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

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.

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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.

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.

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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

Affiliations

Authors

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.

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Competing interests

The authors declare no competing interests.

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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.

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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 (2021). https://doi.org/10.1038/s41593-021-00827-3

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