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.
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.
References:
Mackenzie IR, Rademakers R, Neumann M.
TDP-43 and FUS in amyotrophic lateral sclerosis and frontotemporal dementia.
Lancet Neurol. 2010 Oct;9(10):995-1007.
PubMed.
Gregory RI, Yan KP, Amuthan G, Chendrimada T, Doratotaj B, Cooch N, Shiekhattar R.
The Microprocessor complex mediates the genesis of microRNAs.
Nature. 2004 Nov 11;432(7014):235-40.
PubMed.
Polymenidou M, Lagier-Tourenne C, Hutt KR, Huelga SC, Moran J, Liang TY, Ling SC, Sun E, Wancewicz E, Mazur C, Kordasiewicz H, Sedaghat Y, Donohue JP, Shiue L, Bennett CF, Yeo GW, Cleveland DW.
Long pre-mRNA depletion and RNA missplicing contribute to neuronal vulnerability from loss of TDP-43.
Nat Neurosci. 2011 Apr;14(4):459-68.
PubMed.
Tollervey JR, Curk T, Rogelj B, Briese M, Cereda M, Kayikci M, König J, Hortobágyi T, Nishimura AL, Zupunski V, Patani R, Chandran S, Rot G, Zupan B, Shaw CE, Ule J.
Characterizing the RNA targets and position-dependent splicing regulation by TDP-43.
Nat Neurosci. 2011 Apr;14(4):452-8.
PubMed.
Diaper DC, Adachi Y, Lazarou L, Greenstein M, Simoes FA, Di Domenico A, Solomon DA, Lowe S, Alsubaie R, Cheng D, Buckley S, Humphrey DM, Shaw CE, Hirth F.
Drosophila TDP-43 dysfunction in glia and muscle cells cause cytological and behavioural phenotypes that characterize ALS and FTLD.
Hum Mol Genet. 2013 Jun 25;
PubMed.
Colombrita C, Zennaro E, Fallini C, Weber M, Sommacal A, Buratti E, Silani V, Ratti A.
TDP-43 is recruited to stress granules in conditions of oxidative insult.
J Neurochem. 2009 Nov;111(4):1051-61. Epub 2009 Sep 16
PubMed.
Barmada SJ, Ju S, Arjun A, Batarse A, Archbold HC, Peisach D, Li X, Zhang Y, Tank EM, Qiu H, Huang EJ, Ringe D, Petsko GA, Finkbeiner S.
Amelioration of toxicity in neuronal models of amyotrophic lateral sclerosis by hUPF1.
Proc Natl Acad Sci U S A. 2015 Jun 23;112(25):7821-6. Epub 2015 Jun 8
PubMed.
Jackson KL, Dayton RD, Orchard EA, Ju S, Ringe D, Petsko GA, Maquat LE, Klein RL.
Preservation of forelimb function by UPF1 gene therapy in a rat model of TDP-43-induced motor paralysis.
Gene Ther. 2015 Jan;22(1):20-8. Epub 2014 Nov 6
PubMed.
Barmada SJ, Skibinski G, Korb E, Rao EJ, Wu JY, Finkbeiner S.
Cytoplasmic mislocalization of TDP-43 is toxic to neurons and enhanced by a mutation associated with familial amyotrophic lateral sclerosis.
J Neurosci. 2010 Jan 13;30(2):639-49.
PubMed.
Tatom JB, Wang DB, Dayton RD, Skalli O, Hutton ML, Dickson DW, Klein RL.
Mimicking aspects of frontotemporal lobar degeneration and Lou Gehrig's disease in rats via TDP-43 overexpression.
Mol Ther. 2009 Apr;17(4):607-13.
PubMed.
Huang L, Lou CH, Chan W, Shum EY, Shao A, Stone E, Karam R, Song HW, Wilkinson MF.
RNA homeostasis governed by cell type-specific and branched feedback loops acting on NMD.
Mol Cell. 2011 Sep 16;43(6):950-61.
PubMed.
Yepiskoposyan H, Aeschimann F, Nilsson D, Okoniewski M, Mühlemann O.
Autoregulation of the nonsense-mediated mRNA decay pathway in human cells.
RNA. 2011 Dec;17(12):2108-18. Epub 2011 Oct 25
PubMed.
Brettschneider J, Del Tredici K, Toledo JB, Robinson JL, Irwin DJ, Grossman M, Suh E, Van Deerlin VM, Wood EM, Baek Y, Kwong L, Lee EB, Elman L, McCluskey L, Fang L, Feldengut S, Ludolph AC, Lee VM, Braak H, Trojanowski JQ.
Stages of pTDP-43 pathology in amyotrophic lateral sclerosis.
Ann Neurol. 2013 Jul;74(1):20-38.
PubMed.
Comments
University of Rochester Medical Center
University of Rochester
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.
References:
Mackenzie IR, Rademakers R, Neumann M. TDP-43 and FUS in amyotrophic lateral sclerosis and frontotemporal dementia. Lancet Neurol. 2010 Oct;9(10):995-1007. PubMed.
Gregory RI, Yan KP, Amuthan G, Chendrimada T, Doratotaj B, Cooch N, Shiekhattar R. The Microprocessor complex mediates the genesis of microRNAs. Nature. 2004 Nov 11;432(7014):235-40. PubMed.
Polymenidou M, Lagier-Tourenne C, Hutt KR, Huelga SC, Moran J, Liang TY, Ling SC, Sun E, Wancewicz E, Mazur C, Kordasiewicz H, Sedaghat Y, Donohue JP, Shiue L, Bennett CF, Yeo GW, Cleveland DW. Long pre-mRNA depletion and RNA missplicing contribute to neuronal vulnerability from loss of TDP-43. Nat Neurosci. 2011 Apr;14(4):459-68. PubMed.
Tollervey JR, Curk T, Rogelj B, Briese M, Cereda M, Kayikci M, König J, Hortobágyi T, Nishimura AL, Zupunski V, Patani R, Chandran S, Rot G, Zupan B, Shaw CE, Ule J. Characterizing the RNA targets and position-dependent splicing regulation by TDP-43. Nat Neurosci. 2011 Apr;14(4):452-8. PubMed.
Diaper DC, Adachi Y, Lazarou L, Greenstein M, Simoes FA, Di Domenico A, Solomon DA, Lowe S, Alsubaie R, Cheng D, Buckley S, Humphrey DM, Shaw CE, Hirth F. Drosophila TDP-43 dysfunction in glia and muscle cells cause cytological and behavioural phenotypes that characterize ALS and FTLD. Hum Mol Genet. 2013 Jun 25; PubMed.
Colombrita C, Zennaro E, Fallini C, Weber M, Sommacal A, Buratti E, Silani V, Ratti A. TDP-43 is recruited to stress granules in conditions of oxidative insult. J Neurochem. 2009 Nov;111(4):1051-61. Epub 2009 Sep 16 PubMed.
Barmada SJ, Ju S, Arjun A, Batarse A, Archbold HC, Peisach D, Li X, Zhang Y, Tank EM, Qiu H, Huang EJ, Ringe D, Petsko GA, Finkbeiner S. Amelioration of toxicity in neuronal models of amyotrophic lateral sclerosis by hUPF1. Proc Natl Acad Sci U S A. 2015 Jun 23;112(25):7821-6. Epub 2015 Jun 8 PubMed.
Jackson KL, Dayton RD, Orchard EA, Ju S, Ringe D, Petsko GA, Maquat LE, Klein RL. Preservation of forelimb function by UPF1 gene therapy in a rat model of TDP-43-induced motor paralysis. Gene Ther. 2015 Jan;22(1):20-8. Epub 2014 Nov 6 PubMed.
Barmada SJ, Skibinski G, Korb E, Rao EJ, Wu JY, Finkbeiner S. Cytoplasmic mislocalization of TDP-43 is toxic to neurons and enhanced by a mutation associated with familial amyotrophic lateral sclerosis. J Neurosci. 2010 Jan 13;30(2):639-49. PubMed.
Tatom JB, Wang DB, Dayton RD, Skalli O, Hutton ML, Dickson DW, Klein RL. Mimicking aspects of frontotemporal lobar degeneration and Lou Gehrig's disease in rats via TDP-43 overexpression. Mol Ther. 2009 Apr;17(4):607-13. PubMed.
Huang L, Lou CH, Chan W, Shum EY, Shao A, Stone E, Karam R, Song HW, Wilkinson MF. RNA homeostasis governed by cell type-specific and branched feedback loops acting on NMD. Mol Cell. 2011 Sep 16;43(6):950-61. PubMed.
Yepiskoposyan H, Aeschimann F, Nilsson D, Okoniewski M, Mühlemann O. Autoregulation of the nonsense-mediated mRNA decay pathway in human cells. RNA. 2011 Dec;17(12):2108-18. Epub 2011 Oct 25 PubMed.
Brettschneider J, Del Tredici K, Toledo JB, Robinson JL, Irwin DJ, Grossman M, Suh E, Van Deerlin VM, Wood EM, Baek Y, Kwong L, Lee EB, Elman L, McCluskey L, Fang L, Feldengut S, Ludolph AC, Lee VM, Braak H, Trojanowski JQ. Stages of pTDP-43 pathology in amyotrophic lateral sclerosis. Ann Neurol. 2013 Jul;74(1):20-38. PubMed.
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