Article | Published:

C9ORF72-ALS/FTD-associated poly(GR) binds Atp5a1 and compromises mitochondrial function in vivo

Abstract

The GGGGCC repeat expansion in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). However, it is not known which dysregulated molecular pathways are primarily responsible for disease initiation or progression. We established an inducible mouse model of poly(GR) toxicity in which (GR)80 gradually accumulates in cortical excitatory neurons. Low-level poly(GR) expression induced FTD/ALS-associated synaptic dysfunction and behavioral abnormalities, as well as age-dependent neuronal cell loss, microgliosis and DNA damage, probably caused in part by early defects in mitochondrial function. Poly(GR) bound preferentially to the mitochondrial complex V component ATP5A1 and enhanced its ubiquitination and degradation, consistent with reduced ATP5A1 protein level in both (GR)80 mouse neurons and patient brains. Moreover, inducing ectopic Atp5a1 expression in poly(GR)-expressing neurons or reducing poly(GR) level in adult mice after disease onset rescued poly(GR)-induced neurotoxicity. Thus, poly(GR)-induced mitochondrial defects are a major driver of disease initiation in C9ORF72-related ALS/FTD.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Data availability

The data that support the findings of this study are available from the corresponding author upon request.

Code availability

In this study, we used only publicly available software for data analysis such as ImageJ. Please send a request to the corresponding author for further details.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  1. 1.

    Olney, N. T., Spina, S. & Miller, B. L. Frontotemporal dementia. Neurol. Clin. 35, 339–374 (2017).

  2. 2.

    Hardiman, O. et al. Amyotrophic lateral sclerosis. Nat. Rev. Dis. Primers 3, 17085 (2017).

  3. 3.

    DeJesus-Hernandez, M. et al. Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS. Neuron 72, 245–256 (2011).

  4. 4.

    Renton, A. E. et al. A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD. Neuron 72, 257–268 (2011).

  5. 5.

    Gao, F. B., Almeida, S. & Lopez-Gonzalez, R. Dysregulated molecular pathways in amyotrophic lateral sclerosis-frontotemporal dementia spectrum disorder. EMBO J. 36, 2931–2950 (2017).

  6. 6.

    Mori, K. et al. The C9orf72 GGGGCC repeat is translated into aggregating dipeptide-repeat proteins in FTLD/ALS. Science 1335–1338 (2013).

  7. 7.

    Ash, P. E. et al. Unconventional translation of C9ORF72 GGGGCC expansion generates insoluble polypeptides specific to c9FTD/ALS. Neuron 77, 639–646 (2013).

  8. 8.

    Zu, T. et al. RAN proteins and RNA foci from antisense transcripts in C9ORF72 ALS and frontotemporal dementia. Proc. Natl Acad. Sci. USA 110, E4968–E4977 (2013).

  9. 9.

    Mackenzie, I. R. et al. Dipeptide repeat protein pathology in C9ORF72 mutation cases: clinico-pathological correlations. Acta Neuropathol. 126, 859–879 (2013).

  10. 10.

    Davidson, Y. S. et al. Brain distribution of dipeptide repeat proteins in frontotemporal lobar degeneration and motor neurone disease associated with expansions in C9ORF72. Acta Neuropathol. Commun. 2, 70 (2014).

  11. 11.

    Mackenzie, I. R. et al. Quantitative analysis and clinico-pathological correlations of different dipeptide repeat protein pathologies in C9ORF72 mutation carriers. Acta Neuropathol. 130, 845–861 (2015).

  12. 12.

    Schludi, M. H. et al. Distribution of dipeptide repeat proteins in cellular models and C9orf72 mutation cases suggests link to transcriptional silencing. Acta Neuropathol. 130, 537–555 (2015).

  13. 13.

    Vatsavayai, S. C. et al. Timing and significance of pathological features in C9orf72 expansion-associated frontotemporal dementia. Brain 139, 3202–3216 (2016).

  14. 14.

    Saberi, S. et al. Sense-encoded poly-GR dipeptide repeat proteins correlate to neurodegeneration and uniquely co-localize with TDP-43 in dendrites of repeat-expanded C9orf72 amyotrophic lateral sclerosis. Acta Neuropathol. 135, 459–474 (2018).

  15. 15.

    Sakae, N. et al. Poly-GR dipeptide repeat polymers correlate with neurodegeneration and clinicopathological subtypes in C9ORF72-related brain disease. Acta Neuropathol. Commun. 6, 63 (2018).

  16. 16.

    Cleary, J. D. & Ranum, L. P. New developments in RAN translation: insights from multiple diseases. Curr. Opin. Genet. Dev. 44, 125–134 (2017).

  17. 17.

    Gao, F. B., Richter, J. D. & Cleveland, D. W. Rethinking unconventional translation in neurodegeneration. Cell 171, 994–1000 (2017).

  18. 18.

    Mizielinska, S. et al. C9orf72 repeat expansions cause neurodegeneration in Drosophila through arginine-rich proteins. Science 345, 1192–1194 (2014).

  19. 19.

    Wen, X. et al. Antisense proline-arginine RAN dipeptides linked to C9ORF72-ALS/FTD form toxic nuclear aggregates that initiate in vitro and in vivo neuronal death. Neuron 84, 1213–1225 (2014).

  20. 20.

    Yang, D. et al. FTD/ALS-associated poly(GR) protein impairs the Notch pathway and is recruited by poly(GA) into cytoplasmic inclusions. Acta Neuropathol. 130, 525–535 (2015).

  21. 21.

    Kwon, I. et al. Poly-dipeptides encoded by the C9orf72 repeats bind nucleoli, impede RNA biogenesis, and kill cells. Science 345, 1139–1145 (2014).

  22. 22.

    Tao, Z. et al. Nucleolar stress and impaired stress granule formation contribute to C9orf72 RAN translation-induced cytotoxicity. Hum. Mol. Genet. 24, 2426–2441 (2015).

  23. 23.

    Lopez-Gonzalez, R. et al. Poly(GR) in C9ORF72-related ALS/FTD compromises mitochondrial function and increases oxidative stress and DNA damage in iPSC-derived motor neurons. Neuron 92, 383–391 (2016).

  24. 24.

    Farg, M. A., Konopka, A., Soo, K. Y., Ito, D. & Atkin, J. D. The DNA damage response (DDR) is induced by the C9orf72 repeat expansion in amyotrophic lateral sclerosis. Hum. Mol. Genet. 26, 2882–2896 (2017).

  25. 25.

    Shi, K. Y. et al. Toxic PRn poly-dipeptides encoded by the C9orf72 repeat expansion block nuclear import and export. Proc. Natl Acad. Sci. USA 114, E1111–E1117 (2017).

  26. 26.

    Gascon, E. et al. Alterations in microRNA-124 and AMPA receptors contribute to social behavioral deficits in frontotemporal dementia. Nat. Med. 20, 1444–1451 (2014).

  27. 27.

    Sareen, D. et al. Targeting RNA foci in iPSC-derived motor neurons from ALS patients with a C9ORF72 repeat expansion. Sci. Transl. Med. 5, 208ra149 (2013).

  28. 28.

    Chew, J. et al. Neurodegeneration. C9ORF72 repeat expansions in mice cause TDP-43 pathology, neuronal loss, and behavioral deficits. Science 348, 1151–1154 (2015).

  29. 29.

    Moy, S. S. et al. Sociability and preference for social novelty in five inbred strains: an approach to assess autistic-like behavior in mice. Genes Brain Behav. 3, 287–302 (2004).

  30. 30.

    Forbes, C. E. & Grafman, J. The role of the human prefrontal cortex in social cognition and moral judgment. Annu. Rev. Neurosci. 33, 299–324 (2010).

  31. 31.

    Madabhushi, R., Pan, L. & Tsai, L. H. DNA damage and its links to neurodegeneration. Neuron 83, 266–282 (2014).

  32. 32.

    Zhang, Y. J. et al. Poly(GR) impairs protein translation and stress granule dynamics in C9orf72-associated frontotemporal dementia and amyotrophic lateral sclerosis. Nat. Med. 24, 1136–1142 (2018).

  33. 33.

    Lavie, J. et al. Ubiquitin-dependent degradation of mitochondrial proteins regulates energy metabolism. Cell Rep. 23, 2852–2863 (2018).

  34. 34.

    Franz, A., Kevei, E. & Hoppe, T. Double-edged alliance: mitochondrial surveillance by the UPS and autophagy. Curr. Opin. Cell Biol. 37, 18–27 (2015).

  35. 35.

    Li, Z., Okamoto, K., Hayashi, Y. & Sheng, M. The importance of dendritic mitochondria in the morphogenesis and plasticity of spines and synapses. Cell 119, 873–887 (2004).

  36. 36.

    Pathak, D. et al. The role of mitochondrially derived ATP in synaptic vesicle recycling. J. Biol. Chem. 290, 22325–22336 (2015).

  37. 37.

    Ebrahimi-Fakhari, D. et al. Impaired mitochondrial dynamics and mitophagy in neuronal models of tuberous sclerosis complex. Cell Rep. 17, 1053–1070 (2016).

  38. 38.

    Hong, S., Dissing-Olesen, L. & Stevens, B. New insights on the role of microglia in synaptic pruning in health and disease. Curr. Opin. Neurobiol. 36, 128–134 (2016).

  39. 39.

    Schludi, M. H. et al. Spinal poly-GA inclusions in a C9orf72 mouse model trigger motor deficits and inflammation without neuron loss. Acta Neuropathol. 134, 241–254 (2017).

  40. 40.

    Zhang, Y. J. et al. C9ORF72 poly(GA) aggregates sequester and impair HR23 and nucleocytoplasmic transport proteins. Nat. Neurosci. 19, 668–677 (2016).

Download references

Acknowledgements

We thank A. Tapper for sharing mouse behavioral equipment. This work was supported by the National Institutes of Health (grant no. R01NS093097 to W.-D.Y. and F.-B.G., grant nos. R37NS057553 and R01NS101986 to F.-B.G., and grant nos. P01AG019724 and P50AG023501 to W.W.S.) and by grants from the Packard Center for ALS Research, the Target ALS Foundation and the Muscular Dystrophy Association (F.-B.G.), Frick Foundation for ALS Research, the ALS Association and Angel Fund (S.A.), the Alzheimer’s Association (grant no. 2016-NIRG-396129 to S.A. and grant no. 2018-AARFD-592264 to R.L.-G.), the Tau Consortium and the Bluefield Project to Cure Frontotemporal Dementia (W.W.S.). F.-B.G. thanks the Cellucci family for their inspiration.

Author information

S.Y.C. performed most molecular and mouse behavioral and genetics experiments with some help from R.L.-G. and S.A. G.K. established the poly(GR) ELISA. H.L.P. and W.-D.Y. performed the electrophysiological analysis. A.N.L. and W.W.S. provided human brain tissues. S.Y.C. and F.-B.G. analyzed the data and wrote the manuscript with some input from W.-D.Y. and S.A. F.-B.G. directed the project.

Competing interests

The authors declare no competing interests.

Correspondence to Fen-Biao Gao.

Integrated supplementary information

  1. Supplementary Figure 1 Poly(GR) expression in CamKII;(GR)80 mice.

    a, A representative image showing preferential accumulation of poly(GR) in frontal cortex relative to other cortical regions of line 8 CamKII;(GR)80 mice at 8 months of age, selected from three independent immunostaining experiments. Scale bar: 500 µm. b, Enlarged image of the area highlighted by the red square in Panel a. Scale bar: 50 µm. c, The newly made rabbit polyclonal poly(GR) antibody is specific to (GR)8 and does not react with (GP)8 or (GA)8. The values are mean ± s.d. by one-way ANOVA with Tukey’s post hoc analysis for multiple comparisons: F(2,7) = 1372, P < 0.0001 for (GR)8 vs. (GP)8, P < 0.0001 for (GR)8 vs. (GA)8, P = 0.9924 for (GP)8 vs. (GA)8. (GR)8 = 4118.67 ± 1912.87, n = 3; (GP)8 = 254 ± 41.8, n = 3; (GA)8 = 147.66 ± 36.11 from three independent ELISA experiments. d, A representative z-stack image of poly(GR) nuclear localization in Type B and Type D neurons of CamKII;(GR)80 mice at 8 months of age selected from 5 images. This experiment was repeated three time.

  2. Supplementary Figure 2 Age-dependent behavioral phenotypes of CamKII and CamKII;(GR)80 mice.

    a, The three-chamber social interaction test for CamKII and CamKII;(GR)80 mice at 9 months of age. CamKII:Stranger = 77.43 ± 2.40, n = 11 mice; CamKII;(GR)80:Stranger = 58.16 ± 2.72, n = 10 mice; CamKII:Object = 22.57 ± 2.40, n = 11 mice; CamKII;(GR)80:Object = 41.84 ± 2.72, n = 10 mice; CamKII:Left=355.7 ± 22.78, n = 11 mice; CamKII;(GR)80:Left = 313 ± 14.28, n = 10 mice; CamKII:Center = 74.28 ± 7.59, n = 11 mice; CamKII;(GR)80:Center = 68.30 ± 6.99, n = 10 mice; CamKII:Right = 169.9 ± 20.69, n = 11 mice; CamKII;(GR)80:Right = 218.4 ± 16.81, n = 10 mice. Values are means ± s.e.m. by Student’s t test, two-sided: F(1,19) = 1.17, P < 0.0001 for CamKII:Stranger vs. CamKII;(GR)80:Stranger, F(1,19) = 1.17, P < 0.0001 for CamKII:Object vs. CamKII;(GR)80:Object, F(1, 19) = 2.8, P = 0.1383 for CamKII:Left vs. CamKII;(GR)80:Left, F(1, 19) = 1.3, P = 0.5791 for CamKII:Center vs. CamKII;(GR)80:Center, F(1, 19) = 1.67, P = 0.0882 for CamKII:Right vs. CamKII;(GR)80:Right. b, Schematic of elevated plus maze test. CamKII or CamKII;(GR)80 mice were placed in the center of the elevated plus maze and allowed to explore for 5 min. c, Time spent (left graph) and percentage of entries (right graph) in the open arm of the elevated plus maze by CamKII and CamKII;(GR)80 mice at 3 months of age. CamKII = 23.20 ± 3.78, n = 11 mice; CamKII;(GR)80 = 15.92 ± 3.36 in the left graph, n = 10 mice; CamKII = 14.54 ± 2.22, n = 11 mice; CamKII;(GR)80 = 10.46 ± 2.12 in the right graph, n = 10 mice. Values are mean ± s.e.m. by two-sided Student’s t test: F(1, 19) = 5.98, P = 0.1636 for CamKII vs. CamKII;(GR)80 in the left graph, F(1, 19) = 1.21, P = 0.2008 for CamKII vs. CamKII;(GR)80 in the right graph. d, Time spent (left graph) and percentage of entries (right graph) in the open arm of the elevated plus maze by CamKII and CamKII;(GR)80 mice at 9 months of age. Each dot represents one mouse. CamKII = 14.60 ± 2.67, n = 10 mice; CamKII;(GR)80 = 6.50 ± 2.65 in the left graph, n = 9 mice; CamKII = 10.00 ± 2.21, n = 10 mice; CamKII;(GR)80 = 4.23 ± 1.90 in the right graph, n = 9 mice. Values are mean ± s.e.m. by two-sided Student’s t test: F(1, 17) = 1.2, P = 0.0464 for CamKII vs. CamKII;(GR)80 in the left graph, F(1, 17) = 1.5, P = 0.0196 for CamKII vs. CamKII;(GR)80 in the right graph.

  3. Supplementary Figure 3 Body weight and open-field test of locomotor activity of CamKII and CamKII;(GR)80 mice.

    a, The body weight of five mice of each genotype and sex was measured monthly. There was no difference between CamKII and CamKII;(GR)80 mice. CamKII:male:1 month = 20.50 ± 0.85, n = 6 mice; CamKII;(GR)80:male:1 month = 21.33 ± 0.80, n = 6 mice; CamKII:male:2 months = 23.50 ± 1.02, n = 6 mice; CamKII;(GR)80:male:2 months = 23.83 ± 0.79, n = 6 mice; CamKII:male:3 months = 24.33 ± 1.02, n = 6 mice; CamKII;(GR)80:male:3 months = 24.50 ± 0.92, n = 6 mice; CamKII:male:4 months = 26.80 ± 0.73, n = 5 mice; CamKII;(GR)80:male:4 months = 26.80 ± 1.16, n = 5 mice; CamKII:male:5 months = 27.4 ± 0.93, n = 5 mice; CamKII;(GR)80:male:5 months = 29.00 ± 0.71, n = 4 mice; CamKII:male:6 months = 29.20 ± 1.11, n = 5 mice; CamKII;(GR)80:male:6 months = 31.25 ± 1.03, n = 4 mice; CamKII:male:7 months = 31.00 ± 1.45, n = 5 mice; CamKII;(GR)80:male:7 months = 32.75 ± 1.11, n = 4 mice; CamKII:male:8 months = 32.20 ± 1.83, n = 5 mice; CamKII;(GR)80:male:8 months = 34.00 ± 1.08, n = 4 mice; CamKII:male:9 months = 32.20 ± 1.98, n = 5 mice; CamKII;(GR)80:male:9 months = 35.00 ± 1.47, n = 4 mice; CamKII:male:10 months = 32.40 ± 2.06, n = 5 mice; CamKII;(GR)80:male:10 months = 35.00 ± 1.63, n = 4 mice; CamKII:male:11 months = 33.40 ± 2.23, n = 5 mice; CamKII;(GR)80:male:11 months = 36.25 ± 1.70, n = 4 mice; CamKII:female:1 month = 17.17 ± 0.31, n = 6 mice; CamKII;(GR)80:female:1 month = 1.75 ± 0.34, n = 6 mice; CamKII:female:2 months = 19.00 ± 0.52, n = 6 mice; CamKII;(GR)80:female:2 months = 19.00 ± 0.58, n = 6 mice; CamKII:female:3 months = 20.00 ± 0.52, n = 6 mice; CamKII;(GR)80:female:3 months = 20.17 ± 0.65, n = 6 mice; CamKII:female:4 months = 22.17 ± 0.31, n = 6 mice; CamKII;(GR)80:female:4 months = 21.50 ± 0.56, n = 6 mice; CamKII:female:5 months = 22.83 ± 0.54, n = 6 mice; CamKII;(GR)80:female:5 months = 22.50 ± 0.34, n = 6 mice; CamKII:female:6 months = 22.83 ± 0.70, n = 6 mice; CamKII;(GR)80:female:6 months = 22.83 ± 0.65, n = 6 mice; CamKII:female:7 months = 24.00 ± 0.73, n = 6 mice; CamKII;(GR)80:female:7 months = 23.83 ± 0.65, n = 6 mice; CamKII:female:8 months = 24.67 ± 0.80, n = 6 mice; CamKII;(GR)80:female:8 months = 24.33 ± 0.56, n = 6 mice; CamKII:female:9 months = 24.50 ± 0.85, n = 6 mice; CamKII;(GR)80:female:9 months = 23.83 ± 0.79, n = 6 mice; CamKII:female:10 months = 25.17 ± 0.65, n = 6 mice; CamKII;(GR)80:female:10 months = 24.83 ± 0.87, n = 6 mice; CamKII:female:11 months = 25.50 ± 0.62, n = 6 mice; CamKII;(GR)80:female:11 months = 24.67 ± 1.02, n = 6 mice. Values are mean ± s.e.m. by two-way ANOVA with Bonferroni post hoc test: F(10, 110) = 0.1638, P > 0.9999 for CamKII:male vs. CamKII;(GR)80:male in all months, P > 0.9999 for CamKII:female vs. CamKII;(GR)80:female in all age groups. b–d, Locomotor activity of CamKII and CamKII;(GR)80 mice in the open field was measured for 10 min at 3 (b), 6 (c), and 9 (d) months of age. The frequency of entries into the chamber center, the time spent there, the total distance and velocity of movement were recorded. Each dot represents one mouse. No statistically significant differences were found. The following values are all mean ± s.e.m. and analyzed by two-sided Student’s t test. In the ‘Frequency in Center’ graph in Panel b, CamKII = 34.83 ± 4.24 (n = 12 mice), CamKII;(GR)80=29.25 ± 2.79 (n = 12 mice), F(1, 22) = 2.315, P = 0.2833. In the ‘Duration in Center’ graph Panel b, CamKII = 73.36 ± 11.68 (n = 12 mice),; CamKII;(GR)80 = 83.37 ± 20.06 (n = 12 mice), F(1, 22) = 2.95, P = 0.6704. In the ‘Total Distance’ graph in Panel b, CamKII = 2510 ± 215.30 (n = 12 mice), CamKII;(GR)80 = 2390 ± 202.00 (n = 12 mice), F(1, 22) = 1.14, P = 0.6704. In the ‘Velocity’ graph in Panel b, CamKII = 4.18 ± 0.36 (n = 12 mice), CamKII;(GR)80 = 3.98 ± 0.34 (n=12 mice), F(1, 22) = 1.14, P = 0.69. In the ‘Frequency in Center’ graph Panel c, CamKII = 25.36 ± 4.77 (n=11 mice), CamKII;(GR)80 = 29.56 ± 4.59 (n = 9 mice), F(1, 18) = 1.32, P = 0.5404. In the ‘Duration in Center’ graph Panel c, CamKII = 42.1 ± 9.47 (n = 9 mice), CamKII;(GR)80 = 46.05 ± 7.38 (n = 9 mice), F(1, 18) = 2.01, P = 0.7541. In the ‘Total Distance’ graph in Panel c, CamKII = 2200 ± 231.30 (n=11 mice), CamKII;(GR)80 = 2457 ± 205.1 (n = 9 mice), F(1, 18) = 1.55, P = 0.4267. In the ‘Velocity’ graph in the panel c, CamKII = 3.67 ± 0.39 (n = 11 mice), CamKII;(GR)80 = 4.10 ± 0.34 (n = 9 mice), F(1, 18) = 1.55, P = 0.4265. In the ‘Frequency in Center’ graph in Panel d, CamKII = 26.80 ± 5.62 (n = 10 mice), CamKII;(GR)80 = 26.5 ± 3.22 (n = 8 mice), F(1, 16) = 3.81, P = 0.9660. In the ‘Duration in Center’ graph in Panel d, CamKII = 33.86 ± 9.13, (n = 10 mice); CamKII;(GR)80 = 39.21 ± 15.11 (n = 8 mice), F(1, 16) = 2.19, P = 0.7554. In the ‘Total Distance’ graph in Panel d, CamKII = 2596 ± 248.3 (n = 10 mice), CamKII;(GR)80 = 3035 ± 253.6 (n = 8 mice), P = 0.2387. In the ‘Velocity’ graph in Panel d, CamKII = 4.33 ± 0.41 (n = 10 mice), CamKII;(GR)80 = 5.06 ± 0.42 (n = 8 mice), F(1, 16) = 1.20, P = 0.2387.

  4. Supplementary Figure 4 T-maze working memory test of CamKII and CamKII;(GR)80 mice.

    a, Schematic of the T-maze working memory test. The test mouse was forced into the left arm, where food was placed. After a 10-s delay, the mouse was placed in the center arm and allowed to move into the right or left arm. If the mouse chose the arm where the food was, the event was counted as a success. b, Success rate of CamKII and CamKII;(GR)80 mice at 6–9 months of age in the T-maze test, calculated from 10 trials. Each dot represents one mouse. No statistically significant difference was found. CamKII = 61.11 ± 6.33 (n = 9 mice), CamKII;(GR)80 = 54.00 ± 4.52 (n = 10 mice). Values are mean ± s.e.m., F(1, 17) = 1.77, P = 0.3666, by two-sided Student’s t test.

  5. Supplementary Figure 5 Expression of activated caspase 3 in poly(GR)-expressing neurons of three CamKII;(GR)80 mice.

    Double-immunostaining for poly(GR) and cleaved caspase 3 in the cortex of three 9-month-old CamKII;(GR)80 mice. The immunostaining experiments was repeated three times. Scale bar: 10 µm.

  6. Supplementary Figure 6 Astrogliosis in the cortex of CamKII;(GR)80 mice.

    a, Astrogliosis in CamKII (n = 2) and CamKII;(GR)80 (n = 3) mice at 9 months of age as shown by immunostaining for glial fibrillary acidic protein (Gfap). The lower panels are enlarged images of areas indicated by white boxes in corresponding upper panels. Scale bar: 1 mm in upper panels, 300 µm in lower panels. b, Quantification of relative intensity of Gfap signal in the cortex of 9-month CamKII and CamKII;(GR)80 mice. CamKII = 100 ± 0.08 (n = 3 mice), CamKII;(GR)80 = 134 ± 0.05 (n = 4 mice). Values are mean ± s.e.m., F(1, 5) = 2.4, P = 0.0116, by two-sided Student’s t test.

  7. Supplementary Figure 7 Some known molecular defects in C9ORF72-FTD/ALS are absent in CamKII;(GR)80 mice.

    a, Absence of RanGAP1 aggregates in the poly(GR)-expressing neurons of three 9-month-old CamKII;(GR)80 mice. Three animals per genotype and per age group were analyzed. b, Lack of TDP-43 pathology, shown by immunostaining in the cortex of CamKII and CamKII;(GR)80 mice at 3, 6, and 8 months of age. Three animals per genotype and per age group were analyzed. Scale bars: 20 µm. c, Western blot analysis and quantification show that the p62 level was not affected in 6-month-old CamKII;(GR)80 mice. CamKII = 1.49 ± 0.25 (n = 4 mice), CamKII;(GR)80 = 0.98 ± 0.22 (n = 4 mice). Values are mean ± s.e.m., F(1, 6) = 1.31, P = 0.1760, by two-sided Student’s t test.

  8. Supplementary Figure 8 Increased DNA damage in poly(GR)-expressing neurons of CamKII;(GR)80 mice.

    Double-immunostaining for CamKII and Histone H2AX (DNA damage marker) in the cortex of three 8-month-old CamKII;(GR)80 mice. Because (GR)80 expression is driven by CamKII-tTA, CamKII-positive neurons express (GR)80. Scale bar: 5 µm.

  9. Supplementary Figure 9 Reduced mitochondrial motility in cultured primary neurons of CamKII;(GR)80 mice.

    a, A representative kymograph of mitochondrial movement in neurites of CamKII and CamKII;(GR)80 primary cortical neurons on day 14 in vitro (DIV14). The experiment was performed in three independent cultures from three different animals. Scale bar: 10 µm. b, The velocity of mitochondria in neurite of CamKII and CamKII;(GR)80 primary cortical neurons on DIV14. Each dot represents one mitochondrion. CamKII = 0.15 ± 0.01 (n=76 mitochondria), CamKII;(GR)80 = 0.09 ± 0.01 (n = 47 mitochondria). Values are means ± s.d., F(1, 121) = 1.74, P = 0.0085, by two-sided Student’s t test. c. The percentage of mobile mitochondria in poly(GR)-expressing primary cortical neurons on DIV14. The primary cortical neurons were cultured from three embryos of either CamKII or CamKII;(GR)80 mice, and mitochondrial movement was analyzed on DIV14. CamKII = 29.01 ± 4.80 (n = 3 mice), CamKII;(GR)80 = 13.32 ± 2.29 (n = 3 mice). Values are mean ± s.e.m., F(1, 4) = 4.39, P = 0.0418, by two-sided Student’s t test.

  10. Supplementary Figure 10 Changes in DRP1 and OPA1 levels in neurons of CamKII;(GR)80 mice.

    a, Western blot analysis of DRP1 in the cortex of CamKII mice (n = 3) and CamKII;(GR)80 mice (n = 4) at 9 months of age. b, Quantification of western blot analysis of DRP1 in the cortex of 9-month old CamKII and CamKII;(GR)80 mice. CamKII = 0.78 ± 0.15 (n = 3 mice), CamKII;(GR)80 = 1.51 ± 0.51 (n = 4 mice). Values are mean ± s.e.m., F(1, 5) = 1.49, P = 0.0209, by two-sided Student’s t test. c, Western blot analysis of OPA1 in the cortex of CamKII and CamKII;(GR)80 mice at 9 months of age. d, Quantification of western blot analysis of long and short OPA1 (L-OPA1, S-OPA1) in the cortex of 9-month-old CamKII and CamKII;(GR)80 mice. CamKII:L-OPA1 = 1.00 ± 0.17 (n = 3 mice), CamKII;(GR)80:L-OPA1 = 0.51 ± 0.01 (n = 3 mice), CamKII:S-OPA1 = 1.00 ± 0.14 (n = 3 mice), CamKII;(GR)80:S-OPA1 = 0.60 ± 0.04 (n = 3 mice). Values are mean ± s.e.m., F(1, 4) = 880.7, P = 0.0467 for CamKII:L-OPA1 vs. CamKII;(GR)80:L-OPA1, F(1, 4) = 11.61, P = 0.0483 for CamKII:S-OPA1 vs. CamKII;(GR)80:S-OPA1, by two-sided Student’s t test.

  11. Supplementary Figure 11 Poly(GR) is present inside mitochondria.

    Double-immunostaining for HSP60, a mitochondria-specific marker, and poly(GR) in type C poly(GR)-expressing neurons of two 9-month-old CamKII;(GR)80 mice with high-level of poly(GR) expression (line 8). This immunostaining experiment was repeated three times. Scale bar: 5 µm. Enlarged squares a’, b’ and c’ are shown on the right. Dotted circles indicate the mitochondrial location (from three independently repeated experiments with similar results). Scale bar: 0.5 µm.

  12. Supplementary Figure 12 Binding of poly(GR) to ATP5A1 and the level of Atp5a1 expression in CamKII;(GR)80 mice.

    a, Poly(GR) co-immunoprecipitated with ATP5A1 GFP-(GR)80 overexpressing HEK293 cells was analyzed on western blots (from three independently repeated experiments with similar results). b, Immunostaining for Atp5a1 in cortical neurons expressing poly(GR) (from three independently repeated experiments with similar results). Scale bar: 5 µm. c, Relative expression levels of Atp5a1 mRNA in CamKII and CamKII;(GR)80 mice. CamKII = 1.00 ± 0.03 (n = 6 mice), CamKII;(GR)80 = 1.10 ± 0.02 (n = 6 mice). Values are mean ± s.e.m., F(1, 10) = 1.86, P = 0.1126, by two-sided Student’s t test.

  13. Supplementary Figure 13 The expression of some mitochondrial proteins in cortex of CamKII;(GR)80 mice and C9ORF72 patients.

    a, The western blot analysis of Atp5h in the cortex of CamKII and CamKII;(GR)80 mice at 6-month old of age (from three independently repeated experiments with similar results). b, Quantification of western blot analysis of Atp5h in the cortex of CamKII and CamKII;(GR)80 mice at 6-month old of age. CamKII = 1.00 ± 0.05 (n = 4 mice), CamKII;(GR)80 = 1.03 ± 0.07 (n = 5 mice). Values are mean ± s.e.m., F(1, 7) = 2.16, P = 0.7172, by two-sided Student’s t test. c, Western blot analysis of HSP60 and ATP5H in the prefrontal cortex of C9ORF72 patients (from three independently repeated experiments with similar results). d and e Quantification of western blot in panel c. Values are means ± s.e.m. In the panel d, Control = 1.00 ± 0.10 (n = 3 subjects), C9ORF72 = 0.93 ± 0.07 (n = 4 patients), F(1, 5) = 1.33, P = 0.5919 by two-sided Student’s t test. In the panel e, Control = 1.00 ± 0.05 (n = 3 subjects), C9ORF72 = 0.95 ± 0.22 (n = 4 patients), F(1, 5) = 3.27, P = 0.2939 by two-sided Student’s t test.

  14. Supplementary Figure 14 Suppression of poly(GR) expression prevents behavioral defects of CamKII;(GR)80 mice.

    a, Schematic of doxycycline (DOX) treatment of CamKII;(GR)80 mice from 1–6 months of age. b and c, Results of the three-chamber social interaction test for CamKII and CamKII;(GR)80 mice at 6 months of age. n = 6–10 mice of each genotype at each time point. Each dot represents one mouse. The following values are mean ± s.e.m. and analyzed by two-sided Student’s t test: F(2, 24) = 83.9, P < 0.0001 for CamKII:Stranger vs. CamKII;(GR)80 without DOX:Stranger, F(2, 24) = 83.9, P < 0.0001 for CamKII;(GR)80 without DOX:Stranger vs. CamKII;(GR)80 with DOX:Stranger, F(2, 24) = 83.9, P < 0.0001 for CamKII:Object vs. CamKII;(GR)80 without DOX:Object, F(2, 24) = 83.9, P < 0.0001 for CamKII;(GR)80 without DOX:Object vs. CamKII;(GR)80 with DOX:Object. In Panel b, CamKII:Stranger = 72.83 ± 2.89 (n = 10 mice), CamKII;(GR)80 without DOX:Stranger = 43.27 ± 0.54 (n = 7 mice), CamKII;(GR)80 with DOX:Stranger = 71.53 ± 4.08 (n = 7 mice), CamKII:Object = 27.17 ± 2.89 (n = 10 mice), CamKII;(GR)80 without DOX:Object = 56.73 ± 0.54 (n=7 mice), CamKII;(GR)80 with DOX:Object = 28.47 ± 4.08 (n = 7 mice). In Panel c, CamKII:Left = 376.10 ± 22.99 (n = 10 mice), CamKII;(GR)80 without DOX:Left = 359.29 ± 21.99 (n = 7 mice), CamKII;(GR)80 with DOX:Left = 362.14 ± 24.11 (n = 7 mice), CamKII:Center = 91.15 ± 16.28 (n = 10 mice), CamKII;(GR)80 without DOX:Center = 89.75 ± 15.84 (n = 7 mice), CamKII;(GR)80 with DOX: Center = 91.65 ± 18.25 (n = 7 mice), CamKII:Right = 132.75 ± 21.00 (n = 10 mice), CamKII;(GR)80 without DOX:Right = 150.96 ± 13.9 (n = 7 mice), CamKII;(GR)80 with DOX:Right = 146.21 ± 18.62 (n = 7 mice). d and e, Duration (d) and percentage of entries (e) in the open arm of the elevated plus maze by CamKII and CamKII;(GR)80 mice at 6 months of age. In Panel d, CamKII = 8.82 ± 2.70 (n = 10 mice), CamKII;(GR)80 without DOX = 0.37 ± 0.33 (n = 6 mice), CamKII;(GR)80 with DOX = 6.23 ± 2.30 (n = 6 mice), F(1, 14) = 112.5, P = 0.0317 for CamKII vs. CamKII;(GR)80 without DOX, F(1, 11) = 83.9, P = 0.0394 for CamKII;(GR)80 without DOX vs. CamKII;(GR)80 with DOX, by two-sided Student’s t test. In Panel e, CamKII = 9.35 ± 1.27 (n = 10 mice), CamKII;(GR)80 without DOX = 2.47 ± 1.83 (n = 6 mice), CamKII;(GR)80 with DOX = 9.20 ± 1.82 (n = 7 mice), F(1, 14) = 1.25, P = 0.0066 for CamKII vs. CamKII;(GR)80 without DOX, F(1, 11) = 1.15, P = 0.0249 for CamKII;(GR)80 without DOX vs. CamKII;(GR)80 with DOX, by two-sided Student’s t test.

  15. Supplementary Figure 15 Feeding doxycycline for 2 months reverses increased microgliosis and astrogliosis in 9-month-old CamKII;(GR)80 mice.

    a, Representative images of IbaI-positive cells in the cortex of three CamKII and CamKII;(GR)80 mice of 9-month-old. Scale bar: 25 μm. b, Quantification of IbaI-positive cells in the cortex of 9-month-old CamKII and CamKII;(GR)80 mice. CamKII = 253.09 ± 1.28 (n = 3 mice), CamKII;(GR)80 without DOX = 312.22 ± 5.23 (n = 4 mice), CamKII;(GR)80 with DOX = 250.88 ± 4.98 (n = 3 mice). Values are mean ± s.e.m., F(2, 7) = 1.96, P < 0.0001 for CamKII vs. CamKII;(GR)80 without DOX, P < 0.0001 for CamKII;(GR)80 without DOX vs. CamKII;(GR)80 with DOX, by one-way ANOVA with Tukey’s post hoc analysis for multiple comparisons. c, Western blot analysis of Gfap expression in the cortex of CamKII and CamKII;(GR)80 mice fed normal chow or chow containing doxycycline (DOX). Three mice of each genotype and treatment were analyzed (from three independently repeated experiments with similar results). d, Quantification of Gfap expression level. CamKII = 1.00 ± 0.02 (n = 3 mice) CamKII;(GR)80 without DOX = 1.17 ± 0.05 (n = 3 mice), CamKII;(GR)80 with DOX = 0.97 ± 0.02. Values are mean ± s.e.m., F(2, 6) = 9.68, P = 0.0311 for CamKII vs. CamKII;(GR)80 without DOX, P = 0.0153 for CamKII;(GR)80 without DOX vs. CamKII;(GR)80 with DOX, by two-sided Student’s t test.

Supplementary information

  1. Supplementary Information

    Supplementary Figs. 1–15 and Supplementary Table 1.

  2. Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark
Fig. 1: Age-dependent accumulation of low level of poly(GR) in CamKII;(GR)80 mice.
Fig. 2: CamKII;(GR)80 mice have age-dependent behavioral deficits and synaptic impairment.
Fig. 3: Pathological features in the cortex of CamKII;(GR)80 mice.
Fig. 4: Poly(GR) induces mitochondrial dysfunction in the cortex of CamKII;(GR)80 mice.
Fig. 5: Poly(GR) binds to ATP5A1 and decreases Atp5a1 expression level in the cortex of CamKII;(GR)80 mice.
Fig. 6: Poly(GR) increases ubiquitination and degradation of ATP5A1, and Atp5a1 overexpression rescues neuronal survival of CamKII;(GR)80 neurons.
Fig. 7: Reducing (GR)80 expression in adult mice rescues pre-existing pathological features of CamKII;(GR)80 mice.
Supplementary Figure 1: Poly(GR) expression in CamKII;(GR)80 mice.
Supplementary Figure 2: Age-dependent behavioral phenotypes of CamKII and CamKII;(GR)80 mice.
Supplementary Figure 3: Body weight and open-field test of locomotor activity of CamKII and CamKII;(GR)80 mice.
Supplementary Figure 4: T-maze working memory test of CamKII and CamKII;(GR)80 mice.
Supplementary Figure 5: Expression of activated caspase 3 in poly(GR)-expressing neurons of three CamKII;(GR)80 mice.
Supplementary Figure 6: Astrogliosis in the cortex of CamKII;(GR)80 mice.
Supplementary Figure 7: Some known molecular defects in C9ORF72-FTD/ALS are absent in CamKII;(GR)80 mice.
Supplementary Figure 8: Increased DNA damage in poly(GR)-expressing neurons of CamKII;(GR)80 mice.
Supplementary Figure 9: Reduced mitochondrial motility in cultured primary neurons of CamKII;(GR)80 mice.
Supplementary Figure 10: Changes in DRP1 and OPA1 levels in neurons of CamKII;(GR)80 mice.
Supplementary Figure 11: Poly(GR) is present inside mitochondria.
Supplementary Figure 12: Binding of poly(GR) to ATP5A1 and the level of Atp5a1 expression in CamKII;(GR)80 mice.
Supplementary Figure 13: The expression of some mitochondrial proteins in cortex of CamKII;(GR)80 mice and C9ORF72 patients.
Supplementary Figure 14: Suppression of poly(GR) expression prevents behavioral defects of CamKII;(GR)80 mice.
Supplementary Figure 15: Feeding doxycycline for 2 months reverses increased microgliosis and astrogliosis in 9-month-old CamKII;(GR)80 mice.