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.
Highlights
Energetic tissues such as heart, skeletal muscle, and brain are dependent on efficient energy metabolism for proper function
We examined the metabolic consequences of reduced Mitochondrial DNA (mtDNA) levels in rat striatal neuron-enriched co-cultures (NECos), striatal neurons, and astrocytes using ethidium bromide (EtBr) exposure
Since we have previously reported mtDNA depletion in the putamen of Parkinson’s Disease (PD) patients with L-DOPA Induced Dyskinesia (LID) [43], we sought to determine if this was associated with downregulation of mtCK gene expression, in keeping with our observations in EtBr-treated primary neuronal cultures
Summary
Energetic tissues such as heart, skeletal muscle, and brain are dependent on efficient energy metabolism for proper function. MtDNA depletion decreases neuronal mitochondrial creatine kinase. Vanderbilt High-throughput Screening Core Facility, an institutionally supported core, and was funded by NIH Shared Instrumentation Grant 1S10OD018015. The authors thank Dehui Mi for instrument training and support. Automated imaging using the ImageXpress Micro XL was performed in the Vanderbilt High-throughput Screening Core Facility. The authors thank Joshua Bauer for instrument training and support. The authors thank the Harvard Brain Tissue Resource Center, funded through NIH-NeuroBiobank HHSN271-2013-00030C (NIMH, NINDS, NICHD) and brain donors and their families for the tissue samples used in these studies
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