. NEURODEGENERATION. TDP-43 repression of nonconserved cryptic exons is compromised in ALS-FTD. Science. 2015 Aug 7;349(6248):650-5. PubMed.


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  1. Amyotrophic lateral sclerosis (ALS) occurs as both an inherited disease (~10 percent of cases) and a sporadic disease (~90 percent of cases), implying the contribution of both genetic and environmental/aging-dependent factors to overall risk (Mackenzie et al., 2010). In both forms of the disease, patients display a deposition of TAR DNA-binding protein (TDP)-43 aggregates in the cytoplasm of cells with a concomitant depletion from the nucleus, indicating that TDP-43 protein may be of central importance to disease development. Nevertheless, a main question in the field is whether ALS results from loss of function of wild-type TDP-43 as it is misfolded and incorporated into aggregates, or gain of toxic function from formation of the inclusions. Thus efforts to ameliorate ALS symptoms, as always, must begin with an understanding of TDP-43 function that is illuminated by basic science research. With this in mind, Ling et al. have reported that TDP-43 normally contributes to the fidelity of pre-mRNA splicing.

    TDP-43 is a busy protein, having been ascribed multiple functions that include regulation of microRNA biogenesis (Gregory et al., 2004), splicing and stability of normal transcripts (Polymenidou et al., 2011; Tollervey et al., 2011), localized protein synthesis (Diaper et al., 2013), and formation of stress granules (Colombrita et al., 2009). Ling et al. inducibly ablated TDP-43 expression in mouse ES cells and applied RNA-seq with sufficient read-depth to identify a battery of previously unidentified cryptic exons that were spliced into normal transcripts as a result. Inclusion of cryptic exons was similarly found upon siRNA-mediated depletion of TDP-43 in human HeLa cells, and in both cell types TDP-43 was directly bound at sites adjacent to the cryptic exons (as measured using HITS-CLIP), leading to the hypothesis that normal function of TDP-43 is to repress this process. To test this, an artificial protein was generated comprising the RNA-binding motifs of TDP-43 fused to an unrelated RNA-splicing repressor domain, with the expectation that this should target the fusion protein to the normal binding sites of TDP-43 but use an entirely different splicing repressor to phenocopy the function of endogenous TDP-43. Indeed, expression of this artificial protein rescued cell death associated with TDP-43 depletion and also dampened inclusion of cryptic exons in many of the transcripts previously identified.

    What is the fate of transcripts that contain cryptic exons as a result of TDP-43 depletion? The authors indicate that most are likely eliminated via nonsense-mediated mRNA decay (NMD) because of inclusion of premature termination codons (PTCs). Is the NMD system overwhelmed by inclusion of many diverse transcripts that contain PTCs, and does this stress contribute to cell death? Interestingly, previous links of ALS to NMD have been reported (Barmada et al., 2015; Jackson et al., 2015). Mild overexpression of the RNA helicase UPF1, which is a key NMD factor, partially ameliorates disease symptoms in models where wild-type or mutated TDP-43 are overexpressed but not when TDP-43 is ablated, as was done in Ling et al. Notwithstanding technical caveats, helicase activity of UPF1 is essential for this effect, and a second NMD factor, UPF2, also rescues toxicity. Clearly much remains to be investigated, but these results raise an essential point: How well do TDP-43-based models—either TDP-43 overexpression (Barmada et al., 2010; Tatom et al., 2009) or TDP-43 ablation (as was done by Ling et al)—truly recapitulate the human disease? Superimposed on this is the fact that TDP-43 levels are regulated by a negative feedback loop governed by NMD (Polymenidou et al. 2011), and likewise NMD activity is autoregulated by levels of NMD factors (Huang et al., 2011; Yepiskoposyan et al., 2011), both of which make it necessary to carefully interpret results generated from targeted perturbations in levels of either TDP-43 or NMD-related proteins. The cohort of transcripts found with cryptic exons upon TDP-43 depletion in human and mouse differs, as expected, since the cryptic exons themselves are unlikely to be conserved; thus, the contribution of individual transcripts to disease remains unclear. Anecdotally, SMG5, another NMD factor, appears to contain cryptic exons (Ling et al., Table S1) in mouse, as does UPF2 (Ling et al., Table S3) in humans.

    Overall, Ling et al. report a novel and exciting new function for normal TDP-43: the repression of cryptic exon inclusion in the pool of translation-ready mRNAs. The next challenge is to parse out what contribution this new function makes to disease pathology given the ever-increasing list of jobs already ascribed to TDP-43. More broadly, this study highlights how complicated investigating disease mechanism can be, despite the fact that clinically a single protein is the main feature of TDP-43 proteinopathies and is even used to stage disease progression (Brettschneider et al., 2013). Clearly, a better understating of the disease is a prerequisite for therapeutic intervention.


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