TDP-43 protein variants as biomarkers in amyotrophic lateral sclerosis
© The Author(s) 2017
Received: 10 December 2016
Accepted: 12 January 2017
Published: 25 January 2017
TDP-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.
We 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.
These 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.
KeywordsAmyotrophic lateral sclerosis TDP-43 variants Plasma Biomarker Brain tissue scFv
The progressive loss of motor neurons in regions including the spinal cord and cortex is a general occurrence in the advancement of the neurodegenerative disorder amyotrophic lateral sclerosis (ALS) [1–6]. Approximately 1–2 individuals per 100,000 are affected by ALS per year with an average life expectancy of 3–5 years [7, 8]. ALS cases can be divided into sporadic and familial classifications with the vast majority of the cases in the sporadic category [7, 8]. Mutations in genes including superoxide dismutase 1 (SOD1) and chromosome 9 open reading frame 72 (C9ORF72) have been linked to familial ALS [5, 7, 8]. Alterations to normal expression or structure of TAR DNA-binding protein 43 (TDP-43) have been suggested to play a major role in ALS, occurring in about 97% of cases [1, 3–6, 9, 10]. Normally TDP-43 is predominantly located in the cell nucleus where it participates as an important RNA/DNA binding protein involved in gene splicing and other RNA-related processes [5, 11]. However, during progression of ALS aggregates of TDP-43 accumulate in the cell cytoplasm. Variations of TDP-43 including ubiquitinated, truncated, phosphorylated and oligomeric forms exist in ALS [5, 12–14]. Therefore, reagents that can selectively bind different disease related TDP-43 variants have potential diagnostic and therapeutic applications for ALS.
An association between protein aggregation and disease development is also found in a number of other neurodegenerative disorders including Alzheimer’s (AD) and Parkinson’s (PD) diseases, where aggregates of beta-amyloid and alpha-synuclein, respectively, have been identified [15–31]. We previously demonstrated that we could generate single-chain variable fragments (scFvs) that selectively recognize different oligomeric variants of beta-amyloid, and that these scFvs could readily distinguish between human AD and control brain tissue, cerebrospinal fluid (CSF) and sera samples . Similarly, 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 . These protein variant selective scFvs were isolated using an atomic force microscopy (AFM) based biopanning protocol [15, 20, 24–27]. 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 also utilized to generate scFvs against variants of TDP-43 present in human ALS cases (Stage 1A from Additional file 1: Fig. S1) . We immunoprecipitated TDP-43 proteins from the homogenized motor cortex of ALS and control cases using a commercially available polyclonal anti-TDP-43 antibody as previously described . Since TDP-43 pathology also exists in around 45% of frontotemporal dementia (FTD) cases [5, 33–37], we also included multiple rounds of negative panning against TDP-43 variants immunoprecipitated from the motor cortex of FTD cases. After biopanning, 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) .
Here we further characterized the 23 different scFvs to identify which ones reacted most strongly with ALS samples and to highlight the wide diversity of TDP-43 variants present in human neurodegenerative disease cases. We previously demonstrated a simple capture ELISA that utilizes a phage-displayed detection antibody with sub-femtomolar sensitivity in conjunction with capture scFvs similar to those examined here [28, 38]. Here we generated a phage based detection antibody against TDP-43 for use in a capture ELISA in conjunction with the 23 scFvs (Stages 1B and 2 from Additional file 1: Fig. S1) to analyze sporadic ALS, c9orf72 ALS, FTD and control human samples (Stage 3 from Additional file 1: Fig. S1).
Selection of detection phage for capture ELISA
Binding specificities of the anti-TDP-43 phages
Phage expressing the different anti-TDP-43 scFvs were previously selected for preferential binding to ALS brain tissue samples relative to both control and FTD cases as described . We characterized the binding of 23 different phage-displayed scFvs toward ALS, FTD and healthy human brain tissue samples from the motor cortex using a simple indirect phage ELISA (Additional file 1: Fig. S1, Stage 2). The scFv clones were re-labeled in the current study to facilitate identification and simplify discussion. All scFvs were expressed in cell supernatant (Additional file 2: Fig. S2) and had stronger binding to ALS compared to both FTD and control samples (Additional file 3: Fig. S3) confirming the efficiency of our previously described biopanning protocol for generating reagents that bind disease specific protein variants .
Recognition of multiple TDP-43 variants
Selection of individual ALS cases utilizing human brain tissue
Selection of individual ALS cases utilizing human plasma
While TDP-43 pathology is present in the vast majority of examined ALS cases [1, 3–6, 9, 10, 39], the conformation and location of the TDP-43 aggregates can vary, including oligomeric, truncated, phosphorylated and ubiquitinated configurations [12, 13]. Isolation of reagents that can selectively recognize disease relevant TDP-43 variants could facilitate diagnosis of ALS, particularly if these variants could be detected in blood based samples during early even pre-symptomatic stages of disease progression [40–42]. We previously isolated scFvs with preferential reactivity for TDP-43 immunoprecipitated from ALS human brain tissue relative to TDP-43 immunoprecipitated from healthy and FTD human brain tissue using our AFM based biopanning procedures . Here we further identify a subset of these scFvs with diverse binding specificities that have potential applications to detect blood-based biomarkers for ALS.
We previously identified 23 distinct valid scFv sequences from the ALS TDP-43 positive biopanning and all showed preferential reactivity for homogenized human ALS brain tissue samples compared to FTD and control cases (Additional file 3: Fig. S3). We also generated a phage-displayed scFv that binds all forms of TDP-43 including ALS, FTD and healthy variants (Fig. 2a) to be utilized in our capture ELISA protocol. We applied competition ELISAs to show that the different scFvs generated against the ALS relevant TDP-43 variants were binding different epitopes (Fig. 4a–e). Binding intensity variations between all 23 phages in the indirect phage ELISAs (Additional file 3: Fig. S3) also suggest the presence of multiple TDP-43 variants in ALS.
Using the scFvs in a capture ELISA format, we showed that 12 of the scFvs can statistically distinguish between ALS and healthy human brain tissue samples (Fig. 5a–l). By plotting signal strength as a function of number of standard deviations from the control, we showed that three of the scFvs (ALS-TDP6, ALS-TDP10 and ALS-TDP14) reacted with all five ALS samples studied but none of the controls, while the nine other scFvs selected different combinations of 3–4 of the ALS cases and again none of the controls (Additional file 5: Table S1). The variation in signal intensities for each sample suggests that the different targeted TDP-43 variants are present at different concentrations in the ALS cases. We have previously illustrated successful identification of AD and PD sera cases using multiple scFvs  and expect that such an approach can also be used for diagnosis of ALS.
To determine their potential diagnostic value, we analyzed plasma samples from 4 sporadic ALS cases, 4 c9orf72 ALS cases and 3 controls using the anti-TDP-43 scFvs and identified 9 promising scFvs. All 9 of the scFvs reacted with all 4 sporadic ALS cases (Additional file 6: Table S2). ALS-TDP7 and ALS-TDP10 selected 3 of the 4 c9orf72 ALS cases, 6 of the 9 scFvs recognized 1–2 of the c9orf72 ALS cases and ALS-TDP3 did not select any of the c9orf72 ALS cases. Since the panning protocols included exhaustive negative panning against healthy control human samples, as expected, none of the scFvs reacted with any of the control samples. Since more than 23 scFvs were generated from the TDP-43 panning process, it is possible that screening some of the additional remaining scFvs will identify antibody fragments that select all of the c9orf72 ALS cases. Since plasma samples from different patients show different reactive profiles with the panel of anti-TDP scFvs, each patient may likely have their own personal biomarker profile corresponding to subtly different types of ALS. Utilizing a combination of multiple anti-TDP-43 scFvs may prove valuable in providing a personalized diagnosis for each patient in both sporadic and familial ALS cases. A personalized blood-based diagnosis system could also be very helpful for initiating and monitoring treatment strategies for ALS [41, 42].
Here we characterize the binding specificities of a subset of 23 anti-TDP-43 scFvs we previously generated  with both human brain tissue and plasma samples to illustrate their preferential reactivity with ALS. The scFvs generated by selectively biopanning for TDP-43 variants present in ALS but not FTD brain tissue do preferentially bind ALS compared to both FTD and control samples. These results further support the proficiency of our biopanning process at isolating reagents reactive with target variants of interest. In future studies we intend to screen plasma from larger ALS and FTD sample sets to reaffirm which scFvs are ALS specific and if any also cross-react with FTD samples. Also, since TDP-43 pathology is not unique to ALS and FTD, but has been detected in other neurodegenerative disease including ~ 57% of individuals with AD, 19% of PD cases, 45% of individuals having dementia with Lewy bodies (DLB) and 100% of tested Huntington’s cases [12, 13, 39, 43–47], we also intend to screen the scFvs for cross reactivity with these other diseases. If scFvs can selectively bind specific disease cases, they may be useful reagents along with reagents to other protein variants including beta-amyloid, alpha-synuclein and tau to help improve diagnosis of different neurodegenerative diseases .
We have previously shown that scFvs against alpha-synuclein variants associated with Parkinson’s disease have excellent therapeutic value [49, 50]. In a similar fashion, since our scFvs selectively bind ALS associated variants of TDP-43, in addition to diagnostic value, the scFvs may also have potential therapeutic value.
Our TDP-43 reactive scFvs were selective for individual ALS cases relative to controls utilizing both human brain tissue and plasma samples. The scFvs recognized both sporadic and familial ALS cases with plasma. These results support the potential diagnostic value of our TDP-43 scFvs when employed in our phage capture ELISA system.
Patient cohort demographics
To produce phage particles from the ALS TDP-43 clones isolated earlier, we essentially follow the previously described protocol (http://www.lifesciences.sourcebioscience.com/media/143421/tomlinsonij.pdf) [28, 32, 38]. E. coli TG1 cells containing the plasmids for our clones were cultured in 2xYT containing 100 μg/ml ampicillin and 1% glucose until OD600 was 0.4–0.6. The cells were then incubated with 2 × 1011 of KM13 helper phage or hyperphage (Progen, Germany) for 30 min without shaking, followed by media exchange to 2xYT containing 100 μg/ml ampicillin, 50 μg/ml kanamycin and 0.1% glucose post centrifugation. The cells were then cultured overnight at 30 °C, followed by centrifugation to isolate the supernatant. Polyethylene glycol (PEG)/NaCl was added to the supernatant and it incubated on ice for 1 h. The mixture was then centrifuged and the pellet resuspended in PBS. Following another 1 h incubation on ice, additional cell debris was removed via a last centrifugation step. Their concentrations were estimated using a bicinchoninic acid (BCA) assay (Pierce, USA) and stored at −80 °C.
Biopanning for anti-TDP-43 detection antibody
For the capture ELISA utilized here we require a detection scFv that recognizes all forms of the target antigen, in this case TDP-43. The detection scFv is displayed on the phage surface generating essentially a self-assembling nanoparticle for detection. The detection antibody should bind multiple forms, conformations and variants of the target TDP-43 antigen. To acquire the detection antibody, we utilized our previously described AFM based biopanning protocols . We utilized a combination of three different scFv libraries including the Tomlinson I and J libraries and Sheets library  as our initial scFv pool. A series of negative panning steps were then completed to remove phage binding non-desired targets including bovine serum albumin (BSA) and aggregated synthetic alpha-synuclein (Fig. 1). We then performed a positive panning step using an aliquot of TDP-43 immunoprecipitated from the motor cortex of healthy human brain tissue deposited on a mica substrate (Fig. 1). Bound phages were eluted with glycine and added to a second piece of mica containing an aliquot of TDP-43 immunoprecipitated from ALS human brain tissue. Following glycine elution, phages were then added to a third piece of mica containing an aliquot of TDP-43 immunoprecipitated from FTD human brain tissue. Bound phages were again eluted with glycine and recovered by infection of TG1 cells. We utilized multiple rounds of positive panning with TDP-43 immunoprecipitated from diverse brain homogenate samples to ensure selection of a detection antibody that is reactive with normal and disease associated forms of TDP-43. Eluted phages were then screened using phage ELISAs and the integrity of their DNA sequences verified (Stage 2 from Additional file 1: Fig. S1). Plasmid isolation was accomplished using the Qiagen Miniprep Kit (Valencia, CA, USA). The selected TDP-43 detection phage was then biotinylated using the EZ-Link Pentylamine-Biotinylation kit (Thermo Scientific, USA) as previously described  for use in the capture ELISA.
Indirect ELISA and tissue homogenization were performed as described previously [28, 32, 38]. Briefly, 2–10 μg/ml of homogenized human brain tissue was added to a 96-well ELISA plate and incubated for 1 h at 37 °C. Following three washes with 0.1% PBS-Tween 20, non-specific binding sites were blocked with 2% milk in PBS. Either a 1/100 dilution of phage particles or 1/1000 of rabbit anti-TDP-43 antibody (ProteinTech, IL, USA) was added to the wells followed by anti-M13 HRP (GE Healthcare Life Science, NJ, USA) or goat anti-rabbit IgG HRP (Santa Cruz Biotechnology, Texas, USA), respectively. Enzyme detection was achieved using the SuperSignal ELISA Femto Maximum Sensitivity Substrate kit (Thermo Scientific, USA) and signal intensities quantified using the Perkin Elmer Wallac 1420 Victor2 Multilabel Counter.
ScFv production and purification
ScFvs were expressed and purified as previously described [28, 32, 38]. Briefly, HB2151 cells were cultured for 2–3 h at 37 °C in 2xYT, 0.1% glucose and ampicillin until OD600 was 0.4. Isopropyl β-d-1-thiogalactopyranoside (IPTG) was then added and the temperature reduced to 30 °C. The following day the supernatant was harvested and stored at −20 °C. Purification was completed under native conditions by incubating the supernatant with Ni–NTA agarose beads (Qiagen, CA, USA) for 1–2 h at 4 °C. The mixture was then transferred to a column. Following washing, bound scFvs were eluted using a 250 mM imidazole solution. Dot and Western blotting analyses were used to confirm expression and purification using the C-Terminal c-myc tag on our scFvs.
Phage capture ELISAs
Phage capture ELISAs were performed essentially as described previously . Briefly, unconcentrated supernatant containing the scFvs or Ni–NTA purified scFvs was added to the wells of high binding ELISA plates for 1 h at 37 °C on a shaker. All incubations were performed at 37 °C whilst shaking. After washing with 0.1% PBST three times, the wells were blocked with 2% milk in PBS. Next, human brain tissue (2–10 μg/ml) or sera (1/100 v/v) was added followed by 200 ng/ml of the 40 mmol carboxyl biotinylated phage. After addition of the secondary antibody avidin-HRP (Sigma-Aldrich, USA), enzyme detection was again achieved using the SuperSignal ELISA Femto Maximum Sensitivity Substrate kit.
When screening the potential detection phage using the capture ELISA system, the phage was not biotinylated and therefore detected using the anti-M13 HRP antibody. Similarly, any reactivity with the commercial anti-TDP-43 antibody was recognized with the goat anti-rabbit IgG HRP secondary. Lastly, the quantity of immunoprecipitated proteins utilized in these ELISAs was 20 ng.
Competition ELISAs were carried out using the phage capture ELISA protocol except preceding addition of the brain tissue, the samples were pre-incubated with the different competitive scFvs for ~1 h.
The ratio of each sample reading to the value obtained for PBS was first calculated. The mean of the control and/or FTD cases was then subtracted from each sample. This type of calculation was utilized due to the difficulty in acquiring large purified quantities of the targets recognized by our scFvs in order to generate a standard curve to quantify the reactivity of each sample. We evaluated the reactivity of the test sample relative to the average signal intensity of the control and/or FTD groups by subtracting their average reactivity from that of the ALS group thus setting control values to zero. Statistical significance was calculated using one-way ANOVA and LSD Post-Hoc analyses with significance at p < 0.05. The graphs and statistical analyses were completed using the IBM SPSS Statistics 23 program. In Figs. 5 and 6, to illustrate the intensity difference between the test and control groups, we plotted the number of standard deviations each sample was from the controls by subtracting the mean of the controls from each sample and dividing the result by the standard deviation of the controls. In Additional file 5: Table S1, Additional file 6: Table S2, the level of activity was further highlighted using a “+” sign system. In Additional file 5: Table S1 each “+” sign indicated one standard deviation (SD) increase relative to the mean of the controls. In Additional file 6: Table S2 the first “+” indicated a 1.5 SD increase followed by each additional “+” sign indicating another 1 SD increase. Furthermore, since brain tissue homogenization was conducted at different intervals, technique variation was accounted for in the ELISAs by first dividing by matching controls.
amyotrophic lateral sclerosis
atomic force microscopy
single chain antibody fragment
superoxide dismutase 1
chromosome 9 open reading frame 72
TAR DNA-binding protein 43
bovine serum album
dementia with Lewy bodies
SMW performed most of the experiments described in this study and contributed to the writing of the manuscript. GK completed the immunoprecipitation of TDP-43 variants from human brain tissue. BTH provided the human brain tissue samples and contributed to the writing of the manuscript. JR provided the human plasma samples and contributed to the writing of the manuscript. MRS contributed to the design of the study and the writing of the manuscript. All authors read and approved the final manuscript.
We would like to thank Philip Schulz for his contributions to this study.
The authors declare that they have no competing interests.
Availability of data and material
The datasets generated during the current study are available from the corresponding author on reasonable request.
Consent to publish
Ethics approval and consent to participate
Human brain tissue samples were provided de-identified with regard to patient identifiable information from the Georgetown Brain Bank (Georgetown University Medical Center) and New York Brain Bank (Columbia University) and all tissue/biofluid banks are operating under institutional IRB guidelines. Human plasma samples were provided by Dr. John Ravits (University of California, San Diego School of Medicine) and collected by an Investigational Review Board-compliant process.
This research was supported by a grant from NIH: R21AG042066.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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