P4‐076: Sampling Tube, a Critical Factor in Alzheimer’s Disease Biomarker Standardization

Background: In 2006, frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) are linked by the discovery of the cytoplasmic inclusions of TAR DNA-binding protein-43, TDP-43. TDP-43 is a highly conserved protein comprised of 414 residues with two significant RNA recognition motifs (RRM) and a glycine-rich C-terminal region. It is normally located in the nucleus and involved in gene transcriptions such as suppression of HIV transcription through binding to TAR DNA. The pathogenic mechanism of TDP-43 proteinopathy is largely unknown. The current understanding of TDP-43 proteinopathy involves change of subcellular location from the nucleus to cytoplasm and the formation of TDP-43 inclusions. TDP-43 inclusions isolated from brain tissue of these patients are characterized by ubiquitination, with hyperphosphorylation and N-terminal truncation. Methods: To elucidate the mechanisms of TDP-43 proteinopathies, we generated recombinant full length TDP-43 from E. coil and subjected to analytical size-exclusion chromatography, dynamic light scattering analysis, dot blotting, circular dichroism, DNA binding, and cytotoxicity to characterize the recombinant TDP-43. Results: TDP-43 readily formed large molecular weight aggregates after purification which was eluted in the void volume in the analytical size exclusion chromatography. Dynamic light scattering analysis showed the particle dominantly populated at w30 nm in diameter. Interestingly, the purified TDP-43 from HEK293 cell culture also existed as large oligomers which were able to react with specific anti-amyloid oligomer antibody, indicative of common epitopes between TDP-43 oligomer and amyloid oligomers. These species were resistant to general chemical denaturations and highly helical examined by far-UV circular dichroism spectroscopy. Fluorescence titration studies showed that TDP-43 oligomer was not able to bind to TAR DNA. Under electron and atomic force microscopy, TDP-43 oligomers adopted pore or channel-like structures. The species induced neuritic degeneration in retinoic acid-differentiated Neuro2a cells, suggestive of neurotoxicity. Conclusions: Our data demonstrate that the recombinant full length TDP-43 formed structurally stable and helical amyloid-like oligomers with pore-like morphology. Overall, our results established a possible pathogenic mechanism for TDP-43 proteinopathies which may be related to amyloidosis.