Amyotrophic lateral sclerosis (ALS) is a major neurodegenerative disease caused by the death of motor neurons leading to paralysis. Mechanisms underlying the pathogenesis of the disease remain unknown but with the identification of causative genes from ALS patients, some processes have been linked to the disease. One of these genes is VAPB, a highly conserved protein involved in lipid transfer, vesicle metabolism and synaptic morphology. We modeled in Drosophila the disease-linked P56S mutation (DVAP-P58S) and observed with the expression of this allele neurodegeneration in the eye and loss of motor performance. These phenotypes provide an excellent opportunity to use fly’s genetics to find novel genetic interactors of DVAP and understand ALS pathomechanism. Therefore, we carried out a large scale genetic screen by crossing the ALS model with a collection of P-element overexpression lines. After the analysis of 1183 lines, we obtained 71 modifier lines that suppress DVAP-induced neurodegeneration and 14 lines that enhance this phenotype, decreasing furthermore the eye size and viability of the offspring. To confirm that the effect of modifier lines was caused by a specific gene, we validated them with independent alleles of those genes. Using different sources, we were able to confirm the effect of 63 of the 85 modifiers, providing a strong confirmation of their effect. When we studied the effect of the modifier genes co-expressed with DVAP-P58S in the nervous system, we detected that 46 lines presented the same modifying effect in adult viability and 58 in the motor performance of the adult offspring. Considering the stronger readouts, we obtained 42 genes as novel high confidence DVAP genetic interactors. To understand furthermore the way they are affecting DVAP neurodegeneration, we carried out a series of bioinformatic analyses using Drosophila and human databases. Lipid droplets, vesicle metabolism and cell proliferation appear as the most important categories found in the screen, all processes conserved when analysed with human orthologs of the modifiers. Further characterisation of the endocytosis-linked modifier Rab5 and the predicted DVAP-interactors Rab7 and Rab11, showed that the suppression effect is not only confirmed in vivo but is also conserved in human tissue from ALS patients. These data validate our genetic screen and at the same time open novel opportunities to understand ALS mechanisms and find possible therapeutic targets.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:712274 |
| Date | January 2015 |
| Creators | Sanhueza Cubillos, Mario Andrés |
| Contributors | Pennetta, Giuseppa ; Finnegan, David |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/21080 |
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