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Abstract
In humans, a P56S point mutation in the VAPB/ALS8 MSP domain is associated with amyotrophic lateral sclerosis (ALS) and late-onset spinal muscular atrophy (SMA). The N-terminal MSP domain is cleaved from the C-terminus, and secreted through an unknown, nonconventional manner. The P56S mutation inhibits secretion of the MSP domain. We use Caenorhabditis elegans to study live secretion of VPR-1, as well as to understand vMSP receptor signaling in muscle and gonad.
To study the secretion mechanism of VPR-1 we created a transgenic line of C. elegans with a fluorescently tagged VPR-1. Using this model, we were able to visualize live secretion of N-terminal VPR-1 from neurons. We also observed trafficking of N-terminal VPR-1 along axons. We validated that the fluorescent tags did not drastically alter VPR-1 MSP cleavage through a Western Blot which showed predicted protein bands for the full length fluorescent protein, as well as the cleavage product. This model provided the first insight into live secretion of VPR-1.
We explored the complexing behavior of the three identified vMSP receptors: VAB-1, CLR-1, and ROBO/SAX-3. We found evidence suggesting that VAB-1 and SAX-3 as well as CLR-1 and SAX-3 form heteromeric complexes. vMSP presence appears to increase complexing behavior between CLR-1 and SAX-3. We further investigated vMSP signaling with CLR-1 in muscle and gonad development of C. elegans. We found that vMSP signaling in the gonad is independent of the CLR-1 receptor, and that CLR-1 signaling plays no obvious role in gonad development. We found that vMSP and CLR-1 signaling is important in localizing mitochondria to the I-bands in muscle during the L4/adult stage, and that CLR-1 has an important, and perhaps vMSP independent, signaling role early in muscle development. We also show that endogenous CLR-1 expression is in the somatic gonad and muscle plasma membrane, and expression is independent of vMSP signaling.
A suppressor screen identified survival motor neuron 1 (smn-1 ) as a suppressor of VPR-1 muscle mitochondria defects. We found that smn-1 and arx-2, another identified suppressor of VPR-1 muscle mitochondria defects, colocalize at muscle myofilaments. Overexpression of ARX-2 partially rescues mitochondria defects associated with SMN-1 loss. This presents a possible link between the P56S mutation and ALS and SMA.
