Identification TDP‐43 fragments specific for frontotemporal lobar degeneration with TDP‐43 inclusions

Abstract

AbstractBackgroundAnte‐mortem biomarkers specific for TDP‐43 pathology are highly desired given the challenge in distinguishing frontotemporal lobar degeneration with TDP‐43 pathology (FTLD‐TDP) from phenotypically related disorders. TDP‐43 post‐translational modifications, like C‐terminal fragments, are regarded as disease‐specific TDP‐43 proteoforms; however, the exact structure of these proteoforms remains unclear. This lack of clarity is due in part to the use of instrumentation and techniques with low structural resolution and the study of small sample sizes. With this in mind, we performed high resolution mass spectrometry (HRMS) analysis of brain tissue from cases with and without TDP‐43 proteinopathy to identify TDP‐43 proteoforms unique to FTLD‐TDP.MethodHRMS was used to determine TDP‐43 proteoform composition in insoluble frontal lobe brain tissue from immunohistochemically‐confirmed FTLD‐TDP (n=13), related dementias (i.e., Alzheimer’s disease and FTLD‐tau without TDP‐43 deposits; n=10) and neuropathologically‐unaffected controls (n=3). Brain tissue was fractioned by gel electrophoresis, with HRMS analysis performed on molecular weight regions corresponding to <28 kDa (low molecular weight TDP; L‐TDP), 28‐38 kDa (mid molecular weight TDP; M‐TDP), and 38‐55 kDa (intact TDP; I‐TDP).ResultIn all samples tested, the greatest TDP‐43 sequence coverage was observed for I‐TDP, followed by L‐TDP and then M‐TDP. TDP‐43 peptides from L‐TDP were more frequently detected in the FTLD‐TDP cases compared to both sets of controls, whereas no frequency differences were observed for I‐TDP and M‐TDP. Quantitative analysis revealed peptide concentrations from M‐ and L‐TDP were significantly increased in FTLD‐TDP cases compared to controls. The L‐TDP peptides concentrations differentiated FTLD‐TDP cases from related dementias and unaffected controls with 78% sensitivity and 100% specificity. Further, three in vivo cleavage sites of TDP‐43 were identified, which were unique to FTLD‐TDP cases.ConclusionThis is the largest reported proteomics study to date of histology‐confirmed FTLD‐TDP. This is also the first study to include a large number and range of control tissues, and to provide supporting evidence for in vivo cleavage sites including corroboration of the proteolytic fragment recently found in TDP‐43 fibrils. Clarity and consensus on the sequence of TDP‐43 disease‐specific proteoforms will be helpful in advancing biomarker and drug discovery efforts for TDP‐43 proteopathies.