Estudo do estresse com metilglioxal nas interações TDP-43 - SOD1 em célula H4 como modelo para a esclerose lateral amiotrófica

Abstract

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the loss of motor neurons. Proteopathy is a pathological hallmark of the disease. In approximately 97% of ALS cases, aggregates of TDP-43 are observed in the cytoplasm of motor neurons undergoing neurodegeneration. Additionally, misfolded SOD1 is found in the neurons of patients with both sporadic and familial cases of the disease. TDP-43 is a protein associated with the regulation of transcription, translation, and RNA processing, and is predominantly located in the nucleus, while SOD1 is an antioxidant enzyme present in the cytoplasm, nucleus, and intermembrane space of mitochondria. Previous studies from the group have shown that glycated SOD1 leads to increased phosphorylation of TDP-43, considered a disease biomarker. Furthermore, the literature suggests a potential relationship between SOD1 and calcineurin, a phosphatase responsible for the dephosphorylation of TDP-43. In this context, the objective of the study was to evaluate whether TDP-43 could interact with SOD1 and the effect of methylglyoxal (MGO) and the SOD1G93A mutation, found in ALS patients, on this interaction. Neuroglial (H4) cells were used as a model. The MGO treatment resulted in decreased cell viability, reduced SOD1 activity, and lowered phospho-TDP-43 levels. Cells expressing SOD1G93A showed lower levels of SOD1 activity and phospho-TDP-43 compared to those expressing SOD1WT under control conditions without MGO. The TDP-43–SOD1 interaction was observed using the BiFC (bimolecular fluorescence complementation) system, evidenced by the reconstitution of the Venus protein and fluorescence emission during the physical interaction of the proteins. According to the results, both SOD1WT and the G93A mutant interact with TDP-43. This interaction occurs in various cellular compartments, with a higher proportion of cells exhibiting TDP-43 interacting with SOD1G93A in the cytoplasm. MGO did not alter the cellular compartments of the TDP-43–SOD1WT interaction, only leading to an increase in cytoplasmic inclusions at the 0.4 mM MGO concentration. The observed inclusions did not colocalize with stress granules. Treatment with Cyclosporin A, an inhibitor of the phosphatase calcineurin, reduced the proportion of cells with TDP-43–SOD1WT inclusions and those with TDP-43–SOD1G93A interaction in the cytoplasm. In conclusion, TDP-43 phosphorylation plays an important homeostatic role, and its imbalance leads to altered compartmentalization of TDP-43 and inclusion formation. SOD1 is crucial in maintaining the balance between phosphorylation and dephosphorylation due to its influence on calcineurin activity. This effect is not observed in the G93A mutant, which makes these cells more sensitive to changes in TDP-43 localization, an important marker of ALS.