New blood test pinpoints Parkinson's disease through mitochondrial DNA damage

In a recent study published in the journal Science Translational Medicine, researchers investigate the potential of mitochondrial deoxyribonucleic acid (mtDNA) injury as a serological marker for Parkinson’s disease (PD) using the novel Mito DNA_DX test.

Study: A blood-based marker of mitochondrial DNA damage in Parkinson’s disease. Image Credit: Kateryna Kon / Shutterstock.com

What causes PD?

PD is a neurodegenerative movement disorder with progressive loss of dopaminergic neurons that causes tremors, rigidity, bradykinesia, and postural instability. High-throughput markers are needed to stratify PD patients and ensure the success of disease-modifying therapies.

Mitochondrial dysfunction plays a significant role in PD pathogenesis, with mtDNA damage observed in PD neuronal cultures and animal models. The development of blood-based molecular markers could transform clinical trials and enhance the success of disease-modifying therapies.

About the study

In the present study, researchers investigate whether the polymerase chain reaction (PCR)-based DNA_DX assay could enable accurate quantification of mtDNA damage in real-time.

The DNA_DX assay, which utilizes the deoxyribonucleic acid polymerase enzyme and fluorescent dyes, was used to quantify mtDNA damage in patients with idiopathic PD. The assay involved amplification of PCR fragments targeting the mitochondrial genome, which increases mtDNA injury as compared to controls.

The count of mtDNA lesions depended on the hydrogen peroxide (H₂O₂) concentration. Agarose gel electrophoresis was performed to verify the appropriate size of mitochondrial amplicons.

A semi-automated DNA extraction workflow was used to isolate six specimens within 12 minutes to optimize analytical variables for developing biomarkers and enhancing throughput. The DNA_DX assay results were also compared to those obtained from traditional assays used to quantify DNA injury using enriched mtDNA samples retrieved from H₂O₂-treated human embryonic kidney 293 (HEK293) cells.

The effects of leucine-rich repeat kinase 2 (LRRK2) inhibitors on mtDNA injury were also assessed in cells isolated from the blood of idiopathic PD patients. To address the potential confounding role of PD-related drugs in studies of mtDNA damage, blood and clinical data were gathered from an independent cohort of people with PD during a washout period.

The team investigated whether increases in peripheral mtDNA injury were observed among LRRK2-G2019S mutational carriers with or without a PD diagnosis and compared them with an independent group of idiopathic PD patients. To examine whether mtDNA damage could also be used as a marker for other neurodegenerative diseases, samples obtained from patients with Alzheimer’s disease (AD) were also studied, in addition to age-matched and healthy control individuals recruited from the Memory Disorders Clinic at Duke University.

Study findings

Increased mtDNA damage was observed in PMBCs obtained for idiopathic PD-type patients and those comprising the PD-related LRRK2 amino acid substitution as compared with similarly aged controls. This damage could occur regardless of a PD diagnosis.

Compared to controls, LRRK2 G2019S mutation knock-in murine animals exhibited more mtDNA damage. However, LRRK2 knockout murine animals exhibited fewer mtDNA lesions within the ventral area of the midbrain.

In the PD murine midbrain neuronal model and idiopathic PD patient-derived cells, a small-molecule-type LRRK2 inhibitor reduced mtDNA damage. The mtDNA damage observed in cells derived from individuals with idiopathic PD was mitigated with LRRK2 kinase inhibition.

MLi-2, a high dosage of the LRRK2 kinase inhibitor, repaired mitochondrial damage to the control baseline in idiopathic PD patient-derived LCLs within 24 hours, with no change in mtDNA copy number.

The pathogenic kinase-activating G2019S amino acid substitution impaired basal mitophagy, whereas LRRK2 deficiency enhanced basal mitophagy.

PD patients exhibited elevated amounts of mtDNA damage with or without washout. Increased mtDNA damage was observed among individuals carrying the LRRK2 mutation.

Longitudinal mtDNA lesion frequency was stable in healthy controls, thus indicating that loss of mtDNA maintenance is associated with the disease process rather than due to preanalytical factors associated with sample collection, DNA extraction, or the DNA_DX assay.

Conclusions

LRRK2 contributes to mitochondrial gene homeostasis; therefore, LRRK2 mutation carriers have higher levels of mtDNA damage. In healthy controls, the incidence of mitochondrial DNA lesions remained steady throughout the period, whereas blood-derived cells from individuals with idiopathic PD exhibited increased mtDNA damage.

The DNA_DX assay for assessing mtDNA damage may be a valuable technique for diagnosing PD and measuring the pharmacodynamic response to LRRK2 kinase inhibitors. Moreover, LRRK2 kinase inhibition reduced mtDNA damage in cells obtained from patients with idiopathic PD.