Skip to main content

TDP-43 transports ribosomal protein mRNA to regulate axonal local translation in neuronal axons

Acta Neuropathologica volume 140, pages 695–713 (2020)Cite this article

Abstract

Mislocalization and abnormal deposition of TDP-43 into the cytoplasm (TDP-43 proteinopathy) is a hallmark in neurons of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). However, the pathogenic mechanism of the diseases linked to TDP-43 is largely unknown. We hypothesized that the failure of mRNA transport to neuronal axons by TDP-43 may contribute to neurodegeneration in ALS and FTLD, and sought to examine the function of TDP-43 by identifying its target mRNA for axonal transport. We found that mRNAs related to translational function including ribosomal proteins (RPs) were decreased by shRNA-based TDP-43 knock-down in neurites of cortical neurons. TDP-43 binds to and transports the RP mRNAs through their 5′ untranslated region, which contains a common 5′ terminal oligopyrimidine tract motif and a downstream GC-rich region. We showed by employing in vitro and in vivo models that the RP mRNAs were translated and incorporated into native ribosomes locally in axons to maintain functionality of axonal ribosomes, which is required for local protein synthesis in response to stimulation and stress to axons. We also found that RP mRNAs were reduced in the pyramidal tract of sporadic ALS cases harboring TDP-43 pathology. Our results elucidated a novel function of TDP-43 to control transport of RP mRNAs and local translation by ribosomes to maintain morphological integrity of neuronal axons, and proved the influence of this function of TDP-43 on neurodegeneration in ALS and FTLD associated with TDP-43 proteinopathy.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

USD 39.95

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

References

  1. Akashi K, Kakizaki T, Kamiya H, Fukaya M, Yamasaki M, Abe M et al (2009) NMDA receptor GluN2B (GluR epsilon 2/NR2B) subunit is crucial for channel function, postsynaptic macromolecular organization, and actin cytoskeleton at hippocampal CA3 synapses. J Neurosci 29:10869–10882

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Alami NH, Smith RB, Carrasco MA, Williams LA, Winborn CS, Han SS et al (2014) Axonal transport of TDP-43 mRNA granules is impaired by ALS-causing mutations. Neuron 81:536–543

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Arai T, Hasegawa M, Akiyama H, Ikeda K, Nonaka T, Mori H et al (2006) TDP-43 is a component of ubiquitin-positive tau-negative inclusions in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Biochem Biophys Res Commun 351:602–611

    CAS  PubMed  Google Scholar 

  4. Araki T, Milbrandt J (2003) ZNRF proteins constitute a family of presynaptic E3 ubiquitin ligases. J Neurosci 23:9385–9394

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Araki T, Sasaki Y (2004) Milbrandt, J (2004) Increased nuclear NAD biosynthesis and Sir2 activation prevent axonal degeneration. Science 305:1010–1013

    CAS  PubMed  Google Scholar 

  6. Bannai H, Fukatsu K, Mizutani A, Natsume T, Iemura S, Ikegami T et al (2004) An RNA-interacting protein, SYNCRIP (heterogeneous nuclear ribonuclear protein Q1/NSAP1) is a component of mRNA granule transported with inositol 1,4,5-trisphosphate receptor type 1 mRNA in neuronal dendrites. J Biol Chem 279:53427–53434

    CAS  PubMed  Google Scholar 

  7. Bertrand E, Chartrand P, Schaefer M, Shenoy SM, Singer RH, Long RM (1998) Localization of ASH1 mRNA particles in living yeast. Mol Cell 2:437–445

    CAS  PubMed  Google Scholar 

  8. Caldarola S, De Stefano MC, Amaldi F, Loreni F (2009) Synthesis and function of ribosomal proteins–fading models and new perspectives. Febs J 276:3199–3210

    CAS  PubMed  Google Scholar 

  9. Chu J, Majumder P, Chatterjee B, Huang S, Shen CJ (2019) TDP-43 regulates coupled dendritic mRNA transport-translation processes in co-operation with FMRP and staufen1. Cell Rep 29:3118–3133

    CAS  PubMed  Google Scholar 

  10. Coyne AN, Siddegowda BB, Estes PS, Johannesmeyer J, Kovalik T, Daniel SG et al (2014) Futsch/MAP1B mRNA is a translational target of TDP-43 and is neuroprotective in a Drosophila model of amyotrophic lateral sclerosis. J Neurosci 34:15962–15974

    PubMed  PubMed Central  Google Scholar 

  11. Damgaard CK, Lykke-Andersen J (2011) Translational coregulation of 5'TOP mRNAs by TIA-1 and TIAR. Genes Dev 25:2057–2068

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Dunn KW, Kamocka MM, McDonald JH (2011) A practical guide to evaluating colocalization in biological microscopy. Am J Physiol Cell Physiol 300:C723–742

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Fallini C, Bassell GJ, Rossoll W (2012) The ALS disease protein TDP-43 is actively transported in motor neuron axons and regulates axon outgrowth. Hum Mol Genet 21:3703–3718

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Fallini C, Donlin-Asp PG, Rouanet JP, Bassell GJ, Rossoll W (2016) Deficiency of the survival of motor neuron protein impairs mRNA localization and local translation in the growth cone of motor neurons. J Neurosci 36:3811–3820

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Gazda HT, Preti M, Sheen MR, O'Donohue MF, Vlachos A, Davies SM et al (2012) Frameshift mutation in p53 regulator RPL26 is associated with multiple physical abnormalities and a specific pre-ribosomal RNA processing defect in diamond-blackfan anemia. Hum Mutat 33:1037–1044

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Habuchi S, Tsutsui H, Kochaniak AB, Miyawaki A, van Oijen AM (2008) mKikGR, a monomeric photoswitchable fluorescent protein. PLoS ONE 3:e3944

    PubMed  PubMed Central  Google Scholar 

  17. Hamilton TL, Stoneley M, Spriggs KA, Bushell M (2006) TOPs and their regulation. Biochem Soc Trans 34:12–16

    CAS  PubMed  Google Scholar 

  18. Igaz LM, Kwong LK, Lee EB, Chen-Plotkin A, Swanson E, Unger T et al (2011) Dysregulation of the ALS-associated gene TDP-43 leads to neuronal death and degeneration in mice. J Clin Invest 121:726–738

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Iguchi Y, Katsuno M, Niwa J, Takagi S, Ishigaki S, Ikenaka K et al (2013) Loss of TDP-43 causes age-dependent progressive motor neuron degeneration. Brain 136:1371–1382

    PubMed  Google Scholar 

  20. Intine RV, Tenenbaum SA, Sakulich AL, Keene JD, Maraia RJ (2003) Differential phosphorylation and subcellular localization of La RNPs associated with precursor tRNAs and translation-related mRNAs. Mol Cell 12:1301–1307

    CAS  PubMed  Google Scholar 

  21. Ishiguro A, Kimura N, Watanabe Y, Watanabe S, Ishihama A (2016) TDP-43 binds and transports G-quadruplex-containing mRNAs into neurites for local translation. Genes Cells 21:466–481

    CAS  PubMed  Google Scholar 

  22. Kabashi E, Lin L, Tradewell ML, Dion PA, Bercier V, Bourgouin P et al (2010) Gain and loss of function of ALS-related mutations of TARDBP (TDP-43) cause motor deficits in vivo. Hum Mol Genet 19:671–683

    CAS  PubMed  Google Scholar 

  23. Kabashi E, Valdmanis PN, Dion P, Spiegelman D, McConkey BJ, Vande Velde C et al (2008) TARDBP mutations in individuals with sporadic and familial amyotrophic lateral sclerosis. Nat Genet 40:572–574

    CAS  PubMed  Google Scholar 

  24. Kakegawa T, Ohuchi N, Hayakawa A, Hirata S, Matsuda M, Kogure K et al (2007) Identification of AUF1 as a rapamycin-responsive binding protein to the 5'-terminal oligopyrimidine element of mRNAs. Arch Biochem Biophys 465:274–281

    CAS  PubMed  Google Scholar 

  25. Kawahara Y, Mieda-Sato A (2012) TDP-43 promotes microRNA biogenesis as a component of the Drosha and Dicer complexes. Proc Natl Acad Sci U S A 109:3347–3352

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Kelleher RJ 3rd, Govindarajan A, Tonegawa S (2004) Translational regulatory mechanisms in persistent forms of synaptic plasticity. Neuron 44:59–73

    CAS  PubMed  Google Scholar 

  27. Krichevsky AM, Kosik KS (2001) Neuronal RNA granules: a link between RNA localization and stimulation-dependent translation. Neuron 32:683–696

    CAS  PubMed  Google Scholar 

  28. Lagier-Tourenne C, Polymenidou M, Hutt KR, Vu AQ, Baughn M, Huelga SC et al (2012) Divergent roles of ALS-linked proteins FUS/TLS and TDP-43 intersect in processing long pre-mRNAs. Nat Neurosci 15:1488–1497

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Ling SC, Polymenidou M, Cleveland DW (2013) Converging mechanisms in ALS and FTD: disrupted RNA and protein homeostasis. Neuron 79:416–438

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Liu-Yesucevitz L, Bassell GJ, Gitler AD, Hart AC, Klann E, Richter JD et al (2011) Local RNA translation at the synapse and in disease. J Neurosci 31:16086–16093

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Majumder P, Chu JF, Chatterjee B, Swamy KB, Shen CJ (2016) Co-regulation of mRNA translation by TDP-43 and Fragile X syndrome protein FMRP. Acta Neuropathol 132:721–738

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Mathis AD, Naylor BC, Carson RH, Evans E, Harwell J, Knecht J et al (2017) Mechanisms of in vivo ribosome maintenance change in response to nutrient signals. Mol Cell Proteom 16:243–254

    CAS  Google Scholar 

  33. Meyuhas O (2000) Synthesis of the translational apparatus is regulated at the translational level. Eur J Biochem 267:6321–6330

    CAS  PubMed  Google Scholar 

  34. Neumann M, Sampathu DM, Kwong LK, Truax AC, Micsenyi MC, Chou TT et al (2006) Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Science 314:130–133

    CAS  PubMed  Google Scholar 

  35. Polymenidou M, Lagier-Tourenne C, Hutt KR, Huelga SC, Moran J, Liang TY et al (2011) Long pre-mRNA depletion and RNA missplicing contribute to neuronal vulnerability from loss of TDP-43. Nat Neurosci 14:459–468

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Qiu D, Parada P, Marcos AG, Cardenas D, Remacha M, Ballesta JP (2006) Different roles of P1 and P2 Saccharomyces cerevisiae ribosomal stalk proteins revealed by cross-linking. Mol Microbiol 62:1191–1202

    CAS  PubMed  Google Scholar 

  37. Ratti A, Buratti E (2016) Physiological functions and pathobiology of TDP-43 and FUS/TLS proteins. J Neurochem 138(Suppl 1):95–111

    CAS  PubMed  Google Scholar 

  38. Shigeoka T, Koppers M, Wong HHW, Lin JQ, Cagnetta R, Dwivedy A et al (2019) On-site ribosome remodeling by locally synthesized ribosomal protein in axons. Cell Rep 29:3605–3619

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Slomnicki LP, Pietrzak M, Vashishta A, Jones J, Lynch N, Elliot S et al (2016) Requirement of neuronal ribosome synthesis for growth and maintenance of the dendritic tree. J Biol Chem 291:5721–5739

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Smith BN, Ticozzi N, Fallini C, Gkazi AS, Topp S, Kenna KP et al (2014) Exome-wide rare variant analysis identifies TUBA4A mutations associated with familial ALS. Neuron 84:324–331

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Sreedharan J, Blair IP, Tripathi VB, Hu X, Vance C, Rogelj B et al (2008) TDP-43 mutations in familial and sporadic amyotrophic lateral sclerosis. Science 319:1668–1672

    CAS  PubMed  Google Scholar 

  42. Swarup V, Phaneuf D, Bareil C, Robertson J, Rouleau GA, Kriz J et al (2011) Pathological hallmarks of amyotrophic lateral sclerosis/frontotemporal lobar degeneration in transgenic mice produced with TDP-43 genomic fragments. Brain 134:2610–2626

    PubMed  Google Scholar 

  43. Tollervey JR, Curk T, Rogelj B, Briese M, Cereda M, Kayikci M et al (2011) Characterizing the RNA targets and position-dependent splicing regulation by TDP-43. Nat Neurosci 14:452–458

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Tomono T, Hirai Y, Okada H, Adachi K, Ishii A, Shimada T et al (2016) Ultracentrifugation-free chromatography-mediated large-scale purification of recombinant adeno-associated virus serotype 1 (rAAV1). Mol Ther Methods Clin Dev 3:15058

    PubMed  PubMed Central  Google Scholar 

  45. Tripathi VB, Baskaran P, Shaw CE, Guthrie S (2014) Tar DNA-binding protein-43 (TDP-43) regulates axon growth in vitro and in vivo. Neurobiol Dis 65:25–34

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Twiss JL, Fainzilber M (2009) Ribosomes in axons–scrounging from the neighbors? Trends Cell Biol 19:236–243

    CAS  PubMed  Google Scholar 

  47. van Niekerk EA, Willis DE, Chang JH, Reumann K, Heise T, Twiss JL (2007) Sumoylation in axons triggers retrograde transport of the RNA-binding protein La. Proc Natl Acad Sci U S A 104:12913–12918

    PubMed  PubMed Central  Google Scholar 

  48. Villarreal J Jr, Lee JC (1998) Yeast ribosomal protein L26 is located at the ribosomal subunit interface as determined by chemical cross-linking. Biochimie 80:321–324

    CAS  PubMed  Google Scholar 

  49. Wakatsuki S, Furuno A, Ohshima M, Araki T (2015) Oxidative stress-dependent phosphorylation activates ZNRF1 to induce neuronal/axonal degeneration. J Cell Biol 211:881–896

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Wegorzewska I, Bell S, Cairns NJ, Miller TM, Baloh RH (2009) TDP-43 mutant transgenic mice develop features of ALS and frontotemporal lobar degeneration. Proc Natl Acad Sci U S A 106:18809–18814

    CAS  PubMed  PubMed Central  Google Scholar 

  51. 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 U S A 107:3858–3863

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Wu LS, Cheng WC, Shen CK (2012) Targeted depletion of TDP-43 expression in the spinal cord motor neurons leads to the development of amyotrophic lateral sclerosis-like phenotypes in mice. J Biol Chem 287:27335–27344

    CAS  PubMed  PubMed Central  Google Scholar 

  53. 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

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Yokoseki A, Shiga A, Tan CF, Tagawa A, Kaneko H, Koyama A et al (2008) TDP-43 mutation in familial amyotrophic lateral sclerosis. Ann Neurol 63:538–542

    CAS  PubMed  Google Scholar 

  55. Yoon BC, Jung H, Dwivedy A, O'Hare CM, Zivraj KH, Holt CE (2012) Local translation of extranuclear lamin B promotes axon maintenance. Cell 148:752–764

    CAS  PubMed  PubMed Central  Google Scholar 

  56. Zivraj KH, Tung YC, Piper M, Gumy L, Fawcett JW, Yeo GS et al (2010) Subcellular profiling reveals distinct and developmentally regulated repertoire of growth cone mRNAs. J Neurosci 30:15464–15478

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank Dr. Robert H. Singer (Albert Einstein College of Medicine) and Dr. Katsuhiko Mikoshiba (RIKEN) for providing NLS-MS2-Venus and IP3R 3′UTR-MS2bs plasmids, respectively.

Funding

This work was supported by the Grant-in-Aid for Scientific Research (C) (22590932, 25461302 and 16K09690 to S.N.), the Grant-in-Aid for Scientific Research on Innovative Areas (16H06277 to S.N.), AMED (JP20lm0203007 and JP20ek0109320 to S.N.,  JP18dm0107103 to Y.S.), grants from Japan Foundation for Neuroscience and Mental Health and Strategic Research Program for Brain Sciences (to S.N.), Intramural Research Grant for Neurological and Psychiatric Disorders of NCNP (24-9, 27-7, and 27-9 to S.N. and T.A.), Astellas Research Support, Pfizer Academic Contributions, and Takeda Research Support (T.A.).

Author information

Authors and Affiliations

  1. Department of Peripheral Nervous System Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Japan

    Seiichi Nagano, Megumi Shibata, Shohei Watanabe, Shuji Wakatsuki & Toshiyuki Araki

  2. Department of Neurology, Osaka University Graduate School of Medicine, Osaka, Japan

    Seiichi Nagano, Junki Jinno, Rehab F. Abdelhamid, Yinshi Jin & Hideki Mochizuki

  3. Department of Neurology, China-Japan Union Hospital of Jilin University, Jilin, China

    Yinshi Jin

  4. Department of Molecular Neuroscience, Brain Research Institute, Niigata University, Niigata, Japan

    Sachiko Hirokawa

  5. Department of Neurology, Brain Research Institute, Niigata University, Niigata, Japan

    Masatoyo Nishizawa & Osamu Onodera

  6. Department of Cellular Neurobiology, Brain Research Institute, Niigata University, Niigata, Japan

    Kenji Sakimura

  7. Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Japan

    Hironori Okada

  8. Division of Molecular and Medical Genetics, Center for Gene and Cell Therapy, Institute of Medical Science, University of Tokyo, Tokyo, Japan

    Takashi Okada

  9. Department of Laboratory Medicine, National Center Hospital, National Center of Neurology and Psychiatry, Tokyo, Japan

    Yuko Saito

  10. Department of Neuropathology (Brain Bank for Aging Research), Tokyo Metropolitan Geriatric Hospital and Institute of Gerontology, Tokyo, Japan

    Junko Takahashi-Fujigasaki & Shigeo Murayama

Authors
  1. Seiichi Nagano
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Junki Jinno
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Rehab F. Abdelhamid
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yinshi Jin
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Megumi Shibata
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Shohei Watanabe
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Sachiko Hirokawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Masatoyo Nishizawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Kenji Sakimura
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Osamu Onodera
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Hironori Okada
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Takashi Okada
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Yuko Saito
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Junko Takahashi-Fujigasaki
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Shigeo Murayama
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Shuji Wakatsuki
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Hideki Mochizuki
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. Toshiyuki Araki
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

SN and TA designed the study. SN, JJ, RFA, YJ, MS, SW, SH, MN, KS, OO, HO, TO SW and HM performed experiments. YS, JTF and SM collected and evaluated human samples. SN and TA wrote the manuscript. All authors read and approved the final version of the manuscript.

Corresponding authors

Correspondence to Seiichi Nagano or Toshiyuki Araki.

Ethics declarations

Conflict of interest

Authors declare no conflict of interest.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Movie 1. Walking of TARDBP Flox/Flox, Eno2-Cre(-), Flox/Flox, Eno2-Cre Tg and Flox/+, Eno2-Cre Tg mice at 10 days of age. TARDBP Flox/Flox, Eno2-Cre Tg mice exhibited tremors and a waddling gait. (MP4 11681 kb)

Supplementary file2 (MP4 15348 kb)

Supplementary file3 (MP4 334 kb)

Supplementary file4 (MP4 365 kb)

Movie 2. Movement of Rpl41 mRNA (green) and mCherry-TDP-43 (red) in the axon of live cortical neurons. Both signals in the same granule move simultaneously along the axon (MP4 184 kb)

Supplementary file6 (MP4 11196 kb)

401_2020_2205_MOESM7_ESM.tif

Supplementary Figure 1. The phenotype of neuron-specific TARDBP deficient mice is unrelated to neuronal death. a: Representative photomicrographs showing NeuN immunostaining of cerebral cortices in TARDBP Flox/Flox, Eno2-Cre(-), Flox/Flox, Eno2-Cre Tg and Flox/+, Eno2-Cre Tg mice at 21 days of age. Scale bar, 100 μm. b: The number of NeuN-immunostained neurons in mice from each genotype (n = 3 in each genotype). No significant difference was observed between the different genotypes by one-way ANOVA test. c: The amount of TDP-43 mRNA in the white matter of the mice from each genotype (n = 3 in each genotype). The expression level of TDP-43 mRNA normalized to that of β-actin mRNA was quantified at 21 days of age and values relative to that in TARDBP Flox/Flox, Eno2-Cre(-) mice are shown. The value in TARDBP Flox/Flox, Eno2-Cre Tg mice was significantly lower than that in TARDBP Flox/Flox, Eno2-Cre(-) mice. *P < 0.05 compared with the value in TARDBP Flox/Flox, Eno2-Cre(-) mice by unpaired t-test (TIF 845 kb)

401_2020_2205_MOESM8_ESM.tif

Supplementary Figure 2. Co-localization of RP mRNAs with TDP-43. Double staining of Rplp1 or Rps7 mRNA (green) and TDP-43 protein (red) in neuronal axons by in situ hybridization and immunostaining, respectively. Similar to Rpl41 mRNA, both mRNAs co-localized with TDP-43 in a granular pattern in control axons. Both signals were attenuated in TDP-43 down-regulated axons. Scale bar, 10 μm. (TIF 1399 kb)

401_2020_2205_MOESM9_ESM.tif

Supplementary Figure 3. RP mRNAs are transported into axons as neuronal RNA granules by TDP-43. a: Representative photomicrographs of differential interference contrast (DIC) and tau immunostaining images of neurons cultured in the microfluidic device. Neurites on the right side were regarded as axons and harvested from the area more than 450 μm apart from cell bodies on the left side for the analysis of the expression of RPs and TDP-43. b: Pearson’s R values of co-localization of each RP or GAPDH mRNA by in situ hybridization with antisense or sense probe with TDP-43 protein by immunostaining (n = 17-23 in each set from 3 independent experiments). Note that the value of the RP mRNA by antisense probe was significantly higher than the respective one by sense probe or that of GAPDH mRNA by antisense probe. *P < 1.0 x 10-8 compared with the respective value by sense probe by unpaired t-test, and #P < 0.0001 compared with the value of GAPDH by one-way ANOVA test. c: Percentage of TDP-43 immunoreactivity co-localized with indicated RP mRNA visualized by in situ hybridization in primary cultured mouse cortical neurons. d, e: Quantified fluorescent intensities of TDP-43 (d) and each RP mRNA (e) by immunostaining and in situ hybridization, respectively, in axons of control or TDP-43 shRNA-transduced neurons (n=20-22 in each group from 3 independent experiments). The value of each intensity was normalized to the value of respective protein/mRNA in control shRNA-transduced neurons. *P < 1.0 x 10-6 and **P < 1.0 x 10-8 compared with the value in control shRNA-transduced neurons by unpaired t-test. (TIF 937 kb)

401_2020_2205_MOESM10_ESM.tif

Supplementary Figure 4. The expression of Rpl41 and EGFP-TDP-43-Myc constructs in Neuro2a cells used in the experiment shown in Figure 2b. a: Representative immunoblot analysis showing expression of GFP and GAPDH in Neuro2a cells transfected with mock (control), IP3R (negative control) or Rpl41 full length or Δ5’/3’UTR together with EGFP-TDP-43-Myc. IB - immunoblot. b: The expression level of endogenous GAPDH mRNA and exogenous Rpl41 mRNA sequence in each group (n = 3 in each group from 3 independent experiments). The expression level of each mRNA was quantified and shown normalized to that of GAPDH mRNA relative to that in Neuro2a cells transfected with Rpl41 full length and EGFP-TDP-43-Myc. (TIF 321 kb)

401_2020_2205_MOESM11_ESM.tif

Supplementary Figure 5. Detection of RP mRNA localization by the MS2-MS2 binding RNA sequence system. a: Representative photomicrographs showing co-localization of RP mRNA signals detected by the Venus-labeled MS2-MS2 binding RNA sequence system and in situ hybridization in cell bodies and axons of cortical neurons. Scale bar, 20 μm. b: The number of RP mRNA granules along neurites measured on images by the MS2-MS2 binding RNA sequence system (n = 30-36 in each group from 3 independent experiments). The number of granules of Rpl41 and Rplp1 full length mRNA was significantly more than that of the mock construct or respective mRNA without 5’UTR. *P < 0.0005 compared with the number of the mock construct and #P < 0.05 compared with that of respective mRNA without 5’UTR by Welch’s ANOVA test. c: The number of RP mRNA granules along neurites by the MS2-MS2 binding RNA sequence system with control or TDP-43 shRNA-transduced neurons (n = 35-43 in each group from 3 independent experiments). The number of granules of Rpl41 and Rplp1 full length mRNA in TDP-43 shRNA-transduced neurons was significantly less than that of respective mRNA in control shRNA-transduced neurons. *P < 0.0001 and **P < 0.0005 compared with the number of respective mRNA in control shRNA-transduced neurons by unpaired t-test. d: Pearson’s R values of co-localization of MS2-Venus signals of Rpl41 full length or IP3R mRNA and mCherry-TDP-43 signals in neurites of cortical neurons (n = 23-25 in each group from 3 independent experiments). Co-localization of TDP-43 was higher with Rpl41 mRNA than with IP3R mRNA. *P < 5.0 x 10-8 compared with the value with IP3R mRNA by unpaired t-test. e: Pearson’s R values of co-localization of Rpl41 full length mRNA signals by the MS2-MS2 binding RNA sequence system and FLAG-La, FLAG-TIA-1 or FLAG-AUF1 signals by FLAG immunostaining in neurites of cortical neurons (n = 22-29 in each group from 3 independent experiments). Co-localization of Rpl41 full length mRNA was significantly the highest with FLAG-La among the groups. *P < 0.0001 compared with the value with FLAG-TIA-1 and FLAG-AUF1 by Welch’s ANOVA test. f: Representative photomicrographs showing localization of Rpl41 mRNA and La in the axons of cultured cortical neurons using MS2-MS2 binding RNA sequence system are shown. Cortical neurons were transfected with expression constructs for Rpl41 with 5’UTR bearing MS2 binding RNA sequence, NLS-MS2-Venus, and FLAG-La. Tau is used as an axonal marker. Scale bar, 20 μm. g, h: The median transported distances from the nucleus and the number of RP mRNA granules along neurites by the MS2-MS2 binding RNA sequence system with control or TDP-43 shRNA-transduced neurons (n = 15 in each group). Note that no significant difference of the median transported distance and the number of granules of Rpl41 and Rplp1 mRNA without 5’UTR was observed between the groups by unpaired t-test. (TIF 2033 kb)

401_2020_2205_MOESM12_ESM.tif

Supplementary Figure 6. La enhances expression of RPs. a: Representative photomicrographs showing co-localization of RP mRNAs by in situ hybridization and La by immunocytochemistry in axons of cortical neurons. Scale bar, 10 μm. b: Representative immunoblots showing expression of Rpl26 and Rps6 in the neurite fraction of cortical neurons overexpressing EGFP (control) or La on the chamber insert. c: La expression significantly increased the amount of Rps6, and caused a trend to increase the amount of Rpl26 (n = 3 in each group from 3 independent experiments). The expression level of each RP against that of GAPDH was normalized relative to control neurites. *P < 0.05 compared with the value in control neurites by unpaired t-test. d: The levels of Rpl41 mRNA co-immunoprecipitated with TDP-43. TDP-43 and Rpl41 were expressed together with La in Neuro2a cells and the amount of Rpl41 mRNA co-precipitated with TDP-43 was measured by quantitative PCR. The levels are shown as a value relative to the level of mRNA precipitated with control IgG (n = 3 for each group). *P < 0.001 compared with the negative control (IP3R) by one-way ANOVA test. There was no significant change of binding between Rpl41 mRNA and TDP-43 by La expression. e: The expression levels of indicated genes in neurites of cortical neurons transduced with rAAV1-CAG-FLAG-La relative to the control rAAV1-transduced neurons determined by quantitative RT-PCR (n = 3 in each group). No difference was seen compared with the value of respective mRNA in the control rAAV1-transduced neurons by multiple t-test. (TIF 824 kb)

401_2020_2205_MOESM13_ESM.tif

Supplementary Figure 7. Local translation of RP mRNAs in axons. a: Representative immunoblots of La in control and BDNF-stimulated neurites of cortical neurons on the chamber insert. b: The amount of La was not altered by stimulation with BDNF (n = 3 in each group from 3 independent experiments). The expression level of La was quantified and shown normalized to that of GAPDH relative to the level in control neurites. c: Translation of RPs is enhanced by BDNF in axons independently of that in cell bodies. Expression of Rpl26 detected by immunocytochemistry was quantified in neurites of mouse cortical neurons cultured in the microfluidic device after BDNF application only to the axon compartment, with or without CHX treatment of the cell bodies. Rpl26 expression level in each condition normalized to tau expression relative to the level in untreated axons (control + control) is shown (n = 30 in each group from 3 independent experiments). *P < 0.0001 compared with the value in control + control axons by Welch’s ANOVA test. Note that the expression of Rpl26 was increased significantly by stimulation with BDNF even when cell bodies were treated with CHX. d: The expression levels of indicated genes in neurites of cortical neurons treated with BDNF relative to the control neurons determined by quantitative RT-PCR (n = 3 in each group). No significant difference was observed compared with the value of respective mRNA in the control neurons by multiple t-test. e: Representative immunoblots of TDP-43 and RPs in control and TDP-43-knockdown neurites of cortical neurons on the chamber insert. f: The levels of Rpl26 and Rps6 in neurites were significantly decreased by TDP-43 knockdown (n = 3 in each group from 3 independent experiments). The expression level of each indicated protein normalized to that of GAPDH relative to the level in control neurites is shown. *P < 0.05 compared to the value in control neurites by unpaired t-test. (TIF 323 kb)

401_2020_2205_MOESM14_ESM.tif

Supplementary Figure 8. Expression of HA-tagged RPs in neurons. Representative photomicrographs of immunostaining of cell bodies and axons of cultured cortical neurons transfected with RP-HA including 5’UTR or pcDNA using indicated primary antibodies. Note that proteins translated from RP expression constructs including 5’UTR sequences are detectable in axons. Scale bar, 20 μm. (TIF 877 kb)

401_2020_2205_MOESM15_ESM.xlsx

Supplementary Files 1 and 2 Raw data with graphs of microarray analysis described in Figure 1. Data from experiments with TDP-43 shRNA#1 and #2 are shown in file 1 and 2, respectively. In each experiment, Cy5 and Cy3 labeling show control and shRNA-mediated knock-down condition, respectively. (XLSX 7263 kb)

Supplementary file16 (XLSX 7126 kb)

Supplementary file17 (XLSX 33 kb)

401_2020_2205_MOESM18_ESM.docx

Supplementary File 3 Summary of all the annotated transcripts down-regulated or up-regulated by TDP-43 knock-down identified in the microarray analysis. Among the transcripts that were detected in controls and TDP-43-knock down conditions, the ones down-regulated to less than half or up-regulated to more than 2-fold by both TDP-43 shRNA constructs (#1 and #2) are listed. (DOCX 35 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Nagano, S., Jinno, J., Abdelhamid, R.F. et al. TDP-43 transports ribosomal protein mRNA to regulate axonal local translation in neuronal axons. Acta Neuropathol 140, 695–713 (2020). https://doi.org/10.1007/s00401-020-02205-y

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00401-020-02205-y

Keywords

  • Ribonucleic acid
  • Local translation
  • Ribosome
  • Amyotrophic lateral sclerosis