Regulation of Survival Motor Neuron Gene Expression by Calcium Signaling

Area of Science:

• Neuroscience
• Genetics
• Molecular Biology

Background:

• Spinal muscular atrophy (SMA) is a genetic disorder caused by a deficiency in the survival of motor neuron (SMN) protein, primarily due to homozygous deletion of the SMN1 gene.
• The SMN2 gene, a near-identical duplicate, produces insufficient functional SMN protein due to exon 7 skipping during pre-mRNA splicing, a key factor in SMA pathogenesis.
• Current therapeutic strategies for SMA focus on increasing SMN protein levels, with most drug discovery efforts centered on synthetic compounds, leaving natural products underexplored.

Purpose of the Study:

• To identify novel compounds that can upregulate SMN protein expression from the SMN2 gene in SMA fibroblasts.
• To investigate the mechanism by which identified compounds modulate SMN2 splicing and SMN protein levels.
• To explore the role of intracellular calcium signaling in the regulation of SMN2 splicing.

Main Methods:

• An unbiased, image-based screening of a microbial metabolite library was conducted using SMA fibroblasts and an SMN-specific immunoassay.
• Brefeldin A (BFA), identified as a potent SMN protein inducer, was further analyzed for its effects on SMN2 splicing and intracellular calcium levels.
• The involvement of calcium signaling pathways, including calcium/calmodulin-dependent kinases (CaMKs), and the expression of splicing factors (Tra2-β, SRSF9, PSF, hnRNP M) were investigated.

Main Results:

• Brefeldin A (BFA) was identified as a strong inducer of SMN protein in SMA fibroblasts, significantly increasing functional SMN levels.
• BFA treatment partially rescued the SMN2 pre-mRNA splicing defect, leading to increased exon 7 inclusion.
• The BFA-induced increase in SMN protein and corrected splicing were linked to elevated intracellular calcium concentrations and modulated activity of splicing factors, including reduced Tra2-β and SRSF9 and enhanced binding of PSF and hnRNP M to the SMN2 exon 7 splicing enhancer.

Conclusions:

• Brefeldin A (BFA) represents a promising natural compound for SMA therapy by effectively upregulating SMN protein through the rescue of SMN2 splicing defects.
• Intracellular calcium signaling plays a critical role in regulating SMN2 splicing, likely by influencing the expression and activity of key splicing factors.
• This study highlights a novel mechanism involving calcium-dependent pathways in SMA, opening new avenues for therapeutic development targeting splicing modulation.