Detection and quantification of pathological C-terminal TDP-43 fragments in post mortem brain tissue

Image credit: Hugo Kim, TDP-43 and ubiquitinated cytoplasmic aggregates in sporadic ALS are low frequency and widely distributed in the lower motor neuron columns independent of disease spread


The pathological hallmark of ALS is the nuclear clearance of TDP-43 in neurons and glia, with cytoplasmic inclusions of post-translationally modified and truncated C-terminal TDP-43 fragments (CTF). The reliable measurement of disease-specific TDP-43 in accessible biofluids would have potential as a biomarker of TDP-43 proteinopathies, but results from antibody-based techniques have been inconsistent to date.


To perform absolute quantification of CTFs using targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS).


Biochemical fractionation of post mortem cortical tissue was used to extract soluble and insoluble proteins (urea fraction)(1). Urea fractions were analysed by LC-MS/ MS to detect the peptide coverage of full-length and low molecular weight TDP-43 species. Identified peptide sequences were synthesized with a heavy-label isotope tag for absolute quantification of corresponding endogenous TDP-43 (light) peptides by targeted LC-MS/MS quantitation. Peptide concentrations were determined in ALS-TDP (n=16, with different levels of neuropathological phosphorylated TDP-43 burden), Parkinson’s Disease (PD, n=8), Alzheimer’s Disease (AD, n=8) and healthy control (Ctrl, n=8) urea fractions. The log2 abundance ratio (light peptide/heavy peptide) of healthy controls was compared to all groups using one-way ANOVA Dunnett’s multiple comparison test. CTFs were confirmed by immunoblotting using a C-terminal TDP-43 antibody.


Peptide sequences covering the N- and the C-terminus of TDP-43, as well as its disease-specific truncation sites, were detected in urea fractions enriched for pathological TDP-43. Compared to healthy controls, quantification of peptides in ALS showed an increase for truncation site-specific peptides (p<0.05). In the sub-group of samples of ALS-TDP (n=8) with more severe phosphorylated TDP-43 pathology, the C-terminal-specific peptides were also increased (p<0.05), as expected. At the same time N-terminal-specific TDP-43 peptides were reduced, possibly reflecting the degradation of the N-terminal cleavage products of TDP-43 (p<0.01). Surprisingly, truncation site specific peptides were increased in the AD urea fractions (p<0.001), but not the C-terminal peptides, suggesting pathological TDP-43 processing in AD cases. Truncation site and C-terminal specific peptides were low abundant in PD and Ctrl urea fractions. In samples where LC-MS/MS quantitation showed increased C-terminal specific peptides, the presence of low molecular TDP-43 fragments was confirmed by immunoblotting (25kDa and 35kDa). In ALS urea fractions the 25kDa band was increased reflecting the abundance of CTFs compared to full-length TDP-43 (p<0.05).

Discussion and conclusions: The detection of specific TDP-43 peptides able to quantify the pathological fragments of TDP-43 in post mortem ALS brain tissue, offers the opportunity to develop an in vivo assay to measure pathological TDP-43. This would have major diagnostic, stratification and pharmacodynamic biomarker potential. The finding of CTF in cases labelled as AD may reflect the recent identification of a sub-group labelled Limbic-predominant Agerelated TDP-43 Encephalopathy.