Search

De novo CLTC mutations underlie a spectrum of early-onset neurodevelopmental phenotypes having developmental delay/intellectual disability (ID), epilepsy, and movement disorders (MD) as major clinical features. CLTC encodes the widely expressed heavy polypeptide of clathrin, a major component of the coated vesicles mediating endocytosis, intracellular trafficking, and synaptic vesicle recycling. The underlying pathogenic mechanism is largely unknown. Here, we assessed the functional impact of the recurrent c.2669C > T (p.P890L) substitution, which is associated with a relatively mild ID/MD phenotype. Primary fibroblasts endogenously expressing the mutated protein show reduced transferrin uptake compared to fibroblast lines obtained from three unrelated healthy donors, suggesting defective clathrin-mediated endocytosis. In vitro studies also reveal a block in cell cycle transition from G0/G1 to the S phase in patient’s cells compared to control cells. To demonstrate the causative role of the p.P890L substitution, the pathogenic missense change was introduced at the orthologous position of the Caenorhabditis elegans gene, chc-1 (p.P892L), via CRISPR/Cas9. The resulting homozygous gene-edited strain displays resistance to aldicarb and hypersensitivity to PTZ, indicating defective release of acetylcholine and GABA by ventral cord motor neurons. Consistently, mutant animals show synaptic vesicle depletion at the sublateral nerve cords, and slightly defective dopamine signaling, highlighting a generalized deficit in synaptic transmission. This defective release of neurotransmitters is associated with their secondary accumulation at the presynaptic membrane. Automated analysis of C. elegans locomotion indicates that chc-1 mutants move slower than their isogenic controls and display defective synaptic plasticity. Phenotypic profiling of chc-1 (+/P892L) heterozygous animals and transgenic overexpression experiments document a mild dominant-negative behavior for the mutant allele. Finally, a more severe phenotype resembling that of chc-1 null mutants is observed in animals harboring the c.3146 T > C substitution (p.L1049P), homologs of the pathogenic c.3140 T > C (p.L1047P) change associated with a severe epileptic phenotype. Overall, our findings provide novel insights into disease mechanisms and genotype–phenotype correlations of CLTC-related disorders.

Abstract Chemosensory receptors play a crucial role in distinguishing the wide range of volatile/soluble molecules by binding them with high accuracy. Chemosensation is the main sensory modality in organisms lacking long-range sensory mechanisms like vision/hearing. Despite its low number of sensory neurons, the nematode Caenorhabditis elegans possesses several chemosensory receptors, allowing it to detect about as many odorants as mammals. Here, we show that C. elegans displays attraction towards urine samples of women with breast cancer, avoiding control ones. Behavioral assays on animals lacking AWC sensory neurons demonstrate the relevance of these neurons in sensing cancer odorants: calcium imaging on AWC increases the accuracy of the discrimination (97.22%). Also, chemotaxis assays on animals lacking GPCRs expressed in AWC allow to identify receptors involved in binding cancer metabolites, suggesting that an alteration of a few metabolites is sufficient for the cancer discriminating behavior of C. elegans, which may help identify a fundamental fingerprint of breast cancer.