antioxidative characteristics. We also hypothesized that phosphoric acid would stabilize EGCG and prevent breakdown in an aqueous solution. We performed High Performance Liquid Chromatography–Mass Spectrometry (HPLC-MS) on EGCG from different sources to determine the amount of EGCG degradation in solution. Our analyses showed differential stability in EGCG from different sources or with phosphoric acid. We chose EGCG from three sources to test the hypothesis that these compounds would have differing effects treating bone phenotypes associated with DS. Three-week-old Ts65Dn and control male mice were treated with EGCG for three weeks. At six weeks of age, mice were sacrificed and femurs were extracted. BMD, bone strength, as well as architecture of the femur were assessed. Our results indicate that EGCG from different sources has diverse effects on the correction of bone phenotypes associated with DS. Our work is important to understand how EGCG from different sources may affect DS phenotypes as the EGCG is translated to human use

Evaluation of the Effects of Green Tea Extracts on Bone Homeostasis in the Ts65Dn Down Syndrome Mouse Model

poster abstractDown Syndrome (DS) is a genetic disorder that affects ~1 in 700 live births, caused by trisomy of human chromosome 21 (Hsa21), and results in cognitive impairment, craniofacial abnormalities, low muscle tone, and skeletal deficiencies. To study these phenotypes, we utilized the Ts65Dn mouse model, which contains three copies of approximately half the orthologous found on Hsa21 and exhibits similar phenotypes as found in humans with DS. Individuals with DS and Ts65Dn mice have deficits in bone mineral density (BMD), architecture, and bone strength. Over-expression of DYRK1A, a serine-threonine kinase encoded on Hsa21, has been linked to deficiencies in DS bone homeostasis. Epigallocatechin-3- gallate (EGCG), an aromatic polyphenol found in high concentrations in green tea, is a known inhibitor of Dyrk1a activity. Normalization of Dyrk1a activity by EGCG may have the potential to regulate bone homeostasis and increase BMD and bone strength in individuals with DS. In this study, we hypothesized that EGCG obtained from different sources would have differential effects in correcting bone deficits associated with DS. To test our hypothesis, we performed Liquid chromatography–mass spectrometry (LC-MS) on EGCG and related compounds from different sources. The LC-MS analysis determined the amount of EGCG and the degradation in our stock solution. Next, we treated three-weekold Ts65Dn and control male mice with EGCG for three weeks. At six weeks of age, mice were sacrificed. DXA and micro CT analysis were performed on the femurs and skulls of the mice to assess trabecular and cortical bone structure and BMD. Our results indicate the ability of EGCG to ameliorate skeletal deficiencies and compared pure EGCG with EGCG purchased from commercial vendors in correcting skeletal deficits associated with DS

Can Epigallocatechin gallate (EGCG) Treatment Rescue Hippocampal-Dependent Cognitive Function in a Down Syndrome Mouse Model?

poster abstractDown Syndrome (DS) is caused by the trisomy of human chromosome 21 (Hsa21). Trisomy 21 can cause various behavioral, cognitive, learning and memory deficits. Deficits in hippocampal structure and function have been identified in mouse models of DS and are implicated in cognitive and learning impairments. Mouse models have suggested that deficits in cognitive function are associated with overexpression of Dyrk1a, a gene on Hsa21 found in three copies of individuals with DS. Dyrk1a is a gene that is involved in brain development and function. Ts65Dn DS model mice exhibit trisomy for approximately half of the genes on Hsa21 including Dyrk1a and exhibit cognitive and learning impairments. We are using Ts65Dn mice to test the effects of Epigallocatechin gallate (EGCG), a Dyrk1a inhibitor, on Dyrk1a activity and cognitive function. We hypothesize that EGCG will reduce Dyrk1a activity in the hippocampus and improve hippocampal-dependent spatial learning and memory in the Morris water maze place learning task in Ts65Dn mice. The mice were given daily EGCG treatment (200 mg/kg per day) by means of oral gavage beginning on postnatal day 54 and continuing throughout water maze testing (postnatal days 67-74). Measures of spatial learning included latency and path length to find a submerged platform during acquisition trials (postnatal days 67-73). Memory for the previously learned location of the platform was assessed on a probe trial (postnatal day 74) in which the platform was removed and the amount of time spent swimming in the area of the tank previously containing the platform was measured. These measures allowed us to analyze the mice’s ability to learn and remember the position of the platform and to spatially orient themselves. Preliminary data indicates that EGCG treatment may not be an effective treatment for the spatial learning and memory deficits evident in this mouse model of DS