On the possible role of muscle in the pathogenesis of spinal muscular atrophy
Spinal muscular atrophies (SMA) are characterized by degeneration of motoneurons of the anterior horn of the spinal cord, leading to progressive symmetrical limb and trunk paralysis associated with muscular atrophy. It is a rather frequent inherited disease (1 in 6000 newborns). SMA have been divided into three clinical forms depending on the clinical severity: type I (Werdnig-Hoffmann disease, early onset of weakness, death occuring during the first year of life); type II (intermediate); and type III (Kugelberg-Welander disease, late onset of weakness, patients remaining ambulant).
The gene responsible for SMA has been mapped to 5q11.2–9-q13.3, and subsequently identified as survival motor neuron [SMN]. This region contains an inverted duplication of a 500-kb element, leading to two duplicate inverted copies of SMN, so-called telomeric and centromeric SMN (SMNtel and SMNcent, respectively). The predicted proteins encoded by both genes are identical, although the sequences of their genes differ by 5 nucleotides.
The SMN gene encodes a 294-amino acid protein, which is localized both in the cytoplasm, and in discrete nuclear bodies called gems. In both compartments SMN is part of a large complex that contains several proteins, including Gemin2 and the DEAD box protein Gemin3. In the cytoplasm, the SMN complex is associated with small nuclear ribonucleoproteins (snRNP), Sm core protein and plays a critical role in spliceosomal snRNP assembly. In the nucleus, SMN is required for pre-mRNA as it serves in the regeneration of spliceosomes. Recently, Gemin4, a novel component of SMN complex, has been identified in both gems and nucleoli.
However, why only motoneurons are affected even though all cells display a deficiency in SMN protein remains unexplained at that time.
In 1995, we published results that strongly supported the idea that SMA could result from a muscular defect. Human embryonic myoblasts, or adult muscle satellite cells prepared from muscle fragments, or biopsies when allowed to fuse in vitro into myotubes, can be successfully innervated with rat embryonic spinal cord explants. This system is called a nerve–muscle coculture. When the muscular component of the coculture originated from SMA I, II, but not III, innervated myofibres displayed a marked degeneration with vacuolization, cytoskeletal disorganization and formation of myoballs, which occurred 1.5 (SMA I) to 2.5–3 (SMA II) weeks after innervation. We have speculated that SMA myofibres either produced a diffusible toxic factor or were unable to produce a trophic factor. In order to test this hypothesis, we used multiplate wells mounted with porous membrane inserts that allowed diffusion of soluble molecules between both the bottom wells and the insert wells. Control cocultures were performed on either the bottom wells or insert membranes with the SMA coculture maintened in the counterpart compartment. Under these conditions, control cocultures were unable to prevent SMA coculture degeneration. Conversely, the SMA cocultures were unable to exert any cytotoxic effect on the control cocultures. In addition, brain-derived neurotrophic factor and neurotrophin 3 and 5 were unable to prevent SMA cocultures degeneration. We concluded therefore that the degeneration was not due to the production of a toxic factor or to the lack of secretion of a trophic factor.
The muscular component of our cocultures consists of a mixture of various cell types, including muscle satellite cells, which are predominant, fibroblasts, adipocytes, macrophages, endothelial cells, etc. We have shown that the only cells required for successful innervation were the muscle fibroblasts – involved in the production of a basal lamina surrounding the myofibres – and myogenic cells [myoblasts (foetus) or muscle satellite cells (postnatal)]. Using cloned fibroblasts and myogenic cells derived from healthy or SMA muscle biopsies, we found that fibroblasts from healthy donors and SMA patients contributed equally to the establishment of cocultures and only SMA myogenic cells were responsible for the degeneration of innervated cocultures. Finally, we have shown that degeneration of cocultures derived from cloned SMA muscle satellite cells could be prevented by mixing, before cell fusion, equal amounts of normal cloned satellite cells with SMA satellite cells, to promote the formation of heteromyotubes. We concluded therefore that normal muscle satellite cells provide the heteromyotubes with the signal (s) required for the survival of motor unit.
In a recent paper, Burlet et al. studied SMN protein expression in various human tissues during normal foetal and postnatal development, and have shown a reduction in SMN protein level in the postnatal period. Surprisingly, they observed that in skeletal muscle cells, SMN protein was localized to large cytoplasmic dot-like structures, and was tightly associated with membrane-free heavy sedimenting complexes. Moreover, they noted that the lack, or the marked drop, of SMN in skeletal muscles of type I, II and III foetuses might be related to the pathological defect of muscle fibres in SMA patients, and they stated that their data did not help in deciding whether SMA results from impaired SMN expression in spinal cord, skeletal muscle or both and raised the hypothesis that SMA is an embryo-foetal rather than a postnatal disease.
We present here findings that show that the fusion process of satellite muscle cells, and the expression of SMN mRNA and protein as well as of the myogenic transcription factor, are impaired, which suggest that SMA skeletal muscle cells display an abnormal development. As a result of these findings, we propose an unifying hypothesis to explain SMA pathogenesis.
Filed under: Neuropharmacology