Thesis (PhD (Psychiatry))--Stellenbosch University, 2008. / BACKGROUND: Schizophrenia is a heterogeneous illness resulting from complex gene-environment interplay. The majority of molecular genetic work done has involved Caucasian populations, with studies in these and Asian populations showing 2-32% of sufferers to have chromosomal aberrations. So far the discovery of a specific susceptibility mechanism or gene still eludes us, but the use of endophenotypes is advocated as a useful tool in this search. No cytogenetic studies of this nature have been reported in any African schizophrenia population. AIM: The aim of the study was to combine genotypic and phenotypic data, collected in a homogenous population in a structured manner, with the hope of characterising an endophenotype that could be used for more accurate identification of individuals with possible chromosomal abnormalities. METHODOLOGY: A structured clinical interview was conducted on 112 Xhosa schizophrenia patients. (Diagnostic Interview for Genetic Studies, including Schedules for the Assessment of Negative and Positive Symptoms.) Blood samples (karyotyping and/or FISH analysis) as well as urine samples (drug screening) were obtained and nine head and facial measurements were performed. Descriptive statistics were compiled with reference to demographic, clinical and morphological variables. Comparisons between mean differences for these variables were made.

Human response to ionising radiation (IR) shows a wide variation. This is most clearly seen in the radiation-response of cells as measured by frequencies of chromosomal aberrations. Different frequencies of IR-induced aberrations can be conveniently observed in phytohaemagglutin-stimulated peripheral blood T-lymphocytes from both normal individuals and sporadic cancer cases, in either metaphase chromosomes or as micronuclei in the following cell cycle. Metaphase cells show frequent chromatid breaks, defined as chromatid discontinuities or terminal deletions, if irradiated in the G 2 -phase of the cell cycle. It has been shown that the frequency of chromatid breaks in cells from approximately 40% of sporadic breast cancer patients, are significantly higher than in groups of normal individuals. This suggests that elevated radiation-induced chromatid break frequency may be linked with susceptibility to breast cancer. It is known that chromatid breaks are initiated by a double strand break (DSB), but it appears that the two are linked only indirectly as repair kinetics for DSBs and chromatid breaks do not match. Therefore, the underlying causes of the wide variation in frequencies of chromatid breaks in irradiated T-lymphocytes from different normal individuals and from sporadic breast cancer cases are still unclear but it is unlikely to be linked directly to DSB rejoining. My research has focused on the mechanism through which chromatid breaks are formed from initial DSBs. The lack of a direct association suggested that a signalling process might be involved, connecting the initial DSB and resulting chromatid break. The signal model, suggested that the initial DSB is located within a chromatin loop that leads to an intra- or interchromatid rearrangement resulting in incomplete mis-joining of chromatin ends during the decatenation of chromatids during G 2 . It was therefore proposed that topoisomerase II alpha (topo IIα) might be involved, mainly because of its ability to incise DNA and its role in sister chromatid decatenation. During my PhD research I have used a strategy of altering topo II activity or expression and studying whether this alters IR-induced chromatid break frequency. The first approach involved cell lines that varied in topo IIα expression. The frequency of IR-induced chromatid breaks was found to correlate positively with topo IIα expression level, as measured in three different cell lines by immunoblotting, i.e. two cell lines with lower topo IIα expression exhibited lower chromatid break frequency. Topo II activity in these three cell lines was also estimated indirectly by the ability of a topo IIα poison to activate the G 2 /M checkpoint, and this related well with topo IIα expression. A second approach involved ‘knocking down’ topo IIα protein expression by silencing RNA (siRNA). Lowered topo IIα expression was confirmed by immunoblotting and polymerase chain reaction. SiRNA-lowered topo IIα expression correlated with a decreased IR-induced chromatid break frequency. In a third series of experiments cells were treated with ICRF-193, a topo IIα catalytic inhibitor. It was shown that inhibition of topo IIα also significantly reduced IR-induced chromatid breaks. I also showed that lowered chromatid break frequency was not due to cells with high chromatid break frequencies being blocked in G 2 as the mitotic index was not altered significantly in cells with lowered topo IIα expression or activity. These experiments show that topo IIα is involved in IR-induced chromatid break formation. The final experiments reported here attempted to show how topo II might be recruited in the process of forming IR-induced chromatid breaks. Hydrogen peroxide was used as a source of reactive oxygen species (reported to poison topo IIα) and it was shown that topo IIα under these conditions is involved in the entanglement of metaphase chromosomes and formation of chromatin ‘dots’ as well as chromatid breaks. Experiments using atomic force microscopy attempted to confirm these dots as excised chromatin loops. The possible role of topo IIα in both radiation- and hydrogen peroxide-induced primary DNA damage was also tested. It was shown that topo IIα does not affect radiation-induced DSBs, even though it does affect chromatid break frequency. Also, topo IIα does not affect hydrogen peroxide-induced DNA damage at low doses. The results support the idea that topo IIα is involved in the conversion of DSBs to chromatid breaks after both irradiation and treatment with hydrogen peroxide at a low concentrations. I have demonstrated that topo IIα is involved in forming IR-induced chromatid breaks, most likely by converting the initial DSBs into chromosomal aberrations as suggested by the signal model.

The imprinting status of the SLC22A18AS gene, located in the human chromosome segment 11p15.5, was studied using PCR-SSCP analysis and fetal tissues from a battery of 17 abortuses. This gene showed monoallelic expression (genomic imprinting) in different tissues from two abortuses which were heterozygous for an SNP (c.473G>A) in its coding region. This gene was found to be paternally imprinted (maternally expressed) in five tissues namely lung, liver, brain, kidney and placenta from an abortus. The parental origin of the expressed allele could not be determined in the second abortus as both the mother and the abortus were heterozygous for the SNP. Since paternal blood samples from none of the 17 abortuses could be collected for DNA isolation, the mother's genotype was used to find the origin of the expressed allele. In order to understand the mechanism underlying imprinting of this gene, it was important to understand the nature of the epigenetic marks (imprints) on the two alleles of this gene. Since these epigenetic marks are generally observed in promoters or CpG islands associated with the imprinted genes, the promoters of the SLC22A18AS gene was characterized using transient transfection of putative SLC22A18AS promoter fragments cloned in the pGL3-Basic vector in human cells followed by luciferase reporter assay. Since the promoter of a gene lies upstream to the transcription start site (TSS), TSS of this gene was mapped. In silico approach revealed an EST (CB129046) which had an additional 39 bases upstream to the known mRNA sequence. TSS was then identified by the 5’ primer extension analysis. TSS was found to be 166 bases upstream to the 5’ end of this EST. In order to select cell lines for transient transfection of putative promoter constructs for promoter charaterization, RT-PCR analysis was used to see the expression of this gene in the following available cell lines in the lab: HuH7, HepG2, A549, HeLa, LNCaP and PC3. This gene was found to be maximally expressed in HepG2 cells. Expression of this gene was also observed in A549, HeLa, LNCaP and PC3 cells. HuH7, on the other hand, did not show any detectable expression of this gene. Based on the above data, HepG2 and A549 cells were selected for promoter characterization. Seven putative promoter constructs were transiently transfected in these cells and the promoter activity of different constructs was measured by luciferase assay. The assay identified two promoters for the gene: P1 promoter in a region from -855 to -254 bp and P2 promoter in a region from -1441 to -855. In order to see the presence of putative transcription factor binding sites in the upstream region of the gene, the MatInspector Professional program was used. The gene was found to be devoid of TATA and CCAAT boxes. Most of the putative transcription factor binding sites were present in a region from -855 to -254 bp which spans the P1 promoter, including a binding site for the Sp1 transcription factor. In order to see if Sp1 binds to the promoter of this gene, ChIP assay was performed. Sp1 was shown to bind the region harboring the P1 promoter. In order to see if Sp1 has a role in the regulation of this gene, Sp1 constructs were co-transfected with the SLC22A18AS P1 promoter construct in HepG2 and Sp1-null Drosophila SL2 cells. The results showed that the Sp1 has a positive regulation on the SLC22A18AS promoter activity. As stated earlier, epigenetic marks such as differential methylation of CpG dinucleotides in two alleles are associated with promoters of the genes. Since the promoters for SLC22A18AS were characterized, the presence of allele-specific differentially methylated regions (DMRs) associated with the promoters was investigated. In order to differentiate the two alleles in the promoter regions by SNPs, DNA sequence analysis of the promoter regions was performed in a battery of 17 abortuses to search for SNPs. Abortus no. 3 showed heterozygosity for a C to A change at nucleotide position -445 in the P1 promoter region, while abortus no. 2 showed heterozygosities for G to A and A to G changes at nucleotide positions -919 and -1321 respectively in the P2 promoter region. The alleles in the abortus no. 3 were designated as allele C and allele A. The alleles in abortus no. 2 were designated as allele GG and allele AA. Once the two alleles were differentiated by these SNPs, identification of DMRs was performed using sodium bisulfite genomic DNA sequencing. Genomic DNA from the abortus no. 3 was taken for the identification of DMR in the P1 promoter region, while genomic DNA from abortus no. 2 was taken for the identification of DMR in the P2 promoter region. Sodium bisulfite genomic DNA sequencing of the P1 promoter region showed heavy methylation of both the alleles. No DMR was observed in this region. Sodium bisulfite genomic DNA sequencing of the P2 promoter region using DNA from abortus no. 2 did not show any differential methylation of the two alleles. However, like the P1 promoter region, the P2 promoter region was also heavily methylated. In order to see the methylation status of both the promoter regions in human sperms, sperm DNA from an unrelated healthy volunteer was also subjected to sodium bisulfite genomic sequencing. A dense methylation was observed in both the promoter regions of the gene. Heavy methylation of CpG dinucleotides in these regions corroborates the imprinting result for this gene. Since the methylation epigenetic mark is also known to be associated with CpG islands, CpG Plot/CpG Report analysis was used to identify CpG islands in this gene. The analysis showed the presence of two CpG islands, CpG I and CpG II, in the second intron of the gene. As the CpG I island is known to lack methylated CpGs (Ali et al., unpublished result from our lab), a DMR was sought for the CpG II island region. Heterozygosity was ascertained in this region by sequencing DNA from 17 abortuses. However, none of the abortuses showed heterozygosity. It was reasoned that if there is a differential methylation of the two alleles in this region, half of the clones (alleles) should be unmethylated, and the other half should show methylation. Therefore, DNA from abortus no. 3 was randomly chosen for sodium bisulfite genomic sequence anaylsis to identify DMR. The CpG II island showed heavy methylation. However, a DMR was not identified. In order to see the methylation status of the CpG II island in human sperms, sperm DNA from an unrelated healthy volunteer was also subjected to sodium bisulfite genomic sequencing. Almost all the CpG sites showed methylation. The observation of a dense methylation of both the promoters and CpG II island suggested that methylation has a role in the expression of this gene. In order to confirm this observation, A549 and HuH7 cells were treated with a methyltransferase inhibitor, 5-aza-2’-deoxycytidine. 5-Aza-2’-deoxycytidine treatment in HuH7 cells restored the expression of this gene. Further, the expression of this gene was increased in A549 cells following the drug treatment. These results suggested that DNA methylation has a definite role in the modulation of expression of the SLC22A18AS gene. Histone acetylation is another key epigenetic player which is known to have a role in the expression of genes. In order to study the role of the histone acetylation, HuH7 and A549 cells were treated with TSA, a histone deacetylase inhibitor. Treatment of HuH7 and A549 cells with TSA didn’t have any effect on the expression of this gene. On the other hand, the expression of TPA, a gene shown to be regulated by TSA earlier, increased following the TSA treatment in both cell lines. These results suggested that histone acetylation doesn’t have any effect on the expression of this gene. Based on this observation, it was reasoned that histone acetylation is not associated with the imprinting of this gene. Therefore, we did not look for the allele-specific acetylation of histones in this gene. The SLC22A18AS gene has a weak ORF of 253 amino acids as the translation intiation site does not contain a consensus Kozak sequence for efficient translation. In order to determine if it codes for a protein, Western blot analysis was performed using lysates from A549 cells and human fetal liver tissue, and a polyclonal antibody raised in a rabbit against a bacterially expressed SLC22A18AS protein fragment from amino acids 138 to 245. The Western blot result was negative. It was reasoned that this gene might be expressed at a low level and therefore its expression could not be detected by Western blot analysis. Immunoprecipitation was then performed to enrich the SLC22A18AS protein in the lysates followed by Western blot analysis. SLC22A18AS was shown to be expressed as a 30 kDa band in the immunoprecipiates from A549 cell and human fetal liver tissue lysates. The subcellular localization of this gene was studied by immunofluorescence. The fluorescence immunolocalization was performed on A549 cells with anti-SLC22A18AS antibody. The SLC22A18AS protein was found to be localized in the cytoplasm of A549 cells.

Thesis (doctoral)--Göteborg University, 2005. / Includes bibliographical references (p. 438-449) and index.

Indiana University-Purdue University Indianapolis (IUPUI) / Down syndrome (DS) is caused by trisomy of human chromosome 21 (Hsa21) and occurs in ~1 of every 700 live births. Individuals with DS present craniofacial abnormalities, specifically an undersized, dysmorphic mandible which may lead to difficulty with eating, breathing, and speech. Using the Ts65Dn DS mouse model, which mirrors these phenotypes and contains three copies of ~50% Hsa21 homologues, our lab has traced the mandibular deficit to a neural crest cell (NCC) deficiency in the first pharyngeal arch (PA1 or mandibular precursor) at embryonic day 9.5 (E9.5). At E9.5, the PA1 is reduced in size and contains fewer cells due to fewer NCC populating the PA1 from the neural tube (NT) as well as reduced cellular proliferation in the PA1. We hypothesize that both the deficits in NCC migration and proliferation may cause the reduction in size of the PA1. To identify potential genetic mechanisms responsible for trisomic PA1 deficits, we generated RNA-sequence (RNA-seq) data from euploid and trisomic E9.25 NT and E9.5 PA1 (time points occurring before and after observed deficits) using a next-generation sequencing platform. Analysis of RNA-seq data revealed differential trisomic expression of 53 genes from E9.25 NT and 364 genes from E9.5 PA1, five of which are present in three copies in Ts65Dn. We also further analyzed the data to find that fewer alternative splicing events occur in trisomic tissues compared to euploid tissues and in PA1 tissue compared to NT tissue. In a subsequent study, to test gene-specific treatments to rescue PA1 deficits, we targeted Dyrk1A, an overexpressed DS candidate gene implicated in many DS phenotypes and predicted to cause the NCC and PA1 deficiencies. We hypothesize that treatment of pregnant Ts65Dn mothers with Epigallocatechin gallate (EGCG), a known Dyrk1A inhibitor, will correct NCC deficits and rescue the undersized PA1 in trisomic E9.5 embryos. To test our hypothesis, we treated pregnant Ts65Dn mothers with EGCG from either gestational day 7 (G7) to G8 or G0 to G9.5. Our study found an increase in PA1 volume and NCC number in trisomic E9.5 embryos after treatment on G7 and G8, but observed no significant improvements in NCC deficits following G0-G9.5 treatment. We also observed a developmental delay of embryos from trisomic mothers treated with EGCG from G0-G9.5. Together, these data show that timing and sufficient dosage of EGCG treatment is most effective during the developmental window the few days before NCC deficits arise, during G7 and G8, and may be ineffective or harmful when administered at earlier developmental time points. Together, the findings of both studies offer a better understanding of potential mechanisms altered by trisomy as well as preclinical evidence for EGCG as a potential prenatal therapy for craniofacial disorders linked to DS.

Indiana University-Purdue University Indianapolis (IUPUI) / Down syndrome (DS) is the most common aneuploidy to affect humans and occurs in approximately 1 of 750 live births. Individuals with DS present with a wide range of clinical phenotypes. Common craniofacial phenotypic expressions include a small mandible, protruding tongue, and a flattened nasal bridge. These traits may affect the feeding, breathing, and swallowing of individuals with DS. Because some complications may go unnoticed for longer periods of time, we hypothesize that significant cardiac and GI defects may be indicative of feeding and airway difficulties. In order to better understand the secondary phenotypes resulting from DS, we have implemented a retrospective chart review of 137 infants between zero and six months of age who were evaluated through the Down Syndrome Program at Riley Hospital for Children from August 2005 to August 2008. Data regarding cardiac, gastrointestinal, endocrine, airway, auditory, and feeding abnormalities have been collected and incedences and comorbidites of these traits has been examined. Comprehensive results indicate cardiac abnormalities occur in 80% of infants, 60% experience gastrointestinal complications, feeding difficulties occur in 46%, and airway complications occur in 38% of infants. Infants with DS were found to be breastfed less over time, with an increase in tube feeds. Notably, we have found all infants with videofluoroscopic evaluations had some type of dysphagia. The presence of gastrointestinal abnormalities closely correlate with the need for tube feeds, and the comorbidity between GI anomalies and muscle tone appear to indicate the likelihood of feeding difficulties and need for altered feeding strategies. Comorbidities between feeding difficulties were nearly significant with cardiac defects and significant with GI abnormalities. Identification of such associations will help healthcare providers determine the best course of treatment and recommended feeding methodology for infants with DS. In order to utilize an in vitro model to study the craniofacial dysmorphologies seen in individuals with DS, cranial neural crest cells (NC) have been cultured. With these, we have begun to investigate the mechanisms behind a smaller trisomic mandibular precursor as compared to the euploid. With this in vitro model, we will be able to test proliferation, migration, and senescence of NC in a culture system.

Indiana University-Purdue University Indianapolis (IUPUI) / Down syndrome (DS) is the result of trisomy of human chromosome 21 (Hsa 21) and occurs in approximately 1/700 live births. Mouse models of DS have been crucial in understanding the gene-phenotype relationships that underlie many DS anomalies. The Ts65Dn mouse model, trisomic for half of the Hsa 21 orthologs replicates many DS phenotypes including craniofacial alterations such as a small, dysmorphic mandible, midface, and maxilla. Other mouse models, such as the Ts1Rhr which contains a triplication of 33 Hsa 21 orthologs, have been used to better understand the genes responsible for craniofacial alterations. Our laboratory has demonstrated that the postnatal mandibular phenotype found in Ts65Dn mice can be traced back to an original neural crest cell (NCc) deficit in the developing first pharyngeal arch (PA1) at embryonic day 9.5 (E9.5). Furthermore, evidence suggested that both a proliferation deficit in the PA1 and a migration deficit in the NCC from the neural tube (NT) could be the mechanism behind this deficit. However, the molecular mechanisms behind these deficits remain to be elucidated. Due to the involvement of the Hsa 21 genes DYRK1A and RCAN1 in regulation of signaling pathways including NFATc (NFAT2), a transcription factor known to influence cellular proliferation and, later, bone development, we hypothesized that dysregulation of these genes could underlie the cellular deficit in the PA1. Furthermore, we hypothesized that targeting Dyrk1a by decreasing activity or available protein could ameliorate the established deficits. Through the use of RNA isolation techniques and cell culture systems of cell from the PA1 and NT of E9.5 Ts65Dn, Ts1Rhr, and control embryos, we established that trisomic genes Dyrk1a and Rcan1 ara dysregulated in both structures and that these two genes may interact. Furthermore, we established that a proliferation deficit in the Ts65Dn PA1 and a migration deficit in the Ts65Dn PA1 and NT exists at E9.5 and can be rescued to euploid levels in vitro with the addition of the Dyrk1a inhibitor, EGCG, a green tea polyphenol. We also confirmed that harmine, a more highly studied and specific Dyrk1a inhibitor, is capable of similar effects on proliferation of PA1 cell from E9.5 Ts65Dn embryos. Furthermore, when Ts65Dn pregnant mothers were treated with EGCG in vivo, the cellular deficit found in the developing E9.5 embryonic PA1 was rescued to near euploid volume and NCC number. Treatment with EGCG did not adversely impact litter size or embryonic development. Interestingly, euploid embryonic volume increased with EGCG treatment. Expression analysis of the E9.5 PA1 of EGCG treated Ts65Dn and control embryos revealed dysregulation of several genes involved in craniofacial and developmental pathways including Dyrk1a, Rcan1, Ets2 and members of the sonic hedgehog pathways. Our novel results provide a foundation for better understanding the molecular mechanisms of craniofacial development and may provide evidence-based therapeutic options to improve the quality of life for individuals with DS.

Shared characteristics and predictors of psychological distress are beginning to be identified in research on women seeking genetic testing for BRCA1 and BRCA2 gene mutations. This study further explored patterns of psychological distress for 51 community women waiting to receive such genetic test results. There was no significant relationship between psychological distress and family cancer history, personal cancer history, social support networks, and family environment. Women in this sample tended to rely more on females and relatives for support than males and friends. Social support satisfaction was not related to gender or number of relatives providing support. Thirty-four of the 36 women classified on the family environment type were from Personal Growth-Oriented families. Comparisons with normal and distressed family means revealed increased cohesion and expressiveness with decreased conflict, indicative of supportive family environments. Limitations and implications are discussed.

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.

Indiana University-Purdue University Indianapolis (IUPUI) / Down syndrome (DS) is caused by three copies of human chromosome 21 (Hsa21) and results in an array of phenotypes including intellectual disability. Ts65Dn mice, the most extensively studied DS model, have three copies of ~50% of the genes on Hsa21 and display many phenotypes associated with DS, including cognitive deficits. 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 CNS development and osteoclastogenesis. Epigallocatechin-3-gallate (EGCG), the main polyphenol in green tea, inhibits Dyrk1a activity. We have shown that a three-week EGCG treatment (~10mg/kg/day) during adolescence normalizes skeletal abnormalities in Ts65Dn mice, yet the same dose did not rescue deficits in the Morris water maze spatial learning task (MWM) or novel object recognition (NOR). Others have reported that An EGCG dose of 2-3 mg per day (90mg/ml) improved hippocampal-dependent task deficits in Ts65Dn mice. The current study investigated deficits in a radial arm maze pattern separation task in Ts65Dn mice. Pattern separation requires differentiation between similar memories acquired during learning episodes; distinguishing between these similar memories is thought to depend on distinctive encoding in the hippocampus. Pattern separation has been linked to functional activity of newly generated granule cells in the dentate gyrus. Recent studies in Ts65Dn mice have reported significant reductions in adult hippocampal neurogenesis, and after EGCG treatment, enhanced hippocampal neurogenesis. Thus, it was hypothesized that Ts65Dn mice would be impaired in the pattern separation task, and that EGCG would alleviate the pattern separation deficits seen in trisomic mice, in association with increased adult hippocampal neurogenesis. At weaning, Ts65Dn mice and euploid littermates were randomly assigned to the water control, or EGCG [0.4 mg/mL], with both treatments yielding average daily intakes of ~50 mg/kg/day. Beginning on postnatal day 75, all mice were trained on a radial arm maze-delayed non-matching-to-place pattern separation task. Euploid mice performed significantly better over training than Ts65Dn mice, including better performance at each of the three separations. EGCG did not significantly alleviate the pattern separation deficits in Ts65Dn mice. After the behavioral testing commenced, animals were given ad libitum food access for five days, received a 100mg/kg injection of BrdU, and were perfused two hours later. Coronal sections through the dorsal hippocampus were processed for BrdU labeling, and cells were manually counted throughout the subgranular zone of the dentate gyrus. The euploid controls had significantly more BrdU labeled cells than Ts65Dn mice, however, EGCG does not appear to increase proliferation of the hippocampal neuroprogenitor cells. This is the first report of deficits in Ts65Dn mice on a pattern separation task. To the extent that pattern separation depends on the functional involvement of newly generated neurons in an adult dentate gyrus, this approach in Ts65Dn mice may help identify more targeted pharmacotherapies for cognitive deficits in individuals with DS.