Lanzo, Ambra (2015) Identification of new genes involved in Dopaminergic system using the animal model Caenorhabditis elegans. [Tesi di dottorato]

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Dopamine (DA) is a neurotransmitter that plays a number of very important roles in humans and other animals, such as regulating movement, behavior and cognition. Excess and deficiency of DA have been associated with a number of neurological diseases, including Parkinson's disease and drug addiction. To date the molecular mechanisms and the key elements involved in these disorders have not completely discovered. In C. elegans, studies aimed at understanding the molecular mechanisms regulating the DAergic system have taken advantage either of chemical treatments or of engineered transgenes. Unfortunately these approaches are not feasible to study the loss of function of a gene, while classic genetic mutations can cause pleiotropic effects or lethal phenotypes in mutant animals, hampering the study of the role played by a gene of interest in DAergic system. In this work I used C. elegans as animal model and a powerful variant of RNA-interference approach, to dissect the role played specifically and only in DAergic system by two candidate genes, overcoming the technical limitations previously described for loss of gene function, such as lethality or pleiotropic effects. With this approach I therefore reduced the function of unc-64 gene, the ortholog of the mammalian component of the core synaptic vesicle fusion machinery Syntaxin1A, only in the C. elegans DAergic system. Using a well known behavioral assay in vivo, the SWimming-Induced Paralysis (SWIP), which is known to be regulated by endogenous DA uptake by the DA transporter DAT-1, I confirmed that the presence of unc-64 in DAergic circuit is important to guarantee a correct neurotransmitter reuptake. Moreover I showed that the SWIP effect, produced in the nematode after treatment with a psychostimulant such as Amphetamine (AMPH), strongly depends on the presence of unc-64 specifically in DAergic circuit, together with DAT-1 and DA receptors. This demonstrates that unc-64 is a novel key modulator of DA reuptake mechanism. I then investigated the role of unc-63 gene, the ortholog of the mammalian alpha 6 Cholinergic nicotinic receptor CHRNA6, in the C. elegans DAergic system. Using microscopy analysis I demonstrated a specific expression of unc-63 gene in the nematode DAergic neurons. DAergic related behaviors are not impaired in vivo by loss of unc-63, thus demonstrating that unc-63 does not exert a main role in regulating the DA signaling in this neuronal circuit. Rather, through a well established drug treatment, I showed that unc-63 presence specifically in the DAergic system is necessary to mediate the toxic action produced by the nicotinic agonist DMPP (1,1-dimethyl-4-phenylpiperazinium), during larval development. In conclusion, all these results validated C. elegans as a powerful animal model where to identify new genes playing a role in Daergic neurons and demonstrated all the power of our innovative variant of the RNAi approach, which, by the knock-down a gene of interest only in these neurons, allows to discover new genetic modifiers of Daergic neurotransmission.

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