TDP-43 inclusion bodies formed in bacteria are structurally amorphous, non-amyloid and inherently toxic to neuroblastoma cells

Accumulation of ubiquitin-positive, tau- and a-synuclein-negative intracellular inclusions of TAR DNA binding protein (TDP-43) in the central nervous system represents the major hallmark correlated to amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U). Such inclusions have variably been described as amorphous aggregates or more structured deposits having an amyloid structure. Therefore, it is not yet clear the structure adopted by TDP-43 in such deposits. Following the increasing observations that bacterial inclusion bodies generally consist of amyloid aggregates, We have overexpressed TDP-43 in E. coli cells, purified the resulting TDP-43 containing inclusion bodies (TDP-43 IBs) and subjected them to a number of biophysical analyses to assess their structure and morphology, along with control IBs. We show that the TDP-43 aggregates contained in the bacterial IBs do not bind thioflavin T and Congo red, possess a largely disordered secondary structure, as determined with circular dichroism and infrared spectroscopy, and are highly susceptible to proteinase K digestion, thus possessing none of the distinctive hallmarks for amyloid. In addition, atomic force microscopy imaging revealed that TDP-43 IBs have an irregular structure and a rough surface. However, the TDP-43 IBs were able to severely impair the viability of cultured neuroblastoma cells, when added to their extracellular medium and, even more markedly, when transfected into their cytosol. These data reveal an inherently high propensity of TDP-43 to form amorphous aggregates, which possess, However, an inherently high ability to cause cell dysfunction. This indicates that a gain of toxic function caused by TDP-43 deposits is effective in TDP-43 pathologies, in addition to possible loss of function mechanisms originating from the cellular mistrafficking of the protein.