SURVIVAL MOTOR NEURON PROTEIN (SMN) DEPLETION IN NEURONAL CELLS BY siRNA INHIBITION: AN IN VITRO MODEL FOR SMA

Objectives: Genetic defects in the telomeric survival motor neuron (SMN) gene are responsible for lower motor neuron death and subsequent paralysis of muscles in spinal muscular atrophy (SMA). SMN protein is a component of RNA processing complexes, as well as axonal cones, and recent studies have revealed a correlation between levels of SMN protein and phenotypes of SMA. SMN is required for normal functions of all cells but most cell types except anterior horn cells can tolerate a decrease of intracellular SMN levels. SMN may affect the growth and pathfinding of axons however, the process by which this occurs is not yet clear. We studied the molecular effects of SMN loss in neuronal cell cultures with particular emphasis on axon functions and apoptosis.

Methods: We developed an in vitro neuronal culture system as a model for studying the effect of SMN loss on survival, apoptosis and axonal functions of cells. We utilized a mouse neuroblastoma/spinal cord hybrid cell line (NSC-34), which resembles developing motor neurons. The SMN expression of NSC-34 cells was lowered by small interfering RNA (siRNA) technology. The SMN mRNA, protein levels and nuclear complexes were analyzed at various timepoints.

Results: The maximal decrease (>55%) of SMN mRNA level was obtained at 48 hours after transfection of siRNA. Up to 70% drop in SMN protein was observed at 96 hours. There was a marked change in the mRNA levels of some axonal proteins (e.g. dynactine and tubulin) at early time-points. While molecules participating in cellular apoptosis were affected at late time-points. Conclusion: A reduction of SMN level in neuronal cells resulted in early alterations of axonal proteins. An activation of neuronal apoptosis is a late phenomenon in SMA. Full-scale expression profiling from this cell culture system will provide additional information about the neuronal functions of SMN protein.