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

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.

Therapy Prospects for Mitochondrial DNA Maintenance Disorders

“…Moreover, it improved the cardio-skeletal effects of POLG deficiency. Although it is known that CLO is an antiarrhythmic acting as a potassium channel blocker, its mechanism of action in rescuing mtDNA levels is not known [ 205 ].…”

The Zebrafish, an Outstanding Model for Biomedical Research in the Field of Melatonin and Human Diseases

“…Another advantage over mammals is that zebrafish embryo development is extrauterine, which makes it easy to study and observe the early stages of development, making it an excellent model for the study of developmental biology. These advantages also facilitate the early manipulation of zebrafish larvae through targeted genetic manipulation techniques such as transgenesis, TALEN, CRISPR, RNA overexpression, and others [6][7][8]. In addition, the use of zebrafish is much cheaper than small mammals including mice and rats, because the maintenance of the fish colony comes at a relatively low cost.…”

The Power of Yeast in Modelling Human Nuclear Mutations Associated with Mitochondrial Diseases

“…The clofilim tosylate (CLO), belonging to a class of anti-arrhythmic agents, displayed the best rescuing activity, suppressing the respiratory growth defect and preventing mtDNA loss in all mip1 mutants tested [268]. The rescuing effect of CLO was later validated in two animal disease models, C. elegans and zebrafish [269], as well as in the fibroblasts of a patient carrying compound heterozy-gous POLG mutations. The molecular mechanism by which CLO exerts the rescuing activity is not yet known, however, the successful application in this four-model approach indicates that CLO is acting by a rescuing mechanism conserved through the evolution.…”