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.

Methods
Scans and analysis were performed on right femurs as described (Sloan et al., 2023) using a SkyScan 1172 high-resolution μCT system (Bruker, Kontich, Belgium). Flat field corrections occurred prior to scanning. Hydroxyapatite phantoms (0.25 and 0.75g/cm3 CaHA) were scanned once per week of scanning. Scanning parameters are as follows: 60kV, 12µm resolution, 885ms integration time, 0.7° angular increment, frame averaging of 4. The entire length of the femurs from P12, P15, P18, P24, and P27 were scanned, but due to size constraints, samples from P30 and P42 were scanned from the distal condyles of the femur to at least the third trochanter. Trabecular microarchitecture was analyzed by identifying a trabecular region of interest (ROI) beginning at the proximal end of the distal growth plate and extending proximally based on 10% of the bone length from the proximal end of the distal growth plate to the widest region of the third trochanter using SkyScan CT Analyzer (CTAn) and a custom MATLAB code to exclude the outer cortical bone, using lower greyscale threshold of 45 (P12 mice) or 55 (all other ages) and upper greyscale threshold of 255, as described in previous publications (Goodlett et al., 2020, Stringer et al., 2017a, Thomas et al., 2020, Sloan et al., 2023). The cortical ROI was defined as a region of seven transverse slices at 60% of the overall length of the bone from the proximal end of the distal growth plate and cortical geometry was measured as previously described using MATLAB (Goodlett et al., 2020, Stringer et al., 2017a, Thomas et al., 2020, Sloan et al., 2023).