Genome Wide Analysis Identifies Sphingolipid Metabolism As A New Target Of Valproic Acid

Bipolar disorder (BD), which is characterized by depression and mania, affects about 1% of the total world population. Current treatments are effective in only 40-60% of cases and cause severe side effects. Valproic acid (VPA), a branched short-chain fatty acid, is one of the most widely used drugs for the treatment of BD. Although many hypotheses have been postulated to explain the molecular mechanism of action of this drug in BD, the therapeutic mechanism is not understood. This knowledge gap has hampered the development of new drugs to treat this disorder. To identify candidate pathways affected by VPA, I performed a genome wide expression analysis in yeast cells grown in the presence or absence of the drug. Many genes and pathways showed altered expression in response to VPA. Among these, sphingolipid metabolism genes showed altered expression in response to both chronic and acute VPA treatment. Chronic VPA caused upregulation of FEN1 and SUR4, encoding fatty acid elongases that catalyze the synthesis of very long chain fatty acids (C24 to C26) required for the synthesis of ceramide. Interestingly, fen1Δ and sur4Δ mutants exhibited VPA sensitivity. Consistent with this, VPA increased levels of ceramides, especially those that contain C24 and C26 fatty acids. As expected with an increase in ceramide, VPA decreased the expression of amino acid transporters, increased the expression of ER chaperones, and activated the unfolded protein response element (UPRE), suggesting that VPA induces the UPR pathway. These effects are rescued by supplementation of inositol and are similarly observed in inositol-starved ino1Δ cells. Starvation of ino1Δ cells increased expression of FEN1 and SUR4, increased ceramide levels, decreased expression of nutrient transporters, and induced the UPR. These findings suggest that VPA-mediated inositol depletion induces the UPR by increasing the de novo synthesis of ceramide. In response to acute VPA, the gene that exhibited the highest upregulation was RSB1, which encodes a transporter of the long chain bases (LCBs) dihydrosphingosine (DHS) and phytosphingosine (PHS). In addition to increased mRNA, acute VPA increased Rsb1 protein levels. The rsb1Δ mutant exhibited increased sensitivity to PHS in the presence of VPA, suggesting that VPA increases PHS levels. Consistent with this, acute VPA increased PHS levels, especially in rsb1Δ cells. LCBs are precursors of ceramide synthesis, which begins in the endoplasmic reticulum by the conversion of palmitoyl-CoA to PHS or DHS. These intermediates are converted to ceramide via ceramide synthase by addition of a fatty acid synthesized by the fatty acid elongation pathway. Orm proteins are negative regulators of de novo synthesis of PHS, which was shown to function as a signaling molecule. My findings indicate that acute VPA downregulates ORM and fatty acid elongases FEN1 and SUR4. This leads to increased PHS levels and increased expression of RSB1 as well as genes that transport and metabolize PHS, including YOR1 and DPL1. Inositol starvation of the ino1Δ mutant for 30 minutes increased expression of RSB1 and YOR1 and decreased expression of FEN1, SUR4, ORM1, and ORM2. This study shows for the first time that acute VPA-mediated inositol depletion increases levels of PHS. In summary, I identified sphingolipid metabolism as a new target of VPA. My studies showed that VPA exerts inositol depletion-mediated differential effects on sphingolipid species. Chronic VPA increases ceramide levels and induces the UPR pathway, whereas, acute VPA increases the levels of PHS. These findings suggest that sphingolipid metabolism is a potential target of VPA that could be important for the therapeutic action of this drug