Does New Role for ALS-Linked Protein Help Explain Neurodegeneration?

As the cell processes gene transcripts into mature mRNAs, just a few specific nucleotides mark sites where exons are spliced in, and introns spliced out. What if, by chance, those nucleotide motifs turned up in the middle of an intron? The result is a “cryptic” exon—one that looks to the spliceosome like the real McCoy, but ought never be incorporated into mRNA (see image below). Now, scientists claim that the job of policing these exons wannabes falls to TDP-43, an RNA-binding protein linked to most cases of amyotrophic lateral sclerosis and many instances of frontotemporal dementia and Alzheimer’s disease. Writing in the August 7 Science, researchers led by Philip Wong at Johns Hopkins University School of Medicine in Baltimore report that in cells lacking TDP-43, cryptic exons make their way  into mRNA, and cell death rapidly ensues. Fixing the splice defect saved the cells. “For the first time, we now really understand what TDP-43 does,” Wong said. “I think suppressing cryptic exons is its principal function.”

Extra Exons.

The spliceosome should link exons, cutting out the whole intron, but without TDP-43 cryptic exons are often spliced into mRNA. [Image courtesy of Philip Wong; Science/AAAS.]

Other scientists who spoke with Alzforum were excited about the finding. “Their study revealed a novel function of TDP-43 in splicing,” commented Fen-Biao Gao of the University of Massachusetts Medical School in Worcester. However, he and others said splicing is one of many roles carried out by the protein. “TDP-43 is involved in multiple processes such as RNA splicing, RNA transport, translation, and microRNA biogenesis,” Gao said (see Feb 2014 news; Feb 2012 news; Highley et al., 2014). Robert Baloh of the Cedars-Sinai Medical Center in Los Angeles also noted that recent studies found TDP-43 maintains transcriptome integrity in other ways, by limiting double-stranded RNAs and silencing expression of mobile genetic elements like transposons (see Feb 2013 news; Li et al., 2012). “[Suppressing cryptic exons] is a very cool new function of TDP-43,” Baloh said. “It is hard to say this is the function that is causing disease, but it is a great lead.”

Three loose consensus sequences label an exon for the spliceosome. The key features include an adenine upstream of the splice site, an AG at the 5’ end, and a GU at the 3’ end. Sometimes, Wong said, nucleotides will line up in that pro-splicing pattern by chance. Some researchers call these pseudoexons. Such accidental splice sites are quite common—one study reported 103 of them in a single gene (Sun and Chasin, 2000). Researchers are finding that these accidental exons may contribute to disease (reviewed by Dhir and Burratti, 2010). Splicing mistakes have been linked to neurodegeneration; a mutation that allows an intron to be spliced into the ALS gene SOD1 causes familial disease (Valdmanis et al., 2009). 

Wong and colleagues uncovered cryptic exons never before described. They only make it into mRNA when TDP-43 is missing. These exons occur in otherwise intronic segments, are not conserved between species, and have TDP-43 binding sites nearby. Wong found 50 of them in mouse embryonic stem cells, and 41 in the human HeLa cancer cell line. However, those numbers only reflect genes expressed by those specific cell types. “There could be hundreds of TDP-43 suppressed cryptic exons that we have not discovered yet,” he said. He has a paper under review that details cryptic exons in neurons.

First author Jonathan Ling initially noticed cryptic exons when he sequenced mRNA from a mouse embryonic stem cell line that had a Cre-inducible TDP-43 knockout. These cells die within a few days of TDP-43 loss, but first, they start making transcripts containing cryptic exons. Some of these altered mRNAs will likely acquire stop codons, and Wong presumes the cell destroys them by nonsense-mediated decay. Others may get translated into proteins carrying potentially harmful segments. Each of the 50 fake exons Ling found in these mouse cells occurred near a UG-repeat-rich region—these are known to attract TDP-43.

Ling and Wong hypothesized that if they could re-repress those cryptic exons, they could rescue the TDP-43 knockout cells. They created a hybrid protein comprising the RNA-targeting domains of TDP-43 and the splice-halting segment from a well-characterized splicing repressor, RAVER1. When they transfected their knockout mouse cells with this hybrid, it eliminated the cryptic exons from mature mRNAs and supported normal cell growth and survival (see image below). Wong concluded that TDP-43's normal function is to suppress this wayward splicing.

RAVER1 to the Rescue.

Cells lacking normal TDP-43, but stably transfected with a version containing the splicing repressor RAVER1 (green), grow normally. [Courtesy of Philip Wong; Science/AAAS.]

What about cryptic exons in people? Since they are not normally expressed and there is no evolutionary pressure to retain these cryptic splice sites, they are not conserved among species. To find human versions that are regulated by TDP-43 regulation, Ling sequenced RNA from HeLa cells after silencing TDP-43 expression by RNA interference. He uncovered mRNAs containing 41 cryptic exons. All had a nearby TDP-43 binding site, and none matched the cryptic exons seen in mouse cells. Among them was ATG4B, a protease that participates in autophagy, which Wong noted has been linked to neurodegeneration (see Jul 2015 news; Jul 2014 news; Jul 2010 news). Another was RANBP1, which is involved in nuclear import. Wong speculated loss of RANBP1 might make it harder for the cell to send mislocalized cytoplasmic TDP-43 back to the nucleus.

Next the researchers used PCR to look for these nonconserved cryptic exons in postmortem tissue from people with TDP-43 proteinopathy. They tested brain samples from seven people who had died of FTD, ALS, or ALS-FTD and who had TDP-43 pathology. All contained transcripts with the erroneous splice forms.

How might this particular TDP-43 function be related to disease? The authors propose that in the nucleus, the protein binds the introns of unprocessed mRNAs and suppresses any nearby cryptic exons. If TDP-43 vacates the nucleus, as it does in disease states, it leaves those RNAs unprotected and the spliceosome includes the useless cryptic exons. With the transcriptome and proteome so altered, neurons degenerate, they suggest. The authors are now comparing their lists of mouse and human genes affected by cryptic exons, looking for commonalities that might be involved in neurodegeneration.

The theory lines up with results from a few other labs. Researchers discovered that TDP-43 often attaches to long introns, and Wong’s ideas would explain its presence there (see Mar 2011 news). In addition, researchers have discovered that boosting nonsense-mediated decay protects cells and animal models from TDP-43 proteinopathy (see Jun 2015 news). Sami Barmada of the University of Michigan Medical School in Ann Arbor said Ling’s work provides another reason to think nonsense-mediated decay is important for neuronal health.

Gene Yeo of the University of California in San Diego, one of the scientists who originally profiled TDP-43 RNA binding sites, found the results not entirely surprising. TDP-43 was already understood to regulate splicing of known, annotated exons, he said, so it makes sense that it also manages cryptic ones. While he found the study intriguing, he wondered about its relevance to human disease. Ling’s assay for cryptic splicing in brain tissue was not quantitative, he noted, so it could not tell if those brains contained one or 100,000 mRNAs with the cryptic exons. In addition, he said researchers need to look in cell types more relevant to human disease, not just mouse embryonic stem cells and the transformed HeLa line.

Wong said such work is ongoing; he wants to repeat these experiments in neurons derived from human induced pluripotent stem cells or fibroblasts, starting with healthy donors but eventually looking at disease states, including people with AD. In addition, he wants to test a gene therapy in mice that have no TDP-43 in their forebrain neurons, giving them the RAVER1 hybrid in the hopes of rescuing their defective splicing.

Gao was intrigued by another of Wong’s future projects: using the abnormal proteins made by cryptic transcripts as biomarkers for TDP-43 pathology. Wong’s lab is already designing antibodies to unique peptides that would only occur if cryptic exons are spliced into mRNAs, which would indicate TDP-43 left the nucleus. “If some of this protein gets into cerebrospinal fluid and you could detect it long before terminal neurodegeneration, that would be very cool,” Gao said.—Amber Dance

Comments

  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.

    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.

    View all comments by Lynne Maquat View all comments by Max Popp

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References

News Citations

  1. Escort Service: A Cytoplasmic Role for TDP-43
  2. Slicing and Dicing: TDP-43 Teams Up With Nucleases to Make MicroRNAs
  3. Does ALS Gene Police RNA, Keep Strands From Entangling?
  4. Enhanced Autophagy Protects Against ALS Proteotoxicity
  5. Can Autophagy Protect ALS Cell Models from Mutant TDP-43?
  6. Mutant SOD1 Inflames If Not Quenched by Autophagy
  7. CLIPs of TDP-43 Provide a Glimpse Into Pathology
  8. Scientists Eager to Test ALS Gene Therapy

Paper Citations

  1. Highley JR, Kirby J, Jansweijer JA, Webb PS, Hewamadduma CA, Heath PR, Higginbottom A, Raman R, Ferraiuolo L, Cooper-Knock J, McDermott CJ, Wharton SB, Shaw PJ, Ince PG. Loss of nuclear TDP-43 in amyotrophic lateral sclerosis (ALS) causes altered expression of splicing machinery and widespread dysregulation of RNA splicing in motor neurones. Neuropathol Appl Neurobiol. 2014 Oct;40(6):670-85. PubMed.
  2. Li W, Jin Y, Prazak L, Hammell M, Dubnau J. Transposable elements in TDP-43-mediated neurodegenerative disorders. PLoS One. 2012;7(9):e44099. Epub 2012 Sep 5 PubMed.
  3. Sun H, Chasin LA. Multiple splicing defects in an intronic false exon. Mol Cell Biol. 2000 Sep;20(17):6414-25. PubMed.
  4. Dhir A, Buratti E. Alternative splicing: role of pseudoexons in human disease and potential therapeutic strategies. FEBS J. 2010 Feb;277(4):841-55. Epub 2010 Jan 15 PubMed.
  5. Valdmanis PN, Belzil VV, Lee J, Dion PA, St-Onge J, Hince P, Funalot B, Couratier P, Clavelou P, Camu W, Rouleau GA. A mutation that creates a pseudoexon in SOD1 causes familial ALS. Ann Hum Genet. 2009 Nov;73(Pt 6):652-7. PubMed.

Further Reading

Papers

  1. Chiang PM, Ling J, Jeong YH, Price DL, Aja SM, Wong PC. Deletion of TDP-43 down-regulates Tbc1d1, a gene linked to obesity, and alters body fat metabolism. Proc Natl Acad Sci U S A. 2010 Sep 14;107(37):16320-4. PubMed.
  2. Eom T, Zhang C, Wang H, Lay K, Fak J, Noebels JL, Darnell RB. NOVA-dependent regulation of cryptic NMD exons controls synaptic protein levels after seizure. Elife. 2013 Jan 22;2:e00178. PubMed.
  3. Arnold ES, Ling SC, Huelga SC, Lagier-Tourenne C, Polymenidou M, Ditsworth D, Kordasiewicz HB, McAlonis-Downes M, Platoshyn O, Parone PA, Da Cruz S, Clutario KM, Swing D, Tessarollo L, Marsala M, Shaw CE, Yeo GW, Cleveland DW. ALS-linked TDP-43 mutations produce aberrant RNA splicing and adult-onset motor neuron disease without aggregation or loss of nuclear TDP-43. Proc Natl Acad Sci U S A. 2013 Feb 19;110(8):E736-45. PubMed.
  4. Passoni M, De Conti L, Baralle M, Buratti E. UG repeats/TDP-43 interactions near 5' splice sites exert unpredictable effects on splicing modulation. J Mol Biol. 2012 Jan 6;415(1):46-60. PubMed.
  5. Xiao S, Sanelli T, Dib S, Sheps D, Findlater J, Bilbao J, Keith J, Zinman L, Rogaeva E, Robertson J. RNA targets of TDP-43 identified by UV-CLIP are deregulated in ALS. Mol Cell Neurosci. 2011 Jul;47(3):167-80. PubMed.
  6. Saldi TK, Ash PE, Wilson G, Gonzales P, Garrido-Lecca A, Roberts CM, Dostal V, Gendron TF, Stein LD, Blumenthal T, Petrucelli L, Link CD. TDP-1, the Caenorhabditis elegans ortholog of TDP-43, limits the accumulation of double-stranded RNA. EMBO J. 2014 Dec 17;33(24):2947-66. Epub 2014 Nov 12 PubMed.

Primary Papers

  1. 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.

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