Association between reduced hippocampal neuronal integrity and mtDNA D-loop methylation in a chronic unpredictable mild stress rat model

Introduction

Depression is associated with impaired neuronal integrity in stress-sensitive brain regions, particularly the dentate gyrus of hippocampus (DG). While mitochondrial dysfunction is a central component of depression biology, mitochondrial DNA (mtDNA) has long been considered to lack epigenetic regulation. However, the identification of methylation within the mtDNA D-loop, a critical regulatory region for mitochondrial replication/transcription, suggests that mitoepigenetic mechanisms may contribute to neuronal integrity and survival [1]. NeuN, a nuclear antigen expressed in mature neurons, is widely used to assess neuronal integrity, and reductions in NeuN have been consistently reported in both clinical depression and chronic stress models [2]. This study aimed to determine whether mtDNA D-loop methylation is altered in a rodent model of depression, its relationship with neuronal integrity, and whether antidepressant treatment modulates these associations.

Methods

Male Wistar Albino rats (6–12 weeks) were assigned to Control (n=9), Stress (n=7), and Treatment (n=12) groups. A 35-day Chronic Unpredictable Mild Stress (CUMS) protocol [3] was applied to the Stress and Treatment groups. From day 15 onward, animals received daily intraperitoneal injections of saline (Control and Stress) or fluoxetine (10 mg/kg; Treatment). Behavioral assessments included the Sucrose Preference Test, Open Field Test, and Forced Swim Test, assessing anhedonia-like behavior, locomotor/exploratory activity, and behavioral despair, respectively. Following sacrifice, brain tissues were collected for mtDNA isolation and immunofluorescence staining. MtDNA D-loop CpG1–CpG4 methylation levels were quantified by bisulfite pyrosequencing, and NeuN staining was used to assess NeuN-positive cell proportion and fluorescence intensity in the DG. Total D-loop methylation was calculated as the mean of CpG1–CpG4 methylation percentages. Statistical analyses were conducted in SPSS 28 using Kruskal–Wallis and Spearman correlation tests, with statistical significance set at p<0.05.

Results

Behavioral analyses confirmed the validity of the model. The Stress group showed significantly reduced sucrose preference compared to Controls (p=0.011), while the Treatment group did not differ significantly from either group. In the Open Field Test, locomotor activity and total distance traveled were significantly lower in the Stress group compared to Controls (p=0.003; p=0.033). In the Forced Swim Test, climbing time was increased in the Treatment group compared to both Control and Stress groups (p=0.021; p=0.028). At the molecular level, CpG1 methylation was significantly higher in the Stress group compared to Treatment (p=0.028; U=0.500, p=0.008), with significance maintained after Bonferroni correction. No significant differences were observed between Control and other groups. NeuN-positive cell proportion was reduced in the Stress group, compared to Controls (p=0.015), and NeuN fluorescence intensity was lower in Stress compared to Treatment (p=0.027). Across the full sample, total D-loop methylation was significantly and negatively correlated with NeuN fluorescence intensity (r=−0.712, p=0.014).

Conclusions

These findings demonstrate that mtDNA D-loop methylation is associated with neuronal integrity under chronic stress conditions. Antidepressant treatment was associated with partial restoration of neuronal integrity. The inverse relationship between D-loop methylation and NeuN-based neuronal integrity supports a potential role for mitoepigenetic regulation in stress-related neuronal alterations, warranting further validation in human studies. Future studies should evaluate mtDNA D-loop methylation alongside additional neuroplasticity, neurogenesis, and inflammatory markers.

References

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2. Boldrini, M., Santiago, A. N., Hen, R., Dwork, A. J., Rosoklija, G. B., Tamir, H., Arango, V., & John Mann, J. (2013). Hippocampal granule neuron number and dentate gyrus volume in antidepressanttreated and untreated major depression. Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology, 38(6), 1068–1077. https://doi.org/10.1038/npp.2013.5
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