This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The main objective of this project is to establish the functional connection between folate and sphingolipid pathways and to explore the underlying molecular mechanisms. Specifically, we propose to evaluate the role of sphingolipid signaling in mediation of the effects of folate deficiency and responces to the disturbance of intracellular folate metabolism. At present, there is a lack of knowledge regarding the functional interactions between the folate and sphingolipid pathways, but there are specific indications that the two pathways are functionally connected. (1) They both rely on serine as the principal one-carbon group donor into the folate pool and a substrate for de novo sphingolipid biosynthesis;(2) sphingolipid pathways depend on methylation directly (methylation of phosphatidylethanolamine to phosphatidylcholine) and indirectly (regulation of enzyme expression via DNA/histone methylation), while folates are crucial for cellular methylation;(3) cross talk between the two pathway is suggested by experiments with fumonisine, an inhibitor of seramide synthase, as well as in experiments with atifolates;(4) our studies have demonstrated significant changes in the levels of ceramides upon alterations of intracellular folate metabolism. Therefore we have hypothesized that activation of sphingolipid pathways is the downstream event of the folate stress. The alteration of methylation in spingolipid pathways is proposed as the underlying mechanism in this process. Aims to test this hypothesis are: Aim 1. Determine effects of folate deficiency on sphingolipid pathways. Aim 2. Explore the role of folate-dependent methylation in spingolipid pathways. Aim 3. Determine, whether ceramide signaling is a component of cellular response to impared folate metabolism.