Compromised femoral and lumbovertebral bone in the Dp(16)1Yey Down syndrome mouse model.

Down syndrome (DS), affecting ∼1 in 800 live births, is caused by the triplication of human chromosome 21 (Hsa21). Individuals with DS have skeletal features including craniofacial abnormalities and decreased bone mineral density (BMD). Lowered BMD can lead to increased fracture risk, with common fracture points at the femoral neck and lumbar spine. While the femur has been studied in DS mouse models, there is little research done on the vertebrae despite evidence that humans with DS have affected vertebrae. Additionally, it is important to establish when skeletal deficits occur to find times of potential intervention. The Dp(16)1Yey DS mouse model has all genes triplicated on mouse chromosome 16 orthologous to Hsa21 and displayed deficits in long bone, including trabecular and cortical deficits in male but not female mice, at 12 weeks. We hypothesized that the long bone and lumbovertebral microarchitecture would exhibit sexually dimorphic deficits in Dp(16)1Yey mice compared to control mice and long bone strength would be diminished in Dp(16)1Yey mice at 6 weeks. The trabecular region of the 4th lumbar (L4) vertebra and the trabecular and cortical regions of the femur were analyzed via micro-computed tomography and 3-point bending in 6-week-old male and female Dp(16)1Yey and control mice. Trabecular and cortical deficits were observed in femurs from male Dp(16)1Yey mice, and cortical deficits were seen in femurs of male and female Dp(16)1Yey mice. Male Dp(16)1Yey femurs had more deficits in bone strength at whole bone and tissue-estimate level properties, but female Dp(16)1Yey mice were also affected. Additionally, the L4 of male and female Dp(16)1Yey mice show trabecular deficits, which have not been previously reported in a DS mouse model. Our results indicate that skeletal deficits associated with DS occur early in skeletal development, are dependent on skeletal compartment and site, are sex dependent, and potential interventions should likely begin early in skeletal development of DS mouse models.

Sex specific emergence of trisomic Dyrk1a-related skeletal phenotypes in the development of a Down syndrome mouse model.

Skeletal insufficiency affects all individuals with Down syndrome (DS) or Trisomy 21 (Ts21) and may alter bone strength throughout development due to a reduced period of bone formation and early attainment of peak bone mass compared to typically developing individuals. Appendicular skeletal deficits also appear in males before females with DS. In femurs of male Ts65Dn DS model mice, cortical deficits were pronounced throughout development, but trabecular deficits and Dyrk1a overexpression were transitory until postnatal day (P) 30 when there were persistent trabecular and cortical deficits and Dyrk1a was trending overexpression. Correction of DS-related skeletal deficits by a purported DYRK1A inhibitor or through genetic means beginning at P21 was not effective at P30, but germline normalization of Dyrk1a improved male bone structure by P36. Trabecular and cortical deficits in female Ts65Dn mice were evident at P30 but subsided by P36, typifying periodic developmental skeletal normalizations that progressed to more prominent bone deficiencies. Sex-dependent differences in skeletal deficits with a delayed impact of trisomic Dyrk1a are important to find temporally specific treatment periods for bone and other phenotypes associated with Ts21.

Genetic dissection of triplicated chromosome 21 orthologs yields varying skeletal traits in Down syndrome model mice.

Down syndrome (DS) phenotypes result from triplicated genes, but the effects of three copy genes are not well known. A mouse mapping panel genetically dissecting human chromosome 21 (Hsa21) syntenic regions was used to investigate the contributions and interactions of triplicated Hsa21 orthologous genes on mouse chromosome 16 (Mmu16) on skeletal phenotypes. Skeletal structure and mechanical properties were assessed in femurs of male and female Dp9Tyb, Dp2Tyb, Dp3Tyb, Dp4Tyb, Dp5Tyb, Dp6Tyb, Ts1Rhr and Dp1Tyb;Dyrk1a+/+/- mice. Dp1Tyb mice, with the entire Hsa21 homologous region of Mmu16 triplicated, display bone deficits similar to those of humans with DS and served as a baseline for other strains in the panel. Bone phenotypes varied based on triplicated gene content, sex and bone compartment. Three copies of Dyrk1a played a sex-specific, essential role in trabecular deficits and may interact with other genes to influence cortical deficits related to DS. Triplicated genes in Dp9Tyb and Dp2Tyb mice improved some skeletal parameters. As triplicated genes can both improve and worsen bone deficits, it is important to understand the interaction between and molecular mechanisms of skeletal alterations affected by these genes.

Increased dosage and treatment time of Epigallocatechin-3-gallate (EGCG) negatively affects skeletal parameters in normal mice and Down syndrome mouse models.

Bone abnormalities affect all individuals with Down syndrome (DS) and are linked to abnormal expression of DYRK1A, a gene found in three copies in people with DS and Ts65Dn DS model mice. Previous work in Ts65Dn male mice demonstrated that both genetic normalization of Dyrk1a and treatment with ~9 mg/kg/day Epigallocatechin-3-gallate (EGCG), the main polyphenol found in green tea and putative DYRK1A inhibitor, improved some skeletal deficits. Because EGCG treatment improved mostly trabecular skeletal deficits, we hypothesized that increasing EGCG treatment dosage and length of administration would positively affect both trabecular and cortical bone in Ts65Dn mice. Treatment of individuals with DS with green tea extract (GTE) containing EGCG also showed some weight loss in individuals with DS, and we hypothesized that weights would be affected in Ts65Dn mice after EGCG treatment. Treatment with ~20 mg/kg/day EGCG for seven weeks showed no improvements in male Ts65Dn trabecular bone and only limited improvements in cortical measures. Comparing skeletal analyses after ~20mg/kg/day EGCG treatment with previously published treatments with ~9, 50, and 200 mg/kg/day EGCG showed that increased dosage and treatment time increased cortical structural deficits leading to weaker appendicular bones in male mice. Weight was not affected by treatment in mice, except for those given a high dose of EGCG by oral gavage. These data indicate that high doses of EGCG, similar to those reported in some treatment studies of DS and other disorders, may impair long bone structure and strength. Skeletal phenotypes should be monitored when high doses of EGCG are administered therapeutically.

Skeletal Deficits in Male and Female down Syndrome Model Mice Arise Independent of Normalized Dyrk1a Expression in Osteoblasts.

Trisomy 21 (Ts21) causes alterations in skeletal development resulting in decreased bone mass, shortened stature and weaker bones in individuals with Down syndrome (DS). There is a sexual dimorphism in bone mineral density (BMD) deficits associated with DS with males displaying earlier deficits than females. The relationships between causative trisomic genes, cellular mechanisms, and influence of sex in DS skeletal abnormalities remain unknown. One hypothesis is that the low bone turnover phenotype observed in DS results from attenuated osteoblast function, contributing to impaired trabecular architecture, altered cortical geometry, and decreased mineralization. DYRK1A, found in three copies in humans with DS, Ts65Dn, and Dp1Tyb DS model mice, has been implicated in the development of postnatal skeletal phenotypes associated with DS. Reduced copy number of Dyrk1a to euploid levels from conception in an otherwise trisomic Ts65Dn mice resulted in a rescue of appendicular bone deficits, suggesting DYRK1A contributes to skeletal development and homeostasis. We hypothesized that reduction of Dyrk1a copy number in trisomic osteoblasts would improve cellular function and resultant skeletal structural anomalies in trisomic mice. Female mice with a floxed Dyrk1a gene (Ts65Dn,Dyrk1afl/wt) were mated with male Osx-Cre+ (expressed in osteoblasts beginning around E13.5) mice, resulting in reduced Dyrk1a copy number in mature osteoblasts in Ts65Dn,Dyrk1a+/+/Osx-Cre P42 male and female trisomic and euploid mice, compared with littermate controls. Male and female Ts65Dn,Dyrk1a+/+/+ (3 copies of DYRK1A in osteoblasts) and Ts65Dn,Dyrk1a+/+/Osx-Cre (2 copies of Dyrk1a in osteoblasts) displayed similar defects in both trabecular architecture and cortical geometry, with no improvements with reduced Dyrk1a in osteoblasts. This suggests that trisomic DYRK1A does not affect osteoblast function in a cell-autonomous manner at or before P42. Although male Dp1Tyb and Ts65Dn mice exhibit similar skeletal deficits at P42 in both trabecular and cortical bone compartments between euploid and trisomic mice, female Ts65Dn mice exhibit significant cortical and trabecular deficits at P42, in contrast to an absence of genotype effect in female Dp1Tyb mice in trabecular bone. Taken together, these data suggest skeletal deficits in DS mouse models and are sex and age dependent, and influenced by strain effects, but are not solely caused by the overexpression of Dyrk1a in osteoblasts. Identifying molecular and cellular mechanisms, disrupted by gene dosage imbalance, that are involved in the development of skeletal phenotypes associated with DS could help to design therapies to rescue skeletal deficiencies seen in DS.