PROJECT SUMMARY Brain calcification is a common neuroimaging finding in individuals with a variety of hereditary or nonhereditary syndromes or many other acquired medical conditions. Raine syndrome, a rare autosomal recessive disease, is caused by mutations in the Family with sequence similarity 20, member C (FAM20C) gene. It is characterized by skeletal and dental defects along with brain calcification. Mouse genetic studies have shown that Sox2- cre;Fam20cfl/fl mice, in which Fam20c was deleted in nearly all the body tissues and cells, developed hypophosphatemic rickets/osteomalacia as well as dental abnormalities. FAM20C encodes a Golgi-localized protein kinase that phosphorylates secretory proteins, including osteopontin (OPN) and matrix extracellular phosphoglycoprotein (MEPE) – two members of the Small Integrin-Binding LIgand N-linked Glycoprotein (SIBLING) family. Both OPN and MEPE contain an acidic serine- and aspartate-rich motif (ASARM). Previous studies have demonstrated that OPN and the ASARM peptides derived from OPN and MEPE proteins inhibit mineralization, and such inhibitory effects depend on the extent of their phosphorylation. While significant progresses have been made in understanding the functions of FAM20C in osteogenesis and odontogenesis, the pathogenic mechanisms underlying the brain calcification in Raine syndrome patients remain largely unknown. More recently, the principal investigator (PI) have found that 1) Fam20c is widely expressed in the mouse brain; 2) Sox2-cre;Fam20cfl/fl mice develop apparent brain calcification by the age of postnatal 3 months; and 3) osteopontin (Opn), a member of SIBLING family, is expressed in the mouse brain neurons. These new findings together with the previous findings on FAM20C and the SIBLING proteins lead to the novel hypothesis that the loss of FAM20C function in the brain cells causes the failure of phosphorylation of OPN and MEPE proteins, resulting in brain calcification. Two specific aims are proposed to test this hypothesis: Aim 1 is to determine if the calcification is associated with the loss of FAM20C function in the brain in Sox2-cre;Fam20cfl/fl mice. To complete this aim, the PI will map the locations of the brain calcifications in Sox2-cre;Fam20cfl/fl mice with age and correlate the calcifications with the loss of FAM20C function in each brain structure. Aim 2 is to determine if OPN and MEPE are phosphorylated in the brain tissues of the control mice, and if the phosphorylation of these two proteins is lost in Sox2-cre;Fam20cfl/fl mice. To fulfill this aim, the PI will isolate OPN and MEPE proteins from the brain tissues of Fam20cfl/fl (control) and Sox2-cre;Fam20cfl/fl mice, and then determine and compare the overall phosphorylation levels and phosphorylation sites of these two proteins between Fam20cfl/fl and Sox2-cre;Fam20cfl/fl mice. Successful completion of the proposed studies will provide new insights into our understanding of how ectopic calcification is prevented ...