The lateral meningocele syndrome mutation causes marked osteopenia in mice

Lateral meningocele syndrome (LMS) is a rare genetic disorder characterized by neurological complications and osteoporosis. LMS is associated with mutations in exon 33 of NOTCH3 leading to a truncated protein lacking sequences for NOTCH3 degradation and presumably causing NOTCH3 gain of function. To create a mouse model reproducing human LMS-associated mutations, we utilized CRISPR/Cas9 to introduce a tandem termination codon at bases 6691-6696 (ACCAAG→TAATGA) and verified this mutation (Notch3^tm1.1Ecan ) by DNA sequencing of F1 mice. One-month-old male and female heterozygous Notch3^tm1.1Ecan mice had cancellous and cortical bone osteopenia but exhibited no obvious neurological alterations, and histopathology of multiple organs revealed no abnormalities. Microcomputed tomography of these mutants revealed a 35-60% decrease in cancellous bone volume associated with a reduction in trabecular number and decreased connectivity. During maturation, cancellous and cortical bones were restored in female but not in male mice, which exhibited cancellous bone osteopenia at 4 months. Cancellous bone histomorphometry revealed increased osteoblast and osteocyte numbers and a modest increase in osteoclast surface and bone formation rate. Notch3^tm1.1Ecan calvarial osteoblasts had increased proliferation and increased bone γ-carboxyglutamate protein (Bglap) and TNF superfamily member 11 (Tnfsf11) mRNA levels and lower Tnfrsf11b levels. Tnfsf11 mRNA was increased in osteocyte-rich femora from Notch3^tm1.1Ecan mice. Cultures of bone marrow-derived macrophages from Notch3^tm1.1Ecan mice revealed increased osteoclast formation, particularly in cocultures with osteoblasts from Notch3^tm1.1Ecan mice. In conclusion, the Notch3^tm1.1Ecan mutation causes osteopenia despite an increase in osteoblast proliferation and function and is associated with enhanced Tnfsf11 expression in osteoblasts and osteocytes.