Alternative Splicing Events Are a Late Feature of Pathology in a Mouse Model of Spinal Muscular Atrophy

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.

[1]  C. Lorson,et al.  Survival Motor Neuron Function in Motor Axons Is Independent of Functions Required for Small Nuclear Ribonucleoprotein Biogenesis , 2006, The Journal of Neuroscience.

[2]  B. Wirth,et al.  Different entities of proximal spinal muscular atrophy within one family , 1997, Human Genetics.

[3]  M. Oda,et al.  A study of cell death in Werdnig Hoffmann disease brain , 1998, Neuroscience Letters.

[4]  Yael Mandel-Gutfreund,et al.  Detection and measurement of alternative splicing using splicing-sensitive microarrays. , 2005, Methods.

[5]  S. Hamilton,et al.  The survival motor neuron protein interacts with the transactivator FUSE binding protein from human fetal brain , 2000, FEBS letters.

[6]  B. Wirth.  An update of the mutation spectrum of the survival motor neuron gene (SMN1) in autosomal recessive spinal muscular atrophy (SMA) , 2000, Human mutation.

[7]  Y. Benjamini,et al.  Controlling the false discovery rate: a practical and powerful approach to multiple testing , 1995 .

[8]  R. Myers,et al.  Evolving gene/transcript definitions significantly alter the interpretation of GeneChip data , 2005, Nucleic acids research.

[9]  C. Lorson,et al.  An exonic enhancer is required for inclusion of an essential exon in the SMA-determining gene SMN. , 2000, Human molecular genetics.

[10]  T. Speed,et al.  Summaries of Affymetrix GeneChip probe level data. , 2003, Nucleic acids research.

[11]  Kayoko Saito,et al.  An autopsy case of spinal muscular atrophy type III (Kugelberg‐Welander disease) , 2009, Neuropathology : official journal of the Japanese Society of Neuropathology.

[12]  R. Ward,et al.  Is Werdnig-Hoffmann disease a pure lower motor neuron disorder? , 2004, Acta Neuropathologica.

[13]  Lili Wan,et al.  SMN Deficiency Causes Tissue-Specific Perturbations in the Repertoire of snRNAs and Widespread Defects in Splicing , 2008, Cell.

[14]  K. Talbot,et al.  Pre-symptomatic development of lower motor neuron connectivity in a mouse model of severe spinal muscular atrophy. , 2010, Human molecular genetics.

[15]  K. Talbot,et al.  Selective vulnerability of motor neurons and dissociation of pre- and post-synaptic pathology at the neuromuscular junction in mouse models of spinal muscular atrophy. , 2008, Human molecular genetics.

[16]  Rafael A. Irizarry,et al.  Bioinformatics and Computational Biology Solutions using R and Bioconductor , 2005 .

[17]  J. McPherson,et al.  A single nucleotide difference that alters splicing patterns distinguishes the SMA gene SMN1 from the copy gene SMN2. , 1999, Human molecular genetics.

[18]  Gordon K. Smyth,et al.  limma: Linear Models for Microarray Data , 2005 .

[19]  C. Lorson,et al.  Identification of survival motor neuron as a transcriptional activator-binding protein. , 1999, Human molecular genetics.

[20]  B. Wirth,et al.  Plastin 3 Is a Protective Modifier of Autosomal Recessive Spinal Muscular Atrophy , 2008, Science.

[21]  U. Monani,et al.  The human centromeric survival motor neuron gene (SMN2) rescues embryonic lethality in Smn(-/-) mice and results in a mouse with spinal muscular atrophy. , 2000, Human molecular genetics.

[22]  G. Bernardi,et al.  Mitochondrial damage modulates alternative splicing in neuronal cells: implications for neurodegeneration , 2007, Journal of neurochemistry.

[23]  A. Usiello,et al.  The activity of the spinal muscular atrophy protein is regulated during development and cellular differentiation. , 2005, Human molecular genetics.

[24]  Michael Sendtner,et al.  Specific interaction of Smn, the spinal muscular atrophy determining gene product, with hnRNP-R and gry-rbp/hnRNP-Q: a role for Smn in RNA processing in motor axons? , 2002, Human molecular genetics.

[25]  M. Mann,et al.  A Functional Interaction between the Survival Motor Neuron Complex and RNA Polymerase II , 2001, The Journal of cell biology.

[26]  Michael Sendtner,et al.  Smn, the spinal muscular atrophy–determining gene product, modulates axon growth and localization of β-actin mRNA in growth cones of motoneurons , 2003, The Journal of cell biology.

[27]  W. Schlote,et al.  Classical infantile spinal muscular atrophy with SMN deficiency causes sensory neuronopathy , 2003, Neurology.

[28]  S. Wiese,et al.  Axonal defects in mouse models of motoneuron disease. , 2004, Journal of neurobiology.

[29]  G. Meister,et al.  A multiprotein complex mediates the ATP-dependent assembly of spliceosomal U snRNPs , 2001, Nature Cell Biology.

[30]  A. Smith,et al.  Inactivation of the survival motor neuron gene, a candidate gene for human spinal muscular atrophy, leads to massive cell death in early mouse embryos. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[31]  Arnold Munnich,et al.  Correlation between severity and SMN protein level in spinal muscular atrophy , 1997, Nature Genetics.

[32]  S. Jablonka,et al.  Distinct and overlapping alterations in motor and sensory neurons in a mouse model of spinal muscular atrophy. , 2006, Human molecular genetics.

[33]  Tyler S. Alioto,et al.  U12DB: a database of orthologous U12-type spliceosomal introns , 2006, Nucleic Acids Res..

[34]  G. Meister,et al.  Reduced U snRNP assembly causes motor axon degeneration in an animal model for spinal muscular atrophy. , 2005, Genes & development.

[35]  Kristi L Fox-Walsh,et al.  Splice-site pairing is an intrinsically high fidelity process , 2009, Proceedings of the National Academy of Sciences.

[36]  U. Monani,et al.  SMNDelta7, the major product of the centromeric survival motor neuron (SMN2) gene, extends survival in mice with spinal muscular atrophy and associates with full-length SMN. , 2005, Human molecular genetics.

[37]  Alternative Transcript Analysis Methods for Exon Arrays , 2005 .

[38]  C. Lorson,et al.  A single nucleotide in the SMN gene regulates splicing and is responsible for spinal muscular atrophy. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[39]  J. Yong,et al.  Essential Role for the SMN Complex in the Specificity of snRNP Assembly , 2002, Science.

[40]  G. Meister,et al.  Gene targeting of Gemin2 in mice reveals a correlation between defects in the biogenesis of U snRNPs and motoneuron cell death , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[41]  Y. Sasaki,et al.  A neuropathologic study of Werdnig-Hoffmann disease with special reference to the thalamus and posterior roots , 2004, Acta Neuropathologica.

[42]  T. Gillingwater,et al.  Loss of translation elongation factor (eEF1A2) expression in vivo differentiates between Wallerian degeneration and dying‐back neuronal pathology , 2008, Journal of anatomy.

[43]  J. Weissenbach,et al.  Identification and characterization of a spinal muscular atrophy-determining gene , 1995, Cell.

[44]  L. Pellizzoni.  Chaperoning ribonucleoprotein biogenesis in health and disease , 2007, EMBO reports.

[45]  L. Pellizzoni,et al.  Ribonucleoprotein Assembly Defects Correlate with Spinal Muscular Atrophy Severity and Preferentially Affect a Subset of Spliceosomal snRNPs , 2007, PloS one.

[46]  L. Landmesser,et al.  Reduced SMN protein impairs maturation of the neuromuscular junctions in mouse models of spinal muscular atrophy. , 2008, Human molecular genetics.