A fluid biomarker reveals loss of TDP-43 splicing repression in pre-symptomatic ALS

Abstract

Loss of TAR DNA-binding protein 43 kDa (TDP-43) splicing repression is well-documented in postmortem tissues of amyotrophic lateral sclerosis (ALS), yet whether this abnormality occurs during early-stage disease remains unresolved. Cryptic exon inclusion reflects functional loss of TDP-43, and thus detection of cryptic exon-encoded peptides in cerebrospinal fluid (CSF) could reveal the earliest stages of TDP-43 dysregulation in patients. Here, we use a newly characterized monoclonal antibody specific to a TDP-43-dependent cryptic epitope (encoded by the cryptic exon found in HDGFL2) to show that loss of TDP-43 splicing repression occurs in C9ORF72-associated ALS, including pre-symptomatic mutation carriers. In contrast to neurofilament light and heavy chain proteins, cryptic HDGFL2 accumulates in CSF at higher levels during early stages of disease. Our findings indicate that loss of TDP-43 splicing repression occurs early in disease progression, even pre-symptomatically, and that detection of HDGFL2's cryptic neoepitope may serve as a prognostic test for ALS which should facilitate patient recruitment and measurement of target engagement in clinical trials.

Keywords: ALS; CSF; TDP-43; cryptic exon; pre-symptomatic; prognostic biomarker.

Figure 1:. Identification of human in-frame TDP-43-associated cryptic exons.

(A) UCSC Genome Browser visualization of selected cryptic exons in human motor neurons (Klim, JR et al. 2019) and HeLa cells (Ling, JP et al. 2015) aligned to the GRCh38 assembly. Red tracks indicate TDP-43 knockdown, and blue arrows identify the nonconserved cryptic exons. Gene annotation below the RNA-sequencing tracks indicate the canonical exons (thick lines) and introns (thin lines). (B) Visualization of tissue-type specific gene expression of ACTL6B, AGRN, EPB41L4A, HDGFL2, and SLC24A3. While AGRN and HDGFL2 RNA transcripts are ubiquitously expressed, ACTL6B, EPB41L4A, and SLC24A3 are expressed in a more tissue-specific manner. Normalized area under the curve (NAUC) values from ASCOT (Ling, JP et al. 2020) are used to approximate gene expression levels in different human tissue types. (C) Comparison of wild-type (left) and cryptic (right) HDGFL2 protein structures. Inclusion of the cryptic exon in mRNA leads to the addition of 46 amino acids predicted to form an alpha helix structure (red) between flanking unstructured regions. Both structures are generated using AlphaFold predictions derived from amino acid sequences. Wild-type HDGFL2 protein structure can be found on the AlphaFold protein structure database (UniProt: Q7Z4V5). (D) Alignment of wild-type and cryptic HDGFL2 amino acid sequences. Cryptic inclusion is 46 amino acids long (red) and does not impact flanking amino acids.

Figure 2.. Novel antibody shows specificity for HDGFL2 with cryptic peptide.

(A) TDP-43 is reduced in HeLa treated with TDP-43 siRNA (siTDP). (B) RT-PCR with primers designed to amplify the cryptic exon sequence of HDGFL2 shows a product only in siTDP. (C) Protein extracts as in panel A were subjected to protein blot analysis using an antibody against the native HDGFL2 protein (left) or the novel monoclonal antibody (1–69) against the cryptic sequence in HDGFL2 (right). HDGFL2 harboring the neo-epitope is only detected in siTDP. (D) IP blot using 1–69 cryptic antibody for pulldown and WT HDGFL2 antibody for blotting reveals a band of the expected size only in siTDP.

Figure 3.. Development of an MSD assay specific for cryptic HDGFL2.

(A) Sandwich ELISA using Meso Scale Discovery (MSD) system. (B) Dose-dependent increase of MSD signal in siTDP HeLa lysate compared to control HeLa lysate. MSD signal of control HeLa is subtracted from that of siTDP. (C) Elevated siTDP MSD signal is specific to intact capture and detection antibodies. (D) Average cryptic HDGFL2 signal is elevated in CSF of C9ORF72 ALS compared to controls.

Figure 4.. Cryptic HDGFL2 can be detected by MSD assay in CSF of both pre-symptomatic and symptomatic C9ORF72 mutation carriers.

(A) Elevated cryptic HDGFL2 is detected in pre-symptomatic and symptomatic C9ORF72 mutation carriers compared to controls. (B) Cryptic HDGFL2 detection by our MSD assay in CSF of C9ORF72 mutation carriers diagnosed with ALS, FTD, or ALS-FTD tends to be higher during the earlier stage of symptomatic disease. Pearson correlation, r = −0.30, p = 0.027. (C) ALS staging model (adapted from Benatar et al., 2019) based on the dynamic of neurofilament subunit and cryptic HDGFL2 accumulation in CSF of ALS patients. As our preliminary data indicate that a trend towards higher levels of cryptic HDGFL2 is observed during earlier-stage disease, we hypothesize that while CSF neurofilament subunits rise during the prodromal phase and remain elevated in symptomatic disease, CSF cryptic HDGFL2 would decrease after its peak, which may occur during the prodromal stage. Thus, a ratio of pNfH or NfL to cryptic HDGFL2 of less than 1 or greater than 1 would indicate, respectively, pre-symptomatic or symptomatic phase of disease. (D) Ratios of pNfH/cryptic HDGFL2 and NfL/cryptic HDGFL2 in CSF of presymptomatic and symptomatic C9ORF72 mutation carriers. Using the ratio of pNfH and NfL to cryptic HDGFL2 in CSF, it may be possible to establish an ALS staging scale spanning from pre-symptomatic phase (ratio <1) to symptomatic phase (ratio >1).