Mitochondrial DNA depletion by ethidium bromide decreases neuronal mitochondrial creatine kinase: Implications for striatal energy metabolism

Abstract

Mitochondrial DNA (mtDNA), the discrete genome which encodes subunits of the mitochondrial respiratory chain, is present at highly variable copy numbers across cell types. Though severe mtDNA depletion dramatically reduces mitochondrial function, the impact of tissue-specific mtDNA reduction remains debated. Previously, our lab identified reduced mtDNA quantity in the putamen of Parkinson’s Disease (PD) patients who had developed L-DOPA Induced Dyskinesia (LID), compared to PD patients who had not developed LID and healthy subjects. Here, we present the consequences of mtDNA depletion by ethidium bromide (EtBr) treatment on the bioenergetic function of primary cultured neurons, astrocytes and neuron-enriched cocultures from rat striatum. We report that EtBr inhibition of mtDNA replication and transcription consistently reduces mitochondrial oxygen consumption, and that neurons are significantly more sensitive to EtBr than astrocytes. EtBr also increases glycolytic activity in astrocytes, whereas in neurons it reduces the expression of mitochondrial creatine kinase mRNA and levels of phosphocreatine. Further, we show that mitochondrial creatine kinase mRNA is similarly downregulated in dyskinetic PD patients, compared to both non-dyskinetic PD patients and healthy subjects. Our data support a hypothesis that reduced striatal mtDNA contributes to energetic dysregulation in the dyskinetic striatum by destabilizing the energy buffering system of the phosphocreatine/creatine shuttle.

Prohibitin 1 regulates mtDNA release and downstream inflammatory responses

“…EtBr is widely used to generate ρ0 cell lines with low mtDNA levels for investigation of the roles of mtDNA in inflammasomes and cGAS‐STING axis (Yu et al , 2020; Wang et al , 2021). EtBr treatment also has profound effects on cellular homeostasis, including increased intracellular ROS level, glycolysis, and reduced autophagic level (Nacarelli et al , 2014; Warren et al , 2017). Of note, increased ROS level and glycolysis as well as reduced autophagic level promote the activation of NLRP3‐inflammasome (Zhou et al , 2011; Xie et al , 2016; Zhong et al , 2016), whereas lower mtDNA level blocks NLRP3‐inflammasomes activation (Zhong et al , 2016).…”

Insight into the siRNA transmembrane delivery—From cholesterol conjugating to tagging

“…It was reported that ethidium bromide would not only act as a mutagen in microorganism, but also might induce mutations in higher plants and mammals (Dujardin & Dujon, ; Hayakawa et al, ; Levy & Ashiri, ). Primary cultured neurons treated by ethidium bromide (EtBr) present the consequences of mitochondrial DNA (mtDNA) depletion, which dramatically reduces mitochondrial function and can be used as a method of mouse mtDNA mutagenesis for modeling human pathological conditions (Fayzulin et al, ; Warren, Aicher, Fessel, & Konradi, ; Yu et al, ). Our preliminary experiment indicated that our siRNA tagging technology does not show any cytotoxicity on PC‐3 cell, even at very high tagging concentration (10 μM of the tags).…”

Increased mitochondrial DNA copy number protects hair cells and HEI‑OC1 cells against drug‑induced apoptosis

“…mtDNA copy number varies by cell (10) and by developmental stage (11,12), and depends on the dynamic equilibrium between mtDNA synthesis and degradation. As reported, while a decreased mtDNA copy number or mtDNA depletion may break cellular mitochondrial function and result in many diseases, an increased mtDNA copy number has a protective effect (6,13–15). Given the highly active and vulnerable nature of hair cells, increasing their mtDNA copy number is a potential way to enhance their survival.…”