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Updated: May 11, 2026
CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer
Published on: June 9, 2018
Genome-wide CRISPR/Cas9 screen shows that loss of GET4 increases mitochondria-endoplasmic reticulum contact sites and
Emma L Wilson1,2,3, Yizhou Yu3, Nuno S Leal3
1UK Dementia Research Institute, University of Cambridge, Clifford Albutt building, Cambridge biomedical campus, Cambridge, CB2 0AH, UK.
Abstract:
Organelles form membrane contact sites between each other, allowing for the transfer of molecules and signals. Mitochondria-endoplasmic reticulum (ER) contact sites (MERCS) are cellular subdomains characterized by close apposition of mitochondria and ER membranes. They have been implicated in many diseases, including neurodegenerative, metabolic, and cardiac diseases. Although MERCS have been extensively studied, much remains to be explored. To uncover novel regulators of MERCS, we conducted a genome-wide, flow cytometry-based screen using an engineered MERCS reporter cell line. We found 410 genes whose downregulation promotes MERCS and 230 genes whose downregulation decreases MERCS. From these, 29 genes were selected from each population for arrayed screening and 25 were validated from the high population and 13 from the low population. GET4 and BAG6 were highlighted as the top 2 genes that upon suppression increased MERCS from both the pooled and arrayed screens, and these were subjected to further investigation. Multiple microscopy analyses confirmed that loss of GET4 or BAG6 increased MERCS. GET4 and BAG6 were also observed to interact with the known MERCS proteins, inositol 1,4,5-trisphosphate receptors (IP3R) and glucose-regulated protein 75 (GRP75). In addition, we found that loss of GET4 increased mitochondrial calcium uptake upon ER-Ca2+ release and mitochondrial respiration. Finally, we show that loss of GET4 rescues motor ability, improves lifespan and prevents neurodegeneration in a Drosophila model of Alzheimer's disease (Aβ42Arc). Together, these results suggest that GET4 is involved in decreasing MERCS and that its loss is neuroprotective.
Insights
Researchers identified GET4 and BAG6 as key regulators of mitochondria-endoplasmic reticulum (ER) contact sites (MERCS). Loss of GET4 enhances MERCS, improves mitochondrial function, and shows neuroprotective effects in Alzheimer
Area of Science:
- Cell Biology
- Molecular Biology
- Neuroscience
Background:
- Organelles communicate via membrane contact sites, crucial for cellular functions.
- Mitochondria-endoplasmic reticulum (ER) contact sites (MERCS) are vital for cellular signaling and implicated in various diseases.
- Novel regulators of MERCS are needed to understand their role in health and disease.
Purpose of the Study:
- To identify novel regulators of mitochondria-endoplasmic reticulum contact sites (MERCS).
- To investigate the functional roles of identified MERCS regulators.
- To assess the therapeutic potential of modulating MERCS in neurodegenerative disease models.
Main Methods:
- Genome-wide, flow cytometry-based screen using an engineered MERCS reporter cell line.
- Arrayed screening and validation of candidate genes.
- Confocal microscopy, co-immunoprecipitation, and in vivo Drosophila models of Alzheimer's disease.
Main Results:
- Identified 410 genes promoting MERCS and 230 decreasing MERCS upon downregulation.
- GET4 and BAG6 were validated as top regulators that increase MERCS when suppressed.
- Loss of GET4 enhanced mitochondrial calcium uptake, respiration, and demonstrated neuroprotection in an Alzheimer's disease model.
Conclusions:
- GET4 and BAG6 are novel regulators of MERCS, with GET4 suppressing MERCS formation.
- Modulating GET4 impacts mitochondrial function and calcium signaling.
- Loss of GET4 exhibits neuroprotective effects, suggesting therapeutic potential for neurodegenerative diseases.
