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

Nicola Facchinello,Raquel Brañas Casas,Maria Berica Rasotto,Francesco Vanzi,Andrea Vettori,Giorgia Beffagna,Agnès Delahodde ,Francesco Argenton,Andrea Rasola,Tiziana Lodi,Lisa Locatello,Luisa Dalla Valle,Enrico Baruffini,Chiara Fornetto,Claudio Laquatra,Rudy Celeghin,G Meneghetti ,Massimo Santoro ,Giovanni Risato,Alberto Dinarello,Laura Martorano,Natascia Tiso

doi:10.1038/s41419-020-03359-z

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.

Highlights

In vertebrates, from fish to humans, the mitochondrialDNA is replicated by the DNA polymerase gamma (Polg), encoded by the nuclear genome
Taking advantage of pathway reporter zebrafish lines, we could assess in vivo, at the whole-animal level, if polg deficiency affects metabolic signalling cascades relevant in mitochondria-to-nucleus cross-talk, such as hypoxia-inducible factors (Hif)-Hypoxia and CREB pathways
We found that both Hif-Hypoxia signalling, verified with two independent reporters (Fig. 1H, K, L, P) and CREB signalling (Supplementary Fig. 1A, B, C) were upregulated in polg morphant embryos; a patterning pathway such as Wnt, considered in the analysis, appeared unmodified at the same stages (Fig. 1K, L, Q)

Summary

Introduction

DNA (mtDNA) is replicated by the DNA polymerase gamma (Polg), encoded by the nuclear genome. Mutations in the human POLG gene cause a series of mitochondrial diseases with Mendelian inheritance, collectively named POLG-related disorders, characterized by mtDNA depletion (MDD) or accumulation of multiple deletions. These disorders mainly include Alpers–Huttenlocher syndrome (OMIM 203700), mitochondrial neurogastrointestinal encephalopathy syndrome (MNGIE type) (OMIM 613662), mitochondrial recessive ataxia syndrome (SANDO, SCAE) (OMIM 607459), autosomal recessive progressive external ophthalmoplegia (arPEO) (OMIM 258450) and autosomal dominant progressive external ophthalmoplegia (adPEO) (OMIM 157640). About 300 pathogenic mutations have been reported in the Human DNA Polymerase Gamma Mutation Database (https://tools.niehs.nih.gov// polg/). Clinical phenotypes correlate with the mtDNA genotype

Methods

Results

Conclusion