Thread by @thatdnaguy

#ASHG20 HCM
Increased p4EBP1 underlies ALS pathology associated to P56S mutant VAPB.
Helen Cristina Miranda.

#ASHG20 HCM Amyotrophic lateral sclerosis (ALS). Most common type of adult-onset motor neuron disease. About 50% survive past 3rd year diagnosis. 10% familial. 90% sporadic.

#ASHG20 HCM Involves both upper and lower motor neurons. Many genes associated with ALS. >25 genes associated with familial, sporadic, or both versions.

#ASHG20 HCM VAMP associated protein B (VAPB) highly conserved. Ubiquitous expression. MSP domain is cleaved and secreted. VAPB variants: p.P56S, p.T46I, p.V240I.

#ASHG20 HCM Variants primarily cause ALS type 8. But gene also important in ALS type 1 and sporadic ALS. Decreased VAPB associated with concomitant progression in the SOD1 mouse model. Reduction of VAPB in brain tissue from post-mortem samples of sporadic ALS patients.

#ASHG20 HCM VAPB mediates several intracellular functions. ER stress, lipid transfer, vesicular transport, endosomal trafficking, calcium influx, autophagy, etc.

#ASHG20 HCM Wanted to model the disease using patient iPSCs from familial ALS (affected and unaffected relatives). Use motor neurons and neuronal progenitor cells.

#ASHG20 HCM Wanted to understand the VAPB interactome in humans. IP in these cell lines. Shotgun proteomics. Top pathways: EIF2 signaling, regulation eIF4 and p70S6K, mTOR, cleavage of pre-mRNA.

#ASHG20 HCM Known that mTOR helps control translation through 4EBP1. In ALS type 8 neural progenitor cells (NPCs; differentiated from iPSCs) p4EBP1 is dysregulated.

#ASHG20 HCM 4EBP1 and p4EBP1 are dysregulated in ALS type 8 motor neurons (under normal conditions). Indicates cells might have a direct effect in mRNA translation.

#ASHG20 HCM The ALS type 8 derived motor neurons have altered mRNA translation. Wanted to also test mitochondrial function (mTORC helps control energy balance intracellularly).

#ASHG20 HCM Used Seahorse assay to look. When you add FCCP uncoupling reagent you see lower maximum oxygen consumption rate in the ALS neurons compared to controls.

#ASHG20 HCM Look at the motor neuron activity. Used the Maestro system. Viral transduction to allow direct visualization and sorting if necessary. Plate on electrode grid. See action potential and propagation in the network.

#ASHG20 HCM ALS type 8 motor neurons you see decrease in number of spikes compared to controls. Look over time, initial hyperactivity. Over time they plateau sooner than controls never reaching same level of activity [ in the time frame they looked at ]