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Minor snRNA gene delivery improves the loss of proprioceptive synapses on SMA motor neurons
Erkan Y. Osman, … , Livio Pellizzoni, Christian L. Lorson
Erkan Y. Osman, … , Livio Pellizzoni, Christian L. Lorson
Published June 9, 2020
Citation Information: JCI Insight. 2020;5(12):e130574. https://doi.org/10.1172/jci.insight.130574.
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Research Article Neuroscience

Minor snRNA gene delivery improves the loss of proprioceptive synapses on SMA motor neurons

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Abstract

Spinal muscular atrophy (SMA) is an inherited neuromuscular disorder caused by reduced expression of the survival motor neuron (SMN) protein. SMN has key functions in multiple RNA pathways, including the biogenesis of small nuclear ribonucleoproteins that are essential components of both major (U2-dependent) and minor (U12-dependent) spliceosomes. Here we investigated the specific contribution of U12 splicing dysfunction to SMA pathology through selective restoration of this RNA pathway in mouse models of varying phenotypic severity. We show that virus-mediated delivery of minor snRNA genes specifically improves select U12 splicing defects induced by SMN deficiency in cultured mammalian cells, as well as in the spinal cord and dorsal root ganglia of SMA mice without increasing SMN expression. This approach resulted in a moderate amelioration of several parameters of the disease phenotype in SMA mice, including survival, weight gain, and motor function. Importantly, minor snRNA gene delivery improved aberrant splicing of the U12 intron–containing gene Stasimon and rescued the severe loss of proprioceptive sensory synapses on SMA motor neurons, which are early signatures of motor circuit dysfunction in mouse models. Taken together, these findings establish the direct contribution of U12 splicing dysfunction to synaptic deafferentation and motor circuit pathology in SMA.

Authors

Erkan Y. Osman, Meaghan Van Alstyne, Pei-Fen Yen, Francesco Lotti, Zhihua Feng, Karen K.Y. Ling, Chien-Ping Ko, Livio Pellizzoni, Christian L. Lorson

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Figure 6

AAV9-mediated delivery of minor snRNA genes does not prevent motor neuron degeneration and neuromuscular junction pathology in SMA mice.

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AAV9-mediated delivery of minor snRNA genes does not prevent motor neuro...
(A) ChAT immunostaining of L5 spinal segments at P11 from unaffected control mice and SMA mice that were either untreated or ICV injected with AAV9-U11/U12 and AAV9-U11/U12/U4atac as indicated. In the top panels, dotted circles identify MMC motor neuron pools. Scale bar: 200 μm. Bottom panels display representative images of L5 MMC motor neuron pools from the same treatment groups at higher magnification. Scale bar: 50 μm. (B) AAV9-mediated delivery of minor snRNA genes does not prevent motor neuron degeneration. Total number of L5 MMC motor neurons from the same groups as in A at P11. The scatter plots show individual data points, mean and SEM from 3 mice per group. Statistics were performed with 1-way ANOVA with Tukey’s post hoc test. **P < 0.01; ns, no significance. (C) Images of NMJ innervation in the vulnerable longissimus capitis muscle collected at P12 from unaffected control mice and SMA mice either untreated or injected with AAV9-U11/U12 and AAV9-U11/U12/U4atac. Immunochemistry labeling is as follows: α-bungarotoxin (α-BTX, shown in red), synaptophysin and neurofilament (Syn/NF, shown in green). Scale bar: 40 μm. (D) No improvement in NMJ pathology in SMNΔ7 mice treated with U11, U12, and U4atac minor splicing snRNAs. Bar graph representing the percentage of fully innervated (black), partially innervated (gray stippled), and denervated (white) NMJs from the same treatment groups. NMJ analysis was done by blinded counts for a minimum of 4 fields of view per muscle type from n = 3 animals per treatment. Statistical analysis showed no significant difference between treated and untreated SMA groups using 1-way ANOVA with Tukey’s post hoc test (P = 0.50).

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