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 (Notch3tm1.1Ecan ) by DNA sequencing of F1 mice. One-month-old male and female heterozygous Notch3tm1.1Ecan 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. Notch3tm1.1Ecan 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 Notch3tm1.1Ecan mice. Cultures of bone marrow-derived macrophages from Notch3tm1.1Ecan mice revealed increased osteoclast formation, particularly in cocultures with osteoblasts from Notch3tm1.1Ecan mice. In conclusion, the Notch3tm1.1Ecan mutation causes osteopenia despite an increase in osteoblast proliferation and function and is associated with enhanced Tnfsf11 expression in osteoblasts and osteocytes.