In Vitro Interaction of a C-Terminal Fragment of TDP-43 Protein with Human Serum Albumin Modulates Its Aggregation.

An increased level of naturally occurring anti-TDP-43 antibodies was observed in the serum and cerebrospinal fluid (CSF) of amyotrophic lateral sclerosis patients. Human serum albumin (HSA), the most abundant protein in blood plasma and CSF, is found to interact with pathological proteins like Aß and α-synuclein. Therefore, we examined the effect on the in vitro aggregation of a C-terminal fragment of TDP-43 in the presence of HSA. We found that the lag phase in TDP-432C aggregation is abrogated in the presence of HSA, but there is an overall decreased aggregation as examined by thioflavin-T fluorescence spectroscopy and microscopy. An early onset of TDP-432C oligomer formation in the presence of HSA was observed using atomic force microscopy and transmission electron microscopy. Also, a known chemical inhibitor of TDP-432Caggregation, AIM4, abolishes the HSA-induced early formation of TDP-432C oligomers. Notably, the aggregates of TDP-432C formed in the presence of HSA are more stable against sarkosyl detergent. Using affinity copurification, we observed that HSA can bind to TDP-432C, and biolayer interferometry further supported their physical interaction and suggested the binding affinity to be in sub-micromolar range. Taken together, the data support that HSA can interact with TDP-432C in vitro and affect its aggregation.

Elevated constitutive expression of Hsp40 chaperone Sis1 reduces TDP-43 aggregation-induced oxidative stress in Ire1 pathway dependent-manner in yeast TDP-43 proteinopathy model of amyotrophic lateral sclerosis.

Oxidative stress is a therapeutic target in TDP-43 proteinopathies like amyotrophic lateral sclerosis (ALS) and FTLD-TDP. TDP-43 over-expression causes oxidative stress in yeast model of ALS. Previously, we developed a red/white color conversion reporter assay using ade1 or ade2 mutant yeast to examine oxidative stress induced by expression of amyloidogenic proteins. Also, a previous study showed that overexpression of yeast Hsp40 chaperone Sis1 could mitigate the toxicity and proteosomal blockage induced by TDP-43 over-expression. Here, using the red/white reporter yeast assay and also by CellROX-staining, we found that an elevated expression of Sis1 mitigates the TDP-43-induced oxidative stress. Furthermore, as redox signalling and the ER stress response pathways cross-talk, we checked if the Sis1-mediated mitigation of the TDP-43-induced oxidative stress can also be observed in yeast deleted for ER stress response gene, IRE1. We find that in the yeast deleted for the IRE1 gene, the elevated expression of Sis1 fails to neutralize the TDP-43-induced oxidative stress. Taken together, Hsp40 chaperone modulation can be further examined towards therapeutic research on the TDP-43 proteinopathies.