The objective of the purposed studies is to determine the in vivo function(s) of intact prosaposin, a multifunctional precursor of four lysosomal hydrolase activator proteins, or saposins A,B, C and D. Isolated deficiencies of saposins B and C, or the total deficiency of prosapsoin lead to, respectively, one or several specific glycosphingolipids (GSL) stored in lysosomes of most organs, including the brain. Intact prosaposin has in vitro/ex vivo neurotrophic effects and GSL transport functions. When administered to animals, prosaposin has nerve regenerative properties. A specific 9-12 amino acid sequence of saposin C was identified as critical to the neurotrophic (NT) effects. Using a """"""""knock-out"""""""" (sap-/sap-) mouse, """"""""rescue"""""""" experiments are proposed using prosaposin transgenes designed so that the saposin functions are preserved, bu the in vitro/ex vivo NT functions are destroyed; i.e., the NT sequences will be normal [NT(+)] or mutant [NT(-)]. NT(+) and NT(-) prosaposins or their saposin C counterparts will be expressed heterologously in mammalian and insect cells, or E. coli, respectively. Prosaposins' processing to and turnover of saposins will be evaluated by metabolic labeling analyses. The cellular functions of the resultant saposins will be determined by quantitative GSL turnover, and sulfatide and glucosylceramide loading analyses. Purified prosaposins (insect source) and saposin Cs (E.coli) will be used to evaluate in vitro/ex vivo NT and GSL transport functions prior to making transgenic mice. The sap-/sap- mice expressing NT(+) or NT(-) prosaposins will have extensive histopathologic, biochemical (GSL and Western), immunofluorescence (prosaposin and saposins) and in situ mRNA hybridization analyses to determine the invivo function of the NT sequence. These studies are designed for direct correlations between the in vitro/ex vivo and in vivo functions of the transgenes and to define the minimal NT region necessary to prosaposins' trophic functions. Circumstantial in vitro/ex vivo evidence strongly supports a major role of intact prosaposin as a trophic, particularly neurotrophic, factor. It is the goal of these studies to define this potentially critical function in the only available system for these analyses. The results of these experiments have major import for understanding CNS development, nerve regeneration, GSL metabolism and the pathophysiology of GSL inborn errors of metabolism.
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