TDP-43 protein variants as biomarkers in amyotrophic lateral sclerosis

Stephanie M Williams,Galam Khan,Michael R Sierks,John Ravits,Brent T Harris

doi:10.1186/s12868-017-0334-7

Abstract

BackgroundTDP-43 aggregates accumulate in individuals affected by amyotrophic lateral sclerosis (ALS) and other neurodegenerative diseases, representing potential diagnostic and therapeutic targets. Using an atomic force microscopy based biopanning protocol developed in our lab, we previously isolated 23 TDP-43 reactive antibody fragments with preference for human ALS brain tissue relative to frontotemporal dementia, a related neurodegeneration, and healthy samples from phage-displayed single chain antibody fragment (scFv) libraries. Here we further characterize the binding specificity of these different scFvs and identify which ones have promise for detecting ALS biomarkers in human brain tissue and plasma samples.ResultsWe developed a sensitive capture ELISA for detection of different disease related TDP-43 variants using the scFvs identified from the ALS biopanning. We show that a wide variety of disease selective TDP-43 variants are present in ALS as the scFvs show different reactivity profiles amongst the ALS cases. When assaying individual human brain tissue cases, three scFvs (ALS-TDP6, ALS-TDP10 and ALS-TDP14) reacted with all the ALS cases and 12 others reacted with the majority of the ALS cases, and none of the scFvs reacted with any control samples. When assaying individual human plasma samples, 9 different scFvs reacted with all the sporadic ALS samples and again none of them reacted with any control samples. These 9 different scFvs had different patterns of reactivity with plasma samples obtained from chromosome 9 open reading frame 72 (c9orf72) cases indicating that these familial ALS genetic variants may display different TDP-43 pathology than sporadic ALS cases.ConclusionsThese results indicated that a range of disease specific TDP-43 variants are generated in ALS patients with different variants being generated in sporadic and familial cases. We show that a small panel of scFvs recognizing different TDP-43 variants can generate a neuropathological and plasma biomarker profile with potential to distinguish different TDP-43 pathologies.

Highlights

TAR DNA-binding protein 43 (TDP-43) aggregates accumulate in individuals affected by amyotrophic lateral sclerosis (ALS) and other neurodegenerative diseases, representing potential diagnostic and therapeutic targets
Selection of detection phage for capture ELISA To obtain a detection single chain antibody fragment (scFv) that binds to all TDP-43 variants including healthy and disease related species, we performed a series of atomic force microscopy (AFM) based negative and positive biopanning procedures
We performed sequential rounds of positive panning using TDP-43 immunoprecipitated from the motor cortex of healthy, ALS and frontotemporal dementia (FTD) human brain tissue (Fig. 1)

Summary

Introduction

TDP-43 aggregates accumulate in individuals affected by amyotrophic lateral sclerosis (ALS) and other neurodegenerative diseases, representing potential diagnostic and therapeutic targets. We generated scFvs that selectively bind distinct oligomeric variants of alpha-synuclein, and demonstrated that these scFvs could readily distinguish between human PD and control brain tissue, CSF and sera samples [28] These protein variant selective scFvs were isolated using an atomic force microscopy (AFM) based biopanning protocol [15, 20, 24,25,26,27]. The biopanning protocol utilizes a series of negative panning steps to remove phage particles that bind non-desired targets such as monomeric and fibrillar aggregates prior to completion of the positive panning step This protocol was utilized to generate scFvs against variants of TDP-43 present in human ALS cases (Stage 1A from Additional file 1: Fig. S1) [32]. We identified 23 different complete scFv sequences that all preferentially bound ALS tissue over both FTD and healthy samples using indirect phage ELISAs (Stage 2 from Additional file 1: Fig. S1) [32]

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Conclusion