PROJECT SUMMARY/ABSTRACT Notch receptors play a critical role in cell fate decisions and in the regulation of osteoblast and osteoclast differentiation and function. As a consequence, Notch receptors play an important role in bone remodeling. Whereas this function is unquestionable, most of the investigations have explored the actions of Notch1 and recently Notch2. Notch3 is expressed by skeletal cells, but its role in the fate and function of osteoblasts and osteoclasts is unknown. Lateral Meningocele Syndrome (LMS) is a devastating disease characterized by meningoceles, craniofacial developmental abnormalities and bone loss. LMS is associated with mutations in exon 33 of NOTCH3 upstream of the PEST domain leading to NOTCH3 stabilization and presumably gain-of- function. The aim of the proposed research is to characterize unique and recently created mouse models of LMS to understand the skeletal disease and mechanisms involved in LMS. As a consequence, the function of Notch3 in the skeleton also will be defined. Our specific aims are: Aim 1) To characterize a newly created global Notch3LMS mutant mouse model. The skeletal phenotype of global Notch3LMS mutants will be compared to that of wild type sex-matched littermate mice and determined by contact radiography, densitometry, micro CT scanning and histomorphometry. The biomechanical properties of the skeleton from LMS mutant mice will be analyzed and mechanisms responsible for the phenotype will be explored; Aim 2) To determine cell lineage specific effects of the Notch3LMS mutation. To this end, we created a Notch3LMS conditional by inversion (COIN) model that recreates the LMS mutation following Cre recombination. Our specific goals are to create mutants specific to cells of the osteoblast lineage to determine their contribution to the skeletal manifestations of LMS; and Aim 3) To establish the mechanism responsible for LMS in the skeleton. We will determine whether Notch3 mRNA and protein are stabilized in cells from Notch3LMS mutants explaining the phenotype observed, and whether Notch3 activation is required for the LMS phenotype. We will determine whether the Notch canonical signaling pathway is responsible for the LMS phenotype and whether the Notch3LMS mutant phenotype can be reversed by the administration of anti-Notch3 antibodies.

PROJECT NARRATIVE These investigations will clarify the skeletal manifestations and mechanisms responsible for Lateral Meningocele Syndrome, a devastating disease characterized by a variety of clinical manifestations including bone loss. In addition, the proposed work will serve to establish the function of Notch3 in the skeleton.