Final Report Summary - NEURODOPA DEGEN (Elucidating mechanisms of dopaminergic neuronal degeneration using C. elegans and high-throughput genetic approaches) Dopaminergic neurons, although not the most abundant neuronal type in the brain, are involved in very important biological processes, such as coordination of movement, reward-motivated behaviors, working memory and emotive responses. Their loss or malfunction leads to pathological conditions, most prominent of which is Parkinson’s disease. Thus it is important to understand how these neurons specify in a developing organism, how they maintain their proper function in aging individuals and what goes wrong in cases of disease that results in dopaminergic neurodegeneration.Despite extensive research, our knowledge of the underlying molecular mechanisms that regulate dopaminergic specification or neuronal cell death is incomplete. This lack of understanding hinders the development therapeutic, preventive or replacement strategies in cases of conditions like Parkinson’s disease. The overall objective of this study has been to better understand the molecular mechanisms of dopaminergic neurodegeneration using a Caenorhabditis elegans model in combination with technologically state of the art genetic methodology. The outcome of the project consists of two major breakthroughs that inform on the development as well as the survival of dopaminergic neurons. In addition, further insights were gained into the biology of this neuronal type at the molecular level, which will set the foundation for follow up investigations.The first important outcome of the project has been the discovery of the mechanisms responsible for dopaminergic fate specification in C. elegans. In particular, we uncovered the regulatory mechanisms that coordinate the expression of the “dopamine pathway genes” (which are the genes encoding for five enzymes and transporters involved in the synthesis, trafficking and reuptake of dopamine), which define the identity of all eight dopaminergic neurons in the nervous system of C. elegans. We showed that a cis-regulatory signature, consisting of a previously described Ets, a homeodomain and a Pbx binding sites, is necessary for the coordinated expression of the dopamine pathway genes. Our genetic screens revealed the corresponding transcription factors, in particular the homeodomain transcription factor ceh-43/dlx and the pbx transcription factors ceh-20 and ceh-40 that recognize the corresponding binding sites. These transcription factors act in a cooperative manner together with a previously described Ets transcription factor, to specify dopaminergic fate in C. elegans. Dopaminergic neurons in the mouse olfactory bulb appear to express a similar set of dlx/pbx/ets transcription factors suggesting phylogenetic conservation of dopaminergic regulatory programs. Thus the findings in C. elegans can be relevant for mammalian systems and possibly inform studies aiming at in vitro generation of dopaminergic neurons. The second important finding of the IRG funded project is the isolation and molecular identification of a mutant in which dopaminergic neurons specify normally during development but later on they robustly and progressively degenerate. We showed that a single amino acid substitution in a C. elegans Transient Receptor Potential (TRP) channel, namely trp-4, is a gain-of-function, dominant mutation responsible for the degeneration phenotype. We provided insights on the mode of cell death and the downstream mechanisms of action, implicating involvement of intracellular calcium homeostasis but also exit of calcium from the endoplasmic reticulum in the process of dopaminergic cell death. Our findings have also revealed that not all dopaminergic neurons are equally sensitive to degeneration, which is reminiscent of the selective vulnerability of dopaminergic neurons in the substantia nigra in patients with Parkinson’s disease. In modifier screens, we have isolated a number of ‘suppressor’ mutations, or mutations that partially or fully protect dopaminergic neurons from trp-4-mediated degeneration. Further studies on these mutants will inform on the biology of TRP channels, neuron-specific calcium handling mechanisms and dopaminergic neuroprotection in general.
