The are several advantages in using Caenorhabditis elegans as organism model, among others its small size, the fast vital cycle and the easiness to maintain it in culture. Since between 60 and 80% of human genes have an orthologue in the worm genome, it has already been employed to investigate mitochondrial disorders, neurodegenerative diseases and also to search for neurotoxins and neuroprotective drugs.
During my PhD program I used C. elegans as a model to study genes whose function is unknown or not fully understood and potentially involved in neurometabolic diseases. At this purpose I performed RNA interference (RNAi) experiments and CRISPR/Cas9 genome editing to generate knockdown and knockout (KO)/knock-in worm models, that were then extensively phenotypically characterized.
Cytochrome c oxidase (COX) is the terminal enzymatic complex of the mitochondrial respiratory chain. Mutations in COX genes are responsible for COX deficiency, which is the most frequent cause of mitochondrial encephalomyopathies. COX16 is a COX assembly factor, with a homologue in the worm genome, that is cox-16.
By a COX specific histochemical staining we found that cox-16 is required for COX biogenesis and function, since its knockdown in nematodes causes COX deficiency. These results confirm what was previously obtained in a COX16 KO cellular model, with the advantage of having a multicellular model that could be used for drug screening, since no cure is available so far for COX deficiencies.
MYTHO is a recently identified FOXO-dependent gene which seems to be involved in autophagy and has an orthologue in the C. elegans genome.
Since RNAi did not allow to detect a clearcut phenotype in nematodes, we therefore generated a KO model by CRISPR/Cas9 technology. KO animals did not show a significant reduction in survival after starvation, but manifested a precocious aging phenotype with locomotion impairment and reduced lifespan compared to controls. We are currently performing further experiments to analyze the autophagic flux in absence of MYTHO and characterize the pathway linking this gene to the IGF/Akt/FOXO signaling.
A novel genetic variant has been identified in a gene that belongs to Crescerin1 family of proteins regulating microtubule dynamics, in patients with a Meckel-Gruber-like phenotype. The worm orthologue che-12 is expressed in the cilium of a subset of sensory neurons.
We generated worm lines harboring the novel missense variant found in patients by CRISPR/Cas9 technology. These were then characterized to explore potential effects on behaviors controlled by sensory neurons expressing che-12. We did not observe an impairment in chemotaxis ability on a NaCl gradient, nor a strong reduction of lipophilic dye-uptake frequency, however preliminary results indicate that the cilium of sensory neurons is shortened in che-12 knock-in mutants. The demonstration of the pathogenic effect of the variant could establish an important link between mutations in this gene (that has not been so far associated with a human disease) and ciliopathies
