Biomarkers.

Background: The nuclear clearance and cytoplasmic aggregation of splicing repressor TAR DNA/RNA-binding protein-43 (TDP-43) occur in approximately 50% of Alzheimer's disease (AD) cases and about 45% of frontotemporal dementia (FTD). However, it is not clear how early such mechanism occurs in AD and FTD as there is no method of detecting TDP-43 dysregulation in living individuals. Since the loss of nuclear TDP-43 leads to cryptic exon inclusion, we propose that cryptic exon-encoded peptides may be detected in patient biofluids as biomarkers of TDP-43 loss of function.

Method: We developed antibodies against the TDP-43-dependent cryptic peptide within actin-like protein 6B (ACTL6B) and characterized them through protein blot analysis using TDP-43-knockdown (KD) SH-SY5Y neuroblastoma cell lysates. Next we performed immunofluorescent staining of patient brain tissues to determine whether cryptic ACTL6B can be found in patient neurons. We then developed a Meso Scale Discovery (MSD) ELISA using our cryptic ACTL6B antibody.

Result: While the wild-type (WT) ACTL6B antibody detected an expected band in both WT and KD SH-SY5Y lysates, the cryptic ACTL6B antibody detected a band in only KD SH-SY5Y. Using this antibody, we detected cryptic ACTL6B in FTLD and AD, but not control, brain tissues. The cryptic ACTL6B staining colocalizes with neurons that display TDP-43 nuclear depletion and/or cytoplasmic aggregation. Using transfected SH-SY5Y cells to overexpress either cryptic or WT ACTL6B, the MDS ELISA was sensitive and specific for cryptic ACTL6B, with 0.5 micrograms of lysate from SH-SY5Y overexpressing cryptic ACTL6B showing MSD signal 5-fold higher than 0.5 micrograms of lysate from SH-SY5Y overexpressing WT ACTL6B (1546 vs. 290 units).

Conclusion: The study provides evidence that our cryptic ACTL6B antibody is sensitive and specific for the ACTL6B cryptic peptide. This antibody could be used to determine TDP-43 nuclear clearance/loss of function in patient brain tissues, providing a more sensitive approach to detecting TDP-43 pathology than staining for TDP-43 cytoplasmic aggregates. In the future, we will use our novel MSD ELISA to test the presence of cryptic ACTL6B in patients' CSF and blood, which could aid in early-stage disease detection.