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.
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