Transgenic mice Cre-dependently expressing mutant polymerase-gamma: novel test-system for pharmacological study of mitoprotective drugs

Introduction: PolG-alpha is a nuclear-encoded enzyme which provides replication and repair of mitochondrial DNA. D257A mutation of PolG-alpha leads to change in the N-terminal ”proofreading” domain, which deprives the enzyme of 3′-5′ exonuclease activity, resulting in accumulation of mutations in the mitochondrial genome. Materials and methods: Murine zygotes were microinjected with transgene construction carrying mutant murine Polg coding sequence and GFP coding sequence by a loxP-flanked STOP-cassette. Two Cre-activator strains, CMV-Cre (systemic activation) and Tie2-Cre (endothelial activation), were used for activation of the transgene. To confirm the insertion and Cre-dependent activation of the transgene, genotyping and qPCR copy number measurement of mutant Polg were performed, and GFP fluorescence was assessed. Results: Two primary transgenic animals were used as the founders for two lines with copy numbers of transgene ~7 and ~5. After systemic activation, the number of the transgene copies decreases to ~1.0 while endothelial specific activation does not affect the number of transgene copies in tail tissue. Discussion: A murine model with spatial control of mutant Polg expression has been developed. To our knowledge, this is the first transgenic model of tissue-specific mitochondrial dysfunction. Conclusion: Transgenic mice Cre-dependent expressing mutant polymerase-gamma are a novel test-system for studying mitochondrial biology and efficacy of mitoprotective drugs.

Physical and Functional Interaction of Mitochondrial Single-Stranded DNA-Binding Protein and the Catalytic Subunit of DNA Polymerase Gamma

The maintenance of the mitochondrial genome depends on a suite of nucleus-encoded proteins, among which the catalytic subunit of the mitochondrial replicative DNA polymerase, Pol γα, plays a pivotal role. Mutations in the Pol γα-encoding gene, POLG, are a major cause of human mitochondrial disorders. Here we present a study of direct and functional interactions of Pol γα with the mitochondrial single-stranded DNA-binding protein (mtSSB). mtSSB coordinates the activity of the enzymes at the DNA replication fork. However, the mechanism of this functional relationship is elusive, and no direct interactions between the replicative factors have been identified to date. This contrasts strikingly with the extensive interactomes of SSB proteins identified in other homologous replication systems. Here we show for the first time that mtSSB binds Pol γα directly, in a DNA-independent manner. This interaction is strengthened in the absence of the loop 2.3 structure in mtSSB, and is abolished upon preincubation with Pol γβ. Together, our findings suggest that the interaction between mtSSB and polymerase gamma holoenzyme (Pol γ) involves a balance between attractive and repulsive affinities, which have distinct effects on DNA synthesis and exonucleolysis.

Polymerase Gamma Mitochondrial DNA Depletion Syndrome Initially Presenting as Disproportionate Respiratory Distress in a Moderately Premature Neonate: A Case Report

A 32-week premature infant presented with respiratory failure, later progressing to pulmonary hypertension (PH), liver failure, lactic acidosis, and encephalopathy. Using exome sequencing, this patient was diagnosed with a rare Polymerase Gamma (POLG)-related mitochondrial DNA (mtDNA) depletion syndrome. This case demonstrates that expanding the differential to uncommon diagnoses is important for complex infants, even in premature neonates whose condition may be explained partially by their gestational age (GA). It also shows that patients with complex neonatal diseases with significant family history may benefit from exome sequencing for diagnosis.

PAPOLG is differentially expressed in brain metastatic human breast cancer.

Metastasis to the brain is a clinical problem in patients with breast cancer (1-3). We mined published microarray data (4, 5) to compare primary and metastatic tumor transcriptomes for the discovery of genes associated with brain metastasis in humans with metastatic breast cancer. We found that poly(A) polymerase gamma, encoded by PAPOLG, was among the genes whose expression was most different in the brain metastases of patients with metastatic breast cancer as compared to primary tumors of the breast. PAPOLG mRNA was present at decreased quantities in brain metastatic tissues as compared to primary tumors of the breast. Importantly, expression of PAPOLG in primary tumors was significantly correlated with patient recurrence-free survival. Modulation of PAPOLG expression may be relevant to the biology by which tumor cells metastasize from the breast to the brain in humans with metastatic breast cancer.

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

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.