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Baughn MW, Melamed Z, López-Erauskin J, Beccari MS, Ling K, Zuberi A, Presa M, Gonzalo-Gil E, Maimon R, Vazquez-Sanchez S, Chaturvedi S, Bravo-Hernández M, Taupin V, Moore S, Artates JW, Acks E, Ndayambaje IS, Agra de Almeida Quadros AR, Jafar-Nejad P, Rigo F, Bennett CF, Lutz C, Lagier-Tourenne C, Cleveland DW. Mechanism of STMN2 cryptic splice-polyadenylation and its correction for TDP-43 proteinopathies. Science. 2023 Mar 17;379(6637):1140-1149. Epub 2023 Mar 16 PubMed.
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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:
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