Alternative splicing events are a late feature of pathology in a mouse model of spinal muscular atrophy

Abstract

Spinal muscular atrophy is a severe motor neuron disease caused by inactivating mutations in the SMN1 gene leading to reduced levels of full-length functional SMN protein. SMN is a critical mediator of spliceosomal protein assembly, and complete loss or drastic reduction in protein leads to loss of cell viability. However, the reason for selective motor neuron degeneration when SMN is reduced to levels which are tolerated by all other cell types is not currently understood. Widespread splicing abnormalities have recently been reported at end-stage in a mouse model of SMA, leading to the proposition that disruption of efficient splicing is the primary mechanism of motor neuron death. However, it remains unclear whether splicing abnormalities are present during early stages of the disease, which would be a requirement for a direct role in disease pathogenesis. We performed exon-array analysis of RNA from SMN deficient mouse spinal cord at 3 time points, pre-symptomatic (P1), early symptomatic (P7), and late-symptomatic (P13). Compared to littermate control mice, SMA mice showed a time-dependent increase in the number of exons showing differential expression, with minimal differences between genotypes at P1 and P7, but substantial variation in late-symptomatic (P13) mice. Gene ontology analysis revealed differences in pathways associated with neuronal development as well as cellular injury. Validation of selected targets by RT-PCR confirmed the array findings and was in keeping with a shift between physiologically occurring mRNA isoforms. We conclude that the majority of splicing changes occur late in SMA and may represent a secondary effect of cell injury, though we cannot rule out significant early changes in a small number of transcripts crucial to motor neuron survival.

Figure 1. Phenotype of the SMNΔ7mouse model.

(A) Post-natal weight development of SMA (Smn−/−;SMN2;SMNΔ7) and control (Smn+/+;SMN2;SMNΔ7) mice. Average weight of 4 animals per genotype and time-point; error-bars represent standard deviation of the mean. (B) Representative images of SMA and control mice littermates at P1, P7, and P13. Scale bar 1 cm. Genotypes can be reliably distinguished morphologically from P7 onwards. (C) Western blot of whole spinal cord lysates show markedly reduced SMN levels at all measured time points in the SMA mice. (D) At P13, paucity of large motor neurons in the ventral horn is apparent on H&E stain, with reduced SMN immunoreactivity on immunohistochemistry.

Figure 2. Motor neuron loss in SMA mice.

(A) Cresyl violet stain of spinal cord sections showing the ventral horn in control (top) and SMA mice (bottom), with relative reduction of large Nissl dense cells in SMA mice. (B) Lumbar motor neurons were counted in five animals per genotype at post-natal days P1, P3, P5, P7, P9, and P13. Motor neuron numbers were equal at the pre-symptomatic time points, while motor neuron loss became detectable at P7 followed by further decline to approximately 65% of control animal numbers at late-symptomatic stage. Error bars represent the standard deviation of the mean. (C) At P13, motor neuron loss affects all spinal cord segments, although the absolute number of motor neurons is higher in the cervical and lumbar region, reflecting the innervation of forelimbs and hind limbs.

Figure 3. Global transcriptome changes increase over time.

(A) Number of genes up- or down- regulated more than 1.5 fold in SMA mice compared to control using a p-value threshold of ≤0.05. There is a major increase in gene expression change at late-symptomatic compared to pre-symptomatic and early-symptomatic stages. (B) Gene expression changes in control mice (Smn+/+) between time points are of much larger scale than changes between genotypes.

Figure 4. Exon-level changes are a late occurrence in SMN deficient mice.

(A) Venn diagram depicting potential splicing events as evidenced by a Splicing Index |SI|>0.5. 252 alternative splicing events are present at late-symptomatic SMA mice compared to controls, but only 5 at P7 and 16 at P1. (B) Venn diagram depicting exon-level changes of Ensembl exons between genotypes at each time point investigated. At P1, P7, and P13, each of 211,567 exons was tested for differential expression between SMA and control. 812 exons were associated with disease status at P13, compared to 72 exons at P1 and 66 exons at P7.

Figure 5. Alternative splicing of Chodl.

(A) Graphical output of the exon-level analysis for Chodl. Each column, delineated by bold black lines, corresponds to the preprocessed data from a single Ensembl exon. The vertical axis displays log₂ expression for control (black) and SMA (red) animals, with each point corresponding to an individual animal. Each column is subdivided by vertical dashed lines into time points P1, P7, and P13 (left to right). Orange boxes mark those (exon, time point) combinations that exhibit significant differential expression between cases and controls. Expression of Chodl constitutive exons is reduced progressively from P1 to P13, but there is no difference between SMA and control for the alternative terminal exon ENSMUSE00000556896 indicating an isoform shift towards Chodl-002 (ENSMUST69148) in the SMA mice. Arrows indicate location of qRT–PCR primers for validation. (B) qRT–PCR results at P13 showing marked reduction in Chodl when measured using primers located in the constitutive exons 1–2 and the terminal exon of Chodl-001, while no significant difference of alternative exon ENSMUSE00000556896 exists between control and SMA (*** = p≤1e-3). (C) The differential terminal exon usage is also evident in muscle and kidney in SMA mice, although overall transcript levels are not reduced.

Figure 6. Alternative splicing of Uspl1.

(A) Graphical output of exon-level analysis for Uspl1. Each column, delineated by bold black lines, corresponds to the preprocessed data from a single Ensembl exon. The vertical axis displays log₂ expression for control (black) and SMA (red) animals, with each point corresponding to an individual animal. Each column is subdivided by vertical dashed lines into time points P1, P7, and P13 (left to right). Orange boxes mark those (exon, time point) combinations that exhibit significant differential expression between cases and controls. Expression of Uspl1 is higher in SMA mice for all exons, but this difference is more pronounced for the first exon detected by the array, which corresponds to Uspl1 exon 2 (ENSMUSE00000351955), reflecting a potential alternative splicing event. (B) Validation of the alternative splicing event by RT–PCR. Cartoon depicting primer position in Uspl1 exon 1 and 3. RT–PCR was performed on four biological replicates at P13 showing exon 2 skipping in control mice, and increased exon 2 usage in SMA mice. The difference of the exon 2+/exon 2- ratio between genotypes is tissue specific and most pronounced in muscle and less obvious in kidney. (C) qRT–PCR results for exon 2 showing increased in Uspl1 exon 2 expression in the SMA mice compared to control. The differential expression is more pronounced at symptomatic compared to pre-symptomatic stages.