Transthyretin Inhibits Primary and Secondary Nucleations of Amyloid-β Peptide Aggregation and Reduces the Toxicity of Its Oligomers

Alzheimer's disease is associated with the deposition of the amyloid-β peptide (Aβ) into extracellular senile plaques in the brain. In vitro and in vivo observations have indicated that transthyretin (TTR) acts as an Aβ scavenger in the brain, but the mechanism has not been fully resolved. We have monitored the aggregation process of Aβ40 by thioflavin T fluorescence, in the presence or absence of different concentrations of preformed seed aggregates of Aβ40, of wild-Type tetrameric TTR (WT-TTR), and of a variant engineered to be stable as a monomer (M-TTR). Both WT-TTR and M-TTR were found to inhibit specific steps of the process of Aβ40 fibril formation, which are primary and secondary nucleations, without affecting the elongation of the resulting fibrils. Moreover, the analysis shows that both WT-TTR and M-TTR bind to Aβ40 oligomers formed in the aggregation reaction and inhibit their conversion into the shortest fibrils able to elongate. Using biophysical methods, TTR was found to change some aspects of its overall structure following such interactions with Aβ40 oligomers, as well as with oligomers of Aβ42, while maintaining its overall topology. Hence, it is likely that the predominant mechanism by which TTR exerts its protective role lies in the binding of TTR to the Aβ oligomers and in inhibiting primary and secondary nucleation processes, which limits both the toxicity of Aβ oligomers and the ability of the fibrils to proliferate

A Complex Equilibrium among Partially Unfolded Conformations in Monomeric Transthyretin

Aggregation of transthyretin (TTR) is known to be linked to the development of systemic and localized amyloidoses. It also appears that TTR exerts a protective role against aggregation of the Aβ peptide, a process linked to Alzheimer’s disease. <i>In vitro</i>, both processes correlate with the ability of TTR to populate a monomeric state, yet a complete description of the possible conformational states populated by monomeric TTR <i>in vitro</i> at physiological pH is missing. Using an array of biophysical methods and kinetic tests, we show that once monomers of transthyretin are released from the tetramer, equilibrium is established between a set of conformational states possessing different degrees of disorder. A molten globular state appears in equilibrium with the fully folded monomer, whereas an off-pathway species accumulates transiently during refolding of TTR. These two conformational ensembles are distinct in terms of structure, kinetics, and their pathways of formation. Further subpopulations of the protein fold differently because of the occurrence of proline isomerism. The identification of conformational states unrevealed in previous studies opens the way for further characterization of the amyloidogenicity of TTR and its protective role in Alzheimer’s disease

An in situ and in vitro investigation of cytoplasmic TDP-43 inclusions reveals the absence of a clear amyloid signature

Introduction: Several neurodegenerative conditions are associated with a common histopathology within neurons of the central nervous system, consisting of the deposition of cytoplasmic inclusions of TAR DNA-binding protein 43 (TDP-43). Such inclusions have variably been described as morphologically and molecularly ordered aggregates having amyloid properties, as filaments without the cross-β-structure and dye binding specific for amyloid, or as amorphous aggregates with no defined structure and fibrillar morphology.Aims and Methods: Here we have expressed human full-length TDP-43 in neuroblastoma x spinal cord 34 (NSC-34) cells to investigate the morphological, structural, and tinctorial properties of TDP-43 inclusions in situ. We have used last-generation amyloid diagnostic probes able to cross the cell membrane and detect amyloid in the cytoplasm and have adopted Raman and Fourier transform infrared microspectroscopies to study in situ the secondary structure of the TDP-43 protein in the inclusions. We have then used transmission electron microscopy to study the morphology of the TDP-43 inclusions.Results: The results show the absence of amyloid dye binding, the lack of an enrichment of cross-β structure in the inclusions, and of a fibrillar texture in the round inclusions. The aggregates formed in&nbsp;vitro from the purified protein under conditions in which it is initially native also lack all these characteristics, ruling out a clear amyloid-like signature.Conclusions: These findings indicate a low propensity of TDP-43 to form amyloid fibrils and even non-amyloid filaments, under conditions in which the protein is initially native and undergoes its typical nucleus-to-cell mislocalization. It cannot be excluded that filaments emerge on the long time scale from such inclusions, but the high propensity of the protein to form initially other types of inclusions appear to be an essential characteristic of TDP-43 proteinopathies.KEY MESSAGESCytoplasmic inclusions of TDP-43 formed in NSC-34 cells do not stain with amyloid-diagnostic dyes, are not enriched with cross-β structure, and do not show a fibrillar morphology.TDP-43 assemblies formed in&nbsp;vitro from pure TDP-43 do not have any hallmarks of amyloid