✍🏼 Curran Landry, James P Costanzo, Miguel Mitne-Neto, Mayana Zatz, Ashleigh E Schaffer, Maria Hatzoglou, Alysson R Muotri & Helen C Miranda
🏠 Department of Genetics and Genome Sciences, Case Western Reserve University, Cleveland, OH, USA
📑 EMBO Molecular Medicine (2025)
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Abstract
Vesicle-associated membrane protein-associated protein-B (VAPB) is an endoplasmic reticulum (ER) membrane-bound protein. The P56S mutation in VAPB causes a dominant, familial form of amyotrophic lateral sclerosis (ALS). However, the mechanism by which this mutation leads to motor neuron (MN) degeneration remains unclear. Utilizing inducible pluripotent stem cell (iPSC)-derived MNs expressing either wild-type (WT) or P56S VAPB, we demonstrate that the mutant protein reduces neuronal firing and disrupts ER-mitochondria-associated membranes (ER MAMs), with a time-dependent decline in mitochondrial membrane potential (MMP), hallmarks of MN pathology. These findings were validated in patient-derived iPSC-MNs. Additionally, VAPB P56S MNs show increased susceptibility to ER stress, elevated expression of the Integrated Stress Response (ISR) regulator ATF4 under stress, and reduced global protein synthesis. Notably, pharmacological ISR inhibition using ISRIB rescued ALS-associated phenotypes in both VAPB P56S and patient-derived iPSC-MNs. We present the first evidence that the VAPB P56S mutation activates ISR signaling via mitochondrial dysfunction in human MNs. These findings support ISR modulation as a strategy for ALS intervention and highlight the need for patient stratification in clinical trials.
How the WOLF is used in this study
n this study, the NanoCellect WOLF cell sorter was used to isolate successfully genome-edited patient-derived iPSCs following CRISPR/Cas9 transfection. iPSCs were transfected with a PX458 plasmid encoding Cas9 and a VAPB-targeting guide RNA along with a GFP reporter, enabling identification of edited cells by fluorescence. The WOLF sorter was then employed to select and enrich GFP-positive cells, separating transfected cells from unmodified counterparts while maintaining cell viability. These enriched populations were subsequently expanded into colonies and screened by DNA sequencing to identify clones harboring insertions or deletions at the VAPB exon 2 locus, with successful gene knockout further validated by Western blotting.
