Abstract
Accumulation of ubiquitin-positive, tau- and α-synuclein-negative intracellular inclusions of TDP-43 in the central nervous system represents the major hallmark correlated to amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitin-positive inclusions. Such inclusions have variably been described as amorphous aggregates or more structured deposits having an amyloid structure. Following the observations that bacterial inclusion bodies generally consist of amyloid aggregates, we have overexpressed full-length TDP-43 and C-terminal TDP-43 in E. coli, purified the resulting full-length and C-terminal TDP-43 containing inclusion bodies (FL and Ct TDP-43 IBs) and subjected them to biophysical analyses to assess their structure/morphology. We show that both FL and Ct TDP-43 aggregates contained in the bacterial IBs do not bind amyloid dyes such as thioflavin T and Congo red, possess a disordered secondary structure, as inferred using circular dichroism and infrared spectroscopies, and are susceptible to proteinase K digestion, thus possessing none of the hallmarks for amyloid. Moreover, atomic force microscopy revealed an irregular structure for both types of TDP-43 IBs and confirmed the absence of amyloid-like species after proteinase K treatment. Cell biology experiments showed that FL TDP-43 IBs were able to impair the viability of cultured neuroblastoma cells when added to their extracellular medium and, more markedly, when transfected into their cytosol, where they are at least in part ubiquitinated and phosphorylated. 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.
Highlights
TAR DNA-binding protein 43 (TDP-43) is a multifunctional nuclear protein initially described as a transcription factor [1], but later found to be involved in regulation of RNA splicing, microRNA processing, mRNA transport, stability and translation [2,3]
A similar expression level to that of the glutathione S-transferase (GST)/FL TDP-43 protein was observed by SDS-PAGE, with a band corresponding to a molecular weight of, 46 kDa, that is the sum of the molecular weights of GST (, 26 kDa) and Ct TDP-43 (, 20 kDa) (Fig. 1A)
In 2006 it was reported, for the first time, that ubiquitinpositive, tau- and a-synuclein-negative intracellular inclusions found in the spinal cord motor neurons, in the hippocampus and neocortex of sporadic amyotrophic lateral sclerosis (ALS) and FTLD-U patients, contained the previously unidentified TDP-43 protein or its C-terminal fragments [4]
Summary
TAR DNA-binding protein 43 (TDP-43) is a multifunctional nuclear protein initially described as a transcription factor [1], but later found to be involved in regulation of RNA splicing, microRNA processing, mRNA transport, stability and translation [2,3]. Pathological TDP-43 aggregation is associated with a dislocation of this protein from the nucleus, where the protein normally resides and plays its functions, to the cytoplasm, where the inclusions accumulate [4,6,7]. In such cytoplasmic inclusions TDP-43 is hyperphosphorylated, ubiquitinated and cleaved to form C-terminal fragments [4,6,7], in the spinal cord motor neurons the inclusions consist rather of full-length TDP-43 [8]
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