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

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

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 1

Transgenic expression of minor snRNAs in human cell lines.

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Transgenic expression of minor snRNAs in human cell lines. (A) Schematic...
(A) Schematics of viral constructs harboring cassettes for the expression of human U11, U12, and U4atac snRNAs driven by human U2 promoter with 3′ box at the 3′ end. (B) Semiquantitative RT-PCR analysis of U11, U12, and U4atac snRNA expression 72 hours after transduction of the U11/U12/U4atac lentiviral construct in HeLa, HEK293T, and SMA type I patient fibroblasts (GM03813). U1 and U2 major snRNAs as well as 5S rRNA were used as controls. Lanes that were run on the same gel but were noncontiguous are separated by a vertical line. (C) RT-PCR analysis of the total levels of minor snRNA overexpression in cell extracts from HEK293T cells transiently transfected with the U11/U12/U4atac lentiviral construct for 48 hours relative to mock-transfected cells used as controls. The scatter plot shows the fold change in the relative amounts of U11, U12, and U4atac overexpression over endogenous minor snRNA levels in mock-transfected cells, which were arbitrarily set to 1 (dotted line). Individual data points, mean, and SEM from 3 independent experiments are shown. (D) Equal amounts of cell extract from either U11/U12/U4atac or mock-transfected HEK293T cells as in C were immunoprecipitated with anti-SmB antibodies, followed by RNA purification and RT-PCR analysis. The scatter plot shows the fold change in the amounts of immunoprecipitated minor snRNAs from U11/U12/U4atac-transfected cells relative to the levels from mock-transfected cells, which were arbitrarily set to 1 (dotted line). Individual data points, mean, and SEM from 3 independent experiments are shown.