Increased mitochondrial DNA d-loop methylation in major depressive disorder

Introduction

Major Depressive Disorder (MDD) is a multifactorial psychiatric disorder in which mitochondrial dysfunction, particularly impaired bioenergetics and oxidative stress, plays a key role. Despite growing evidence, mitochondrial DNA (mtDNA) epigenetics remains poorly understood (1). The D-loop region, which regulates mtDNA replication and transcription, is a critical target for epigenetic investigation; however, prior studies have yielded inconsistent results (2-3). This study examined D-loop methylation using sequencing in neuron-derived extracellular vesicles (NDEVs), offering a proxy for central processes.

Methods

The study included 54 participants, comprising 30 patients with DSM-IV–confirmed MDD and 24 age- and sex-matched healthy controls, all aged 18–45 years and medication-free at baseline. Individuals with potential confounding conditions, including active infection, obesity, inflammatory diseases, or severe neurological disorders, were excluded. Depression severity was assessed using the Hamilton Depression Rating Scale (HDRS). Fasting venous blood samples were collected under standardized conditions. Plasma-derived extracellular vesicles were isolated using sequential centrifugation and ultracentrifugation, and NDEVs were enriched via NCAM/CD56 immunocapture. Vesicle characterization was performed using nanoparticle tracking analysis, transmission electron microscopy, and Western blotting for extracellular vesicles and neuronal markers. mtDNA was extracted from NDEVs, followed by bisulfite conversion and targeted sequencing of eight CpG sites within the D-loop region. Methylation levels were analyzed both individually and as a global D-loop methylation factor (MF) derived via principal component analysis. Patients were reassessed after an 8-week follow-up period to evaluate longitudinal changes. Statistical analyses included covariate-adjusted group comparisons, correlation, and multivariable regression analyses with correction for multiple testing.

Results

No significant differences were observed between groups in demographic variables. Patients with MDD exhibited significantly higher HDRS scores compared to healthy controls. The global D-loop methylation factor was significantly elevated in the MDD group relative to controls (p=0.020). At the CpG-specific level, significant hypermethylation was detected at CpG6 (p < 0.001) and CpG8 (p = 0.007), whereas no significant differences were observed at other CpG sites. Hypermethylation at CpG6 and CpG8 remained significant after BH correction across the eight CpG sites (FDR-adjusted p=0.008 and 0.028, respectively). Importantly, global D-loop methylation demonstrated a significant positive correlation with depression severity (Spearman’s ρ = 0.480, p < 0.001). In multivariable regression analysis, HDRS score emerged as the only independent predictor of D-loop methylation (β = 0.359, p = 0.010). Longitudinal analyses revealed no significant changes in D-loop methylation levels over the 8-week follow-up period.

Variable	HC (n=24)	MDD(n=31)	p
Sex (Female)	17(70.8%)	21(67.7%)	0.806
Smoking(Yes)	8(33.3%)	19(61.3%)	0.040
Exercise (Yes)	11(45.8%)	20(64.5%)	0.050
Alcohol (Yes)	16(66.7%)	19(61.3%)	0.425
Marital (Married)	5(20.8%)	12(38.7%)	0.209
Working Status(Working)	13(54.2%)	16(51.6%)	0.346

Scale		HC (M±SD)	MDD (M±SD)	p
HDRS		0.65 ± 1.43	23.26 ± 7.66	<0.001
Perceived Stress Scale		21.55 ± 7.51	32.50 ± 6.40	<0.001
Health Promoting Lifestyle Profile		134.48 ± 23.62	110.26 ± 20.85	0.002

Conclusions

These findings indicate that mtDNA D-loop methylation in NDEVs is associated with depression severity and may serve as biomarker of mitochondrial dysfunction in MDD. This approach may also provide a minimally invasive strategy to capture central molecular alterations and support future biomarker-driven stratification and treatment monitoring in depression.

References

1. D. Ceylan Arat-Çelik HE, Aksahin IC (2024). Integrating mitoepigenetics into research in mood disorders: a state-of-the-art review. Front. Physiol, Sec. Mitochondrial Research Volume 15 – 202
2. D. Ceylan, Karaçiçek B, Tufekci KU, Akşahin IC, Genç S. Mitochondrial DNA Oxidation, Methylation, and Copy Number Alterations in Major and Bipolar Depression. Front. Psychiatry Sec. Molecular Psychiatry. Volume 14 - 2023
3. Chung, J. K., Lee, S. Y., Park, M., Joo, E. J., Kim, S. A. 2019a. "Investigation of mitochondrial DNA copy number in patients with major depressive disorder". Psychiatry Research, 282.