PUBLICATION

CRISPR/Cas9-Mediated Constitutive Loss of VCP (Valosin-Containing Protein) Impairs Proteostasis and Leads to Defective Striated Muscle Structure and Function In Vivo

Authors

Voisard, P., Diofano, F., Glazier, A.A., Rottbauer, W., Just, S.

ID

ZDB-PUB-220625-16

Date

2022

Source

International Journal of Molecular Sciences 23(12): (Journal)

Registered Authors

Diofano, Federica, Just, Steffen, Rottbauer, Wolfgang

Keywords

CRISPR/Cas9, VCP, VCPopathies, disease modeling, protein homeostasis, zebrafish

MeSH Terms

Cell Cycle Proteins/genetics
Cell Cycle Proteins/metabolism
Zebrafish/genetics
Zebrafish/metabolism
Frontotemporal Dementia*/genetics
Frontotemporal Dementia*/metabolism
CRISPR-Cas Systems
Animals
Mutation
Muscle, Striated*/metabolism
Adenosine Triphosphatases/genetics
Adenosine Triphosphatases/metabolism
Myositis, Inclusion Body*/genetics
Myositis, Inclusion Body*/metabolism
Muscle, Skeletal/metabolism
Proteostasis/genetics
Valosin Containing Protein/genetics
Valosin Containing Protein/metabolism

(all 18)

PubMed

35743185 Full text @ Int. J. Mol. Sci.

Abstract

Valosin-containing protein (VCP) acts as a key regulator of cellular protein homeostasis by coordinating protein turnover and quality control. Mutations in VCP lead to (cardio-)myopathy and neurodegenerative diseases such as inclusion body myopathy with Paget's disease of the bone and frontotemporal dementia (IBMPFD) or amyotrophic lateral sclerosis (ALS). To date, due to embryonic lethality, no constitutive VCP knockout animal model exists. Here, we generated a constitutive CRISPR/Cas9-induced vcp knockout zebrafish model. Similar to the phenotype of vcp morphant knockdown zebrafish embryos, we found that vcp-null embryos displayed significantly impaired cardiac and skeletal muscle function. By ultrastructural analysis of skeletal muscle cells and cardiomyocytes, we observed severely disrupted myofibrillar organization and accumulation of inclusion bodies as well as mitochondrial degeneration. vcp knockout was associated with a significant accumulation of ubiquitinated proteins, suggesting impaired proteasomal function. Additionally, markers of unfolded protein response (UPR)/ER-stress and autophagy-related mTOR signaling were elevated in vcp-deficient embryos, demonstrating impaired proteostasis in VCP-null zebrafish. In conclusion, our findings demonstrate the successful generation of a stable constitutive vcp knockout zebrafish line that will enable characterization of the detailed mechanistic underpinnings of vcp loss, particularly the impact of disturbed protein homeostasis on organ development and function in vivo.

Genes / Markers

Figures

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Expression

Gene	Antibody	Fish	Conditions	Stage	Qualifier	Anatomy	Assay	Figure
vcp		vcp^{ulm105/ulm105}	standard conditions	Protruding-mouth		whole organism	RTPCR	Fig. 1
vcp		WT	standard conditions	Protruding-mouth		whole organism	RTPCR	Fig. 1

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Phenotype

Fish	Conditions	Stage	Phenotype	Figure
WT + MO3-vcp	control	Protruding-mouth	myotome muscle cell disorganized, abnormal	Fig. 2
vcp^{ulm105/ulm105}	standard conditions	Prim-5	whole organism morphology, normal	Fig. 1
vcp^{ulm105/ulm105}	standard conditions	Long-pec	heart heart contraction normal process quality, normal	Fig. 3
vcp^{ulm105/ulm105}	standard conditions	Long-pec	myotome muscle cell organized, normal	Fig. 2
vcp^{ulm105/ulm105}	standard conditions	Long-pec	whole organism morphology, normal	Fig. 1
vcp^{ulm105/ulm105}	standard conditions	Protruding-mouth	cardiac muscle cell mitochondrion morphology, abnormal	Fig. 3
vcp^{ulm105/ulm105}	standard conditions	Protruding-mouth	heart cardiac muscle cell disorganized, abnormal	Fig. 3
vcp^{ulm105/ulm105}	standard conditions	Protruding-mouth	heart heart contraction decreased process quality, abnormal	Fig. 3
vcp^{ulm105/ulm105}	standard conditions	Protruding-mouth	muscle cell inclusion body increased amount, abnormal	Fig. 2
vcp^{ulm105/ulm105}	standard conditions	Protruding-mouth	muscle cell mitochondrion morphology, abnormal	Fig. 2

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Mutations / Transgenics

Allele	Construct	Type	Affected Genomic Region
ulm105		Indel	vcp

Human Disease / Model

Sequence Targeting Reagents

Target	Reagent	Reagent Type
vcp	CRISPR1-vcp	CRISPR
vcp	MO3-vcp	MRPHLNO

Fish

Fish
vcp^{ulm105/ulm105}
WT
WT + MO3-vcp

Antibodies

Name	Type	Antigen Genes	Isotypes	Host Organism
Ab1-rps6	polyclonal			Rabbit
Ab2-vcp	monoclonal		IgG2a	Mouse
Ab4-ub	monoclonal		IgG1	Mouse
Ab12-hspa5	polyclonal	hspa5	IgG	Rabbit
Ab-MF20	monoclonal		IgG2b	Mouse
Ab-S46	monoclonal	myh6	IgG1	Mouse

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Orthology

Engineered Foreign Genes

Mapping

Voisard et al., 2022 ZDB-PUB-220625-16 PMID:35743185

CRISPR/Cas9-Mediated Constitutive Loss of VCP (Valosin-Containing Protein) Impairs Proteostasis and Leads to Defective Striated Muscle Structure and Function In Vivo

Voisard et al., 2022

ZDB-PUB-220625-16
PMID:35743185