Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Gain of toxic function by long-term AAV9-mediated SMN overexpression in the sensorimotor circuit

Nature Neuroscience volume 24, pages 930–940 (2021)Cite this article

Subjects

Abstract

The neurodegenerative disease spinal muscular atrophy (SMA) is caused by deficiency in the survival motor neuron (SMN) protein. Currently approved SMA treatments aim to restore SMN, but the potential for SMN expression beyond physiological levels is a unique feature of adeno-associated virus serotype 9 (AAV9)-SMN gene therapy. Here, we show that long-term AAV9-mediated SMN overexpression in mouse models induces dose-dependent, late-onset motor dysfunction associated with loss of proprioceptive synapses and neurodegeneration. Mechanistically, aggregation of overexpressed SMN in the cytoplasm of motor circuit neurons sequesters components of small nuclear ribonucleoproteins, leading to splicing dysregulation and widespread transcriptome abnormalities with prominent signatures of neuroinflammation and the innate immune response. Thus, long-term SMN overexpression interferes with RNA regulation and triggers SMA-like pathogenic events through toxic gain-of-function mechanisms. These unanticipated, SMN-dependent and neuron-specific liabilities warrant caution on the long-term safety of treating individuals with SMA with AAV9-SMN and the risks of uncontrolled protein expression by gene therapy.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Long-term AAV9-mediated SMN overexpression induces sensorimotor toxicity in WT and SMA mice.
Fig. 2: AAV9-SMN induces late-onset deafferentation of motor neurons in WT and SMA mice.
Fig. 3: AAV9-SMN induces late-onset neurodegeneration in WT mice.
Fig. 4: AAV9 drives long-term transgene overexpression in motor neurons and proprioceptive neurons but not the liver of WT mice.
Fig. 5: AAV9-SMN induces cytoplasmic aggregation of SMN and SmB proteins.
Fig. 6: Long-term AAV9-mediated SMN overexpression induces widespread transcriptome alterations in DRGs.

Similar content being viewed by others

Data availability

The data supporting the findings of this study are available in this paper or the Supplementary Information. Sequencing data have been deposited in the Gene Expression Omnibus database under accession number GSE149391. Source data are provided with this paper. Any other raw data that support the findings of this study are available from the corresponding author upon reasonable request.

References

  1. Burghes, A. H. M. & Beattie, C. E. Spinal muscular atrophy: why do low levels of survival motor neuron protein make motor neurons sick? Nat. Rev. Neurosci. 10, 597–609 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Tisdale, S. & Pellizzoni, L. Disease mechanisms and therapeutic approaches in spinal muscular atrophy. J. Neurosci. 35, 8691–8700 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Lefebvre, S. et al. Identification and characterization of a spinal muscular atrophy-determining gene. Cell 80, 155–165 (1995).

    Article  CAS  PubMed  Google Scholar 

  4. Lorson, C. L., Hahnen, E., Androphy, E. J. & Wirth, B. A single nucleotide in the SMN gene regulates splicing and is responsible for spinal muscular atrophy. Proc. Natl Acad. Sci. USA 96, 6307–6311 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Hua, Y. et al. Peripheral SMN restoration is essential for long-term rescue of a severe spinal muscular atrophy mouse model. Nature 478, 123–126 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Porensky, P. N. et al. A single administration of morpholino antisense oligomer rescues spinal muscular atrophy in mouse. Hum. Mol. Genet. 21, 1625–1638 (2012).

    Article  CAS  PubMed  Google Scholar 

  7. Naryshkin, N. A. et al. Motor neuron disease. SMN2 splicing modifiers improve motor function and longevity in mice with spinal muscular atrophy. Science 345, 688–693 (2014).

    Article  CAS  PubMed  Google Scholar 

  8. Palacino, J. et al. SMN2 splice modulators enhance U1-pre-mRNA association and rescue SMA mice. Nat. Chem. Biol. 11, 511–517 (2015).

    Article  CAS  PubMed  Google Scholar 

  9. Foust, K. D. et al. Rescue of the spinal muscular atrophy phenotype in a mouse model by early postnatal delivery of SMN. Nat. Biotechnol. 28, 271–274 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Passini, M. A. et al. CNS-targeted gene therapy improves survival and motor function in a mouse model of spinal muscular atrophy. J. Clin. Invest. 120, 1253–1264 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Dominguez, E. et al. Intravenous scAAV9 delivery of a codon-optimized SMN1 sequence rescues SMA mice. Hum. Mol. Genet. 20, 681–693 (2011).

    Article  CAS  PubMed  Google Scholar 

  12. Valori, C. F. et al. Systemic delivery of scAAV9 expressing SMN prolongs survival in a model of spinal muscular atrophy. Sci. Transl. Med. 2, 35ra42 (2010).

    Article  PubMed  Google Scholar 

  13. Finkel, R. S. et al. Nusinersen versus sham control in infantile-onset spinal muscular atrophy. N. Engl. J. Med. 377, 1723–1732 (2017).

    Article  CAS  PubMed  Google Scholar 

  14. Finkel, R. S. et al. Treatment of infantile-onset spinal muscular atrophy with nusinersen: a phase 2, open-label, dose-escalation study. Lancet 388, 3017–3026 (2016).

    Article  CAS  PubMed  Google Scholar 

  15. Mercuri, E. et al. Nusinersen versus sham control in later-onset spinal muscular atrophy. N. Engl. J. Med. 378, 625–635 (2018).

    Article  CAS  PubMed  Google Scholar 

  16. Mendell, J. R. et al. Single-dose gene-replacement therapy for spinal muscular atrophy. N. Engl. J. Med. 377, 1713–1722 (2017).

    Article  CAS  PubMed  Google Scholar 

  17. Al-Zaidy, S. et al. Health outcomes in spinal muscular atrophy type 1 following AVXS-101 gene replacement therapy. Pediatr. Pulmonol. 54, 179–185 (2019).

    Article  PubMed  Google Scholar 

  18. Dhillon, S. Risdiplam: first approval. Drugs 80, 1853–1858 (2020).

  19. Mercuri, E., Pera, M. C., Scoto, M., Finkel, R. & Muntoni, F. Spinal muscular atrophy—insights and challenges in the treatment era. Nat. Rev. Neurol. https://doi.org/10.1038/s41582-020-00413-4 (2020).

  20. Hinderer, C. et al. Severe toxicity in nonhuman primates and piglets following high-dose intravenous administration of an adeno-associated virus vector expressing human SMN. Hum. Gene Ther. 29, 285–298 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. National Library of Medicine. Study of intrathecal administration of onasemnogene abeparvovec-xioi for spinal muscular atrophy (STRONG). ClinicalTrials.gov https://clinicaltrials.gov/ct2/show/NCT03381729 (2017).

  22. European Medicines Agency. Zolgensma: European public assessment report. https://www.ema.europa.eu/en/documents/assessment-report/zolgensma-epar-public-assessment-report_en.pdf (2020).

  23. Passini, M. A. et al. Translational fidelity of intrathecal delivery of self-complementary AAV9-survival motor neuron 1 for spinal muscular atrophy. Hum. Gene Ther. 25, 619–630 (2014).

    Article  CAS  PubMed  Google Scholar 

  24. Gray, S. J. & Samulski, R. Vector design and considerations for CNS applications. in Gene Vector Design and Application to Treat Nervous System Disorders (ed. Glorioso. J.) 7–15 (Society for Neuroscience, 2011).

  25. Taylor, M. D. et al. Postnatal regulation of limb proprioception by muscle-derived neurotrophin-3. J. Comp. Neurol. 432, 244–258 (2001).

    Article  CAS  PubMed  Google Scholar 

  26. Simon, C. M. et al. Stasimon contributes to the loss of sensory synapses and motor neuron death in a mouse model of spinal muscular atrophy. Cell Rep. 29, 3885–3901 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Mentis, G. Z. et al. Early functional impairment of sensorimotor connectivity in a mouse model of spinal muscular atrophy. Neuron 69, 453–467 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Fletcher, E. V. et al. Reduced sensory synaptic excitation impairs motor neuron function via Kv2.1 in spinal muscular atrophy. Nat. Neurosci. 20, 905–916 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Simon, C. M. et al. Converging mechanisms of p53 activation drive motor neuron degeneration in spinal muscular atrophy. Cell Rep. 21, 3767–3780 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Li, D. K., Tisdale, S., Lotti, F. & Pellizzoni, L. SMN control of RNP assembly: from post-transcriptional gene regulation to motor neuron disease. Semin. Cell Dev. Biol. 32, 22–29 (2014).

    Article  CAS  PubMed  Google Scholar 

  31. Ruggiu, M. et al. A role for SMN exon 7 splicing in the selective vulnerability of motor neurons in spinal muscular atrophy. Mol. Cell. Biol. 32, 126–138 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Van Alstyne, M. et al. Dysregulation of Mdm2 and Mdm4 alternative splicing underlies motor neuron death in spinal muscular atrophy. Genes Dev. 32, 1045–1059 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  33. Saltzman, A. L., Pan, Q. & Blencowe, B. J. Regulation of alternative splicing by the core spliceosomal machinery. Genes Dev. 25, 373–384 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  34. Tenner, A. J., Stevens, B. & Woodruff, T. M. New tricks for an ancient system: physiological and pathological roles of complement in the CNS. Mol. Immunol. 102, 3–13 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Vukojicic, A. et al. The classical complement pathway mediates microglia-dependent remodeling of spinal motor circuits during development and in SMA. Cell Rep. 29, 3087–3100 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Lucin, K. M. & Wyss-Coray, T. Immune activation in brain aging and neurodegeneration: too much or too little? Neuron 64, 110–122 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Sleigh, J. N. et al. Chondrolectin affects cell survival and neuronal outgrowth in in vitro and in vivo models of spinal muscular atrophy. Hum. Mol. Genet. 23, 855–869 (2014).

    Article  CAS  PubMed  Google Scholar 

  38. Nussbacher, J. K., Tabet, R., Yeo, G. W. & Lagier-Tourenne, C. Disruption of RNA metabolism in neurological diseases and emerging therapeutic interventions. Neuron 102, 294–320 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Monani, U. R. The human centromeric survival motor neuron gene (SMN2) rescues embryonic lethality in Smn−/− mice and results in a mouse with spinal muscular atrophy. Hum. Mol. Genet. 9, 333–339 (2000).

    Article  CAS  PubMed  Google Scholar 

  40. Gavrilina, T. O. et al. Neuronal SMN expression corrects spinal muscular atrophy in severe SMA mice while muscle-specific SMN expression has no phenotypic effect. Hum. Mol. Genet. 17, 1063–1075 (2008).

    Article  CAS  PubMed  Google Scholar 

  41. Lorson, C. L. et al. SMN oligomerization defect correlates with spinal muscular atrophy severity. Nat. Genet. 19, 63–66 (1998).

    Article  CAS  PubMed  Google Scholar 

  42. Pellizzoni, L., Charroux, B. & Dreyfuss, G. SMN mutants of spinal muscular atrophy patients are defective in binding to snRNP proteins. Proc. Natl Acad. Sci. USA 96, 11167–11172 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Bühler, D., Raker, V., Lührmann, R. & Fischer, U. Essential role for the tudor domain of SMN in spliceosomal U snRNP assembly: implications for spinal muscular atrophy. Hum. Mol. Genet. 8, 2351–2357 (1999).

    Article  PubMed  Google Scholar 

  44. U.S. Food and Drug Administration. AveXis. Zolgensma (onasemnogene abeparvovec-xioi) package insert. https://www.fda.gov/vaccines-blood-biologics/zolgensma (2019).

  45. Hordeaux, J. et al. The neurotropic properties of AAV-PHP.B are limited to C57BL/6J mice. Mol. Ther. 26, 664–668 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Schuster, D. J. et al. Biodistribution of adeno-associated virus serotype 9 (AAV9) vector after intrathecal and intravenous delivery in mouse. Front. Neuroanat. 8, 42 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  47. DiDonato, C. J. et al. Cloning, characterization and copy number of the murine survival motor neuron gene homolog of the spinal muscular atrophy-determining gene. Genome Res. 7, 339–352 (1997).

    Article  CAS  PubMed  Google Scholar 

  48. Lorson, M. A., Spate, L. D., Prather, R. S. & Lorson, C. L. Identification and characterization of the porcine (Sus scrofa) survival motor neuron (SMN1) gene: an animal model for therapeutic studies. Dev. Dyn. 237, 2268–2278 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Wu, J., Anczuków, O., Krainer, A. R., Zhang, M. Q. & Zhang, C. OLego: fast and sensitive mapping of spliced mRNA-seq reads using small seeds. Nucleic Acids Res. 41, 5149–5163 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Yan, Q. et al. Systematic discovery of regulated and conserved alternative exons in the mammalian brain reveals NMD modulating chromatin regulators. Proc. Natl Acad. Sci. USA 112, 3445–3450 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Benjamini, Y. & Hochberg, Y. Controlling the false discovery rate—a practical and powerful approach to multiple testing. J. R. Stat. Soc. Ser. B 57, 289–300 (1995).

    Google Scholar 

  52. Robinson, M. D., Mccarthy, D. J. & Smyth, G. K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010).

    Article  CAS  Google Scholar 

  53. Huang, D. W., Sherman, B. T. & Lempicki, R. A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 4, 44–57 (2009).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We are grateful to S. Jaffrey for comments and critical reading of the manuscript. This work was supported by NIH grants R01NS102451 (to L.P.), R21NS099977 (to L.P.), R01NS078375 (to G.Z.M.) and R01AA027079 (to G.Z.M.). High-performance computation was supported by NIH grants S10OD012351 and S10OD021764. This research was funded in part through the NIH/NCI Cancer Center Support Grant P30CA013696 and used the Genomics and High-Throughput Screening Shared Resource.

Author information

Authors and Affiliations

  1. Center for Motor Neuron Biology and Disease, Columbia University, New York, NY, USA

    Meaghan Van Alstyne, Ivan Tattoli, Nicolas Delestrée, Yocelyn Recinos, Eileen Workman, Chaolin Zhang, George Z. Mentis & Livio Pellizzoni

  2. Department of Pathology and Cell Biology, Columbia University, New York, NY, USA

    Meaghan Van Alstyne, Ivan Tattoli, Nicolas Delestrée, Eileen Workman, George Z. Mentis & Livio Pellizzoni

  3. Department of Neurology, Columbia University, New York, NY, USA

    Meaghan Van Alstyne, Nicolas Delestrée, George Z. Mentis & Livio Pellizzoni

  4. Department of Systems Biology, Columbia University, New York, NY, USA

    Yocelyn Recinos & Chaolin Zhang

  5. Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA

    Yocelyn Recinos & Chaolin Zhang

  6. Neuroscience Therapeutic Area, Sanofi, Framingham, MA, USA

    Lamya S. Shihabuddin

Authors
  1. Meaghan Van Alstyne
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ivan Tattoli
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Nicolas Delestrée
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yocelyn Recinos
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Eileen Workman
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Lamya S. Shihabuddin
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Chaolin Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. George Z. Mentis
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Livio Pellizzoni
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

L.P. designed and supervised the study. M.V.A. performed the experiments and analyzed the data except as noted below. I.T. and E.W. contributed to behavioral studies. N.D. performed electrophysiological studies and data analysis. Y.R. and C.Z. carried out bioinformatic analysis of RNA-seq data. L.S.S. provided AAV9 vectors. G.Z.M. contributed to the design, analysis and interpretation of functional studies. M.V.A. and L.P. wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Livio Pellizzoni.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Neuroscience thanks the anonymous reviewers 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

Extended Data Fig. 1 Long-term AAV9-mediated SMN overexpression decreases sensory-motor synaptic transmission of proprioceptive neurons.

a-c, Representative EMG recordings from the footpad of uninjected (a) and either AAV9-GFP (5) (b) or AAV9-SMN (5) (c) treated WT mice at P300 using a stimulation frequency of 0.1 Hz. This analysis captures both proprioceptive synaptic function (H-reflex) and motor neuron function (M-response), and the H/M ratio can determine a preferential deficit in either response. Arrows point to the stimulus artifact. d, Amplitude of the M-response from the same groups as shown in (a-c). The box-and-whiskers graph shows the median, interquartile range, minimum and maximum (n = 8 animals). Statistics were performed with one-way ANOVA with Tukey’s post hoc test. (ns) no significance. P = 0.9741, F2, 21 = 0.02629. e, Amplitude of H-reflex from the same groups as shown in (a-c). The box-and-whiskers graph shows the median, interquartile range, minimum and maximum (n = 8 animals). Statistics were performed with one-way ANOVA with Tukey’s post-hoc test. (*) P < 0.05. WT vs SMN(5): P = 0.0420, q = 3.684, d.f.=21. f, Ratio of H-reflex and M-response from the same groups as shown in (a-c). The box-and-whiskers graph shows the median, interquartile range, minimum and maximum (n = 8 animals). Statistics were performed with one-way ANOVA with Tukey’s post hoc test. (*) P < 0.05. WT vs SMN(5): P = 0.0103, q = 4.592, df=21. g, Repetitive nerve stimulation at different frequencies from 0.1 to 50 Hz does not change the amplitude of the M-response recorded from the footpad of uninjected and either AAV9-GFP (5) or AAV9-SMN (5) treated WT mice at P300. The box-and-whiskers graph shows the median, interquartile range, minimum and maximum from the following number of animals: WT (0.1-10 Hz n = 8; 20-50 Hz n = 6); WT + AAV-GFP(5) (n = 8); WT + AAV-SMN(5) (0.1-10 Hz n = 8; 20 Hz n = 7; 50 Hz n = 6). Statistics were performed with one-way ANOVA with Tukey’s post hoc test. (ns) no significance. WT: P = 0.9938, F4, 31 = 0.05630; WT + AAV-GFP(5): P = 0.6199, F4, 35 = 0.06660; WT + AAV-SMN(5): P = 0.5344, F4, 32 = 0.07997.

Extended Data Fig. 2 Effects of AAV9-SMN on motor neuron survival, C-boutons and NMJ innervation in WT mice.

a, ChAT immunostaining of L5 spinal segments from uninjected, AAV9-GFP (5) and AAV9-SMN (5) injected WT mice at P150. Scale bar=100 µm. b, Soma size of MNs from the same groups as in (a) at P150. The violin plot shows the median (solid line) and interquartile range (dotted lines) from the following number of MNs (WT, n = 227 neurons; WT + GFP(5), n = 234 neurons; WT + SMN(5), n = 234 neurons) from 3 animals per group. Statistics were performed with one-way ANOVA with Tukey’s post hoc test. (ns) no significance. P = 0.3083, F3, 449 = 1.180. c, Percentage of the number of L5 LMC MNs per 75 µm section relative to WT in the same groups as in (a) at P150. The box-and-whiskers graph shows the median, interquartile range, minimum and maximum from the following number of sections (WT, n = 36 sections; WT + GFP(5), n = 31 sections; WT + SMN(5), n = 43 sections) from 3 animals per group. Statistics were performed with one-way ANOVA with Tukey’s post hoc test. (*) P < 0.05; (**) P < 0.01. WT vs SMN(5): P = 0.0284, q = 3.678, df=107; GFP(5) vs SMN(5): P = 0.0064, q = 4.420, df=107. d, ChAT immunostaining of L5 LMC MNs from uninjected, AAV9-GFP (5) and AAV9-SMN (5 and 10) injected WT mice at P300. Scale bar=25 µm. e, Total number of ChAT+ C-boutons on L5 LMC MNs from the same groups as in (d) at P150 and P300. The box-and-whiskers graph shows the median, interquartile range, minimum and maximum from the following numbers of neurons and animals at P150 (WT, n = 39 neurons, n = 3 animals; WT + GFP(5), n = 37 neurons, n = 3 animals; WT + SMN(5), n = 49 neurons, n = 3 animals) and P300 (WT, n = 28 neurons, n = 3 animals; WT + GFP(5), n = 31 neurons, n = 3 animals; WT + SMN(5), n = 36 neurons, n = 3 animals; WT + SMN(10), n = 14 neurons, n = 4 animals). Statistics were performed with one-way ANOVA with Tukey’s post hoc test. (ns) no significance. For MNs at P150, P = 0.9855, F2, 122 = 0.01456. For MNs at P300, P = 0.0651, F3, 105 = 2.481. f, NMJ immunostaining of EDL muscles with Neurofilament-M (NF-M), Synaptophysin (SYP) and bungarotoxin (BTX) from the same groups as in (a) at P300. Scale bar=25 µm. g, Percentage of innervated, partially innervated and denervated NMJs in the EDL muscle from the same groups as in (a) at P150 and P300. Data represent mean and SEM (n = 3 animals). Statistics were performed with two-way ANOVA with Tukey’s post hoc test. (*) P < 0.05. For innervated NMJs: SMN(5) P150 vs SMN(5) P300: P = 0.0346, q = 4.588, df=42; SMN(5) P150 vs SMN(10) P300: P = 0.0317, q = 4.636, df=42.

Extended Data Fig. 3 Effects of AAV9-SMN on motor neuron survival, soma size and NMJ innervation in SMA mice.

a, ChAT immunostaining of L2 MNs from untreated WT (SMNΔ7) mice and either AAV9-GFP (5) or AAV9-SMN (5) treated SMA mice at the indicated times. Scale bars=75 µm (P11) and 100 µm (P190). b, NMJ immunostaining with Neurofilament-M (NF-M), Synaptophysin (SYP) and bungarotoxin (BTX) of QL muscles from the same groups as in (a). Scale bar=25 µm (P11) and 50 µm (P190). c, Soma size of MNs from the same groups as in (a) at P11 and P190. The violin plot shows the median (solid line) and interquartile range (dotted lines) from the following number of MNs at P11 (WT (SMNΔ7), n = 230 neurons; SMA + GFP(5), n = 185 neurons; SMA + SMN(5), n = 175 neurons) and P190 (WT (SMNΔ7), n = 231 neurons; SMA + SMN(5), n = 164 neurons) from 3 animals per group. Statistics were performed with one-way ANOVA with Tukey’s post hoc test. (**) P < 0.01; (****) P < 0.0001. WT (SMNΔ7) P11 vs SMA + GFP(5) P11: P < 0.0001, q = 7.696, df=980; SMA + GFP(5) P11 vs SMA + SMN(5) P11: P = < 0.0001, q = 7.714, df=980; WT (SMNΔ7) P190 vs SMA + SMN(5) P190: P = 0.0058, q = 4.841, df=980. d, Percentage of the number of MNs per 75 µm section relative to WT (SMNΔ7) in the same groups as in (a) at P11 and P190. The box-and-whiskers graph shows the median, interquartile range, minimum and maximum from the following number of sections and animals at P11 (WT (SMNΔ7), n = 48 sections, n = 3 animals; SMA + GFP(5), n = 58 sections, n = 6 animals; SMA + SMN(5), n = 56 sections n = 6 animals) and P190 (WT (SMNΔ7), n = 42 sections, n = 3 animals; SMA + SMN(5), n = 41 sections, n = 3 animals). Statistics were performed with one-way ANOVA with Tukey’s post hoc test. (**) P < 0.01; (***) P < 0.001; (ns) no significance. WT (SMNΔ7) P11 vs SMA + GFP(5) P11: P < 0.0001, q = 13.58, df=241; WT (SMNΔ7) P11 vs SMA + SMN(5) P11: P < 0.0001, q = 8.359, df=241; SMA + GFP(5) P11 vs SMA + SMN(5) P11: P = 0.0018, q = 5.364, df=241; WT (SMNΔ7) P190 vs SMA + SMN(5) P190: P < 0.0001, q = 8.108, df=241; SMA + SMN(5) P11 vs SMA + SMN(5) P190: P = 0.9927, q = 0.6110, df=241. e, Percentage of innervated, partially innervated and denervated NMJs relative to WT (SMNΔ7) in the QL muscle from the same groups as in (b) at P11 and P190. Data represent mean and SEM (n = 3 animals). Statistics were performed with two-way ANOVA with Tukey’s post hoc test. (****) P < 0.0001; (ns) no significance. For innervated NMJs: WT (SMNΔ7) P11 vs SMA + GFP(5) P11: P < 0.0001, q = 10.72, df=30; SMA + GFP(5) P11 vs SMA + SMN(5) P11: P < 0.0001, q = 7.789, df=30; SMA + SMN(5) P11 vs SMA + SMN(5) P190: P = 0.9999, q = 0.2233, df=30. f, Total number of PV+ PNs in L2 DRGs from untreated WT (SMNΔ7) mice and AAV9-SMN (5) treated SMA mice at P190. Data represent mean and SEM (n = 3 DRGs from n = 3 animals). Statistics were performed with unpaired Student’s t-test. (ns) no significance. P = 0.9106, t = 0.1196, df=4.

Extended Data Fig. 4 Efficiency of AAV9 transduction and long-term transgene expression in tissues of WT mice.

a, Percentage of GFP+ L5 LMC MNs from uninjected and AAV9-GFP (5) injected WT mice at P300. Data represent mean and SEM (n = 3 animals). b, Percentage of GFP+ PNs in L5 DRGs from the same groups as in (a) at P300. Data represent mean and SEM (n = 3 animals). c, RT-qPCR analysis of GFP mRNA in spinal cord, DRG and liver from WT mice injected with AAV9-GFP (5) at P11 and P300. Data represent mean and SEM (n = 3 animals) normalized to Gapdh mRNA and expressed relative to levels at P11 (set as 1). d, RT-qPCR analysis of full-length human SMN (hSMN-FL) mRNA in spinal cord, DRG and liver from WT mice injected with AAV9-SMN (5) at P11 and P300. Data represent mean and SEM (n = 3 animals) normalized to Gapdh mRNA and expressed relative to levels at P11 (set as 1).

Extended Data Fig. 5 AAV9-SMN leads to persistent SMN overexpression in the spinal cord and DRGs but not the liver of SMA mice.

a-c, RT-qPCR analysis of hSMN-FL mRNA levels in spinal cord (a), DRG (b) and liver (c) of uninjected WT (SMNΔ7) mice and SMA mice injected with either AAV9-GFP (5) or AAV9-SMN (5) at the indicated times. Data represent mean and SEM (n = 3 animals) normalized to Gapdh mRNA and expressed relative to levels in WT (SMNΔ7) mice (set as 1). Statistics were performed with one-way ANOVA with Tukey’s post hoc test. (*) P < 0.05; (****) P < 0.0001; (ns) no significance. For spinal cord: SMA + SMN(5) P11 vs SMA + SMN(5) P190: P = 0.5887, q = 2.111, df=10. For DRG: SMA + SMN(5) P11 vs SMA + SMN(5) P190: P = 0.0396, q = 4.866, df=10. For liver: SMA + SMN(5) P11 vs SMA + SMN(5) P190: P < 0.0001, q = 16.28, df=10. d-e, Western blot analysis of spinal cord (d) and liver (e) from the same groups as in (a). SMN expression was analyzed with antibodies that specifically detect only human SMN (hSMN) or both human and mouse SMN (pan-SMN). Cropped images are shown. f, Quantification of total SMN protein levels from the Western blot analysis in (d). Data represent mean and SEM (n = 3 animals) normalized to Gapdh and expressed relative to levels in WT (SMNΔ7) mice (set as 1). Statistics were performed with one-way ANOVA with Tukey’s post hoc test. (****) P < 0.0001; (***) P < 0.001. WT (SMNΔ7) P11 vs SMA + GFP(5) P11: P < 0.0001, q = 24.80, df=10; WT (SMNΔ7) P11 vs WT (SMNΔ7) P190: P = 0.0001, q = 11.12, df=10; WT (SMNΔ7) P11 vs SMA + SMN(5) P190: P = 0.0003, q = 9.975, df=10; SMA + SMN(5) P11 vs SMA + SMN(5) P190: P < 0.0001, q = 11.23, df=10. g, Quantification of human SMN protein levels from the Western blot analysis in (d). Data represent mean and SEM (n = 3 animals) normalized to Gapdh and expressed relative to levels in WT (SMNΔ7) mice (set as 1). Statistics were performed with one-way ANOVA with Tukey’s post hoc test. (****) P < 0.0001; (***) P < 0.001; (**) P < 0.01; (*) P < 0.05. WT (SMNΔ7) P11 vs SMA + GFP(5) P11: P = 0.0165, q = 5.671, df=10; WT (SMNΔ7) P11 vs SMN(5) P11: P < 0.0001, q = 25.08, df=10; WT (SMNΔ7) P11 vs SMA + SMN(5) P190: P = 0.0009, q = 8.513, df=10. SMA + SMN(5) P11 vs SMA + SMN(5) P190: P < 0.0001, q = 16.57, df=10. h, Quantification of total SMN protein levels from the Western blot analysis in (e). Data represent mean and SEM (n = 3 animals) normalized to Gapdh and expressed relative to levels in WT (SMNΔ7) mice (set as 1). Statistics were performed with one-way ANOVA with Tukey’s post hoc test. (****) P < 0.0001. WT (SMNΔ7) P11 vs SMA + SMN(5) P11: P < 0.0001, q = 13.08, df=10; SMA + SMN(5) P11 vs SMA + SMN(5) P190: P < 0.0001, q = 15.56, df=10.

Source data

Extended Data Fig. 6 AAV9-SMN drives cytoplasmic aggregation of overexpressed SMN in motor circuit neurons of mouse models.

a, ChAT and SMN immunostaining of L5 LMC MNs from uninjected and either AAV9-GFP (5) or AAV9-SMN (5) injected WT mice at P11 and P150. Scale bar=10 µm. b, Percentage of L5 LMC MNs with SMN aggregates in the same groups as in (a) at P300. Data represent mean and SEM (n = 3 animals). c, PV and SMN immunostaining of L5 DRG PNs from uninjected and either AAV9-GFP (5) or AAV9-SMN (5) injected WT mice at P11 and P150. Scale bar=10 µm. d, Percentage of L5 DRG PNs with SMN aggregates in the same groups as in (c) at P300. Data represent mean and SEM (n = 3 animals). e, ChAT and SMN immunostaining of L2 MNs from WT (SMNΔ7) mice and either AAV9-GFP (5) or AAV9-SMN (5) injected SMA mice at the indicated time points. Scale bar=10 µm. f, Percentage of L2 MNs with SMN aggregates in the same groups as in (e) at P190. Data represent mean and SEM (n = 3 animals). g, PV and SMN immunostaining of L2 DRG PNs from the same groups as in (e). Scale bar=10 µm. h, Percentage of L2 PNs with SMN aggregates in the same groups as in (g) at P190. Data represent mean and SEM (n = 3 animals).

Extended Data Fig. 7 AAV9-mediated SMN overexpression induces cytoplasmic aggregation of SmB in motor circuit neurons of mouse models.

a, ChAT and SmB immunostaining of L5 LMC MNs from uninjected and either AAV9-GFP (5) or AAV9-SMN (5) injected WT mice at P11 and P150. Scale bar=10 µm. b, PV and SmB immunostaining of L5 DRG PNs from the same groups as in (a). Scale bar=10 µm. c, ChAT and SmB immunostaining of L2 MNs from WT (SMNΔ7) mice and either AAV9-GFP (5) or AAV9-SMN (5) injected SMA mice at the indicated time points. Scale bar=10 µm. d, PV and SmB immunostaining of L2 PNs from the same groups as in (c). Scale bar=10 µm.

Extended Data Fig. 8 Immunohistochemical analysis of cytoplasmic SMN aggregates in motor neurons.

a-d, Immunostaining of L5 LMC MNs from WT mice injected with AAV9-SMN (5) at P300 with DAPI, ChAT, SMN and RNA-binding proteins KSRP (a), FUS (b), hnRNPA1 (c) or HuR (d) as indicated. Scale bar=5 µm. e-f, Immunostaining with DAPI, ChAT and RNA transport factors Ran (e) and Crm1 (f) in the same MNs as in (a). Scale bar=5 µm. g-h, Immunostaining with DAPI, ChAT, SMN and either GW182 to label P-bodies (g) or TIA-1 to label stress granules (h) in the same MNs as in (a). Scale bar=5 µm. i-j, Immunostaining with DAPI, ChAT, SMN and either ubiquitin (i) or the autophagy marker LC3B (j) in the same MNs as in (a). Scale bar=5 µm.

Extended Data Fig. 9 Viral-mediated overexpression of human SMN induces cytoplasmic aggregation and sequestration of SmB in human cells.

HeLa cells were untreated (mock) or transduced with lentivirus expressing either GFP (Lenti-GFP) or human SMN (Lenti-SMN). Immunostaining with DAPI, GFP, SMN and SmB was performed five days post transduction. Scale bar=10 µm.

Extended Data Fig. 10 Model of the toxic effects of long-term AAV9-mediated SMN overexpression in neurons.

Schematic depicting the gain of toxic function mechanisms induced by long-term overexpression of SMN in the sensory-motor circuit of mouse models (see text for further details).

Supplementary information

Reporting Summary

Supplementary Tables 1–5

Supplementary tables and descriptive legends.

Supplementary Video 1

Clasping phenotype of WT mice injected with AAV9-SMN.

Supplementary Video 2

Hind-limb paralysis of a P300 WT mouse injected with a high dose (1 × 1011 vg g−1) of AAV9-SMN.

Source data

Source Data Fig. 4

Unprocessed western blots.

Source Data Extended Data Fig. 5

Unprocessed western blots.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Van Alstyne, M., Tattoli, I., Delestrée, N. et al. Gain of toxic function by long-term AAV9-mediated SMN overexpression in the sensorimotor circuit. Nat Neurosci 24, 930–940 (2021). https://doi.org/10.1038/s41593-021-00827-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41593-021-00827-3

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing