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Isolation and characterization of antibody fragments selective for human FTD brain derived TDP-43 variants.

ABSTRACT: BACKGROUND:Frontotemporal dementia (FTD) is the second leading cause of early onset dementia following Alzheimer's disease. It involves atrophy of the frontal and temporal regions of the brain affecting language, memory, and behavior. Transactive response DNA-binding protein 43 (TDP-43) pathology is found in most FTD and ALS cases. It plays a role in transcription, translation and serves as a shuttle between the nucleus and cytoplasm. Prior to its aggregation, TDP-43 exists as polyubiquitinated, hyperphosphorylated C-terminal fragments that correlate well with FTD disease progression. Because of the importance of TDP-43 in these diseases, reagents that can selectively recognize specific toxic TDP variants associated with onset and progression of FTD can be effective diagnostic and therapeutic tools. RESULTS:We utilized a novel atomic force microscopy (AFM) based biopanning protocol to isolate single chain variable fragments (scFvs) from a phage display library that selectively bind TDP variants present in human FTD but not cognitively normal age matched brain tissue. We then used the scFvs (FTD-TDP1 through 5) to probe post-mortem brain tissue and sera samples for the presence of FTD related TDP variants. The scFvs readily selected the FTD tissue and sera samples over age matched controls. The scFvs were used in immunohistochemical analysis of FTD and control brain slices where the reagents showed strong staining with TDP in FTD brain tissue slice. FTD-TDP1, FTD-TDP2, FTD-TDP4 and FTD-TDP5 all protected neuronal cells against FTD TDP induced toxicity suggesting potential therapeutic value. CONCLUSIONS:These results show existence of different disease specific TDP variants in FTD individuals. We have identified a panel of scFvs capable of recognizing these disease specific TDP variants in postmortem FTD tissue and sera samples over age matched controls and can thus serve as a biomarker tool.

SUBMITTER: Venkataraman L

PROVIDER: S-EPMC7472585 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Isolation and characterization of antibody fragments selective for human FTD brain derived TDP-43 variants.

Venkataraman Lalitha L He Ping P Khan Galam G Harris Brent T BT Sierks Michael R MR

BMC neuroscience 20200904 1

<h4>Background</h4>Frontotemporal dementia (FTD) is the second leading cause of early onset dementia following Alzheimer's disease. It involves atrophy of the frontal and temporal regions of the brain affecting language, memory, and behavior. Transactive response DNA-binding protein 43 (TDP-43) pathology is found in most FTD and ALS cases. It plays a role in transcription, translation and serves as a shuttle between the nucleus and cytoplasm. Prior to its aggregation, TDP-43 exists as polyubiqui ...[more]

PMID: 32887544

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Project description:The pathological hallmark of amyotrophic lateral sclerosis (ALS) is the presence of cytoplasmic inclusions, containing C-terminal fragments of the protein TDP-43. Here, we tested the hypothesis that highly sensitive mass spectrometry with parallel reaction monitoring (MS-PRM) can generate a high-resolution map of pathological TDP-43 peptide ratios to form the basis for quantitation of abnormal C-terminal TDP-43 fragment enrichment. Human cortex and spinal cord, microscopically staged for the presence of p-TDP-43, p-tau, alpha-synuclein, and beta-amyloid pathology, were biochemically fractionated and analyzed by immunoblot and MS for the detection of full-length and truncated (disease-specific) TDP-43 peptides. This informed the synthesis of heavy isotope-labeled peptides for absolute quantification of TDP-43 by MS-PRM across 16 ALS, 8 Parkinson's, 8 Alzheimer's disease, and 8 aged control cases. We confirmed by immunoblot the previously described enrichment of pathological C-terminal fragments in ALS-TDP urea fractions. Subsequent MS analysis resolved specific TDP-43 N- and C-terminal peptides, including a novel N-terminal truncation site-specific peptide. Absolute quantification of peptides by MS-PRM showed an increased C:N-terminal TDP-43 peptide ratio in ALS-TDP brain compared to normal and disease controls. A C:N-terminal ratio >1.5 discriminated ALS from controls with a sensitivity of 100% (CI 79.6-100) and specificity of 100% (CI 68-100), and from Parkinson's and Alzheimer's disease with a sensitivity of 93% (CI 70-100) and specificity of 100% (CI 68-100). N-terminal truncation site-specific peptides were increased in ALS in line with C-terminal fragment enrichment, but were also found in a proportion of Alzheimer cases with normal C:N-terminal ratio but coexistent limbic TDP-43 neuropathological changes. In conclusion this is a novel, sensitive, and specific method to quantify the enrichment of pathological TDP-43 fragments in human brain, which could form the basis for an antibody-free assay. Our methodology has the potential to help clarify if specific pathological TDP-43 peptide signatures are associated with primary or secondary TDP-43 proteinopathies.

Dataset Information

Isolation and characterization of antibody fragments selective for human FTD brain derived TDP-43 variants.

Publications

Isolation and characterization of antibody fragments selective for human FTD brain derived TDP-43 variants.

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