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Measuring Single-Cell Mitochondrial DNA Copy Number and Heteroplasmy Using Digital Droplet Polymerase Chain Reaction
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Decrease of MtDNA copy number affects mitochondrial function and involves in the pathological consequences of

Zhaojing Zhang1, Dongzhi Yang2, Baixue Zhou1

  • 1Department of Medical Genetics & Cell Biology, School of Basic Medical Sciences, Zhengzhou University, Zhengzhou, China.

Journal of Cellular and Molecular Medicine
|July 6, 2022
PubMed
Summary
This summary is machine-generated.

Mitochondrial DNA (mtDNA) copy number reduction, linked to D-loop mutations and TFAM gene dysfunction, impairs nerve cell mitochondrial function and metabolism, contributing to ischemic stroke (IS) pathology.

Keywords:
TFAMischaemic strokemitochondrial functionmtDNA

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Area of Science:

  • Mitochondrial biology
  • Neuroscience
  • Genetics

Background:

  • Mitochondrial DNA (mtDNA) copy number influences mitochondrial function and disease development.
  • The mechanisms underlying mtDNA copy number variation and its role in ischemic stroke (IS) are poorly understood.
  • Investigating mtDNA copy number variation is crucial for understanding IS pathogenesis.

Purpose of the Study:

  • To investigate the relationship between mtDNA copy number and ischemic stroke (IS).
  • To explore the impact of D-loop variations and TFAM gene on mtDNA copy number in IS.
  • To elucidate the effect of mtDNA copy number variation on mitochondrial function in nerve cells.

Main Methods:

  • Quantitative real-time PCR (qRT-PCR) to measure mtDNA copy number in 101 IS patients and 101 controls.
  • Construction of a TFAM gene knockdown-overexpression (KD-OE) PC12 cell model.
  • Transfection of HEK 293T cells with TFAM gene knockdown (Sh-TFAM) and over-expression (OE-TFAM) plasmids.

Main Results:

  • IS patients exhibited significantly lower mtDNA copy numbers compared to normal controls.
  • TFAM gene mRNA expression was significantly reduced in oxygen-glucose deprivation/reperfusion (OGD/R) treated cells.
  • TFAM knockdown decreased mtDNA copy number and ATP production, while TFAM overexpression increased them.

Conclusions:

  • Mitochondrial D-loop mutations and TFAM gene dysfunction contribute to decreased mtDNA copy number in IS.
  • Reduced mtDNA copy number negatively affects mitochondrial metabolism and nerve cell function.
  • These alterations play a role in the pathological mechanisms underlying ischemic stroke (IS).