PUBLICATION

Efficient clofilium tosylate-mediated rescue of POLG-related disease phenotypes in zebrafish

Authors

Facchinello, N., Laquatra, C., Locatello, L., Beffagna, G., Brañas Casas, R., Fornetto, C., Dinarello, A., Martorano, L., Vettori, A., Risato, G., Celeghin, R., Meneghetti, G., Santoro, M.M., Delahodde, A., Vanzi, F., Rasola, A., Dalla Valle, L., Rasotto, M.B., Lodi, T., Baruffini, E., Argenton, F., Tiso, N.

ID

ZDB-PUB-210121-11

Date

2021

Source

Cell Death & Disease 12: 100 (Journal)

Registered Authors

Argenton, Francesco, Beffagna, Giorgia, Dalla Valle, Luisa, Facchinello, Nicola, Fornetto, Chiara, Risato, Giovanni, Santoro, Massimo, Tiso, Natascia, Vanzi, Francesco, Vettori, Andrea

Keywords

none

MeSH Terms

Phenotype
Mitochondrial Diseases/genetics*
Quaternary Ammonium Compounds/metabolism*
Disease Models, Animal
Zebrafish
Animals

(all 6)

PubMed

33469036 Full text @ Cell Death Dis.

Abstract

The DNA polymerase gamma (Polg) is a nuclear-encoded enzyme involved in DNA replication in animal mitochondria. In humans, mutations in the POLG gene underlie a set of mitochondrial diseases characterized by mitochondrial DNA (mtDNA) depletion or deletion and multiorgan defects, named POLG disorders, for which an effective therapy is still needed. By applying antisense strategies, ENU- and CRISPR/Cas9-based mutagenesis, we have generated embryonic, larval-lethal and adult-viable zebrafish Polg models. Morphological and functional characterizations detected a set of phenotypes remarkably associated to POLG disorders, including cardiac, skeletal muscle, hepatic and gonadal defects, as well as mitochondrial dysfunctions and, notably, a perturbed mitochondria-to-nucleus retrograde signaling (CREB and Hypoxia pathways). Next, taking advantage of preliminary evidence on the candidate molecule Clofilium tosylate (CLO), we tested CLO toxicity and then its efficacy in our zebrafish lines. Interestingly, at well tolerated doses, the CLO drug could successfully rescue mtDNA and Complex I respiratory activity to normal levels, even in mutant phenotypes worsened by treatment with Ethidium Bromide. In addition, the CLO drug could efficiently restore cardio-skeletal parameters and mitochondrial mass back to normal values. Altogether, these evidences point to zebrafish as a valuable vertebrate organism to faithfully phenocopy multiple defects detected in POLG patients. Moreover, this model represents an excellent platform to screen, at the whole-animal level, candidate molecules with therapeutic effects in POLG disorders.

Genes / Markers

Figures

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Expression

Gene	Fish	Conditions	Stage	Anatomy	Assay	Figure
fosab	polg^{sa9574/sa9574}	standard conditions	Day 4	whole organism	RTPCR	Fig. 5
fosab	polg^{sa9574/sa9574}	chemical treatment by environment: Clofilium	Days 7-13	whole organism	RTPCR	Fig. 6
fosab	polg^{sa9574/sa9574}	standard conditions	Days 7-13	whole organism	RTPCR	Fig. 6
fosab	WT	chemical treatment by environment: Clofilium	Days 7-13	whole organism	RTPCR	Fig. 6
fosab	WT	standard conditions	Days 7-13	whole organism	RTPCR	Fig. 6
fosab	WT	standard conditions	Day 4	whole organism	RTPCR	Fig. 5
hbbe3	polg^{sa9574/sa9574}	standard conditions	Days 7-13	whole organism	RTPCR	Fig. 6
hbbe3	polg^{sa9574/sa9574}	chemical treatment by environment: Clofilium	Days 7-13	whole organism	RTPCR	Fig. 6
hbbe3	polg^{sa9574/sa9574}	standard conditions	Day 4	whole organism	RTPCR	Fig. 5
hbbe3	WT	standard conditions	Day 4	whole organism	RTPCR	Fig. 5

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Phenotype

Fish	Conditions	Stage	Phenotype	Figure
WT	chemical treatment by environment: Clofilium	Days 7-13	whole organism mitochondrial chromosome increased amount, abnormal	Fig. 6
WT	chemical treatment by environment: Clofilium	Adult	skeletal muscle NADH dehydrogenase (ubiquinone) activity magnitude, normal	Fig. 6
WT	chemical treatment by environment: Clofilium, chemical treatment by environment: ethidium bromide	Days 7-13	whole organism mt-nd1 expression increased amount, abnormal	Fig. 6
WT	chemical treatment by environment: Clofilium, chemical treatment by environment: ethidium bromide	Days 7-13	whole organism mitochondrial chromosome increased amount, abnormal	Fig. 6
WT + MO1-polg	standard conditions	Prim-5	whole organism polg expression decreased amount, abnormal	Fig. 1
WT + MO1-polg	standard conditions	Long-pec	atrium increased size, abnormal	Fig. 1
WT + MO1-polg	standard conditions	Long-pec	skeletal muscle mitochondrion fused with skeletal muscle mitochondrion, abnormal	Fig. 1
WT + MO1-polg	standard conditions	Long-pec	skeletal muscle mitochondrion swollen, abnormal	Fig. 1
WT + MO1-polg	standard conditions	Long-pec	whole organism polg expression decreased amount, abnormal	Fig. 1
WT + MO1-polg	standard conditions	Long-pec	whole organism decreased size, abnormal	Fig. 1

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Mutations / Transgenics

Allele	Construct	Type	Affected Genomic Region
gz15Tg	Tg(ela3l:EGFP) Tg(fabp10a:DsRed)	Transgenic Insertion
ia21Tg	Tg(4xHRE-TATA:EGFP)	Transgenic Insertion
ia300Tg	Tg(tg:EGFP,myl7:EGFP)	Transgenic Insertion
ia301Tg	Tg(Hsa.Cox8a:MLS-EGFP)	Transgenic Insertion
ia302		Small Deletion	polg
sa9574		Point Mutation	polg
zf3275Tg	Tg(6xCre:EGFP)	Transgenic Insertion

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Human Disease / Model

Sequence Targeting Reagents

Target	Reagent	Reagent Type
polg	CRISPR2-polg	CRISPR
polg	MO1-polg	MRPHLNO
polg	MO2-polg	MRPHLNO

Fish

Fish
ia21Tg + MO1-polg
ia300Tg + MO1-polg
polg^ia302/ia302
polg^{sa9574/sa9574}
polg^{sa9574/sa9574}
polg^{sa9574/sa9574}; gz15Tg
polg^{sa9574/sa9574}; ia301Tg
WT
WT + MO1-polg
zf3275Tg + MO1-polg

Antibodies

Orthology

Engineered Foreign Genes

Marker	Marker Type	Name
DsRed	EFG	DsRed
EGFP	EFG	EGFP

Mapping

Facchinello et al., 2021 ZDB-PUB-210121-11 PMID:33469036

Efficient clofilium tosylate-mediated rescue of POLG-related disease phenotypes in zebrafish

Facchinello et al., 2021

ZDB-PUB-210121-11
PMID:33469036