2.
ROLE OF TDP-43 AGGREGATION IN NEURODEGENERATION: A DROSOPHILA MELANOGASTER DISEASE MODEL AND INNOVATIVE THERAPEUTIC APPROACHESLucía Cragnaz, 2016, doctoral dissertation
Abstract: TDP-43 inclusions are important histopathological features of various neurodegenerative disorders, including Amyotrophic Lateral Sclerosis. However, the relation of these inclusions with the pathogenesis of the disease is still unclear. Various hypotheses have been proposed. For instance, it was suggested that the inclusions are (1) primary toxic species, (2) part of the normal cellular protective response to toxic intermediates and (3) responsible for the nuclear depletion of TDP-43. Understanding the relationship between TDP-43 aggregation and neurodegeneration is crucial for the eventual management of the disease.
TDP-43 is a protein that has a marked tendency to unfold and become insoluble. In particular, its C-terminal end has a so-called “prion-like domain”, a sequence rich in Glutamine (Q) and Asparagine (N) that is involved in both the interactions with other proteins and the self-aggregation process. A cellular model of aggregation has been previously developed by our group, using the TDP-43 Q/N rich amino acid sequence repeated 12 times (12xQ/N) fused to EGFP reporter. The EGFP-12xQ/N cellular inclusions are capable of sequestering wild type TDP-43 both in non-neuronal and neuronal cells.
In this study we went further with ALS modeling, creating a Drosophila model with EGFP-12xQ/N-induced aggregates. We show here that Drosophila melanogaster TDP-43 ortholog (TBPH) overexpression in Drosophila eye using GMR-Gal4 driver, is neurotoxic and causes necrosis and loss of function of the eye. More important, the neurotoxicity of TBPH can be abolished by its incorporation to the insoluble aggregates induced by EGFP-12xQ/N. This data indicates that aggregation is not toxic per se and instead has a protective role, modulating the functional TBPH available in the tissue.
Notwithstanding the fact that aggregation is protective in presence of an excess of TBPH, we wanted to further understand the role of the aggregates in an environment where just the endogenous TBPH is present. For this purpose, we induced EGFP-12xQ/N transgene constitutively in CNS using ELAV-Gal4 driver. The flies were born and went through the larval stage without differing from control flies in any significant feature of their
8
phenotype. However, during aging the locomotion ability and survival rate of EGFP-12xQ/N flies were impaired. Interestingly, the climbing deficit was correlated with a physiological reduction in the endogenous TBPH levels. Thus, the aggregation, when coupled with low TDP-43 levels generates phenotypic consequences in our Drosophila model, probably due to a TDP- 43 loss of function.
In sum, these data suggest that although the aggregates may be a result of neuroprotection in a context where TBPH is in excess, at a certain stage they become responsible for the pathology, likely due to the TBPH loss of function. If we consider that TDP-43/TBPH inclusions act as a sink for the newly formed soluble TDP-43/TBPH, the modulation of these inclusions could be used as a potential therapeutic approach, as this would restore the normal levels of TDP-43/TBPH and its function. Consequently, in the last part of the study we were interested to understand if the clearance of TDP- 43/TBPH aggregates could be an effective strategy to treat ALS, by recovering TDP-43/TBPH function. For this purpose, using the previously established cell-based TDP-43 aggregation models we analyzed aggregate clearance after treatment with several FDA approved drugs. Three of these drugs were found to significantly reduce aggregation through the proteasome pathway. Furthermore, one of the drugs (nortriptyline) was shown to rescue EGFP-12xQ/N dependent locomotor dysfunction in the Drosophila model. Altogether these data indicate that the clearance of TDP-43 aggregates may be a novel therapeutic strategy for ALS treatment.
Keywords: TDP-43/TBPH, aggregation, Drosophila melanogaster, ALS
Published in RUNG: 03.03.2016; Views: 5449; Downloads: 85
Full text (53,65 MB)
