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

Abstract

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 Caenorhabditis elegans. A previous genome-wide RNAi screen for suppressors of clozapine-induced developmental delay and lethality revealed 40 candidate genes, including sms-1, which encodes a sphingomyelin synthase. One sms-1 isoform is expressed in the C. elegans pharynx, and its transgene rescues the sms-1 mutant phenotype. We examined pharyngeal pumping and observed that clozapine-induced inhibition of pharyngeal pumping requires sms-1, 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 observing the 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 α-synuclein, PolyQ protein, and α-1-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. Neuropsychopharmacology (2017) 42, 951-962;

The marginal cells of the Caenorhabditis elegans pharynx scavenge cholesterol and other hydrophobic small molecules

“…6d, e). We infer that the remaining signal is either background staining from the SM probe and/or SM that is produced by other SMSs 27 . We conclude that sms-5 mutants produce less SM in the anterior pharynx compared to wild-type animals.…”

The Marginal Cells of theCaenorhabditis elegansPharynx Scavenge Cholesterol and Other Hydrophobic Small Molecules

“…The sms-5 null mutant shows a significant decrease of fluorescent signal in the anterior pharynx (Fig 5d and 5e). We infer that the remaining signal is either background staining from the sphingomyelin probe and/or sphingomyelin that is produced by other sphingomyelin synthases [27]. We conclude that sms-5 mutants produce less sphingomyelin in the anterior pharynx compared to wild type animals.…”

Pharming for Genes in Neurotransmission: Combining Chemical and Genetic Approaches in Caenorhabditis elegans

“…Further experiments provided evidence that clozapine activates sms-1, which boosts glucosylceramide levels which in turn reduce cellular protein aggregates by inducing autophagy. 75 Analysis of a null mutation in the cation-selective transient potential receptor channel gtl-2 that suppresses larval arrest provided evidence for multiple tissues involved in clozapine action, as restoring gtl-2 expression only in the excretory canal cell is sufficient to rescue suppression. 76 Although still unclear, suppression appears to be mediated through regulation of Mg 2+ homeostasis specifically in this cell.…”

“…Further experiments provided evidence that clozapine activates sms-1 , which boosts glucosylceramide levels which in turn reduce cellular protein aggregates by inducing autophagy. 75…”