Irwin KE, Jasin P, Braunstein KE, Sinha I, Bowden KD, Moghekar A, Oh ES, Raitcheva D, Bartlett D, Berry JD, Traynor B, Ling JP, Wong PC. A fluid biomarker reveals loss of TDP-43 splicing repression in pre-symptomatic ALS. bioRxiv. January 24, 2023 bioRxiv

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  1. This is a remarkable feat of science by a fantastic group of experts from great research centers around the world. It relates to a field of work that has been percolating along for years, and this may represent a significant step forward.

    The orientation of the study is toward ALS, which would be interesting and perhaps clinically useful, but the relevance may also exist for LATE-NC, which is 100-fold more common than ALS, and a context where a biomarker is particularly urgently needed.

    These studies beget some obvious questions:

    1. Can we find some of these markers in CSF from persons with LATE-NC?
    2. Can some of these pathology-signaling results also manifest in blood/plasma for a more generalizable biomarker application?
    3. Are there differences between subtypes of TDP-43 proteinopathy diseases?

    View all comments by Peter Nelson

  2. I was really impressed by this work by Seddighi and colleagues for several reasons.

    First, this is one of the clearest demonstrations that changes at the RNA level in models of disease have substantial and consequential impacts at the protein level.

    Second, this work confirms that most TDP-43-related mis-splicing events result in the degradation or instability of affected RNA and proteins. However, in a minority of cases the mis-splicing events create new proteins or proteins with novel sequences that can have unanticipated effects. The authors convincingly show this for one candidate, HDGFL2, and similar findings are likely to affect many other candidates.

    Third, while many of the candidates identified in this work may be important for pathogenesis, it is clear that at the very least they will be highly relevant as outcome measures of TDP-43-related function. This, in turn, could be incredibly valuable for the design of fluid biomarkers for patient stratification (for instance, distinguishing FTLD-TDP from FTLD-Tau), target engagement, prognosis and diagnosis.

    These biomarkers were investigated in CSF, but a more accessible fluid type (i.e. serum) is a possibility that can be pursued in future studies. It's also essential to examine these same markers in ALS/FTD cases vs. controls, and to examine these processes in disorders with predominant TDP-43 pathology (i.e., FTLD-GRN).

    Fourth, this is a beautiful demonstration of how cultured human neurons can recapitulate TDP-43 biology seen in vivo, including even in people.

    View all comments by Sami Barmada

  3. Following up on the seminal discovery that loss of TDP-43 results in the production of cryptic exons, the authors demonstrate that some of these exons can be translated into new polypeptides, thereby altering their interactions with other proteins, and possibly the functions of host proteins.

    This interesting finding complements earlier reports that cryptic exons lead to truncation of some full-length host proteins and further supports the notion that correcting multiple TDP-43 targets simultaneously may offer a better chance of success in clinical settings (Halim and Gao, 2022). A major challenge for the future is to identify the subset of TDP-43 targets, if they exist, that may be responsible for the bulk of the consequences of loss of TDP-43 function during disease progression.

    Equally important is the finding that these de novo polypeptides can be detected in cerebrospinal fluid of patients with ALS, raising the exciting prosect that novel biomarkers can be developed and, if they are sensitive enough, used to monitor TDP-43 pathology in living patients, which would facilitate clinical trials.

    References:

    Halim D, Gao FB. RNA targets of TDP-43: Which one is more important in neurodegeneration?. Transl Neurodegener. 2022 Feb 25;11(1):12. PubMed.

    View all comments by Fen-Biao Gao

  4. I think it is quite an interesting concept to use the expression of aberrant exons as a biomarker for ALS. It potentially takes advantage of the fact that abnormalities in TDP-43 localization are seen in up to 98 percent of ALS patients, and nuclear depletion of TDP-43 is associated with the inclusion of cryptic exons.

    A prevailing idea in the ALS field is that abnormalities with TDP-43 are actually pathogenic for ALS, and so a biomarker that is potentially linked to that mechanism could be quite valuable.

    View all comments by Steven Finkbeiner

  5. In the past year the ALS field has been altered by the realization that expression of cryptic exons (CE) under conditions of TDP-43 mis-localization, links together several strands of research, including investigations into ALS-associated genetic variants within UNC13A, which is itself a target of TDP-43 CE splicing. Evidence to date suggests that inclusion of CE within a transcript typically leads to nonsense-mediated decay and reduced expression of the host gene/protein with toxic consequences. However, an interesting question arises over the loss of TDP-43 splicing repression and if it is always pathogenic; after all, as Seddighi and colleagues point out, TDP-43 mis-localization is part of the normal cellular stress response. Another interesting question, which is answered in this preprint, is whether the CE-containing transcripts are ever translated into proteins.

    This work is primarily based in iPSC-derived neurons, in which the authors have performed CRISPRi knockdown of TDP-43 expression. Under these conditions they were able to confirm expression of CE-containing transcripts and proteins. Given that many stages of cellular quality control can intervene to prevent translation of unwanted proteins, the key step here was the validation, using antibodies and/or shotgun proteomics, that CE-containing proteins are expressed. While this validation was achieved in the iPSC-derived neurons, the authors relied on transcript expression data for postmortem brain tissue. The most exciting part of the study concerned the detection of CE-containing proteins in patient CSF. Here the authors used PRM mass spectrometry proteomics to show that they were able to detect 18 peptides derived from 13 genes in CSF from 15 ALS patients.

    Seddighi et al. have developed the CE story by showing that a subset of CE-containing transcripts are actually translated into proteins. The authors propose that these proteins may be toxic—either directly or indirectly via the immune response—or could serve as biomarkers of disease activity, e.g., via detection in CSF. Both of these proposals portend to the question about the normal role of TDP-43 mis-localization in the cellular stress response. A biomarker is most effective if it is disease-specific and more work remains to quantify CE-containing transcripts and proteins in normal controls, and patients with other diseases, such as Alzheimer's. An obvious prediction is that patients with rapidly progressive disease will have more TDP-43 mis-localization, but this analysis was not included in this preprint. The authors state that their assay is subject to technical artefacts, which means that it is only semi-quantitative, but maybe more of these questions will be answered in the final manuscript.

    View all comments by Johnathan Cooper-Knock

  6. We initially discovered the fundamental role of TDP-43 in repressing non-conserved cryptic exons and suggested that such function is lost in ALS-FTD (Ling et al., 2015); subsequently we also showed this to occur in AD with TDP-43 proteinopathy (Sun et al., 2017) and in Inclusion Body Myositis (Britson et al., 2022). 

    While recent studies confirm the idea that most of the TDP-43-dependent cryptic exon inclusion would lead to depletion of mRNAs (and their respective proteins), such as STMN2 (Klim et al., 2019; Melamed et al., 2019; Prudencio et al., 2020) and UNC13A (Ma et al., 2022; Brown et al., 2022), this new study by Michael Ward and colleagues focuses on TDP-43 targets with in-frame cryptic exon inclusion (lacking a termination codon), such that the "cryptic" protein can be more easily detected in cells or in biofluid of patients. For example, HDGFL2 is one such TDP-43 target. In fact, we have taken a complementary approach to generate monoclonal antisera directed against this neo-epitope found in HDGFL2. By developing a highly sensitive MSD sandwich ELISA assay, we now show in a large cohort of C9ORF72 patients that loss of TDP-43 splicing repression occurs during early stage disease, including those pre-symptomatic ones.

    References:

    Ling JP, Pletnikova O, Troncoso JC, Wong PC. NEURODEGENERATION. TDP-43 repression of nonconserved cryptic exons is compromised in ALS-FTD. Science. 2015 Aug 7;349(6248):650-5. PubMed.

    Sun M, Bell W, LaClair KD, Ling JP, Han H, Kageyama Y, Pletnikova O, Troncoso JC, Wong PC, Chen LL. Cryptic exon incorporation occurs in Alzheimer's brain lacking TDP-43 inclusion but exhibiting nuclear clearance of TDP-43. Acta Neuropathol. 2017 Jun;133(6):923-931. Epub 2017 Mar 22 PubMed.

    Britson KA, Ling JP, Braunstein KE, Montagne JM, Kastenschmidt JM, Wilson A, Ikenaga C, Tsao W, Pinal-Fernandez I, Russell KA, Reed N, Mozaffar T, Wagner KR, Ostrow LW, Corse AM, Mammen AL, Villalta SA, Larman HB, Wong PC, Lloyd TE. Loss of TDP-43 function and rimmed vacuoles persist after T cell depletion in a xenograft model of sporadic inclusion body myositis. Sci Transl Med. 2022 Jan 19;14(628):eabi9196. PubMed.

    Klim JR, Williams LA, Limone F, Guerra San Juan I, Davis-Dusenbery BN, Mordes DA, Burberry A, Steinbaugh MJ, Gamage KK, Kirchner R, Moccia R, Cassel SH, Chen K, Wainger BJ, Woolf CJ, Eggan K. ALS-implicated protein TDP-43 sustains levels of STMN2, a mediator of motor neuron growth and repair. Nat Neurosci. 2019 Feb;22(2):167-179. Epub 2019 Jan 14 PubMed.

    Melamed Z, López-Erauskin J, Baughn MW, Zhang O, Drenner K, Sun Y, Freyermuth F, McMahon MA, Beccari MS, Artates JW, Ohkubo T, Rodriguez M, Lin N, Wu D, Bennett CF, Rigo F, Da Cruz S, Ravits J, Lagier-Tourenne C, Cleveland DW. Premature polyadenylation-mediated loss of stathmin-2 is a hallmark of TDP-43-dependent neurodegeneration. Nat Neurosci. 2019 Feb;22(2):180-190. Epub 2019 Jan 14 PubMed.

    Prudencio M, Humphrey J, Pickles S, Brown AL, Hill SE, Kachergus JM, Shi J, Heckman MG, Spiegel MR, Cook C, Song Y, Yue M, Daughrity LM, Carlomagno Y, Jansen-West K, de Castro CF, DeTure M, Koga S, Wang YC, Sivakumar P, Bodo C, Candalija A, Talbot K, Selvaraj BT, Burr K, Chandran S, Newcombe J, Lashley T, Hubbard I, Catalano D, Kim D, Propp N, Fennessey S, NYGC ALS Consortium, Fagegaltier D, Phatnani H, Secrier M, Fisher EM, Oskarsson B, van Blitterswijk M, Rademakers R, Graff-Radford NR, Boeve BF, Knopman DS, Petersen RC, Josephs KA, Thompson EA, Raj T, Ward M, Dickson DW, Gendron TF, Fratta P, Petrucelli L. Truncated stathmin-2 is a marker of TDP-43 pathology in frontotemporal dementia. J Clin Invest. 2020 Nov 2;130(11):6080-6092. PubMed.

    Ma XR, Prudencio M, Koike Y, Vatsavayai SC, Kim G, Harbinski F, Briner A, Rodriguez CM, Guo C, Akiyama T, Schmidt HB, Cummings BB, Wyatt DW, Kurylo K, Miller G, Mekhoubad S, Sallee N, Mekonnen G, Ganser L, Rubien JD, Jansen-West K, Cook CN, Pickles S, Oskarsson B, Graff-Radford NR, Boeve BF, Knopman DS, Petersen RC, Dickson DW, Shorter J, Myong S, Green EM, Seeley WW, Petrucelli L, Gitler AD. TDP-43 represses cryptic exon inclusion in the FTD-ALS gene UNC13A. Nature. 2022 Mar;603(7899):124-130. Epub 2022 Feb 23 PubMed.

    Brown AL, Wilkins OG, Keuss MJ, Hill SE, Zanovello M, Lee WC, Bampton A, Lee FC, Masino L, Qi YA, Bryce-Smith S, Gatt A, Hallegger M, Fagegaltier D, Phatnani H, NYGC ALS Consortium, Newcombe J, Gustavsson EK, Seddighi S, Reyes JF, Coon SL, Ramos D, Schiavo G, Fisher EM, Raj T, Secrier M, Lashley T, Ule J, Buratti E, Humphrey J, Ward ME, Fratta P. TDP-43 loss and ALS-risk SNPs drive mis-splicing and depletion of UNC13A. Nature. 2022 Mar;603(7899):131-137. Epub 2022 Feb 23 PubMed.

    View all comments by Philip Wong

  7. This is great work; however, it is not the first time proteins derived from cryptic exons have been described in ALS. We reported a peripherin splice variant retaining intron 4 and encoding Per 61 protein in mutant SOD1 mice (Robertson et al., 2003), and Per 28 retaining introns 3 and 4 in ALS tissues (Xiao et al., 2008). We also suggested that these isoforms could be used as biomarkers to differentiate between disease/injury paradigms (McLean et al., 2010).

    References:

    Robertson J, Doroudchi MM, Nguyen MD, Durham HD, Strong MJ, Shaw G, Julien JP, Mushynski WE. A neurotoxic peripherin splice variant in a mouse model of ALS. J Cell Biol. 2003 Mar 17;160(6):939-49. PubMed.

    Xiao S, Tjostheim S, Sanelli T, McLean JR, Horne P, Fan Y, Ravits J, Strong MJ, Robertson J. An aggregate-inducing peripherin isoform generated through intron retention is upregulated in amyotrophic lateral sclerosis and associated with disease pathology. J Neurosci. 2008 Feb 20;28(8):1833-40. PubMed.

    McLean J, Liu HN, Miletic D, Weng YC, Rogaeva E, Zinman L, Kriz J, Robertson J. Distinct biochemical signatures characterize peripherin isoform expression in both traumatic neuronal injury and motor neuron disease. J Neurochem. 2010 Aug;114(4):1177-92. PubMed.

    View all comments by Janice Robertson

  8. These two preprints beautifully outline the potential for RNA splicing changes related to TDP-43 proteinopathy to be harnessed as biomarkers, through their translation into more readily tested cryptic peptides. This represents a significant conceptual leap, analogous to the translation of the intronic C9orf72 repeat expansion into dipeptide repeats.

    Taken together, Seddighi et al. and Irwin et al. present compelling evidence that the cryptic peptide of HDGFL2 can distinguish C9orf72-linked cases from controls and predict symptom onset, particularly when combined with neurofilament H or L. It will be of great interest to see how this biomarker performs in larger cohorts of sporadic cases and controls. Combining a panel of these novel cryptic peptide antibodies may improve case/control discrimination against heterogeneous genetic and clinical backgrounds.

    In our own preprint, deposited this week (Cao et al., 2023), we depleted TARDBP from human brain-derived pericytes, generating one of the few non-reprogrammed human TDP-43 loss-of-function models. Combined with previous studies (Cao and Scotter, 2022; Brown et al., 2022; Klim et al., 2019; Ling et al., 2015; Ma et al., 2022; Melamed et al., 2019; Shiga et al., 2012), we identified TDP-43-related splicing changes in human ALS motor cortex tissue. Indeed, combining a panel of such splicing changes discriminated ALS tissue from controls.

    Splicing changes and cryptic peptides are probably more specific and proximal reporters of TDP-43 loss of function than differential gene expression, and the detection of cryptic peptides in the CSF is hugely exciting. We look forward to the application of these findings to better understanding TDP-43 dysfunction, including in non-neuronal cell types, and to improving therapeutic options for people living with ALS and FTD.

    References:

    Brown AL, Wilkins OG, Keuss MJ, Hill SE, Zanovello M, Lee WC, Bampton A, Lee FC, Masino L, Qi YA, Bryce-Smith S, Gatt A, Hallegger M, Fagegaltier D, Phatnani H, NYGC ALS Consortium, Newcombe J, Gustavsson EK, Seddighi S, Reyes JF, Coon SL, Ramos D, Schiavo G, Fisher EM, Raj T, Secrier M, Lashley T, Ule J, Buratti E, Humphrey J, Ward ME, Fratta P. TDP-43 loss and ALS-risk SNPs drive mis-splicing and depletion of UNC13A. Nature. 2022 Mar;603(7899):131-137. Epub 2022 Feb 23 PubMed.

    Cao MC, Scotter EL. Transcriptional targets of amyotrophic lateral sclerosis/frontotemporal dementia protein TDP-43 - meta-analysis and interactive graphical database. Dis Model Mech. 2022 Sep 1;15(9) Epub 2022 Sep 13 PubMed.

    Cao MC, Ryan B, Wu J, Curtis MA, Faull RL, Dragunow M, Scotter EL. Differential exon usage and cryptic exon profiles of TDP-43 loss of function in amyotrophic lateral sclerosis brain tissue. bioRxiv. February 4, 2023 bioRxiv

    Klim JR, Williams LA, Limone F, Guerra San Juan I, Davis-Dusenbery BN, Mordes DA, Burberry A, Steinbaugh MJ, Gamage KK, Kirchner R, Moccia R, Cassel SH, Chen K, Wainger BJ, Woolf CJ, Eggan K. ALS-implicated protein TDP-43 sustains levels of STMN2, a mediator of motor neuron growth and repair. Nat Neurosci. 2019 Feb;22(2):167-179. Epub 2019 Jan 14 PubMed.

    Ling JP, Pletnikova O, Troncoso JC, Wong PC. NEURODEGENERATION. TDP-43 repression of nonconserved cryptic exons is compromised in ALS-FTD. Science. 2015 Aug 7;349(6248):650-5. PubMed.

    Ma XR, Prudencio M, Koike Y, Vatsavayai SC, Kim G, Harbinski F, Briner A, Rodriguez CM, Guo C, Akiyama T, Schmidt HB, Cummings BB, Wyatt DW, Kurylo K, Miller G, Mekhoubad S, Sallee N, Mekonnen G, Ganser L, Rubien JD, Jansen-West K, Cook CN, Pickles S, Oskarsson B, Graff-Radford NR, Boeve BF, Knopman DS, Petersen RC, Dickson DW, Shorter J, Myong S, Green EM, Seeley WW, Petrucelli L, Gitler AD. TDP-43 represses cryptic exon inclusion in the FTD-ALS gene UNC13A. Nature. 2022 Mar;603(7899):124-130. Epub 2022 Feb 23 PubMed.

    Melamed Z, López-Erauskin J, Baughn MW, Zhang O, Drenner K, Sun Y, Freyermuth F, McMahon MA, Beccari MS, Artates JW, Ohkubo T, Rodriguez M, Lin N, Wu D, Bennett CF, Rigo F, Da Cruz S, Ravits J, Lagier-Tourenne C, Cleveland DW. Premature polyadenylation-mediated loss of stathmin-2 is a hallmark of TDP-43-dependent neurodegeneration. Nat Neurosci. 2019 Feb;22(2):180-190. Epub 2019 Jan 14 PubMed.

    Shiga A, Ishihara T, Miyashita A, Kuwabara M, Kato T, Watanabe N, Yamahira A, Kondo C, Yokoseki A, Takahashi M, Kuwano R, Kakita A, Nishizawa M, Takahashi H, Onodera O. Alteration of POLDIP3 splicing associated with loss of function of TDP-43 in tissues affected with ALS. PLoS One. 2012;7(8):e43120. PubMed.

    View all comments by Emma Scotter View all comments by Maize Cao

  9. Using sophisticated, highly sensitive, albeit different approaches, these two papers show that “cryptic” protein sequences can be detected in CSF from ALS/FTD patients. These elusive protein sequences have long been predicted to exist by Dr. Phil Wong and others in the field; however, their presence has only now been demonstrated, for the first time.

    These elegant new detection methods will allow researchers and clinicians to better predict disease progression, which is especially important for asymptomatic mutation carriers, and to better evaluate the outcomes of clinical trials. While the presence of “cryptic” protein sequences reveals an additional layer of complexity in the pathophysiology of ALS/FTD, the real game-changer here lies in the ability of the ”cryptic” protein sequences to serve as much-awaited and much-needed biomarkers for ALS/FTD.

    View all comments by Daniela Zarnescu

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