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

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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.

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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.).

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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

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  1. Seiichi Nagano
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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.

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Correspondence to Seiichi Nagano or Toshiyuki Araki.

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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)

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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)

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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)

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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)

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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)

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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)

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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)

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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)

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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)

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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)

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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

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

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