Analysis of the substrate recognition state of TDP-43 to single-stranded DNA using fluorescence correlation spectroscopy.

Normal function and abnormal aggregation of transactivation response (TAR) DNA/RNA-binding protein 43 kDa (TDP-43) are directly associated with the lethal genetic diseases: cystic fibrosis, amyotrophic lateral sclerosis (ALS), and frontotemporal lobar degeneration (FTLD). The binding of TDP-43 to single-stranded DNA (ssDNA) or RNA is involved in transcriptional repression, regulation of RNA splicing, and RNA stabilization. Equilibrium dissociation constants (Kd) of TDP-43 and ssDNA or RNA have been determined using various methods; however, methods that can measure Kd with high sensitivity in a short time using a small amount of TDP-43 in solution would be advantageous. Here, in order to determine the Kd of TDP-43 and fluorescence-labeled ssDNA as well as the binding stoichiometry, we use fluorescence correlation spectroscopy (FCS), which detects the slowed diffusion of molecular interactions in solution with single-molecule sensitivity, in addition to electrophoretic mobility shift assay (EMSA). Using tandem affinity chromatography of TDP-43 dually tagged with glutathione-S-transferase and poly-histidine tags, highly purified protein was obtained. FCS successfully detected specific interaction between purified TDP-43 and TG ssDNA repeats, with a Kd in the nanomolar range. The Kd of the TDP-43 mutant was not different from the wild type, although mutant oligomers, which did not bind ssDNA, were observed. Analysis of the fluorescence brightness per dimerized TDP-43/ssDNA complex was used to evaluate their binding stoichiometry. The results suggest that an assay combining FCS and EMSA can precisely analyze ssDNA recognition mechanisms, and that FCS may be applied for the rapid and quantitative determination of the interaction strength between TDP-43 and ssDNA or RNA. These methods will aid in the elucidation of the substrate recognition mechanism of ALS- and FTLD-associated variants of TDP-43.

Interaction of RNA with a C-terminal fragment of the amyotrophic lateral sclerosis-associated TDP43 reduces cytotoxicity.

A hallmark of amyotrophic lateral sclerosis (ALS), a devastating neurodegenerative disease, is formation of inclusion bodies (IBs) from misfolded proteins in neuronal cells. TAR RNA/DNA-binding protein 43 kDa (TDP43) is an ALS-causative protein forming IBs in ALS patients. The relation between localization of the IBs and neurotoxicity remains largely unknown. We characterized aggregation of fluorescently tagged TDP43 and its carboxyl-terminal fragments (CTFs) by analytical fluorescence imaging techniques. Quantitative time-lapse analysis in individual live cells showed that fluorescent-protein-tagged TDP43 was cleaved and a 35 kDa TDP43 CTF (TDP35) formed ubiquitin (Ub)-negative cytoplasmic IBs. Although TDP35 formed mildly toxic Ub-negative IBs in the cytoplasm, TDP25, another type of a TDP43 CTF, efficiently formed sufficiently toxic Ub-positive IBs. One- or two-color fluorescence correlation spectroscopy (FCS/FCCS) revealed that coaggregation of TDP25 with TDP43 was initiated by depletion of the RNA that binds to TDP25. Moreover, nuclear localization tagging TDP25 reduced the rate of neuronal cell death. These observations point to the need to elucidate the novel sequestration mechanism and details of the toxicity of the misfolded and aggregation-prone TDP43 CTFs (as well as the RNA binding and nuclear retention) in order to identify possible preventive interventions against ALS.