The effect of TDP-43 antibodies on TDP-43 aggregation and toxicity

Abstract

Over the last years antibody-based therapies have become increasingly important as novel therapeutic options in many different neurodegenerative diseases like Alzheimer’s and Parkinson’s. With protein aggregation being a key pathophysiological feature of amyotrophic lateral sclerosis (ALS), we hypothesized that antibodies targeted against TDP-43 may also be beneficial in inhibiting protein aggregation, propagation and ultimately associated cellular toxicity. Therefore, we chose commercially available antibodies against different parts of TDP-43 that had been reported to play a role in TDP-43 aggregation and solubility (a broadly binding c-terminal antibody, a broadly binding n-terminal antibody and an antibody against the RRM of TDP-43) as well as an antibody specifically binding to phosphorylated TDP-43 (phosphorylated Serin at 409 and 410). This modification is only found in TDP-43 clustered in pathological aggregates but not in the physiological form of the protein. As a readout, we used a luciferase-based protein complementation assay for TDP-43 that had been previously established. Both broadly binding antibodies, the c-terminal one as well as the n-terminal antibody, did show a significant reduction of TDP-43 aggregation (~9%). When additionally adding oxidative stress to the cells, this positive effect was lost for the n-terminal antibody, while the c-terminal antibody did show an even greater reduction in aggregation of TDP-43 (~17%). To further investigate this aspect, we performed a Urea/RIPA solubility assay with the same antibodies und conditions of oxidative stress. Interestingly, not only the c-terminal, n-terminal but the pTDP-s409/410 antibody as well were all able to greatly reduce insoluble TDP-43 by up to 66% with the greatest effects detected for the pTDP-s409/410 antibody. An antibody that had only shown a trend towards reducing aggregation before (reduction of ~6%; p=0,08). However, while the RRM2 domain had previously been linked to aggregation, the tested RRM2 antibody was not able to reduce either aggregation nor did it promote changes in TDP-43 solubility and was hence ruled out for further testing. Further, we wanted to test if our antibodies were also able to reduce cellular toxicity associated with TDP-43 overexpression and the addition of cellular stress. However, only the pathology specific pTDP-s409/410 antibody, was able to reduce toxicity, and only without the addition of oxidative stress to the cells. Lastly, we tested the ability of our antibodies to inhibit cellular propagation of TDP-43, a mechanism that is hypothesized to play a major role in the spread of TDP-43 associated diseases. The c-terminal antibody was able to reduce the uptake of TDP-43 from the media into cells but did not reduce toxicity in this setting compared to our control. All other antibodies, including our antibody control, were even slightly increasing TDP-43 uptake compared to the untreated control. With further research still being necessary to understand the treatment possibilities of antibodies in TDP-43-associated disease fully, we could show that a treatment of TDP-43 targeting antibodies can have an impact on TDP-43 aggregation, solubility propagation and even toxicity in cell culture. Therefore, the results gained in this study hint towards a possible benefit of antibody-based therapeutics in ALS and other TDP-43 proteinopathies, similar to Parkinson’s and Alzheimer’s disease, as has been suggested during the last couple of years.