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. 2014 Mar 31:2:33.
doi: 10.1186/2051-5960-2-33.

Novel monoclonal antibodies to normal and pathologically altered human TDP-43 proteins

Linda K Kwong, David J Irwin, Adam K Walker, Yan Xu, Dawn M Riddle, John Q Trojanowski, Virginia M Y Lee  1
Affiliations

Novel monoclonal antibodies to normal and pathologically altered human TDP-43 proteins

Linda K Kwong et al. Acta Neuropathol Commun. .

Abstract

The RNA/DNA-binding protein, TDP-43, is the key component of ubiquitinated inclusions characteristic of amyotrophic lateral sclerosis (ALS) and the majority of frontotemporal lobar degeneration (FTLD-TDP) referred to collectively as TDP-43 proteinopathies. To further elucidate mechanisms of pathological TDP-43 processing and identify TDP-43 epitopes that could be useful as potential biomarkers of TDP-43 proteinopathies, we developed a panel of novel monoclonal antibodies (MAbs) directed at regions extending across the length of TDP-43. Here, we confirm previous observations that there is no or minimal accumulation of TDP-43 N-terminal domains in neocortical inclusions in human TDP-43 proteinopathy tissues and we identify a subset of these MAbs that are specific for human versus mouse TDP-43. Notably, one of these MAbs recognized an epitope that preferentially detected pathological TDP-43 inclusions with negligible reactivity for normal nuclear TDP-43 resembling anti-phospho-TDP-43 specific antibodies that only bind pathological TDP-43. Hence, we infer that this new MAb recognizes a phosphorylation independent but disease-specific pathologic conformation in abnormal TDP-43. These data suggest that the novel MAbs reported here will be useful for patient-oriented research as well as for studies of animal and cell-based models of TDP-43 proteinopathies including ALS and FTLD-TDP.

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Figures

Figure 1
Figure 1
Schematic representation of TDP-43 domains containing epitopes recognized by the MABs reported here. The TDP-43 domains harboring the epitopes recognized by 45 newly generated MAbs were mapped to regions along the entire length of TDP-43 (A-F) by immunoblot and indirect peptide ELISA and the putative amino acid stretches included in these epitopes is depicted in the table to the right of the schematic. RRM: RNA-recognition motif; NLS: bipartite nuclear localization signal; NES: nuclear export signal.
Figure 2
Figure 2
Representative IHC staining patterns and immunoblot profiles of selected MAbs that recognize human- or human- and mouse-TDP-43. MAb 5195 detects normal nuclear TDP-43 in (a) human and (b) mouse hippocampus dentate gyrus. In contrast, MAb 5095 detects (c) human but not (d) mouse TDP-43, which is representative of 7 MAbs that preferentially recognize human TDP-43. Scale bar = 50 μm. (e) All the MAbs here show strong immunoreactivity with a 43 kDa band from human cell lysates, but 7 MAbs preferentially recognize human TDP-43 with little or no detection of mouse TDP-43. MAb clone numbers are shown above each immunoblot, and MAb 5195 is representative of 8 MAbs that detect human and mouse TDP-43 with equal affinity. H – human, M – mouse.
Figure 3
Figure 3
IHC and IB properties of new N- and C-terminal specific anti-TDP-43 MAbs. N-terminal specific MAb 5031 reacts with spinal cord ALS TDP-43 pathology (a) with minimal detection of FTLD cortical TDP-43 pathology (arrow, c), while MAb 5056 shows robust reactivity for both spinal cord (b) and cortical TDP-43 inclusions (arrows, d). Consistent with previous studies [13], immunoblot analysis shows that MAb 5031 detects full-length but not the CTF of TDP-43 (e), while C-terminal specific MAb 5056 recognizes both (f). Scale bar = 100 μm.
Figure 4
Figure 4
Preferential recognition of pathological TDP-43 by MAb 138. MAb 138 robustly detects pathological TDP-43 inclusions (arrows) with no or very minimal nuclear staining in FTLD temporal cortex (a) and little recognition of normal nuclear TDP-43 in human control dentate gyrus (e), similar to previously described antibodies specific for phospho TDP-43 (b, f). In contrast MAb 5060, the epitope for which lies within aa 182–261, and MAb 5195 which detects an epitope contained within aa 394–414, recognize both pathological TDP-43 inclusions (arrows) and normal nuclear TDP-43 (asterisks) in FTLD temporal cortex (c,d) and control dentate gyrus (g,h). Scale bar = 50 μm.
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