CRISPR/Cas9-engineered Drosophila knock-in models to study VCP diseases

Abstract

Valosin containing protein (VCP) is a hexameric type II AAA ATPase required for several cellular processes including ER-associated degradation, organelle biogenesis, autophagy and membrane fusion. VCP contains three domains: a regulatory N-terminal domain and two ATPase domains (D1 and D2). Mutations in the N-terminal and D1 domains are associated with several degenerative diseases, including Multisystem Proteinopathy (MSP-1) and ALS. However, patients with VCP mutations vary widely in their pathology and clinical penetrance, making it difficult to devise effective treatment strategies. Having a deeper understanding of how each mutation affects VCP function could enhance the prediction of clinical outcomes and design of personalized treatment options. Over-expressing VCP patient mutations in Drosophila has been shown to mimic many pathologies observed in human patients. The power of a genetically tractable model organism coupled with well-established in vivo assays and a relatively short life cycle make Drosophila an attractive system to study VCP disease pathogenesis and novel treatment strategies. Using CRISPR/Cas9, we have generated individual Drosophila knock-in mutants that include nine hereditary VCP disease mutations. We validate that these models display many hallmarks of VCP-mediated degeneration, including progressive decline in mobility, protein aggregate accumulation and defects in lysosomal and mitochondrial function. We also made some novel and unexpected findings, including laminopathies and sex-specific phenotypic differences in several mutants. Taken together, the Drosophila VCP disease models we have generated in this study will be useful for studying the etiology of individual VCP patient mutations and for testing potential genetic and/or pharmacological therapies.

Novel VCP activator reverses multisystem proteinopathy nuclear proteostasis defects and enhances TDP-43 aggregate clearance

“…The presence of ubiquitin-positive intranuclear inclusions in FTLD-TDP type D, inclusion body myopathy, and Paget’s disease of bone suggested that these diseases are associated with a loss of nuclear proteostasis. Prior overexpression studies have suggested that pathogenic VCP variants are partially excluded from the nucleus (29,30). To determine whether pathogenic VCP variants result in changes in the subcellular localization of endogenous VCP, CRISPR-Cas9 was used to edit the endogenous VCP locus by knocking-in two different pathogenic VCP variants into HeLa cells: p.A232E (the variant associated with the highest ATPase activity in vitro) and p.R155H (the most common pathogenic MSP variant).…”

VCP acts downstream of tTAFs to downregulate mono-ubiquitinated H2A and promote spermatocyte differentiation inDrosophila

“…The following fly strains were used in this study: w 1118 , VCP-GFP (Wall, et al, 2021), UAS- VCP -RNAi (VDRC #24354), BamGal4 (Doug Harrison, Univ. Kentucky), sa-GFP (Chen, et al, 2005), UAS- bam -RNAi (BDSC #33631), UAS- sa -RNAi (BDSC #36730), UAS- mia -RNAi (BDSC #57790), UAS- sce -RNAi (BDSC #67924), UAS-ufd1- RNAi (BDSC #41823), UAS- rpt2 -RNAi (BDSC #34795), sce KO (BDSC #80157), UAS- GFP nls (lab stock), VasaGal4; hsFLP.D5 , UAS- GFP /cyo; FRT82B , tubGal80/TM6B (Butsch, et al, 2022), EyaGal4 (Leatherman and DiNardo, 2008), UASp-FRT-H2A-eGFP-PolyA-FRT-H2A-mCherry (Kahney, et al, 2021), tubGal80 AID (McClure, et al, 2022) (BDSC #92470), and UAS-VCP K2A (Chang, et al, 2021).…”

The Cure VCP Scientific Conference 2021: Molecular and clinical insights into neurodegeneration and myopathy linked to multisystem proteinopathy-1 (MSP-1)

“…Dr. Johnson presented preliminary data suggesting that flies harboring VCP patient mutations show TL disruption as well as many of the same defects found in VCP disease including locomotor defects, proteostasis decline, accumulation of TDP-43, and defective nuclear morphology ( Wall et al, 2021 ). Interestingly, overexpression of SVIP in R152H/C (R155H in humans) mutant flies was able to rescue the TL network and improve locomotor defects.…”