✍🏼 Curran Landry, James Costanzo, Miguel Mitne-Neto, Mayana Zatz, Ashleigh Schaffer, Maria Hatzoglou, Alysson Muotri, Helen Cristina Miranda
🏠 Department of Genetics and Genome Sciences, Case Western Reserve University, Cleveland, OH, USA
📑 bioRxiv 2024
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Abstract
Vesicle-associated membrane protein-associated protein-B (VAPB) is an ER membrane bound protein. VAPB P56S causes a dominant, familial form of amyotrophic lateral sclerosis (ALS), however, the mechanism through which this mutation causes motor neuron (MN) disease remains unknown. Using inducible wild type (WT) and VAPB P56S expressing iPSC-derived MNs we show that VAPB P56S, but not WT, protein decreased neuronal firing and mitochondrial-ER contact (MERC) with an associated age-dependent decrease in mitochondrial membrane potential (MMP); all typical characteristics of MN-disease. We further show that VAPB P56S expressing iPSC-derived MNs have enhanced age-dependent sensitivity to ER stress. We identified elevated expression of the master regulator of the Integrated Stress Response (ISR) marker ATF4 and decreased protein synthesis in the VAPB P56S iPSC-derived MNs. Chemical inhibition of ISR with the compound, ISRIB, rescued all MN disease phenotype in VAPB P56S MNs. Thus, our results not only support ISR inhibition as a potential therapeutic target for ALS patients, but also provides evidence to pathogenesis.
How the WOLF was used in this study
The WOLF cell sorter was used to enrich live, high‑quality single cells prior to single‑cell RNA sequencing. After generating single‑cell suspensions from tongue tissues of treated and control mice, cells were stained with viability and phenotypic markers and then sorted on the WOLF to remove dead cells and debris while isolating defined populations for downstream single‑cell transcriptomic profiling. This sorting step was essential for preparing samples with high viability and purity for subsequent 10× Genomics single‑cell sequencing, enabling accurate dissection of the tumor microenvironment’s cellular composition and its remodeling in response to PD‑1 blockade, photodynamic therapy, or their combination.
