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By Jamshed Arslan Pharm.D.
Motor neurons control our ability to speak, walk, swallow, and even, breath. People with a motor neuron disease called amyotrophic lateral sclerosis (ALS) cannot perform these primal functions optimally. A point mutation (P56S) in VAMP associated protein isoform B (VAPB), a protein associated with the membrane around the endoplasmic reticulum, causes late-onset familial ALS8 by prompting VAPB to aggregate and lose its job of preventing buildup of abnormal proteins. To study ALS8, C. elegans is one of the animals of choice due to its simple neurophysiology: a single worm has only 302 neurons and 113 of them are associated with locomotion. To elucidate the role of VAPB in motor neurons during ALS8 pathogenesis, researchers from University of Ottawa, Canada, disrupted VAPB functions in DA neurons. They concluded that both under- and over-expression of VAPB can have deleterious effects, and inhibiting phosphatidylinositol-4 (PtdIns 4)-kinase activity can restore a loss of VAPB function in C. elegans model of ALS8.
Orthogonal Strategies Validation. Immunocytochemistry/Immunofluorescence: VAP-B Antibody [NBP1-89112] - Staining of human cell line U-2 OS shows positivity in endoplasmic reticulum. Antibody staining is shown in green
To characterize VAPB functions, the team generated two models: worms expressing human VAPB (either wild type or P56S), and worms with the knockdown of vpr-1 (a VAPB ortholog). They selected two kinds of easily-visible cholinergic motor neurons, dorsal A (DA) and dorsal B (DB), that innervate a worm’s dorsal muscles to control its backward (9 DA neurons) and forward (7 DB neurons) locomotion. The overexpression of VAPB under DA-specific unc-4 promoter led to a backward locomotion defect in VAPB worms that worsened with age. Crossing these strains with a GFP-expressing reporter strain showed that this backward locomotion defect was associated with axonal misguidance and degeneration (loss of GFP signal) of DA motor neurons.
However, this axonal misguidance phenotype is not observed in ALS8 patients. Moreover, P56S promotes loss of function phenotype and a decreased VAPB level has been found in the spinal cord of ALS patients. So, the investigators used a second model (vpr-1 knockdowns) to properly emulate human ALS.
Since vpr1 knockout is embryonically lethal, researchers generated worms in which RDE-1 (an argonaut protein for RNAi activity) could be reconstituted upon heating in DA motor neurons to knockdown vpr1. To avoid developmental defects and death associated with vpr1 knockout, these heat-inducible vpr1-knockdowns were heat shocked at alternating days. As expected, vpr1 knockdowns exhibited significantly slower rate of backward locomotion after heat shock and an uncoordinated phenotype (partial body movements or coiling etc.) that worsened with age. Likewise, crossing these worms with GFP-expressing reporter strain indicated an age-dependent neuronal loss in heat-inducible vpr1-knockdowns.
Since high PtdIns4-phosphate is known to be associated with ALS-related loss of VAPB phenotype, researchers then investigated if the neuronal loss in vpr1 knockdowns can be mitigated by PtdIns4-kinase inhibition.
PIK-93 was able to partially rescue the uncoordinated phenotype and backward locomotion defect in vpr1-knockdowns. Interestingly, a continuous administration of PIK-93 was necessary for DA neuronal survival since even the temporary removal of PIK-93 led to a significantly higher DA neuronal loss. In short, PIK-93 regimen could restore the defects in vpr1-knockdowns.
This study shows the importance of optimal levels of VAPB for normal physiology: both overexpression (first model) and lower expression (second model) exhibit ALS-related defects. By shedding light on the ALS pathophysiology, the researchers provided a target (PtdIns4-kinase inhibition) to rescue defects associated with the disease.
Explore antibodies for C. elegans research
Jamshed Arslan, Pharm D.
University of Alabama at Birmingham, School of Medicine
Dr. Arslan studies cell signaling in mitochondrial defects in C. elegans
and transgenic mice.
References
Zhang, Wendy, Antonio Colavita, and Johnny K. Ngsee. "Mitigating Motor Neuronal Loss in C. elegans Model of ALS8."Scientific Reports, vol. 7, 2017, n. pag. doi:10.1038/s41598-017-11798-6
