Light-strand bias and enriched zones of embedded ribonucleotides are associated with DNA replication and transcription in the human-mitochondrial genome.

Abundant ribonucleoside-triphosphate (rNTP) incorporation into DNA by DNA polymerases in the form of ribonucleoside monophosphates (rNMPs) is a widespread phenomenon in nature, resulting in DNA-structural change and genome instability. The rNMP distribution, characteristics, hotspots and association with DNA metabolic processes in human mitochondrial DNA (hmtDNA) remain mostly unknown. Here, we utilize the ribose-seq technique to capture embedded rNMPs in hmtDNA of six different cell types. In most cell types, the rNMPs are preferentially embedded on the light strand of hmtDNA with a strong bias towards rCMPs; while in the liver-tissue cells, the rNMPs are predominately found on the heavy strand. We uncover common rNMP hotspots and conserved rNMP-enriched zones across the entire hmtDNA, including in the control region, which links the rNMP presence to the frequent hmtDNA replication-failure events. We show a strong correlation between coding-sequence size and rNMP-embedment frequency per nucleotide on the non-template, light strand in all cell types, supporting the presence of transient RNA-DNA hybrids preceding light-strand replication. Moreover, we detect rNMP-embedment patterns that are only partly conserved across the different cell types and are distinct from those found in yeast mtDNA. The study opens new research directions to understand the biology of hmtDNA and genomic rNMPs.

Is Drp1 a link between mitochondrial dysfunction and inflammation in Alzheimer's disease?

Recent advances highlight that inflammation is critical to Alzheimer Disease (AD) pathogenesis. Indeed, several diseases characterized by inflammation are considered risk factors for AD, such as type 2 diabetes, obesity, hypertension, and traumatic brain injury. Moreover, allelic variations in genes involved in the inflammatory cascade are risk factors for AD. AD is also characterized by mitochondrial dysfunction, which affects the energy homeostasis of the brain. The role of mitochondrial dysfunction has been characterized mostly in neuronal cells. However, recent data are demonstrating that mitochondrial dysfunction occurs also in inflammatory cells, promoting inflammation and the secretion of pro-inflammatory cytokines, which in turn induce neurodegeneration. In this review, we summarize the recent finding supporting the hypothesis of the inflammatory-amyloid cascade in AD. Moreover, we describe the recent data that demonstrate the link between altered mitochondrial dysfunction and the inflammatory cascade. We focus in summarizing the role of Drp1, which is involved in mitochondrial fission, showing that altered Drp1 activation affects the mitochondrial homeostasis and leads to the activation of the NLRP3 inflammasome, promoting the inflammatory cascade, which in turn aggravates Amyloid beta (Ab) deposition and tau-induced neurodegeneration, showing the relevance of this pro-inflammatory pathway as an early event in AD.

Mitochondrial DNA Repair in Neurodegenerative Diseases and Ageing.

Mitochondria are the only organelles, along with the nucleus, that have their own DNA. Mitochondrial DNA (mtDNA) is a double-stranded circular molecule of ~16.5 kbp that can exist in multiple copies within the organelle. Both strands are translated and encode for 22 tRNAs, 2 rRNAs, and 13 proteins. mtDNA molecules are anchored to the inner mitochondrial membrane and, in association with proteins, form a structure called nucleoid, which exerts a structural and protective function. Indeed, mitochondria have evolved mechanisms necessary to protect their DNA from chemical and physical lesions such as DNA repair pathways similar to those present in the nucleus. However, there are mitochondria-specific mechanisms such as rapid mtDNA turnover, fission, fusion, and mitophagy. Nevertheless, mtDNA mutations may be abundant in somatic tissue due mainly to the proximity of the mtDNA to the oxidative phosphorylation (OXPHOS) system and, consequently, to the reactive oxygen species (ROS) formed during ATP production. In this review, we summarise the most common types of mtDNA lesions and mitochondria repair mechanisms. The second part of the review focuses on the physiological role of mtDNA damage in ageing and the effect of mtDNA mutations in neurodegenerative disorders such as Alzheimer's and Parkinson's disease. Considering the central role of mitochondria in maintaining cellular homeostasis, the analysis of mitochondrial function is a central point for developing personalised medicine.

DNA Repair Protein APE1 Degrades Dysfunctional Abasic mRNA in Mitochondria Affecting Oxidative Phosphorylation.

APE1 is a multifunctional protein which plays a central role in the maintenance of nuclear and mitochondrial genomes repairing DNA lesions caused by oxidative and alkylating agents. In addition, it works as a redox signaling protein regulating gene expression by interacting with many transcriptional factors. Apart from these canonical activities, recent studies have shown that APE1 is also enzymatically active on RNA molecules. The present study unveils for the first time a new role of the mitochondrial form of APE1 protein in the metabolism of RNA in mitochondria. Our data demonstrate that APE1 is associated with mitochondrial messenger RNA and exerts endoribonuclease activity on abasic sites. Loss of APE1 results in the accumulation of damaged mitochondrial mRNA species, determining impairment in protein translation and reduced expression of mitochondrial-encoded proteins, finally leading to less efficient mitochondrial respiration. Altogether, our data demonstrate that APE1 plays an active role in the degradation of the mitochondrial mRNA and has a profound impact on mitochondrial well-being.

Mitochondrial Oxidative Stress Induces Rapid Intermembrane Space/Matrix Translocation of Apurinic/Apyrimidinic Endonuclease 1 Protein through TIM23 Complex.

Mitochondria are essential cellular organelles that import the majority of proteins to sustain their function in cellular metabolism and homeostasis. Due to their role in oxidative phosphorylation, mitochondria are constantly affected by oxidative stress. Stability of mitochondrial DNA (mtDNA) is essential for mitochondrial physiology and cellular well-being and for this reason mtDNA lesions have to be rapidly recognized and repaired. Base excision repair (BER) is the main pathway responsible for repairing non-helix distorting base lesions both into the nucleus and in mitochondria. Apurinic/Apyrimidinic Endonuclease 1 (APE1) is a key component of BER pathway and the only protein that can recognize and process an abasic (AP) site. Comprehensions of the mechanisms regulating APE1 intracellular trafficking are still fragmentary. In this study we focused our attention on the mitochondrial form of APE1 protein and how oxidative stress induces its translocation to maintain mtDNA integrity. Our data proved that: (i) the rise of mitochondrial ROS determines a very rapid translocation of APE1 from the intermembrane space (IMS) into the matrix; and (ii) TIM23/PAM machinery complex is responsible for the matrix translocation of APE1. Moreover, our data support the hypothesis that the IMS, where the majority of APE1 resides, could represent a sort of storage site for the protein.

Mitochondrial apurinic/apyrimidinic endonuclease 1 enhances mtDNA repair contributing to cell proliferation and mitochondrial integrity in early stages of hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is the leading cause of primary liver cancers. Surveillance of individuals at specific risk of developing HCC, early diagnostic markers, and new therapeutic approaches are essential to obtain a reduction in disease-related mortality. Apurinic/apyrimidinic endonuclease 1 (APE1) expression levels and its cytoplasmic localization have been reported to correlate with a lower degree of differentiation and shorter survival rate. The aim of this study is to fully investigate, for the first time, the role of the mitochondrial form of APE1 in HCC. METHODS: As a study model, we analyzed samples from a cohort of patients diagnosed with HCC who underwent surgical resection. Mitochondrial APE1 content, expression levels of the mitochondrial import protein Mia40, and mtDNA damage of tumor tissue and distal non-tumor liver of each patient were analyzed. In parallel, we generated a stable HeLa clone for inducible silencing of endogenous APE1 and re-expression of the recombinant shRNA resistant mitochondrially targeted APE1 form (MTS-APE1). We evaluated mtDNA damage, cell growth, and mitochondrial respiration. RESULTS: APE1's cytoplasmic positivity in Grades 1 and 2 HCC patients showed a significantly higher expression of mitochondrial APE1, which accounted for lower levels of mtDNA damage observed in the tumor tissue with respect to the distal area. In the contrast, the cytoplasmic positivity in Grade 3 was not associated with APE1's mitochondrial accumulation even when accounting for the higher number of mtDNA lesions measured. Loss of APE1 expression negatively affected mitochondrial respiration, cell viability, and proliferation as well as levels of mtDNA damage. Remarkably, the phenotype was efficiently rescued in MTS-APE1 clone, where APE1 is present only within the mitochondrial matrix. CONCLUSIONS: Our study confirms the prominent role of the mitochondrial form of APE1 in the early stages of HCC development and the relevance of the non-nuclear fraction of APE1 in the disease progression. We have also confirmed overexpression of Mia40 and the role of the MIA pathway in the APE1 import process. Based on our data, inhibition of the APE1 transport by blocking the MIA pathway could represent a new therapeutic approach for reducing mitochondrial metabolism by preventing the efficient repair of mtDNA.