Repository of University of Nova Gorica

Show document
A+ | A- | SLO | ENG

Title:ROLE OF TDP-43 AGGREGATION IN NEURODEGENERATION: A DROSOPHILA MELANOGASTER DISEASE MODEL AND INNOVATIVE THERAPEUTIC APPROACHES
Authors:Cragnaz, Lucía (Author)
Baralle, Marco (Mentor) More about this mentor... New window
Files:.pdf Lucia_Cragnaz.pdf (53,65 MB)
 
Language:English
Work type:Doctoral dissertation (mb31)
Tipology:2.08 - Doctoral Dissertation
Organization:FPŠ - Graduate School
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
Year of publishing:2016
Source:Nova Gorica
COBISS_ID:4156667 Link is opened in a new window
URN:URN:SI:UNG:REP:TMBK4YKZ
Views:3067
Downloads:36
Metadata:XML RDF-CHPDL DC-XML DC-RDF
Categories:Document is not linked to any category.
:
  
Average score:(0 votes)
Your score:Voting is allowed only for logged in users.

Hover the mouse pointer over a document title to show the abstract or click on the title to get all document metadata.

Secondary language

Language:Slovenian
Title:VLOGA KOPIČENJA TDP-43 PRI NEVRODEGENERACIJI: DROSOPHILA MELANOGASTER KOT MODEL BOLEZNI IN INOVATIVNI TERAPEVTSKI PRISTOPI
Abstract:Vključki proteinov TDP-43 so pomembne histopatološke značilnosti različnih nevrodegenerativnih bolezni, vključno z amiotrofično lateralno sklerozo (ALS), kljub temu pa je njihova povezava s temi boleznimi še vedno nejasna. Postavljene so bile mnoge hipoteze. Med drugim je bilo predlagano, da so tovrstni vključki (1) primarne toksične strukture, (2) del običajnega celičnega obrambnega odziva proti toksičnim intermediatom in (3) so odgovorni za odsotnost TDP-43 v jedrih celic. Razumevanje odnosa med kopičenjem TDP-43 v citoplazmi in nevrodegeneracijo je ključnega pomena za morebitno vodenje bolezni. TDP-43 je protein z izrazito težnjo po porušenju strukture in pojavu netopnosti. Njegov C-terminalni konec ima tako imenovano “prionu podobno domeno”, ki je zaporedje bogato z glutaminom (Q) in asparaginom (N) in je vpleteno v interakcije z drugimi proteini in procesom kopičenja. Naša skupina je predhodno razvila model celične agregacije z uporabo TDP-43 Q/N bogatega aminokislinskega zaporedja, ki se ponovi 12-krat (12xQ/N) in je priključeno na EGFP reporter. EGFP-12xQ/N celični vključki so sposobni sekvestriranja TDP-43 v ne-nevronskih in nevronskih celicah. Za proučevanje ALS smo v tej študiji ustvarili model vinske mušice z kopičenjem TDP-43, ki v temelji na EGFP-12xQ/N vključkih. Pokazali smo, da je povečano izražanje TBPH v očeh vinske mušice z GMR-Gal4 nevrotoksično ter povzroči nekrozo in izgubo funkcije očesa. Še pomembneje, nevrotoksičnost TBPH je lahko odpravljena z vključitvijo le- tega v netopne agregate. Ti podatki kažejo, da kopičenje ni nevarno samo po sebi in ima namesto tega zaščitno vlogo, in sicer s spreminjanjem prisotnosti delujočega TBPH v tkivu. Ne glede na dejstvo, da ima kopičenje zaščitno vlogo v prisotnosti prebitka TBPH, smo želeli bolje razumeti vlogo vključkov v okolju, kjer je prisoten samo endogeni TBPH. Za ta namen smo konstitutivno izrazili EGFP-12xQ/N v CŽS z uporabo ELAV-Gal4. Vinske mušice so se rodile brez fenotipskih sprememb. Kasneje so se kljub temu pojavile nepravilnosti v gibalnosti ter krajše preživetje EGFP-12xQ/N mušic. Zanimivo je, da so bile pomanjkljivosti v plezanju v korelaciji s fiziološkim znižanjem nivoja endogenega TBPH. 10 Kopičenje z združenimi znižanimi vrednostmi TDP-43 tako povzroči spremembe v fenotipu vinskih mušic, po vsej verjetnosti zaradi izgube funkcije TDP-43. Ti podatki kažejo, da so vključki kljub možnim nevroprotektivnim lastnostim kadar je TBPH v prebitku, še vedno vzrok patoloških stanj, kot je npr. izguba funkcije TBPH. Če upoštevamo, da TDP-43/TBPH vključki delujejo kot vaba za topne TDP-43/TBPH, se bi spreminjanje teh vključkov lahko uporabilo kot potencialni terapevtski pristop, saj bi s tem povrnili normalno raven TDP-43/TBPH in njegovo funkcijo. Zato smo v zadnjem delu študije raziskovali ali je uničenje TDP-43/TBPH agregatov lahko učinkovita strategija za zdravljenje ALS zaradi povrnitve funkcije TDP-43/TBPH. Za ta namen smo uporabili predhodno pripravljene celične modele kopičenja TDP- 43, ki smo jih tretirali z FDA-potrjenimi zdravili. Tri od teh zdravil so bistveno zmanjšale kopičenje. Poleg tega je eno od zdravil (nortriptilin) povzročilo povrnitev gibalnih sposobnosti na modelu vinske mušice. V študiji smo torej pokazali, da je uničenje TDP-43 agregatov lahko nova terapevtska strategija za zdravljenje ALS.
Keywords:TDP-43/TBPH, vključki, Drosophila melanogaster, ALS


Back