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Browsing by Author "Blazek, Joshua D."
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Item Abnormal mineralization of the Ts65Dn Down syndrome mouse appendicular skeleton begins during embryonic development in a Dyrk1a-independent manner(Elsevier, 2015-05) Blazek, Joshua D.; Malik, Ahmed M.; Tischbein, Maeve; Arbones, Maria L.; Moore, Clara S.; Roper, Randall J.; Biology, School of ScienceThe relationship between gene dosage imbalance and phenotypes associated with Trisomy 21, including the etiology of abnormal bone phenotypes linked to Down syndrome (DS), is not well understood. The Ts65Dn mouse model for DS exhibits appendicular skeletal defects during adolescence and adulthood but the developmental and genetic origin of these phenotypes remains unclear. It is hypothesized that the postnatal Ts65Dn skeletal phenotype originates during embryonic development and results from an increased Dyrk1a gene copy number, a gene hypothesized to play a critical role in many DS phenotypes. Ts65Dn embryos exhibit a lower percent bone volume in the E17.5 femur when compared to euploid embryos. Concomitant with gene copy number, qPCR analysis revealed a ~1.5 fold increase in Dyrk1a transcript levels in the Ts65Dn E17.5 embryonic femur as compared to euploid. Returning Dyrk1a copy number to euploid levels in Ts65Dn, Dyrk1a+/− embryos did not correct the trisomic skeletal phenotype but did return Dyrk1a gene transcript levels to normal. The size and protein expression patterns of the cartilage template during embryonic bone development appear to be unaffected at E14.5 and E17.5 in trisomic embryos. Taken together, these data suggest that the dosage imbalance of genes other than Dyrk1a is involved in the development of the prenatal bone phenotype in Ts65Dn embryos.Item EGCG from different sources: differential stability and effects on treating bone phenotypes related to Down syndrome(Office of the Vice Chancellor for Research, 2014-04-11) Thomas, Jared R.; Abeysekera, Irushi S.; Blazek, Joshua D.; Roper, Randall J.Down 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.Item EMBRYONIC BONE DEVELOPMENT AND NFAT EXPRESSION IN THE TS65DN MOUSE MODEL FOR DOWN SYNDROME(Office of the Vice Chancellor for Research, 2012-04-13) Malik, Ahmed M.; Blazek, Joshua D.; Roper, Randall J.Down syndrome (DS) is a common genetic disorder that occurs in ap-proximately 1 out of every 750 live births. DS phenotypes include cognitive deficits, altered craniofacial features, muscle hypotonia, heart defects, and abnormal bone structure. The Ts65Dn mouse model is the most common or-ganismal model used to study DS phenotypes. This model exhibits a number of phenotypic traits comparable to those of humans with DS, including bone anomalies. Past studies have shown that Ts65Dn mice exhibit weaker tra-becular bone due to less trabeculae. They have also been shown to have less bone mineral density and bone mineral content at 6 weeks of age when compared to their euploid counterparts, with the severity of these defects lessening by 16 weeks. No studies of bone development have yet decisively identified the origin of these defects. We hypothesized that abnormal endochondral ossification is responsible for the presence of these deficien-cies in bone mineral content and bone mineral density. Aberrant expression of Nfat has been implicated as the molecular cause of many DS-related phe-notypes, and activity of Nfat can be determined based upon its localization. Specifically, Nfat has been shown to control many aspects of bone develop-ment, which makes it of special interest to this research. To test our hypoth-esis of a bone deficit present during embryonic development of Ts65Dn em-bryos, we are comparing cartilaginous template characteristics, progression of the mineralization front, osteoclast activity, percent bone volume, and Nfat localization in euploid and trisomic mouse femurs at embryonic day 17.5. Our preliminary data show lower percent bone volumes in trisomic fe-murs, suggesting that endochondral ossification in Ts65Dn mice lags behind that of their euploid counterparts. These results indicate that DS bone phe-notypes do indeed originate during embryonic development and create a foundation for future work on their treatment. Supported by: National Science Foundation GK-12 Fellowship; Jerome Lejeune FoundationItem Evaluation of the Effects of Green Tea Extracts on Bone Homeostasis in the Ts65Dn Down Syndrome Mouse Model(Office of the Vice Chancellor for Research, 2013-04-05) Abeysekera, Irushi S.; Thomas, Jared R.; Blazek, Joshua D.; Roper, Randall J.Down 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.Item Influence of prenatal EGCG treatment and Dyrk1a dosage reduction on craniofacial features associated with Down syndrome(Oxford Academic, 2016-11-15) McElyea, Samantha D.; Starbuck, John M.; Tumbleson-Brink, Danika M.; Harrington, Emily; Blazek, Joshua D.; Ghoneima, Ahmed; Kula, Katherine; Roper, Randall J.; Biology, School of ScienceTrisomy 21 (Ts21) affects craniofacial precursors in individuals with Down syndrome (DS). The resultant craniofacial features in all individuals with Ts21 may significantly affect breathing, eating and speaking. Using mouse models of DS, we have traced the origin of DS-associated craniofacial abnormalities to deficiencies in neural crest cell (NCC) craniofacial precursors early in development. Hypothetically, three copies of Dyrk1a (dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A), a trisomic gene found in most humans with DS and mouse models of DS, may significantly affect craniofacial structure. We hypothesized that we could improve DS-related craniofacial abnormalities in mouse models using a Dyrk1a inhibitor or by normalizing Dyrk1a gene dosage. In vitro and in vivo treatment with Epigallocatechin-3-gallate (EGCG), a Dyrk1a inhibitor, modulated trisomic NCC deficiencies at embryonic time points. Furthermore, prenatal EGCG treatment normalized some craniofacial phenotypes, including cranial vault in adult Ts65Dn mice. Normalization of Dyrk1a copy number in an otherwise trisomic Ts65Dn mice normalized many dimensions of the cranial vault, but did not correct all craniofacial anatomy. These data underscore the complexity of the gene–phenotype relationship in trisomy and suggest that changes in Dyrk1a expression play an important role in morphogenesis and growth of the cranial vault. These results suggest that a temporally specific prenatal therapy may be an effective way to ameliorate some craniofacial anatomical changes associated with DS.Item MOLECULAR MECHANISMS ALTERING SKELETAL DEVELOPMENT AND HOMEOSTASIS IN TS65NDN DOWN SYNDROME MICE(Office of the Vice Chancellor for Research, 2012-04-13) Blazek, Joshua D.; Malik, Ahmed; Roper, Randall J.Down syndrome (DS) is caused by three copies of human chromosome 21 (HSA21) and results in abnormal craniofacial and appendicular bone phe-notypes. The Ts65Dn mouse model of DS contains three copies of nearly half of the genes found on HSA21, and exhibits craniofacial skeletal phenotypes similar to those observed in humans with DS. We recently demonstrated ab-normalities in the development and homeostasis of the appendicular skele-ton of Ts65Dn mice. Femurs from trisomic mice exhibit alterations in trabec-ular bone architecture and overall bone strength. Furthermore, bone for-mation rates were found to be significantly reduced, suggesting trisomy im-pacts bone development and maintenance in Ts65Dn mice, and by extension humans with DS. DYRK1A is triplicated in both humans with DS and Ts65Dn mice and its protein acts as a kinase critical during development. Dyrk1A negatively regulates the nuclear localization and activation of Nfatc, a tran-scription factor critical to signaling pathways associated with cell proliferation and bone development, and is overexpressed in the E9.5 Ts65Dn mandible precursor. We hypothesize that the previously documented Ts65Dn bone phenotype originates during embryonic development, and the presence of an extra copy of Dyrk1a contributes to the abnormal bone phenotype observed in Ts65Dn mice and humans with DS. To test our first hypothesis, analysis of the cartilage template and early bone precursor is being conducted on the femurs from embryonic day 17.5 trisomic and euploid embryos. To implicate the involvement of Dyrk1a in the DS bone phenotype, Ts65Dn mice are be-ing treated with a known Dyrk1a inhibitor, EGCG, to determine if correcting the functional expression of Dyrk1a impacts the development of the Ts65Dn postnatal bone phenotype. Understanding the molecular mechanisms under-lying DS bone phenotypes may help improve the quality of life for individuals with DS and provide viable options for the treatment of osteoporosis.Item Rescue of the abnormal skeletal phenotype in Ts65Dn Down syndrome mice using genetic and therapeutic modulation of trisomic Dyrk1a(Oxford, 2015) Blazek, Joshua D.; Abeysekera, Irushi; Li, Jiliang; Roper, Randall J.; Biology, School of ScienceTrisomy 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.