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1.
ROLE OF TDP-43 AGGREGATION IN NEURODEGENERATION: A DROSOPHILA MELANOGASTER DISEASE MODEL AND INNOVATIVE THERAPEUTIC APPROACHES
Lucía 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.
Found in: ključnih besedah
Keywords: TDP-43/TBPH, aggregation, Drosophila melanogaster, ALS
Published: 03.03.2016; Views: 3066; Downloads: 36
.pdf Fulltext (53,65 MB)

2.
Dissecting the role of REEP1 in preventing Tau-mediated neurodegeneration in a D.melanogaster Alzheimer's disease model
Alessio Guglielmi, 2019, doctoral dissertation

Abstract: Tau is natively an unfolded protein that promotes the assembly and the stability of the axonal microtubules in the central nervous system. Increased formation of Tau protein aggregates has been causatively implicated in several neurodegenerative diseases called tauopathies. In the present study, we used the Drosophila melanogaster system to express the longest isoform of human Tau (2N4R) in the nervous system of adult flies, recreating the main features of the human pathology. Herein, this Tau-mediated neurodegeneration model was used as a platform to perform genetic screenings to identify putative modifiers of Tau toxicity. Our strategy exploited the modulation of genes considered as risk factors of Alzheimer’s disease (AD), Frontotemporal Dementias and other neurodegenerative diseases by RNA interference in vivo. This approach allowed us to identify a new gene which participates in the neuronal response against Tau induced neurotoxicity in Drosophila: D-Reep1, homologue of human REEP1 gene (h-Reep1). D-Reep1 knockout flies showed no apparent phenotypes in physiological growing and developmental conditions, however, they showed peculiar sensitivity to stress conditions. In addition, D-Reep1 knockout enhanced the neurodegeneration mediated by Tau expression in Drosophila eyes. On the contrary, the overexpression of UAS-D-Reep1 and UAS-h-Reep1 abolished the typical rough eye phenotype induced by the presence of Tau. The Co-expression of D-Reep1 in Tau backgrounds did not alter the phosphorylation pattern of this protein while, the presence of D-Reep1 seemed to prevent the formation of Tau aggregates in vivo. Thus, the data support the idea that D-Reep1 exerts a protective role on Tau induced toxicity which is independent of its phosphorylation status. In this work, I analysed the mechanisms behind the neuroprotective role of D-Reep1 and, in particular, I found that REEP1 is involved in the regulation of the unfolded protein response (UPR) through the PERK-ATF4 cascade within the ER. By the activation of this pathway, the neurotoxic aggregates of Tau are removed from Drosophila neuronal tissues rescuing the normal characteristics of the affected tissues. Evidences also suggest that the activation of autophagy was behind the removal of Tau aggregates, providing new molecular information about the physiological role of D Reep1 in the nervous system.
Found in: ključnih besedah
Summary of found: ...In the present study, we used the Drosophila melanogaster system to express the longest isoform...
Keywords: AD Alzheimer Disease APP Amyloid precursor protein CNS Central Nervous System DM Drosophila melanogaster HSP Hereditary Spastic Paraplegia LN Lewy’s neurite MT Microtubule MAP Microtubule associated protein MT Microtubule/s MTBD Microtubule binding domain NFT Neurofibrillary tangle NP Neuritic plaques PHF Paired helical filament PS1 Presenilin 1 PS2 Presenilin 2 SPG Spastic Paraplegia ThS Thioflavin S
Published: 06.12.2019; Views: 809; Downloads: 33
.pdf Fulltext (2,59 MB)

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