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Uvod: Amiotrofična lateralna skleroza (ALS) je smrtonosna napredujoča nevrodegenerativna bolezen, za katero so značilni citoplazemski skupki proteinov v prizadetih nevronih. Glavna komponenta teh inkluzij je ubikvitiniran, fosforiliran in cepljen protein TDP-43. Primerov ALS z mutacijami v genu je TDP-43 zelo malo, kar pomeni, da ima TDP-43 v patoloških agregatih pri večini pacientov povsem običajno zaporedje. Vedno več raziskav nakazuje, da imajo citoplazemski skupki TDP-43 lastnosti podobne prionskim proteinom. Zanje je značilno sejanje agregatov in njihovo širjenje med celicami in vitro in in vivo, novejše študije pa so identificirale tudi različne tipe agregatov pri bolnikih z ALS in frontotemporalno demenco (FTD). Alternativne patološke konformacije agregatov bi lahko podobno kot sevi prionskih proteinov, bile osnova za raznolikost proteinopatij TDP-43 in heterogenost bolezni. Raziskovalne skupine so poleg TDP-43 identificirale že več kot 50 genov, ki povzročajo ali vplivajo na nastanek ALS in so vključeni v dve skupini celičnih procesov: metabolizem RNA in razgradnja proteinov. Pri tem pa vloga drugih genov, povezanih z ALS in njihovih mutacij na agregacijo TDP-43, ostaja neznanka. Namen dela in hipotezi: V pričujoči študiji smo primerjali vpliv več mutiranih in nemutiranih genov, povezanih z ALS, na agregacijo TDP-43 in biokemijske lastnosti agregatov in vitro. Hipotezi, na katerih sloni raziskava, se osredotočata na dva vidika agregacije proteina TDP-43 - na vpliv genov povezanih z ALS na agregacijo TDP-43 in na citoplazemsko lokalizacijo agregatov TDP-43: Izražanje z ALS povezanih genov (UBQLN2, MATR3, VCP, hnRNPA1) vpliva na biokemijske lastnosti in/ali celično lokalizacijo proteina TDP-43. Modeliranje okvar jedrno-citoplazemskega transporta v celicah zaostri patološke spremembe, ki so posledica izražanja posamezne mutacije povezane z ALS. Metode dela: Da bi raziskali oba vidika nastanka agregatov TDP-43 smo najprej razvili nov in vitro model agregacije TDP-43 v celični liniji SH-SY5Y. Okvare jedrno-citoplazemskega transporta TDP-43 smo modelirali z eliminacijo signala za jedrno lokalizacijo (NLS) in s tem premaknili TDP-43 iz jedra v citoplazmo. Nato smo postopoma skrajševali LCD proteina in s tem zmotili fiziološko konformacijo dimerov TDP-43. Pri ko-transfekcijskih eksperimentih, s katerimi smo želeli preveriti vpliv prej naštetih mutiranih in nemutiranih z ALS-povezanih genov na agregacijo TDP-43, smo uporabili celoten TDP-43 z odstranjenim NLS (dNLS) in TDP-43 brez NLS in IDR2 (dNLSd343). Nastanek agregatov v kotransfeciranih celicah smo kvantificirali s konfokalno mikroskopijo in nadalje analizirali s programom ImageJ z dodanim vtičnikom Shape Descriptors. Topnost oziroma netopnost agregatov v pufrih RIPA in UREA smo analizirali s prenosom western. Rezultati in razprava: Pokazali smo, da krajšanje domene LCD vodi do različnih vzorcev agregacije TDP-43. S ko-transfekcijo konstruktov dNLS in dNLSd343 z mutiranimi in nemutiranimi z ALS-povezanimi geni smo pokazali, da ima vsak izmed testiranih genov edinstven vpliv na vzorec agregacije TDP-43 in na topnost agregatov. Te učinke smo preučevali v kontekstu okvarjenega nukleocitoplazemskega transporta, ki je zaostril patološko agregacijo v celicah ko-transfeciranih z mutiranimi ali nemutiranimi z ALS-povezanimi geni. Nekateri geni, kot sta na primer mutirani in nemutirani hnRNPA1 so zmanjšali število agregatov in povečali njihovo topnost, drugi pa so povečali število agregatov in/ali njihovo velikost in/ali zmanjšali njihovo topnost. Pokazali smo tudi, da vsak izmed genov edinstveno vpliva tako na dNLS kot na dNLSd343, kar kaže na pomembno vlogo skrajnega C-končnega dela domene LCD pri nastanku in maturaciji agregatov. Izsledki naše študije tako potrjujejo, da vsak izmed z ALS-povezanih genov, ki smo jih raziskovali, edinstveno vpliva na procese agregacije TDP-43, kar vodi do nastanka različnih »tipov agregatov,« ki bi lahko predstavljali temelj za heterogenost bolezni ALS. Zaključki in znanstveno-raziskovalni pomen študije: Študija, predstavljena v tej doktorski disertaciji je prva, ki primerja vpliv z ALS-povezanih genov na agregacijo proteina TDP-43. V sklopu študije smo tudi razvili model agregacije proteina TDP-43, s katerim bi lahko v prihodnosti raziskovali zmožnost potencialnih terapevtskih učinkovin za zmanjšanje ali eliminacijo agregatov TDP-43 in s tem izboljšanje patoloških sprememb v prizadetih celicah bolnikov z ALS. Naši rezultati potrjujejo potrebo po primerjalnih raziskavah na področju ALS in razkrivajo, kako različni mehanizmi interagirajo na molekulskem nivoju in vodijo v nevrodegeneracijo, kar se bo morda izkazalo kot ključnega pomena za uspešen razvoj terapevtikov. Introduction: ALS is a fatal progressive neurodegenerative disease pathologically characterized by cytoplasmic deposits of misfolded proteins in the affected neurons. The main component of these inclusions is ubiquitinated, phosphorylated and cleaved TDP-43 protein. Mutated TDP-43 is very rare, meaning that most of the patients with TDP-43 positive aggregates do not carry any mutation in this protein. Mounting evidence suggests that cytoplasmic accumulations of TDP-43 exhibit prion-like characteristics. In addition to seeding and intercellular propagation of TDP-43 aggregation between cells in vitro and in vivo, different types of the TDP-43 aggregates in ALS and FTD diseased brains have been identified. It has been proposed, that alternate pathological conformations may form the basis for the diversity of TDP-43 proteinopathies and disease heterogeneity, reminiscent of prion strains. So far, more than 50 potentially causative or ALS-modifying genes have been identified, mainly involved in two cellular processes: RNA metabolism and quality control of protein metabolism. However, the role of ALS-associated genes, the possible impact of their mutations on TDP-43 aggregate behavior and properties remains largely unknown. Aim and Hypotheses: In this study, we sought to compare the impact of several wild-type and mutated ALS-associated genes on TDP-43 aggregation in vitro. Our hypotheses focused on two aspects of TDP-43 aggregation. On the impact, ALS-associated genes display on TDP-43 aggregation and on the cytoplasmic localization of TDP-43 aggregates. Expression of genes (UBQLN2, MATR3, VCP and hnRNPA1) linked to ALS affects biochemical properties and/or cellular localization of protein TDP-43. Nucleo-cytoplasmic transport defect modelling exacerbates pathological changes resulting from the expression of a certain ALS mutation. Methods: To explore both aspects of TDP-43 aggregation, we first developed a novel in vitro TDP-43 aggregation model in neuroblastoma SH-SY5Y cell line. To model defects of nucleo-cytoplasmic transport and achieve TDP-43 cytoplasmic localization, we eliminated NLS from the full-length TDP-43 sequence. Then we stepwise shortened its LCD, to disrupt physiological conformation of TDP-43 dimers. Full-length TDP-43 lacking only NLS (dNLS) and TDP-43 without NLS and IDR2 (dNLSd343) constructs were used in co-transfection experiments to assess the impact of pre-selected wild-type and mutant ALS-associated genes on TDP-43 aggregate behavior. Aggregate formation in co-transfected cells was quantified by confocal microscopy and further analyzed by ImageJ software with Shape Descriptors plugin. Solubility and insolubility of the aggregates in UREA and RIPA buffers was analyzed by western blotting. Results and Discussion: We demonstrated that shortening of the LCD domain leads to distinct patterns of TDP-43 aggregation. By co-transfecting dNLS and dNLSd343 constructs with wild-type and mutant ALS-associated genes, we demonstrated that each of the tested genes has a unique influence on TDP-43 aggregation pattern and solubility of the aggregates. These effects were studied in the context of disrupted nucleocytoplasmic transport, which caused exacerbation of pathological aggregation following co-transfection with wild-type or mutant ALS-associated genes. While some of the ALS-associated genes, such as wt and mut hnRNPA1 reduced the number of aggregates and increased their solubility, others increased the number of aggregates and/or their size and/or decreased their solubility. We also show that each of the genes has a unique influence on both dNLS and dNLSd343, suggesting that the extreme C-terminus has a prominent role in initiation and maturation of the aggregates. Considering all this data, we propose that each ALS-linked gene we studied uniquely affects TDP-43 aggregation processes, leading to the formation of distinct “aggregate types,” which could represent the basis for the disease heterogeneity. Conclusions: The study presented in this doctoral dissertation is first to provide an insight into the impact ALS-associated genes have on TDP-43 aggregation. Additionally, as a part of our research, we developed a TDP-43 aggregation model, which could be used in future studies of therapeutic compounds with a potential to reduce or eliminate TDP-43 aggregates and thus reverse pathological changes in the affected cells of ALS patients. Our results also emphasize the significance of comparative research in the ALS field and provide an understanding on how different mechanisms interact at the molecular level and lead to neurodegeneration that may reveal crucial in the future for successful development of therapeutics. |
