Amyotrophic lateral sclerosis (ALS) is a rapidly progressing neurodegenerative disease that is characterized by motor neuron loss and that leads to paralysis and death 2–5 years after disease onset1. Nearly all patients with ALS have aggregates of the RNA-binding protein TDP-43 in their brains and spinal cords2, and rare mutations in the gene encoding TDP-43 can cause ALS3. There are no effective TDP-43-directed therapies for ALS or related TDP-43 proteinopathies, such as frontotemporal dementia. Antisense oligonucleotides (ASOs) and RNA-interference approaches are emerging as attractive therapeutic strategies in neurological diseases4. Indeed, treatment of a rat model of inherited ALS (caused by a mutation in Sod1) with ASOs against Sod1 has been shown to substantially slow disease progression5. However, as SOD1 mutations account for only around 2–5% of ALS cases, additional therapeutic strategies are needed. Silencing TDP-43 itself is probably not appropriate, given its critical cellular functions1,6. Here we present a promising alternative therapeutic strategy for ALS that involves targeting ataxin-2. A decrease in ataxin-2 suppresses TDP-43 toxicity in yeast and flies7, and intermediate-length polyglutamine expansions in the ataxin-2 gene increase risk of ALS7,8. We used two independent approaches to test whether decreasing ataxin-2 levels could mitigate disease in a mouse model of TDP-43 proteinopathy9. First, we crossed ataxin-2 knockout mice with TDP-43 (also known as TARDBP) transgenic mice. The decrease in ataxin-2 reduced aggregation of TDP-43, markedly increased survival and improved motor function. Second, in a more therapeutically applicable approach, we administered ASOs targeting ataxin-2 to the central nervous system of TDP-43 transgenic mice. This single treatment markedly extended survival. Because TDP-43 aggregation is a component of nearly all cases of ALS6, targeting ataxin-2 could represent a broadly effective therapeutic strategy.
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This work was supported by NIH grants R01NS065317, R01NS09386501, R01NS073660 and R35NS097263 (10) (A.D.G.), NIH grant R35NS097974 (J.P.T), HHMI (J.P.T), NIH grants R21NS081182 and R37NS033123 (S.M.P), the National Science Foundation Graduate Research Fellowship (L.A.B.), the Robert Packard Center for ALS Research at Johns Hopkins (A.D.G.), Target ALS (A.D.G.), the Glenn Foundation (A.D.G.), and the DFG grant AU96/13-1 (G.A.). We thank L. Petrucelli and V. Lee for sharing TDP-43 antibodies, J. Shorter and L. Petrucelli for comments on the manuscript and discussions, A. Olsen and the Stanford Neuroscience Microscopy Service, supported by a grant from NIH (NS069375), for help with the confocal images, Y. Zuber (Stanford Veterinary Service Center) for mouse husbandry advice and support, Stanford’s Human Immune Monitoring Center (HIMC) for performing the Luminex assays.
P.J.-N., A.S. and F.R. are employed by Ionis Pharmaceuticals, a for-profit company that develops ASO therapies.
Reviewer Information Nature thanks R. L. Juliano, J. Rothstein and T. Siddique for their contribution to the peer review of this work.
Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
Extended Data Figure 1 Reduction of ataxin-2 using two independent Atxn2 knockout mice lines extends lifespan of TDP-43 transgenic mice.
a–d, Lines A and B were generated using Atxn2+/− mice from a congenic C57BL/6 and a hybrid B6.129S background, respectively. Within line A, TDP-43Tg/TgAtxn2+/− (light blue, n = 7) (a) and TDP-43Tg/TgAtxn2−/− (dark blue, n = 7) (c) mice lived significantly longer than TDP-43Tg/TgAtxn2+/+ mice (red, n = 16). Within line B, TDP-43Tg/TgAtxn2+/− (n = 13) (b) and TDP-43Tg/TgAtxn2−/− (n = 11) (d) mice also lived significantly longer than TDP-43Tg/TgAtxn2+/+ mice (n = 12). Curves were compared by log-rank test and effect size estimated by a Cox proportional hazards model. HR, hazard ratio. Ticks indicate mice that were euthanized for tissue collection before reaching the humane euthanasia end point or that were still alive at the time of submission (see Methods). e, After taking genotype into account, the line that the mice came from, but not the sex of the mice, significantly affected lifespan. A Cox proportional hazards likelihood ratio test was used to compare the null model including only genotype to an alternative model including genotype and line or genotype and sex. f, We found evidence for two groups of responders (strong and weak) in the TDP-43Tg/TgAtxn2−/− population (parametric bootstrap P = 0.02, see Methods). The Kaplan–Meier curve of all TDP-43Tg/TgAtxn2−/− mice from both lines is plotted, and the one and two group models are shown. g, Knockout of Atxn2 did not affect weight in non-transgenic or TDP-43Tg/Tg adolescents. Data are mean ± s.e.m.
a, Representative NeuN stains of a sagittal section through the cortex. Layer V is marked by a green bar. b, c, Layer V neurons were 30% less numerous (b) and had smaller cell bodies (c) in TDP-43Tg/TgAtxn2+/+ mice. These phenotypes were significantly ameliorated in TDP-43Tg/TgAtxn2−/− mice. Four mice were quantified per genotype, and the values for individual brain sections are plotted. Genotype groups were compared using linear mixed models with a random effect to appropriately account for the multiple measurements per mouse (see Methods). d, Representative NeuN stains of L5 lumbar ventral horn showing large lower motor neurons. e, Quantification of motor neuron cell bodies present in the ventral horn of the lumbar enlargement at levels L3–L6. There was a 27% decrease in motor neurons on average in TDP-43Tg/TgAtxn2+/+ compared to wild-type mice. Six P23 animals were used per genotype. Two-tailed t-tests were performed between groups of interest. Data are mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant.
Extended Data Figure 3 Lowering ataxin-2 levels does not affect expression of the human TDP-43 transgene.
a, Atxn2 mRNA levels were decreased in the Atxn2+/− mouse brain by approximately 50% and completely absent in Atxn2−/− mice. b, Among TDP-43Tg/Tg mice, Atxn2 reduction did not significantly affect levels of the human TDP-43 (hTDP) transgene by ordinary one-way ANOVA. Samples were collected at P21. Data are mean ± s.e.m.
Extended Data Figure 4 Protein levels of TDP-43 are not significantly affected by ataxin-2 reduction in TDP-43Tg/Tg mice.
a, Nucleocytoplasmic fractioning of mouse brain tissue segregated the nuclear marker lamin A/C from the cytoplasmic marker GAPDH. b–d, Nuclear levels of human TDP-43 (hTDP-43; b) or total full-length TDP-43 (c) were not altered among the genotypes, although nuclear TDP-43 CTFs were slightly elevated in TDP-43Tg/TgAtxn2+/+ mice (d). e–g, Cytoplasmic hTDP-43 (e) and total TDP-43 CTFs (g) were also unaltered among the genotypes, although cytoplasmic full-length TDP-43 (f) seemed slightly elevated in TDP-43Tg/TgAtxn2+/+ mice. Samples were collected at P21. Data are mean ± s.e.m. Gel source data can be found in Supplementary Fig. 1.
a–c, Two different phosphorylation-specific TDP-43 antibodies (a, b) and a C-terminal epitope TDP-43 antibody (c) readily stained stress granules, indicated by the stress granule markers EIF3η and ataxin-2.
a–d, m–p, None of the TDP-43 antibodies tested recognized inclusions in wild-type mice. e, f, h, q–t, One of the three phosphorylated TDP-43 (pTDP-43)-specific antibodies (e) and TDP-43 antibodies that were not phosphorylation-specific (f, h, q–t) recognized spherical, predominantly nuclear inclusions. g, The other two pTDP-43-specific antibodies (only one is shown) recognized smaller cytoplasmic and nuclear inclusions (g). i, j, l, The first type of inclusion was very rare in TDP-43Tg/TgAtxn2−/− mice. k, The second type of inclusion appeared smaller and reduced in number in TDP-43Tg/TgAtxn2−/− mice. q–t, Nuclear inclusions were effectively stained with total TDP-43 (tTDP-43), human-specific TDP-43 (hTDP-43), and mouse-specific TDP-43 (mTDP-43) antibodies. s, Diffuse mTDP-43 is greatly decreased in TDP-43Tg/Tg mice, an expected outcome of TDP-43 autoregulation. u, Levels of diffuse nuclear tTDP-43 were quantified by immunofluorescence microscopy in wild-type neurons or TDP-43Tg/Tg neurons with or without inclusions. These three groups were compared in a pairwise fashion using linear mixed models with a term to appropriately account for multiple measurements per mouse (n = 3 mice per genotype, see Methods). Median and minimum to maximum are plotted. Images were taken of cervical spinal cord. Samples were collected at P21. Scale bars, 10 μm.
Extended Data Figure 7 An ASO that targets Atxn2 is able to successfully reduce mRNA levels throughout the central nervous system.
a, ICV injection at P1 of an ASO that targets Atxn2 was able to successfully reduce levels of Atxn2 mRNA in the spinal cord by approximately 75% when assessed at P28. b, c, Atxn2 reduction was also seen in the cortex (b) and cerebellum (c). d, Grip strength of wild-type mice was not effected by injection of control (Ctrl) or Atxn2 ASOs (n = 16 mice per treatment). Data are mean ± s.e.m. e–h, Genetic markers of gliosis, Aif1 and Gfap, were not altered in the spinal cord (e, f) or cortex (g, h) after ASO injection. a–c, e–h, Biological replicates and mean are shown. i, Using a Luminex 38-plex assay, we could not detect a significant difference in inflammatory markers among uninjected wild-type mice (n = 5) and wild-type mice treated with the Atxn2 (n = 4) or control (n = 5) ASOs (two-way ANOVA treatment group factor P = 0.32). However, the ASO-treated animals had a small increase in 1 of the 38 markers, VEGF. Multiplicity-adjusted pairwise tests revealed that this difference was not significant for mice treated with the Atxn2 ASO (P = 0.17), but was for mice treated with control ASO (P = 0.006). Minimum to maximum are shown.
This file contains the uncropped western blots. (PDF 1256 kb)
This mouse was given a gait impairment score of 0. (MOV 11869 kb)
This mouse was given a gait impairment score of 1. (MOV 18138 kb)
This mouse was 21 days old and given a gait impairment score of 3 as it had very limited hindlimb joint movement and falls over. (MOV 25860 kb)
This mouse was 22 days old and given a gait impairment score of 4 (humane euthanasia endpoint) because it was unable to right itself within 30 seconds of falling on its side on all 3 of 3 trials. (MOV 14070 kb)
This TDP-43Tg/TgAtxn2–/– mouse was at 61 days old and still had no overt motor impairment. It was therefore given a gait impairment score of 0. (MOV 19391 kb)
Examples of three P20 TDP-43Tg/Tg that received intracerebroventricular (ICV) administration of either the control ASO or the Atxn2 ASO at P1. The two severely impaired mice (gait impairment score of 4), which were unable to right themselves, received the control ASO whereas the one unimpaired mouse received the Atxn2 ASO. (MOV 11199 kb)
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Becker, L., Huang, B., Bieri, G. et al. Therapeutic reduction of ataxin-2 extends lifespan and reduces pathology in TDP-43 mice. Nature 544, 367–371 (2017). https://doi.org/10.1038/nature22038
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