Splicing repression is a major function of TDP-43 in motor neurons
Nuclear depletion of TDP-43, an essential RNA binding protein, may underlie neurodegeneration in amyotrophic lateral sclerosis (ALS). As several functions have been ascribed to this protein, the critical role(s) of TDP-43 in motor neurons that may be compromised in ALS remains unknown. We show here that TDP-43 mediated splicing repression, which serves to protect the transcriptome by preventing aberrant splicing, is central to the physiology of motor neurons. Expression in Drosophila TDP-43 knockout models of a chimeric repressor, comprised of the RNA recognition domain of TDP-43 fused to an unrelated splicing repressor, RAVER1, attenuated motor deficits and extended lifespan. Likewise, AAV9-mediated delivery of this chimeric rescue repressor to mice lacking TDP-43 in motor neurons delayed the onset, slowed the progression of motor symptoms, and markedly extended their lifespan. In treated mice lacking TDP-43 in motor neurons, aberrant splicing was significantly decreased and accompanied by amelioration of axon degeneration and motor neuron loss. This AAV9 strategy allowed long-term expression of the chimeric repressor without any adverse effects. Our findings establish that splicing repression is a major function of TDP-43 in motor neurons and strongly support the idea that loss of TDP-43-mediated splicing fidelity represents a key pathogenic mechanism underlying motor neuron loss in ALS.
KeywordsAmyotrophic lateral sclerosis Cryptic exon Drosophila Motor neuron Mouse TDP-43
We thank L. Martin, C. Sumner, and T. Lloyd for thoughtful comments and V. Nehus and H. Wu for technical assistance.
PCW, LC, AD and MS designed the experiments. JPL designed and cloned the constructs. AD and BP performed ICV injections. AD, BP, XW, and XC performed behavioral analyses in mice, and MS and LC in Drosophila. AD performed histological and RNA analysis in mice, and MS and LC in Drosophila. AD and KEB analyzed and interpreted data. AD, MS, LC, and PCW wrote the manuscript with the approval of all authors.
This work was supported by the NIH Grant No. R01 NS095969 (PCW), McKnight Memory and Cognitive Disorders Award (PCW and LC), Robert Packard Center for ALS Research (LC and PCW) and the Amyotrophic Lateral Sclerosis Association (PCW). JPL is a recipient of a Johns Hopkins Kavli Neuroscience Discovery Institute fellowship award.
Compliance with ethical standards
Conflict of interest
Authors declare no competing interests.
Movie S1. Representative video of p90 untreated ChAT-IRES-Cre;tardbpF/F mice (left cage) and a CTR-treated ChAT-IRES-Cre;tardbpF/F mouse (right cage). At the end of the video, a representative ChAT-IRES-Cre;TardbpF/+ mouse is shown as well. The improvement in motor function in CTR-treated Tardbp knockout mice is evident by the increased locomotion and hindlimb mobility
- 9.Chew J, Gendron TF, Prudencio M, Sasaguri H, Zhang YJ, Castanedes-Casey M et al (2015) Neurodegeneration. C9ORF72 repeat expansions in mice cause TDP-43 pathology, neuronal loss, and behavioral deficits. Science 348:1151–1154. https://doi.org/10.1126/science.aaa9344 CrossRefPubMedPubMedCentralGoogle Scholar
- 29.LaClair KD, Donde A, Ling JP, Jeong YH, Chhabra R, Martin LJ et al (2016) Depletion of TDP-43 decreases fibril and plaque beta-amyloid and exacerbates neurodegeneration in an Alzheimer’s mouse model. Acta neuropathologica. https://doi.org/10.1007/s00401-016-1637-y (SQSTM1/p62859–62873) CrossRefPubMedPubMedCentralGoogle Scholar
- 38.McGurk L, Gomes E, Guo L, Mojsilovic-Petrovic J, Tran V, Kalb RG et al (2018) Poly(ADP-Ribose) prevents pathological phase separation of TDP-43 by promoting liquid demixing and stress granule localization. Mol Cell 71(703–717):e709. https://doi.org/10.1016/j.molcel.2018.07.002 CrossRefGoogle Scholar
- 44.Neumann M, Kwong LK, Truax AC, Vanmassenhove B, Kretzschmar HA, Van Deerlin VM et al (2007) TDP-43-positive white matter pathology in frontotemporal lobar degeneration with ubiquitin-positive inclusions. J Neuropathol Exp Neurol 66:177–183. https://doi.org/10.1097/01.jnen.0000248554.45456.58 CrossRefPubMedGoogle Scholar
- 48.Renton AE, Majounie E, Waite A, Simon-Sanchez J, Rollinson S, Gibbs JR, Schymick JC, Laaksovirta H et al (2011) A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD. Neuron 72:257–268. https://doi.org/10.1016/j.neuron.2011.09.010 CrossRefPubMedPubMedCentralGoogle Scholar
- 53.Schmid B, Hruscha A, Hogl S, Banzhaf-Strathmann J, Strecker K, van der Zee J et al (2013) Loss of ALS-associated TDP-43 in zebrafish causes muscle degeneration, vascular dysfunction, and reduced motor neuron axon outgrowth. Proc Natl Acad Sci USA 110:4986–4991. https://doi.org/10.1073/pnas.1218311110 CrossRefPubMedGoogle Scholar
- 57.Sun M, Bell W, LaClair KD, Ling JP, Han H, Kageyama Y et al (2017) Cryptic exon incorporation occurs in Alzheimer’s brain lacking TDP-43 inclusion but exhibiting nuclear clearance of TDP-43. Acta Neuropathol 133:923–931. https://doi.org/10.1007/s00401-017-1701-2 CrossRefPubMedPubMedCentralGoogle Scholar
- 64.Wils H, Kleinberger G, Janssens J, Pereson S, Joris G, Cuijt I et al (2010) TDP-43 transgenic mice develop spastic paralysis and neuronal inclusions characteristic of ALS and frontotemporal lobar degeneration. Proc Natl Acad Sci USA 107:3858–3863. https://doi.org/10.1073/pnas.0912417107 CrossRefPubMedGoogle Scholar
- 67.Xu YF, Gendron TF, Zhang YJ, Lin WL, D’Alton S, Sheng H et al (2010) Wild-type human TDP-43 expression causes TDP-43 phosphorylation, mitochondrial aggregation, motor deficits, and early mortality in transgenic mice. J Neurosci 30:10851–10859. https://doi.org/10.1523/JNEUROSCI.1630-10.2010 CrossRefPubMedPubMedCentralGoogle Scholar