Aggregation studies on the transactive response DNA binding protein of 43 KDA (TDP-43)

The pathological deposition of the transactive response DNA-binding protein of 43 kDa (TDP-43) occurs in the majority (97%) of amyotrophic lateral sclerosis (ALS) and in around 45% of frontotemporal lobar degeneration cases (FTLD). ALS and FTLD clinically overlap, presenting a continuum of phenotypes. Both ALS and FTLD lack treatments able to interfere with the underlying pathological process and early detection of TDP-43 pathology would facilitate the development of disease modifying drugs. The Real Time Quaking Induced Conversion reaction (RT-QuIC) showed the ability to detect prions in several peripheral tissues of patients with different forms of prion and prion-like diseases. Despite TDP-43 displays prion-like properties, to date the RT-QuIC technology has not yet been adapted to this protein. The main aim of this study was to adapt the RT-QuIC technique for the TDP-43 substrate and to exploit the intrinsic ability of this technology to amplify minutes amount of misfolded proteins for the detection of pathological TDP-43 species in the CSF of ALS and FTLD patients. As a second objective, we aimed at performing a preliminary evaluation of the seeding properties of the in vitro obtained TDP-43 synthetic aggregates, in order to evaluate their ability to recapitulate ALS/FTD TDP-43 pathology. First, we adapted the RT-QuIC technique for the aggregation of the TDP-43 substrate and then we optimized it with synthetic TDP-43 preformed aggregates and with autopsy-verified brain homogenate samples. After this optimization, we analyzed CSF samples from ALS and FTLD patients and controls. TDP-43 RT-QuIC was able to detect as little as 15 picograms of TDP-43 aggregates, discriminating between a cohort of subjects affected by ALS and FTLD and age-matched controls, with a total sensitivity of 94% and a specificity of 85%. Our preliminary analysis of HuTDP-43(263-414) fibrils showed that they were readily internalized and actively phosphorylated in SH-SY5Y cells. Furthermore, they were able to recruit the full-length endogenous protein in the formation of intra-cellular aggregates composed of hyperphosphorylated forms of the protein. We observed that the formation of TDP-43 intra-cellular aggregates resulted in a severe reduction of cell vitality which was not linked to a TDP-43 loss of function, but potentially related to a maintained RNA-binding capacity of TDP-43 aggregates. In conclusion, our data represent a proof-of-concept of TDP-43 RT-QuIC potential for the detection of TDP-43 pathological aggregates. Together with prion, amyloid beta, tau and α-syn RT-QuIC assays, a further optimization of the presented TDP-43 RT-QuIC protocol, would increase the opportunity to perform the earliest and most accurate diagnosis at a single patient level. Furthermore, we show that CSF detection of TDP-43 pathological aggregates may be exploited as a disease biomarker for ALS and FTLD. Our preliminary results regarding the seeding properties of TDP-43 synthetic aggregates suggest that TDP-43 RT-QuIC could be exploited not only as a powerful drug screening and diagnostic tool but could also serve as a very helpful instrument to further elucidate TDP-43 prion-like features.