Clozapine Modulates Glucosylceramide, Clears Aggregated Proteins, and Enhances ATG8/LC3 in Caenorhabditis elegans

Defining the mechanisms of action of the antipsychotic drug (APD), clozapine, is of great importance, as clozapine is more effective and has therapeutic benefits in a broader range of psychiatric disorders compared with other APDs. Its range of actions have not been fully characterized. Exposure to APDs early in development causes dose-dependent developmental delay and lethality in Caenorhabditrs elegans. A previous genome-wide RNAi screen for suppressors of clozapine-induced developmental delay and lethality revealed 40 candidate genes, including sms-I, which encodes a sphingomyelin synthase. One sms-I isoform is expressed in the C. elegans pharynx, and its transgene rescues the sms-I mutant phenotype. We examined pharyngeal pumping and observed that clozapine-induced inhibition of pharyngeal pumping requires sms-I, a finding that may explain the role of the gene in mediating clozapine-induced developmental delay/lethality. By analyzing multiple enzymes involved in sphingolipid metabolism, and by observingthe effect of addition of various lipids directly to the worms, we suggest that glucosylceramide may be a key mediator of the effects of clozapine. We further observed that clozapine clears protein aggregates, such as a-synuclein, PolyQ protein, and alpha-I-antitrypsin mutant protein. In addition, it enhances ATG8/LC3. We conclude that clozapine appears to affect the development and induce lethality of worms, in part, through modulating glucosylceramide. We discuss the possible connections among glucosylceramide, protein aggregate clearance, and autophagy. Interactions, including mechanistic pathways involving these elements, may underlie some of the clinical effects of clozapine.