Introduction: POLG-related disorders are a group of multi-organ pathologies characterized by the dysfunction of the mitochondrial DNA polymerase gamma, encoded by the nuclear gene POLG and essential for mtDNA replication, repair and stability. As with all mitochondrial disorders, there is so far no cure for POLG-related diseases. Moreover, the paucity of animal models for these pathologies is hindering the set up of a pharmacological screen. Methods: The recent innovations in the field of transient gene perturbation and stable genome editing, based on morpholino (MO) antisense and CRISPR/Cas9 technologies, respectively, point to the small teleost zebrafish (Danio rerio) as an excellent candidate organism where to rapidly perform POLG knock-down, as well as targeted mutagenesis, followed by phenotype rescue via large-scale drug screen. Preliminary results: We have performed MO-based transient knock-down of the zebrafish polg gene, validating the splice blocking MO activity and obtaining a phenotype characterized by homogeneous developmental delay without obvious malformations, except for the observation of enlarged cardiac chambers, associated with increased heart rate. Of note, dilated cardiomyopathy is included among POLG-associated signs. Moreover, taking advantage of signaling pathway reporter lines, available in our laboratory, we have found that Hypoxia and CREB pathways are up-regulated in zebrafish polg morphants, while Wnt signaling appears unmodified. Discussion and conclusion: Our preliminary results on polg transient inactivation support the zebrafish as a suitable model to perform Crispr/Cas9- mediated mutagenesis of DNA polymerase gamma. The evidence of Hypoxia reporter activation under polg knock-down conditions suggests the existence of cross-talk mechanisms sensing mitochondrial dysfunction and triggering Hypoxia signaling even in a normoxic environment. Moreover, results from signaling pathway analysis in polg morphant embryos point to Hypoxia and CREB as candidate pathways for hypothesis-driven screening of molecules with therapeutic effect on POLG-related phenotypes. This work is supported by AFM-Telethon (18572 - POLYGON), EU ZF-HEALTH, Italian Ministry of Health (RF-2010-2309484 - TRAMBIGEN) and UniPD (CPDA128151 - OPTOZEN).

A zebrafish model to study mitochondrial DNA polymerase gamma deficiency in vertebrates

TISO, NATASCIA;BUSOLIN, GIORGIA;EK, OLIVIER FREDERIC D;ASTONE, MATTEO;FACCHINELLO, NICOLA;GIULIODORI, ALICE;MILANETTO, MARTINA;PERON, MARGHERITA;SCHIAVONE, MARCO;VETTORI, ANDREA;ARGENTON, FRANCESCO
2015

Abstract

Introduction: POLG-related disorders are a group of multi-organ pathologies characterized by the dysfunction of the mitochondrial DNA polymerase gamma, encoded by the nuclear gene POLG and essential for mtDNA replication, repair and stability. As with all mitochondrial disorders, there is so far no cure for POLG-related diseases. Moreover, the paucity of animal models for these pathologies is hindering the set up of a pharmacological screen. Methods: The recent innovations in the field of transient gene perturbation and stable genome editing, based on morpholino (MO) antisense and CRISPR/Cas9 technologies, respectively, point to the small teleost zebrafish (Danio rerio) as an excellent candidate organism where to rapidly perform POLG knock-down, as well as targeted mutagenesis, followed by phenotype rescue via large-scale drug screen. Preliminary results: We have performed MO-based transient knock-down of the zebrafish polg gene, validating the splice blocking MO activity and obtaining a phenotype characterized by homogeneous developmental delay without obvious malformations, except for the observation of enlarged cardiac chambers, associated with increased heart rate. Of note, dilated cardiomyopathy is included among POLG-associated signs. Moreover, taking advantage of signaling pathway reporter lines, available in our laboratory, we have found that Hypoxia and CREB pathways are up-regulated in zebrafish polg morphants, while Wnt signaling appears unmodified. Discussion and conclusion: Our preliminary results on polg transient inactivation support the zebrafish as a suitable model to perform Crispr/Cas9- mediated mutagenesis of DNA polymerase gamma. The evidence of Hypoxia reporter activation under polg knock-down conditions suggests the existence of cross-talk mechanisms sensing mitochondrial dysfunction and triggering Hypoxia signaling even in a normoxic environment. Moreover, results from signaling pathway analysis in polg morphant embryos point to Hypoxia and CREB as candidate pathways for hypothesis-driven screening of molecules with therapeutic effect on POLG-related phenotypes. This work is supported by AFM-Telethon (18572 - POLYGON), EU ZF-HEALTH, Italian Ministry of Health (RF-2010-2309484 - TRAMBIGEN) and UniPD (CPDA128151 - OPTOZEN).
2015
9th European Zebrafish Meeting
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11577/3194973
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