Cryptic exon mis-splicing due to loss of nuclear TDP-43 function has emerged as one of the hallmarks in TDP-43 proteinopathies. One previously identified target is STMN2 RNA, which is particularly interesting due to the crucial role of STMN2 in axonal regeneration. The study by Baughn et al. deciphers in detail the molecular mechanism through which TDP-43 regulates STMN2 pre-mRNA processing and identified antisense oligonucleotides (ASOs) capable of restoring normal stathmin-2 protein and RNA levels within the mammalian nervous system. I congratulate the authors on this exciting and very thoroughly performed study.
Obviously, that cryptic mis-splicing, per se, can be targeted with ASOs is very exciting and promising from a therapeutic point of view. Although, as pointed out by the authors, it remains to be seen if targeting only one of the many mis-spliced RNA targets due to nuclear TDP-43 loss will be sufficient to modulate the clinical disease course of TDP-43 proteinopathies.
This is compelling work, for a number of reasons. First, it is somewhat surprising that the majority of the TDP-43 protein is not required for proper STMN2 splicing. As long as something (MS2 coat protein, CasRx, an ASO) is sitting on the same locus, splicing of STMN2 exon 2A will be repressed. This is incredibly important for the design of therapeutic modalities that act on this locus.
Just as important, the authors found that restoration of STMN2 protein to 25 percent of normal is sufficient to rescue phenotypes such as axonal regeneration. This means that therapies may not have to completely replace STMN2, as effects may be seen with as little as a quarter of the normal STMN2 levels.
The positional effects of ASOs are intriguing. The most effective splice repressors were not those that closely overlap TDP-43 binding sites. Rather, binding downstream of the TDP-43 binding site produces the most impressive effects on STMN2 RNA and protein.
Although the authors demonstrated restoration of STMN2 levels in vivo (in humanized mice) through injection of rASOs, there were no clear symptoms to monitor in these animals. This makes it difficult to gauge the potential therapeutic impact of this strategy.
Original studies hinted at STMN2 loss with TDP43 overexpression, in addition to loss of function. In this study, however, expression of a mutant (Q331K) TDP-43 that remains in the nucleus had little to no effect on STMN2 processing in STMN2 humanized mice. This result argues against STMN2 mis-splicing in TDP-43 transgenic and/or gain-of-function models.
Baughn et al. address the molecular mechanism of splicing repression by TDP-43 by focusing on the inclusion of a non-conserved cryptic exon embedded within intron 1 of STMN2 (Klim et al., 2019; Melamed et al., 2019) and the identification of ASOs to restore its repression as a potential therapeutic strategy for ALS. Using a clever gene-editing approach, the authors showed that TDP-43 binding to UG-rich repeats within this cryptic exon blocks recognition of the 3' splice site in intron 1 to allow proper splicing of STMN2 pre-mRNA.
While this is an attractive mechanism of splicing repression by TDP-43, it is not clear how ALS-linked missense mutant TDP-43 (occurs mostly within C-terminal prion-like domain of TDP-43) apparently fails to repress splicing of this STMN2 cryptic exon (Klim et al., 2019; Melamed et al., 2019) How would one amino acid substitution within the C-terminal domain of TDP-43 release this steric blockage of the recognition of the 3' splice site in intron 1 of STMN2? One resolution is if there exists an instructive repression signal directed to the spliceosome conferred by the C-terminal domain of TDP-43; it is possible that such a signal can be disrupted by missense mutations within this domain. Structural/functional analysis may be necessary to resolve this issue.
Because TDP-43-dependent cryptic exons, including that of STMN2, are non-conserved (Ling et al., 2015), Baughn et al. partially humanized the mouse Stmn2 to validate a therapeutic ASO designed to restore repression of STMN2 cryptic exon. While their data support such ASOs for clinical testing, which recently has already begun (see clinicaltrials.gov), an outstanding question is whether restoring STMN2 cryptic exon repression is sufficient to attenuate neuron loss within the context of neurodegeneration. That genetic knockout/knockdown studies of Stmn2 in mice, which exhibit some, but not all, key features of motor neuron disease, namely motor neuron loss leading to denervation muscle atrophy and paralysis (Guerra San Juan et al., 2022; Lopez-Erauskin et al., 2022), suggests that targeting other TDP-43 dependent cryptic exons, such as UNC13A (Ma et al., 2022; Brown et al., 2022) and other emerging sites, would be required.
If multiple TDP-43-dependent cryptic exons are required to be repressed, the N-terminal half of TDP-43 (containing its RNA binding domains—RRM1 and RRM2) fused to the repressor domain of RAVER1 (Ling et al., 2015; Donde et al., 2019) might prove to be beneficial. Since it is now recognized that loss of TDP-43 splicing repression can occur during presymptomatic stage of ALS-FTD (Seddighi et al., 2023; Irwin et al., 2023), early intervention to complement such loss would be desirable. This unique repressor, shown to complement the loss of TDP-43 splicing repression, when delivered early by an adeno-associated viral vector, would hold promise for the treatment of ALS-FTD.
References:
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.
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.
Guerra San Juan I, Nash LA, Smith KS, Leyton-Jaimes MF, Qian M, Klim JR, Limone F, Dorr AB, Couto A, Pintacuda G, Joseph BJ, Whisenant DE, Noble C, Melnik V, Potter D, Holmes A, Burberry A, Verhage M, Eggan K.
Loss of mouse Stmn2 function causes motor neuropathy.
Neuron. 2022 May 18;110(10):1671-1688.e6. Epub 2022 Mar 15
PubMed.
Lopez-Erauskin J, Bravo-Hernandez M, Presa M, Baughn MW, Melamed Z, Beccari MS, AgradeAlmeidaQuadros AR, Zuberi A, Ling K, Platoshyn O, Nino-Jara E, Ndayambaje IS, Arnold-Garcia O, McAlonis-Downes M, Cabrera L, Artates JW, Ryan J, Bennett F, Jafar-nejad P, Rigo F, Marsala M, Lutz CM, Cleveland DW, Lagier-Tourenne C.
Stathmin-2 loss leads to neurofilament-dependent axonal collapse driving motor and sensory denervation.
2022 Dec 12 10.1101/2022.12.11.519794
(version 1)
bioRxiv.
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.
Correction.
Donde A, Sun M, Ling JP, Braunstein KE, Pang B, Wen X, Cheng X, Chen L, Wong PC.
Splicing repression is a major function of TDP-43 in motor neurons.
Acta Neuropathol. 2019 Nov;138(5):813-826. Epub 2019 Jul 22
PubMed.
Seddighi S, Qi YA, Brown AL, Wilkins OG, Bereda C, Belair C, Zhang Y, Prudencio M, Keuss MJ, Khandeshi A, Pickles S, Hill SE, Hawrot J, Ramos DM, Yuan H, Roberts J, Sacramento EK, Shah SI, Nalls MA, Colon-Mercado J, Reyes JF, Ryan VH, Nelson MP, Cook C, Li Z, Screven L, Kwan JY, Shantaraman A, Ping L, Koike Y, Oskarsson B, Staff N, Duong DM, Ahmed A, Secrier M, Ule J, Jacobson S, Rohrer J, Malaspina A, Glass JD, Ori A, Seyfried NT, Maragkakis M, Petrucelli L, Fratta P, Ward ME.
Mis-spliced transcripts generate de novo proteins in TDP-43-related ALS/FTD.
bioRxiv. January 23, 2023
bioRxiv
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
Comments
University of Tübingen and DZNE AG Neumann
Cryptic exon mis-splicing due to loss of nuclear TDP-43 function has emerged as one of the hallmarks in TDP-43 proteinopathies. One previously identified target is STMN2 RNA, which is particularly interesting due to the crucial role of STMN2 in axonal regeneration. The study by Baughn et al. deciphers in detail the molecular mechanism through which TDP-43 regulates STMN2 pre-mRNA processing and identified antisense oligonucleotides (ASOs) capable of restoring normal stathmin-2 protein and RNA levels within the mammalian nervous system. I congratulate the authors on this exciting and very thoroughly performed study.
Obviously, that cryptic mis-splicing, per se, can be targeted with ASOs is very exciting and promising from a therapeutic point of view. Although, as pointed out by the authors, it remains to be seen if targeting only one of the many mis-spliced RNA targets due to nuclear TDP-43 loss will be sufficient to modulate the clinical disease course of TDP-43 proteinopathies.
View all comments by Manuela NeumannThis is compelling work, for a number of reasons. First, it is somewhat surprising that the majority of the TDP-43 protein is not required for proper STMN2 splicing. As long as something (MS2 coat protein, CasRx, an ASO) is sitting on the same locus, splicing of STMN2 exon 2A will be repressed. This is incredibly important for the design of therapeutic modalities that act on this locus.
Just as important, the authors found that restoration of STMN2 protein to 25 percent of normal is sufficient to rescue phenotypes such as axonal regeneration. This means that therapies may not have to completely replace STMN2, as effects may be seen with as little as a quarter of the normal STMN2 levels.
The positional effects of ASOs are intriguing. The most effective splice repressors were not those that closely overlap TDP-43 binding sites. Rather, binding downstream of the TDP-43 binding site produces the most impressive effects on STMN2 RNA and protein.
Although the authors demonstrated restoration of STMN2 levels in vivo (in humanized mice) through injection of rASOs, there were no clear symptoms to monitor in these animals. This makes it difficult to gauge the potential therapeutic impact of this strategy.
Original studies hinted at STMN2 loss with TDP43 overexpression, in addition to loss of function. In this study, however, expression of a mutant (Q331K) TDP-43 that remains in the nucleus had little to no effect on STMN2 processing in STMN2 humanized mice. This result argues against STMN2 mis-splicing in TDP-43 transgenic and/or gain-of-function models.
View all comments by Sami BarmadaJohns Hopkins
Baughn et al. address the molecular mechanism of splicing repression by TDP-43 by focusing on the inclusion of a non-conserved cryptic exon embedded within intron 1 of STMN2 (Klim et al., 2019; Melamed et al., 2019) and the identification of ASOs to restore its repression as a potential therapeutic strategy for ALS. Using a clever gene-editing approach, the authors showed that TDP-43 binding to UG-rich repeats within this cryptic exon blocks recognition of the 3' splice site in intron 1 to allow proper splicing of STMN2 pre-mRNA.
While this is an attractive mechanism of splicing repression by TDP-43, it is not clear how ALS-linked missense mutant TDP-43 (occurs mostly within C-terminal prion-like domain of TDP-43) apparently fails to repress splicing of this STMN2 cryptic exon (Klim et al., 2019; Melamed et al., 2019) How would one amino acid substitution within the C-terminal domain of TDP-43 release this steric blockage of the recognition of the 3' splice site in intron 1 of STMN2? One resolution is if there exists an instructive repression signal directed to the spliceosome conferred by the C-terminal domain of TDP-43; it is possible that such a signal can be disrupted by missense mutations within this domain. Structural/functional analysis may be necessary to resolve this issue.
Because TDP-43-dependent cryptic exons, including that of STMN2, are non-conserved (Ling et al., 2015), Baughn et al. partially humanized the mouse Stmn2 to validate a therapeutic ASO designed to restore repression of STMN2 cryptic exon. While their data support such ASOs for clinical testing, which recently has already begun (see clinicaltrials.gov), an outstanding question is whether restoring STMN2 cryptic exon repression is sufficient to attenuate neuron loss within the context of neurodegeneration. That genetic knockout/knockdown studies of Stmn2 in mice, which exhibit some, but not all, key features of motor neuron disease, namely motor neuron loss leading to denervation muscle atrophy and paralysis (Guerra San Juan et al., 2022; Lopez-Erauskin et al., 2022), suggests that targeting other TDP-43 dependent cryptic exons, such as UNC13A (Ma et al., 2022; Brown et al., 2022) and other emerging sites, would be required.
If multiple TDP-43-dependent cryptic exons are required to be repressed, the N-terminal half of TDP-43 (containing its RNA binding domains—RRM1 and RRM2) fused to the repressor domain of RAVER1 (Ling et al., 2015; Donde et al., 2019) might prove to be beneficial. Since it is now recognized that loss of TDP-43 splicing repression can occur during presymptomatic stage of ALS-FTD (Seddighi et al., 2023; Irwin et al., 2023), early intervention to complement such loss would be desirable. This unique repressor, shown to complement the loss of TDP-43 splicing repression, when delivered early by an adeno-associated viral vector, would hold promise for the treatment of ALS-FTD.
References:
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.
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.
Guerra San Juan I, Nash LA, Smith KS, Leyton-Jaimes MF, Qian M, Klim JR, Limone F, Dorr AB, Couto A, Pintacuda G, Joseph BJ, Whisenant DE, Noble C, Melnik V, Potter D, Holmes A, Burberry A, Verhage M, Eggan K. Loss of mouse Stmn2 function causes motor neuropathy. Neuron. 2022 May 18;110(10):1671-1688.e6. Epub 2022 Mar 15 PubMed.
Lopez-Erauskin J, Bravo-Hernandez M, Presa M, Baughn MW, Melamed Z, Beccari MS, AgradeAlmeidaQuadros AR, Zuberi A, Ling K, Platoshyn O, Nino-Jara E, Ndayambaje IS, Arnold-Garcia O, McAlonis-Downes M, Cabrera L, Artates JW, Ryan J, Bennett F, Jafar-nejad P, Rigo F, Marsala M, Lutz CM, Cleveland DW, Lagier-Tourenne C. Stathmin-2 loss leads to neurofilament-dependent axonal collapse driving motor and sensory denervation. 2022 Dec 12 10.1101/2022.12.11.519794 (version 1) bioRxiv.
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. Correction.
Donde A, Sun M, Ling JP, Braunstein KE, Pang B, Wen X, Cheng X, Chen L, Wong PC. Splicing repression is a major function of TDP-43 in motor neurons. Acta Neuropathol. 2019 Nov;138(5):813-826. Epub 2019 Jul 22 PubMed.
Seddighi S, Qi YA, Brown AL, Wilkins OG, Bereda C, Belair C, Zhang Y, Prudencio M, Keuss MJ, Khandeshi A, Pickles S, Hill SE, Hawrot J, Ramos DM, Yuan H, Roberts J, Sacramento EK, Shah SI, Nalls MA, Colon-Mercado J, Reyes JF, Ryan VH, Nelson MP, Cook C, Li Z, Screven L, Kwan JY, Shantaraman A, Ping L, Koike Y, Oskarsson B, Staff N, Duong DM, Ahmed A, Secrier M, Ule J, Jacobson S, Rohrer J, Malaspina A, Glass JD, Ori A, Seyfried NT, Maragkakis M, Petrucelli L, Fratta P, Ward ME. Mis-spliced transcripts generate de novo proteins in TDP-43-related ALS/FTD. bioRxiv. January 23, 2023 bioRxiv
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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