Abstract
Spinal muscular atrophy (SMA), the leading genetic cause of infant mortality, is caused by reduced levels of functional survival motor neuron (SMN) protein. To identify therapeutic agents for SMA, we established a versatile SMN2-GFP reporter line by targeting the human SMN2 gene. We then screened a compound library and identified Z-FA-FMK as a potent candidate. Z-FA-FMK, a cysteine protease inhibitor, increased functional SMN through inhibiting the protease-mediated degradation of both full-length and exon 7-deleted forms of SMN. Further studies reveal that CAPN1, CAPN7, CTSB, and CTSL mediate the degradation of SMN proteins, providing novel targets for SMA. Notably, Z-FA-FMK mitigated mitochondriopathy and neuropathy in SMA patient-derived motor neurons and showed protective effects in SMA animal model after intracerebroventricular injection. E64d, another cysteine protease inhibitor which can pass through the blood-brain barrier, showed even more potent therapeutic effects after subcutaneous delivery to SMA mice. Taken together, we have successfully established a human SMN2 reporter for future drug discovery and identified the potential therapeutic value of cysteine protease inhibitors in treating SMA via stabilizing SMN proteins.
Highlights
Spinal muscular atrophy (SMA), the leading genetic cause of infant mortality, is characterized by the specific degeneration of spinal motor neurons caused by the mutation in the SMN1 gene (Pearn et al, 1978; Pearn, 1980; Burglen et al, 1995; Lefebvre et al, 1995)
Given that spinal motor neurons degenerate in SMA patients, we examined the effect of Z-FA-FMK on the expression of survival motor neuron (SMN) in SMA patient–derived spinal motor neurons
SMA spinal motor neurons degenerated; as we reported before, there was a significant increase in the caspase 3/7 activity, revealing increased apoptosis caused by the deficiency of functional SMN in long-term cultures
Summary
Spinal muscular atrophy (SMA), the leading genetic cause of infant mortality, is characterized by the specific degeneration of spinal motor neurons caused by the mutation in the SMN1 gene (Pearn et al, 1978; Pearn, 1980; Burglen et al, 1995; Lefebvre et al, 1995). Humans are unique in that they have two SMN genes, SMN1 and SMN2 (Lorson et al, 1999; Monani et al, 1999; Rochette et al, 2001). The SMN1 gene generates full-length transcripts (SMN-FL) and functional survival motor neuron (SMN) protein. Human SMN2 gene reporters have been generated to screen for candidate drugs, most of these studies used mini-genes that do not have the full expression, splicing, or regulatory elements (Andreassi et al, 2001; Zhang et al, 2001; Lunn et al, 2004; Morse et al, 2012). Building human SMN2 reporters that contain all the elements of the human SMN2 gene would be essential to identify therapeutic agents that can effectively rescue motor neuron degeneration in SMA patients
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