Dissecting the role of cytoskeleton remodelling in a Drosophila model of TTR-induced neurodegeneration

FCUP/ICBAS DISSECTING THE ROLE OF CYTOSKELETON REMODELLING IN A DROSOPHILA MODEL OF TTR-INDUCED NEURODEGENERATION xi Familial Amyloid Polyneuropathy (FAP) is a neurodegenerative disease characterized by the deposition of mutated transthyretin (TTR) in the form amyloid fibrils, particularly in the peripheral nervous system. The most common pathogenic substitution is Val30Met and as a consequence of TTR deposition there is axonal degeneration that results in neuronal death with disease progression. Although therapeutic strategies have been implemented, none targets neurodegeneration, which remains irreversible, suggesting that further characterization of cellular pathways and identification of new drug targets involved in FAP are required. Preliminary in vitro data from our laboratory suggested that cytoskeleton defects are involved in FAP pathogenesis. Aiming at determining whether cytoskeleton remodelling is a target for TTR-induced neurodegeneration in vivo we used a previously reported Drosophila model for FAP. The fly model for FAP is based on the expression of human TTRV30M in differentiated cells of the fly developing eye (photoreceptor cells and accessory cells). Expression of TTR transgenes led to roughening of the eye and degeneration, when compared with wild-type flies. We also detected reduced lifespan and impairment of climbing ability in TTR expressing flies. The effect of TTR in the parameters described above is gene dosage and temperature dependent, and appears consistently more severe in TTRV30M expressing flies. In order to determine whether a defect in axonal cytoskeleton precedes neurodegeneration, we evaluated the cytoskeleton organization of photoreceptor cells using different markers, as Chaoptin, a photoreceptor cell-specific membrane glycoprotein, Futsch, a microtubule associated protein and Lifeact, a construct that labels F-actin. The evaluation was performed in larval stage and we found defects in target and organization of the photoreceptor projecting axons. Moreover, we detected an abnormal distribution of F-actin in the growth cones of photoreceptor cells. The defects found were always more severe in larvae expressing TTRV30M when compared to WT TTR expression. The evaluation of glial cells proposed that they are not affected upon expression of TTR transgenes. In addition, we performed a genetic screen to identify enhancers and suppressors of TTRV30M-induced rough eye phenotype. Flies expressing TTRV30M were crossed with RNAi fly lines for candidate genes whose functions are associated with cytoskeleton dynamics. We found mainly suppressors of the phenotype promoted by TTRV30M expression and one enhancer. The results of the screen suggest a major involvement of the cytoskeleton regulators in the TTRV30M-induced phenotype, with a particular role of Rho GTPases proteins in the modulation of the phenotype. FCUP/ICBAS DISSECTING THE ROLE OF CYTOSKELETON REMODELLING IN A DROSOPHILA MODEL OF TTR-INDUCED NEURODEGENERATION xii In summary, this work highlighted that cytoskeleton defects underlie TTR-induced neurodegeneration and pinpointed novel TTR interactors.