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LONRF2 is a protein quality control ubiquitin ligase whose deficiency causes late-onset neurological deficits

Nature Aging volume 3, pages 1001–1019 (2023)Cite this article

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Abstract

Protein misfolding is a major factor of neurodegenerative diseases. Post-mitotic neurons are highly susceptible to protein aggregates that are not diluted by mitosis. Therefore, post-mitotic cells may have a specific protein quality control system. Here, we show that LONRF2 is a bona fide protein quality control ubiquitin ligase induced in post-mitotic senescent cells. Under unperturbed conditions, LONRF2 is predominantly expressed in neurons. LONRF2 binds and ubiquitylates abnormally structured TDP-43 and hnRNP M1 and artificially misfolded proteins. Lonrf2−/− mice exhibit age-dependent TDP-43-mediated motor neuron (MN) degeneration and cerebellar ataxia. Mouse induced pluripotent stem cell-derived MNs lacking LONRF2 showed reduced survival, shortening of neurites and accumulation of pTDP-43 and G3BP1 after long-term culture. The shortening of neurites in MNs from patients with amyotrophic lateral sclerosis is rescued by ectopic expression of LONRF2. Our findings reveal that LONRF2 is a protein quality control ligase whose loss may contribute to MN degeneration and motor deficits.

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Fig. 1: LONRF2 regulates protein aggregation in senescent human fibroblasts.
Fig. 2: LONRF2 is a bona fide protein quality control ubiquitin ligase.
Fig. 3: Lonrf2 predominantly expresses in neurons and ubiquitylates misfolded TDP-43 and hnRNP M1.
Fig. 4: Lonrf2−/− mice showed late-onset neurological deficits.
Fig. 5: Accumulation of stress granules and pTDP-43 foci in spinal cord and cortex of Lonrf2−/− mice.
Fig. 6: Ectopic expression of LONRF2 restores abnormalities in MNs derived from Lonrf2−/− iPS cells and ALS hiPS cells.
Fig. 7: Identification of a loss-of-function variant in sporadic ALS but not in control cohorts.

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Data availability

RNA-seq data have been deposited in GEO under accession no. GSE179465. Access to DNA sequencing data of patients or healthy individuals is not available to entities without mutual research agreement, as consent for data provision has not been obtained from the individuals. All other data needed to evaluate the conclusions presented here are included in the paper and/or the supplementary materials. All data are available from the corresponding authors.

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Acknowledgements

We are grateful to M. Nakanishi (Tokiwa-Bio) for generation of iPS cells and Y. Chiba, M. Ishiura, T. Ando, M. Iwai, M. Kikuchi, S. Omori (University of Tokyo) and S. Nakamura, F. Ozawa, C. Kato (Keio University) and N. Shimakura (Tohoku University) for technical assistance. We also thank H. Inoue (Kyoto University) for kindly donating the hiPS clone (A3411). Histological sections were performed with the help of the Pathology Core Laboratory (University of Tokyo). Confocal microscopy services were provided by the Imaging Core Laboratory (University of Tokyo). Computational resources were provided by the supercomputer system SHIROKANE at the Human Genome Center (University of Tokyo). This study was supported by AMED under grant nos. 21zf0127003 (M.N.), 21cm0106175 (M.N.) and 21gm5010001 (M.N.), 21bm0804027 (H.O.), 22bm0804003 (H.O.), 21ek0109492 (H.O.), 21wm0425009 (H.O.), 214600040 (Y.J.) and by MEXT/JSPS KAKENHI under grant nos. 20H00514 (M.N.), 19H05740 (M.N.), JP18H05026m (Y.J.), JP16H06148 (Y.J.), JP16K15238 (Y.J.), JP22K15736 (S.M.), JP21H05273 (S.M.), JP20H00485 (H.O.) and by the Princess Takamatsu Cancer Research Fund (M.N.), the Uehara Memorial Foundation (S.M.), the Yukihiko Miyata Memorial Trust For Research (S.M.) and Yoshio Koide Grant, Japan ALS Association (S.M.).

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Authors and Affiliations

  1. Division of Cancer Cell Biology, The University of Tokyo, Tokyo, Japan

    Dan Li, Yoshikazu Johmura, Chieko Konishi, Narumi Suzuki, Atsuya Nishiyama, Alexander Stephen Harris & Makoto Nakanishi

  2. Division of Cancer and Senescence Biology, Cancer Research Institute, Institute for Frontier Science Initiative, Kanazawa University, Kanazawa, Japan

    Yoshikazu Johmura

  3. Department of Physiology, Keio University School of Medicine, Tokyo, Japan

    Satoru Morimoto & Hideyuki Okano

  4. Department of Neuroscience and Cell Biology, Osaka University Graduate School of Medicine, Osaka, Japan

    Miyuki Doi & Shoichi Shimada

  5. Department of Pediatrics, Central Hospital, and Department of Disease Model, Institute for Developmental Research, Aichi Developmental Disability Center, Kasugai, Japan

    Keiko Nakanishi

  6. Laboratory of Reproductive Systems Biology, The University of Tokyo, Tokyo, Japan

    Manabu Ozawa

  7. Division of Molecular and Medical Genetics, Center for Gene and Cell Therapy, The University of Tokyo, Tokyo, Japan

    Yuji Tsunekawa & Takashi Okada

  8. Division of Genetics, The University of Tokyo, Tokyo, Japan

    Akane Inoue-Yamauchi & Yuji Yamanashi

  9. Department of Neurology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan

    Hiroya Naruse, Takashi Matsukawa, Shoji Tsuji & Tatsushi Toda

  10. Department of Neurology and Clinical Neuroscience, Yamaguchi University Graduate School of Medicine, Yamaguchi, Japan

    Yukio Takeshita

  11. Department of Neurology, Tohoku University Graduate School of Medicine, Sendai, Japan

    Naoki Suzuki, Masashi Aoki & Ayumi Nishiyama

  12. Laboratory of Functional Analysis in silico, Human Genome Center, The University of Tokyo, Tokyo, Japan

    Xin Zeng & Kenta Nakai

  13. Division of Clinical Genome Research, The University of Tokyo, Tokyo, Japan

    Mariko Morita, Kiyoshi Yamaguchi & Yoichi Furukawa

  14. Division of Stem Cell Biology, Center for Stem Cell Biology and Regenerative Medicine, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan

    Satoshi Yamazaki

  15. Laboratory of Stem Cell Therapy, Faculty of Medicine, University of Tsukuba, Ibaraki, Japan

    Satoshi Yamazaki

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M.N. and Y.J. conceived the idea of the project, D.L., Y.J., T.T. and M.N. planned the experiments, D.L., Y.J,. S.M., M.D., K.N., M.O., Y.T., A.I.Y., H.N,. T.M., Y.T., N.S., M.A., A.N., X.Z., C.K., N.S., A.N., A.S.H., M.M., K.Y. and S.Y. performed the experiments, D.L., Y.J., S.M., M.D., K.N., M.O., A.I.-Y., H.N,. T.M., X.Z., N.S., A.N., M.M., K.Y., Y.F., K.N., S.T., S.Y., Y.Y., S.S., T.O., H.O., T.T. and M.N. analyzed the results, Y.T, N.S., M.A. and A.Y. established iPS cells from patients with ALS and M.N. wrote the manuscript with editing by all the other authors.

Corresponding authors

Correspondence to Yoshikazu Johmura or Makoto Nakanishi.

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Competing interests

M.N. is a scientific advisor to Airweave and reverSASP Therapeutics and a shareholder of reverSASP Therapeutics. All other authors declare no competing interests.

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Nature Aging thanks the anonymous reviewers for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Lonrf2, but not Lonrf1 or Lonrf3, is predominantly expressed in senescent cells.

(a) Structure of LONRF proteins of Homo sapiens (human). Domain structures are indicated. TPR is a protein-protein interaction domain, the RING finger is a domain that binds ubiquitination enzymes and their substrates and functions as a ligase, and the LON substrate binding domain is related to turnover of many misfolded or denatured proteins. (b) qPCR analysis of the indicated genes in uninduced (low passage) or senescent HCA2 cells induced by 9 mM of RO3306 for 24 hrs, 5 mM of nutlin3a for 48 hrs, and then cultured with medium containing 100 nM of BI2536 for 18 days (n-Sen). n = 3. (c) qPCR analysis of the indicated genes in uninduced (low passage) or senescent HCA2 cells induced by 100 nM of doxorubicin for 24 hrs, and then cultured with medium containing 100 nM of BI2536 for 20 days (d-Sen).n = 3. (d) Lysates of d-Sen cells expressing Flag-Lonrf2 in the presence or absence of doxycycline (1 mg/ml) were subjected to immunoblotting using the indicated antibodies. SYPRO orange staining (e) and thioflavin-T (ThT) (10μM) staining (f), of d-Sen cells treated in (d), followed by quantification of fluorescence intensity in the right panel (f) (n = 3). qPCR analysis of LONRF2 (g), SYPRO orange staining (top panel) (h), thioflavin-T (ThT) (10μM) staining (i) in uninduced or d-Sen cells expressing control or LONRF2 shRNA (shLONRF2-1, 2) in the presence of doxycycline (1 μg/ml) (n = 3). Lysates of d-Sen cells in (g) were subjected to immunoblotting with the indicated antibody (bottom panel). Quantification of fluorescence intensity in the right panel (n = 3) (i). (j) HeLa cells were transfected with the indicated plasmids, lysed under denaturing conditions, and subjected to immunoprecipitation with an anti-FLAG M2 affinity gel, followed by immunoblotting with the indicated antibodies. Scale bar: 50 μm (f, i). Data are presented as means ±s.d. of three independent experiments, error bars show s.d. One-way ANOVA with Dunnett’s multiple comparisons post hoc test (g, i) and unpaired two-tailed Student’s t-test (b, c, f). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

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Extended Data Fig. 2 LONRF2 is required for the effective disassembly of SGs and reduction in the level of misfolded proteins.

(a) Immunoblotting of lysates from MCF7 and AsPC-1 cells expressing the indicated Dox (1 mg/ml)-inducible shRNA for 48 hrs using the indicated antibodies. (b) MCF7 and (c) AsPC-1 cells as in (a) were treated with NaAsO2 (1 mM) for 30 min, washed with PBS, and then subjected to immunofluorescent analysis using anti-G3BP1 antibody and Hoechst33342. Relative populations of stress granule-positive cells (cells with more than five G3BP1-positive foci) were determined (n = 200). (d) Immunoblotting of lysates from A549 cells expressing Dox-inducible Lonrf2 shRNA together with shRNA-resistant Lonrf2-WT or its mutants. (e) A549 cells as in (d) were treated with NaAsO2 (1 mM) for 30 min, washed with PBS, and then subjected to immunohistochemical analysis using anti-G3BP1 antibody and Hoechst33342 at 0 and 120 min after PBS washout. Relative population of stress granule-positive cells (cells with more than five G3BP1-positive foci) were determined (n = 200). (f and g) A549 cells expressing Dox-inducible Lonrf2 shRNA were treated with H2O2 (1 mM) for 60 min (f) or heat shock (43 °C, for 60 min) (g), were recovered, and were then subjected to immunofluorescent analysis using anti-G3BP1 antibody and Hoechst33342 at the indicated times after recovery. Relative population of stress granule-positive cells (cells with more than five G3BP1-positive foci) were determined (n = 200). (h and i) A549 cells treated as in (f and g) were subjected to a pull-down assay with FLAG-LONRF2 affinity beads, followed by immunoblotting using the indicated antibodies. Quantification of the intensity is shown in the right panels. Data are presented as means ±s.d. of three independent experiments, error bars show s.d. Two-tailed one-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons post hoc test (b-c, e-i). **P < 0.01, ***P < 0.001.

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Extended Data Fig. 3 Lonrf2−/− mice showed a shorter lifespan than Lonrf2+/+ mice.

(a) Sequence alignment of Lonrf2-WT (+/+) and Lonrf2-KO (−/−) alleles. The 5 depleted base pairs are shown in red. (b) Body weights of Lonrf2+/+ and Lonrf2−/− mice at 21 months of age, (n = 8: 6 males and 2 femals). (c) Representative pictures of Lonrf2+/+ and Lonrf2−/− mice at 21 months of age (d) Survival rates of Lonrf2+/+ (n = 14, 8 males and 6 females), Lonrf2+/− (n = 11, 6 males and 5 females), and Lonrf2−/− mice (n = 11, 4 males and 7 females), data are shown as a percentage of the original mice. Statistical significance was determined with a log-rank (Mantel-Cox) test (p<0.01). Data are presented as means ±s.d. of the indicated independent experiments, error bars show s.d. Unpaired two-tailed Student’s t-test (b).

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Extended Data Fig. 4 Lonrf2 is predominantly expressed in neurons.

(a) UMAP clustering of the adult mouse spinal cord (upper) from 43,890 single-cell transcriptomes, visualization of ChAT (middle) and Lonrf2 (lower) expressed populations. (b) Violin plot of Lonrf2 expressed populations in young (16,028 single-cell transcriptomes from 8 young mouse brains) and old (21,041 single-cell transcriptomes from 8 old mouse brains) mice. (c) Violin plot of Lonrf2 expressed populations of cerebellar cortex in 780,553 single nuclei transcriptomes (530,063 profiles from male donors, 4 males at P60, 1 male at E18, 1 male at P0, 2 males at P12; 250,490 profiles from female donors, 2 females of P60, 1 female at P4, 1 female at P8, 2 females at P16). Expression level represents Log-Normalized UMI counts. (d) Left and right figures showed primary motor cortex of 7-week-old mice and 97-week-old mice, respectively. In situ hybridization with antisense probe detected Lonrf2 positive cells in the primary motor cortex of both 7-week-old mice and 97-week-old mice. Sense probe did not yield any specific staining. Small square images showed the higher magnification of the boxed areas. Arrowheads indicated representative mRNA signals. Cortical layers were shown as L1, L2/3, L5, and L6. Scale bars: 100 and 10 mm. Three independent experiments were performed.

Extended Data Fig. 5 Lonrf2−/− mice showed neurodegenerative phenotypes in the cortex.

Immunofluorescent analyses of NeuN (magenta) (a) and Fluoro Jade C (green) (b) -positive cells counterstained with Hoechst or DAPI (blue) using cortical brain from Lonrf2+/+ or Lonrf2−/− mice at 21 months of age. Representative images are shown (left panels). Scale bars: 50 mm. Quantification of NeuN- and Fluoro Jade C-positive cells per square millimeter (right figure) (n = 6, males). Immunofluorescence staining of cortical brain from male (black) and female (red) Lonrf2+/+ or Lonrf2−/− mice at 21 months of age using anti-Ataxin2 (green) (c), anti-G3BP1 (green) (d), or anti-pTDP-43 (green) (e) antibodies, co-staining with anti-NeuN antibodies (magenta) and Hoechst (blue). Representative images are shown (left panels). Scale bars: 10 mm. Quantification of the percentages of Ataxin2, G3BP1 or pTDP-43-positive cells in NeuN-positive cells are shown on the right (n = 8, 6 males and 2 females). (f) Quantification of NeuN-positive, Fluoro Jade C-positive, and percentages of Ataxin-2-positive cells in the striatum of Lonrf2+/+ or Lonrf2−/− mice at 21 months of age are shown (n = 6, males). Data are presented as means ±s.d. of the indicated independent experiments, error bars show s.d. Unpaired two-tailed Student’s t-test (a-f). ***P < 0.001, ****P < 0.0001.

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Extended Data Fig. 6 Lonrf2−/− mice showed neurodegenerative phenotypes in the cerebellum.

Immunofluorescent analysis of NeuN (red) (a), Fluoro Jade C (green) (b)-positive cells counterstained with Hoechst or DAPI (blue) using cerebellum from Lonrf2+/+ or Lonrf2−/− mice at 21 months of age. Representative images are shown (left panels). Scale bars: 50 mm. Quantification of NeuN- and Fluoro Jade C-positive cells per square millimeter (right panels) (n = 6, male). Immunofluorescent analysis of Ataxin-2 (red) (c), G3BP1 (green) (d), and pTDP-43 (green)(e) -positive cells counterstained with Hoechst or DAPI (blue) using cerebellum from male (black) and female (red) Lonrf2+/+ or Lonrf2−/− mice at 21 months of age. Representative images are shown (left panels). Scale bars: 10 mm. Percentages of Ataxin-2, G3BP1, and pTDP-43-positive cells in NeuN-positive cells are shown in the right figures, (n = 8, 6 male and 2 female). (f) Representative images of Purkinje cells stained by Calbindin in the cerebellum from Lonrf2+/+ (left panels) or Lonrf2−/− (right panels) mice at 3 months of age (upper panels) and 21 months of age (lower panels). Scale bars: 50 mm. (g) Quantification of Purkinje cells per millimeter of the Purkinje layer in (f), (n = 6, males). (h) Measurement of granular and molecular layer thicknesses in the cerebellum from Lonrf2+/+ or Lonrf2−/− mice at 21 months of age. Data are presented as means ±s.d. of three independent experiments, error bars show s.d. Unpaired two-tailed Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

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Extended Data Fig. 7 Neurite length shortening in Lonrf2−/− iPS cell-derived neurons is restored by ectopic expression of Lonrf2.

(a) Lonrf2+/+ (left) and Lonrf2−/− (right) mouse iPS cells were positive for alkaline phosphatase staining. Scale bar: 20 mm. (b) Growth curves of established Lonrf2+/+, Lonrf2+/−, and Lonrf2−/− mouse iPS cells. (c) Differentiation rates of Lonrf2+/+, Lonrf2+/−, and Lonrf2−/− neuronal precursors to motor neurons. (d) Representative images of the results shown in Fig. 6f. Anti-Tuj1 antibody (green), anti-ChAT (red) and Hoechst33342 (blue). Scale bar: 50 mm. (e) Representative images of the results shown in Fig. 6f. Cells were infected with AAV-FLAG-Lonrf2 vector (5 × 105 GC/mL) for 12 hrs and cultured for an additional 0 or 14 days. Anti-Tuj1 antibody (green), anti-Flag (gray), anti-ChAT (red), and Hoechst33342 (blue). Scale bar: 50 mm. Data are presented as means ±s.d. of three independent experiments, error bars show s.d.

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Extended Data Fig. 8 Lonrf2−/− iPS cell-derived neurons show reduced survival, accumulation of pTDP-43 and G3BP1 aggregates.

Representative images of the results shown in Fig. 6g(a), 6h (b), and 6i (c). TUNEL (green), anti-pTDP-43 (green), anti-G3BP1 (green) and anti-ChAT (red) antibodies. Scale bar: 50 mm (a), 10 mm (b, c).

Extended Data Fig. 9 Ectopic expression of LONRF2 restores mortality and suppresses accumulation in pTDP-43 of Lonrf2−/− iPS cell-derived neurons.

(a) Representative images of the results shown in Fig. 6f. Motor neurons derived from Lonrf2+/+ and Lonrf2−/− iPS cells were stained with anti-LONRF2 (green), anti-FLAG (grey), and anti-ChAT (red) antibodies, counterstained with Hoechst (blue). Representative images of the results shown in Fig. 6g(b) and 6h (c). Cells were infected with AAV-FLAG-Lonrf2 vector (5 × 105 GC/mL) for 12 hrs and cultured for an additional 0 or 14 days. TUNEL (green), anti-pTDP-43 (green), anti-Flag (gray), and anti-ChAT (red). Scale bar: 50 mm (a, b), 10 mm (c).

Extended Data Fig. 10 Lonrf2 is expressed primarily in mouse motor neurons, while both Lonrf1 and Lonrf2 are expressed in human motor neurons derived from iPS cells.

For the human motor neuron differentiation, hiPSCs were transferred to a chemically transitional embryoid-body-like state (CTraS) in AK02N medium (Ajinomoto, Japan) using SB431542, dorsomorphine, and CHIR99021, trypsinized into single cells, and plated on poly-L-lysin and matrigel-coated plates in MN medium (KBM Neural Stem Cell Medium, Kohjin Bio, Japan) with B-27 supplement and Y-27632. Simultaneously, the iPSCs were infected with Sendai virus including Lhx3, Ngn2, and Islet1 (multiplicity of infection = 5, ID Pharma, Tokyo, Japan) on day 0. The medium was changed to motor neuron medium without Y-27632 on day 1 and mRNA of human motor neurons was collected at day 14. Normalized maximum neurite length (a) of motor neurons derived from normal human (201B7 cell line) and two ALS patients with an M337V mutation of TARDBP gene (A3411 cell line) and G4C2 repeat expansion in the C9orf72 gene (C9 148-1) (C9rof72 148-1 cell line) were measured during the experimental course. n = 1, data are presented as the mean relative neurite length in 5 fields/well, error bars show SEM. Mouse motor neurons were induced as shown in Fig. 6 a, mRNA was collected on day of 14 of the assay. qPCR analysis (b) with the indicated primers using RNAs from mouse or human iPS-induced motor neurons. Expression level of LONRF1, 2, 3 was normalized using GAPDH. Set expression level of LONRF1 as 1, relative expression level of LONRF2 and LONRF3 were calculated. The relative expressions are shown. Data are presented as means ±s.d. of three independent experiments, error bars show s.d. Two-tailed one-way analysis with Dunnett’s (a) or Tukey (b) multiple comparisons post hoc test, *P < 0.05, **P < 0.01, ***P < 0.001.

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Supplementary information

Reporting Summary

Supplementary Video 1

Ledge test for WT mouse.

Supplementary Video 2

Ledge test for Lonrf2-KO mouse.

Supplementary Video 3

Hind limb test for WT mouse.

Supplementary Video 4

Hind limb test for Lonrf2-KO mouse.

Supplementary Video 5

Gait test for WT mouse.

Supplementary Video 6

Gait test for Lonrf2-KO mouse.

Supplementary Table 1

Information of primers used in this study.

Supplementary Table 2

Information of antibodies used in this study.

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Li, D., Johmura, Y., Morimoto, S. et al. LONRF2 is a protein quality control ubiquitin ligase whose deficiency causes late-onset neurological deficits. Nat Aging 3, 1001–1019 (2023). https://doi.org/10.1038/s43587-023-00464-4

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