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. Author manuscript; available in PMC: 2025 Oct 1.
Published in final edited form as: J Affect Disord. 2024 Jun 28;362:217–224. doi: 10.1016/j.jad.2024.06.092

Exosome-associated mitochondrial DNA in Late-Life Depression: implications for cognitive decline in older adults

Ana Paula Mendes-Silva a,b,c,*, Yuliya S Nikolova a,d, Tarek K Rajji d,e, James L Kennedy a,b,d, Breno S Diniz f, Vanessa F Gonçalves a,b,d,1, Erica L Vieira a,d,e,1
PMCID: PMC11316645  NIHMSID: NIHMS2007494  PMID: 38945405

Abstract

Background:

Disrupted cellular communication, inflammatory responses and mitochondrial dysfunction are consistently observed in late-life depression (LLD). Exosomes (EXs) mediate cellular communication by transporting molecules, including mitochondrial DNA (EX-mtDNA), playing critical role in immunoregulation alongside tumor necrosis factor (TNF). Changes in EX-mtDNA are indicators of impaired mitochondrial function and might increase vulnerability to adverse health outcomes. Our study examined EX-mtDNA levels and integrity, exploring their associations with levels of TNF receptors I and II (TNFRI and TNFRII), and clinical outcomes in LLD.

Methods:

Ninety older adults (50 LLD and 40 controls (HC)) participated in the study. Blood was collected and exosomes were isolated using size-exclusion chromatography. DNA was extracted and EX-mtDNA levels and deletion were assessed using qPCR. Plasma TNFRI and TNFRII levels were quantified by multiplex immunoassay. Correlation analysis explored relationships between EX-mtDNA, clinical outcomes, and inflammatory markers.

Results:

Although no differences were observed in EX-mtDNA levels between groups, elevated levels correlated with poorer cognitive performance (r=−.328, p=0.002) and increased TNFRII levels (r=.367, p=0.004). LLD exhibited higher deletion rates (F(83,1)=4.402, p=0.039), with a trend remaining after adjusting for covariates (p=0.084). Deletion correlated with poorer cognitive performance (r=−.335, p= 0.002). No other associations were found.

Limitation:

Cross-sectional study with a small number of participants from a specialized geriatric psychiatry treatment center.

Conclusion:

Our findings suggest that EX-mtDNA holds promise as an indicator of cognitive outcomes in LLD. Additional research is needed to further comprehend the role of EX-mtDNA levels/integrity in LLD, paving the way for its clinical application in the future.

Keywords: exosomes, mitochondrial DNA, late-life depression, cognitive function, inflammation

Introduction

Late-life depression (LLD) is a serious condition that is associated with medical and psychiatric comorbidity, impaired functioning, intense use of healthcare resources, and increased mortality (Alexopoulos, 2005; Diniz et al., 2013; Herrman et al., 2022). Despite the high prevalence (Beekman et al., 1999; Zenebe et al., 2021), the underlying causes for the pathophysiology of LLD remain unknown, and various factors can influence its presentation (Vaughan et al., 2015; Xue et al., 2024). Consistent findings in LLD include disturbances in cellular communication (Brites and Fernandes, 2015), pro-inflammatory cascades (Alexopoulos and Morimoto, 2011; Charlton et al., 2018; Saraykar et al., 2018), and malfunctioning of mitochondria (Głombik et al., 2021; Lorenzo et al., 2023; Mastrobattista et al., 2023). Increasing attention is being directed toward understanding the complex interplay among these biological features, which may be influenced by the content and function of exosomes (Kalluri and LeBleu, 2020; Ridder et al., 2014).

Exosomes (EXs) are the best characterized subtype of extracellular vesicles, ranging in size from 40 to 160 nm in diameter (Colombo et al., 2014). EXs are formed in the endosomal system within the cytoplasm and released into the extracellular space (Kalluri and LeBleu, 2020). They are released from the cell and target other cells in neighboring or distant tissues, carry proteins, lipids and nucleic acids (e.g. mitochondrial DNA – mtDNA), serving as important intercellular communication factors (Gurung et al., 2021; Miliotis et al., 2019; van Niel et al., 2018). Studies have shown that the quantity and content of exosomes depend on environmental conditions, cellular stress, and parent cell maturity (De Maio, 2011; Miliotis et al., 2019). Exosome cargo molecules modulate immune responses which might also involve the presentation of immunoregulatory molecules such as tumor necrosis factor (TNF). The TNF signaling pathways, involving the two independent receptors (TNFRI and TNFRII), are thought to contribute to inflammation and inhibit mitophagy, ultimately leading to altered mitochondrial function, metabolic abnormalities, and elevated levels of mtDNA in the cytosol (Lin et al., 2022; Willemsen et al., 2021).

The mitochondria play a crucial role in several critical biological processes, such as energy and reactive oxygen species (ROS) production, calcium regulation, inflammation, and apoptosis (Visentin et al., 2020). The imbalance between ROS production and the cellular antioxidant capacity can lead to oxidative damage to the mtDNA (Pizzino et al., 2017). The imbalance between ROS production and the cellular antioxidant capacity can lead to metabolic oxidative stress, genomic instability (e.g. mutations and deletions), less energy production and cellular injury (Guo et al., 2013; Kowaltowski et al., 2009; Pizzino et al., 2017; Voets et al., 2012). Deletions are notably prevalent in the major arc of mitochondrial DNA (mtDNA), encompassing genes such as MT-ND4, whereas deletions are rare in the minor arc, containing genes like MT-ND2 (Chen et al., 2011; Lott et al., 2013; Phillips et al., 2014). The mtDNA instability increases susceptibility to fragmentation, facilitating it is released from mitochondria, and in the cytosol can be expelled from cells as free molecules or encapsulated within exosomes (EX-mtDNA) (Vaidya et al., 2022). The cytosolic and EX-mtDNA can serve as a part of the damage-associated molecular pattern (DAMP), enhancing pro-inflammatory responses through the toll-like receptor (TLR) pathway or inflammasome activation (Park and Hayakawa, 2021; Todkar et al., 2021). Moreover, the EX-mtDNA is found to carry disease-specific mutations and deletions resulting from disease progression and response to treatment (Sansone et al., 2017). These findings suggest that mitochondrial components may be a significant functional cargo within EXs. Consequently, it is hypothesized that mitochondria and mtDNA may be packaged and transferred within EX to support inflammatory responses.

Although EXs cargo have been implicated previously in cancer (Guescini et al., 2010; Sansone et al., 2017) and neurodegenerative diseases (Picca et al., 2020; Tamboli et al., 2010), their involvement in mental disorders such as LLD remains understudied. There is also a limited understanding of whether qualitative and quantitative changes in mtDNA (amount of mtDNA, and the rate of mutations and deletions) within EXs vary depending on the disease, its specific stage, progression, and response to therapy. Therefore, this study aimed to provide insight into the changes in specific features of EX-mtDNA, amount and integrity, in individuals with LLD compared to healthy controls (HC). Additionally, we examined whether EX-mtDNA levels and deletion rate were associated with plasma TNFRI and TNFRII levels, as well as with clinical outcomes, encompassing measures of depressive symptoms, health status, and cognitive performance.

Methods

Recruitment of Participants

Ninety participants (50 LLD and 40 healthy older adults (HC)) were recruited at the Centre for Addiction and Mental Health (CAMH), Toronto, Canada. Ethical approval was granted by the local research board and all individuals provided written informed consent for participation in this study.

Semi-structured diagnostic interviews were conducted with all participants using the Diagnostic and Statistical Manual of Mental Disorders, 5th edition (DSM-V)(2013). The Mini International Psychiatric interview (MINI) (Sheehan et al., 1998) was also administered to confirm the diagnosis of a major depressive episode or to exclude a lifetime history of psychiatric disorder. Inclusion criteria for the control group consisted of older adults ≥ 60 years, with no history of major depressive episodes, other psychiatric disorders, or substance abuse. Exclusion criteria for all participants included a prior diagnosis of dementia, major neurological disorders (e.g. bipolar disorder, post-traumatic disorder, schizophrenia), history of stroke, inflammatory disorders, use of immunomodulators, presence of current unstable medical conditions, such as recent cancer (except non-melanoma skin cancer), and history of alcohol or recreational drug abuse in the past six months. Subjects with comorbid anxiety disorders (e.g., phobias) were not excluded due to significant overlap with major depressive disorders. Participants with LLD were not under antidepressant treatment and immunomodulators for a minimum of three months prior to undergoing the research assessment and blood collection.

All participants underwent assessment of depression severity with Montgomery-Åsberg Depression Rating Scale (MADRS) (Montgomery and Asberg, 1979). Moreover, the Montreal Cognitive Assessment (MoCA) (Nasreddine et al., 2005) was used to assess global cognitive performance by screening various cognitive domains, including attention, memory, language, visuospatial abilities, executive functions, and orientation. Out of the total participants, six individuals scored below 22 on the MoCA assessment, suggesting potential cognitive impairment. A sensitivity analysis removing these participants did not significantly change the study findings. The Cumulative Illness Rating Scale (CIRS-G) (Miller et al., 1992) was used to evaluate the overall health status of the participants. The CIRS-G considers a wide array of comorbidities commonly encountered in older adults, ranging from cardiac and respiratory disorders to neurological and psychiatric conditions.

Samples

Peripheral blood was drawn from all participants via venipuncture in sodium citrate tubes following an overnight fasting, typically between 9 to 11 am. Sample processing was performed within 3 hours of collection. In brief, sodium citrate tubes with blood were centrifuged at 2500g for 15 min at 18° C to separate platelet-poor plasma. Subsequently, a second centrifugation step at 13000g for 5 minutes at 18° C was conducted to obtain platelet-free plasma. After each centrifugation step, only 2/3 of the samples were collected. The aliquoted samples were then stored at −80°C until laboratory analysis.

Exosomes isolation and DNA extraction

The total exosomes were isolated using size exclusion chromatography, following the manufacturer’s instructions (Cell Guidance Systems, St. Louis, MO, USA). Briefly, 180 μl of the platelet-free plasma samples were thawed and centrifuged at 5000 × g for 10 minutes at room temperature. Subsequently, the sample were loaded into the size exclusion chromatography columns previously equilibrated with PBS. The initial flow-through was then discarded to remove microvesicles and proteins. Exosomes were eluted with 180 μL of PBS by centrifugation at 100 × g for 30 minutes at room temperature. The size of the exosomes was confirmed using nanoparticle tracking analysis conducted with the NanoSight NS300 instrument (Malvern Panalytical, Malvern, UK). The recovered eluate was then stored at −20°C until DNA extraction.

Prior to DNA extraction, the samples underwent DNase treatment (Lucigen, Cat: DB0715K) to degrade any DNA material present on the outside of the exosomes. Samples were treated with 6.5 μl of DNase Reaction Buffer, and 5 U of DNase per reaction at 37°C for 30 minutes. The reaction was then stopped by adding 6.5 μl DNase Stop Solution per reaction at 65°C for 10 minutes. Following this, each sample was adjusted to 200 μl with sterile PBS, and then manufacturer’s DNA extraction protocol for the QIAmp 96 DNA Blood kit (Qiagen, Valence, USA) was followed. The eluted DNA (~50 μl) was stored at −20°C before a qPCR assay.

Assessment of EX-mtDNA levels and deletion rate by qPCR

The real-time quantitative polymerase chain reaction (qPCR) method employed in this study was modified from a previously published method (Phillips et al., 2014). To evaluate the overall abundance of EX-mtDNA, we targeted the mitochondrial gene MT-ND2 (mitochondrial encoded NADH: Ubiquinone Oxidoreductase Core Subunit 2), located in the minor arc where large deletions are rare, if not absent. This selection enabled us to evaluate EX-mtDNA levels without the interference of deletions. On the other hand, to estimate the occurrence of deletions within the mitochondrial DNA, we targeted a second mitochondrial gene MT-ND4 (mitochondrial encoded NADH: Ubiquinone Oxidoreductase Core Subunit 4), situated in the major arc where approximately 84% of the large deletions are common.After confirming a positive correlation between their levels in this cohort, we further evaluate the relative integrity of the EX-mtDNA by dividing the copy number of MT-ND2 by MT-ND4 (Phillips et al., 2014). As the ratio between the minor and major arc should be consistent in the absence of deletions, utilizing the ratio allows us to estimate deletions within the major arc in patients with LLD compared to controls.

The primers employed were as follows: MT-ND2 – Forward 5’-CACACTCATCACAGCGCTAA-3’ and Reverse 5’-GGATTATGGATGCGGTTGCT-3’ (Life Technology, Paisley, UK); MT-ND4 - Forward 5’-ACCTTGGCTATCATCACCCGAT −3’ and Reverse 5’-AGTGCGATGAGTAGGGGAAGG-3’ (Life Technology, Paisley, UK).

The qPCR was conducted using the CFX96 Touch Real-Time PCR Detection System (Bio-rad, Hercules, California, USA). Each reaction, with a total volume of 20μL, comprised 50 ng of DNA template, 1μL of each primer (10μM), 10μL SYBR MIX (2x Sensifast, Bioline, London, UK), and nuclease-free water. For each primer pair, we performed an eight-point standard curve using the same pooled DNA sample (combined plasma DNA samples). All reactions were performed in triplicate. The PCR program consisted of an initial denaturation at 95°C for 2 min, followed by 45 cycles comprising 95°C in 5 sec (melting), annealing at 60–65°C for 10 sec, and extension at 72°C for 10sec (extension). The program concluded with a melting curve analysis, with fluorescence measured continuously from 60°C to 95°C.

The relative concentration of the EX-mtDNA was determined by comparing crossing-point values from the testing samples with the standard curve. The number of EX-mtDNA units per sample was calculated using the formula: DNA amount (g μl−1) divided by the size of the PCR-fragment (MT-ND2: 161 bp; MT-ND4–107 bp) and the molar mass per base pair (g mol−1), then multiplied by Avogadro’s constant. The EX-mtDNA levels and deletion rate were reported on a logarithmic scale of units per microliter (units/μL).

Plasma TNFRI and TNFRII levels

Blood was collected by venipuncture in EDTA tubes. Plasma was obtained from the blood by centrifugation at 3,000g for 10 min at 4 °C, aliquoted, and stored at − 80°C freezer until laboratory analyses. Plasma samples were analyzed using LUMINEX 100/200 (Luminex Corp., Austin, TX) and a customized multiplex immunoassay containing tumor necrosis factor receptor I and II (TNFRI and TNFRII, respectively) analytes from R&D/Biotechne (Minneapolis, MN). All laboratory analyses were carried out according to the manufacturer’s instructions in two different batches with LLD and HC samples randomly distributed between these batches.

Statistical analysis

The Shapiro-Wilk test was used to check the normality of the data. Categorical variables were compared using the chi-square test. The estimated deletion rate and levels of Ex-mtDNA, TNFRI and TNFRII were previously log-transformed for the subsequent analysis. We carried out Student’s t-test or analysis of variance (ANOVA) to test for differences between LLD and controls for continuous variables with non-normal or normal distribution, respectively. Analyses of covariates were performed using age, sex, tobacco (pack per year) and body mass index (BMI). Correlations analysis utilizing Spearman’s (rho) or Pearson (r) test was performed in R (v.4.2.3) to explore associations between EX-mtDNA levels and estimated deletion rate with demographics, clinical measures, and plasma TNFRI and TNFRII levels. All other statistical analyses were conducted using SPSS software (IBM SPSS Statistics 25). Bonferroni adjustment was applied to account for multiple correlated tests across 10 variables, setting the significance threshold at padjusted = 0.005. For the group comparisons regarding the integrity and levels of Ex-mtDNA, TNFRI, and TNFRII, a significance threshold of p ≤ 0.05 was applied for statistical significance.

Results

Demographic and clinical characteristics

Demographic and clinical information are summarized in Table 1. There was a significant difference in age (t(88,1)=2.142, p=0.036), BMI (t(83,1)=−2.121, p=0.037) and years of education (t(88,1)=2.093, p=0.039) between groups, while no significant difference was observed in sex or tobacco consumption (pack per year). The scores on the MADRS were significantly higher in the LLD group (t(88,1)=−15.70, p<0.001) when compared to HC. Moreover, there was a significant difference in burden status measured by CIRS-G score between groups (t(87,1)=−5.292, p<0.001). Furthermore, the analysis indicated that no statistically significant disparities in global cognitive performance, as evaluated using MOCA was observed when comparing the HC and LLD groups.

Table 1 –

Baseline demographic, clinical, EX-mtDNA levels and inflammatory markers characteristics.

Demographics HC (n=40) LLD (n=50) Statistic p-value
Age, mean ± SD 71.15 ± 7.31 68.04 ± 5.74 t(88,1)=2.142 0.036 *
Sex (Female %) 20 (50) 35 (70) χ2(88,1) =3.740 0.053
Years of education, mean ± SD 15.45 ± 1.77 14.52 ± 2.44 t(88,1)=2.020 0.046 *
Tobacco (pack per year), mean ± SD 5.19 ± 16.31 9.04 ± 16.42 t(88,1)=−1.107 0.27
BMI, mean ± SD 26.33 ± 3.59 28.51 ± 5.86 t(83,1)=−2.121 0.037 *
Clinical
MADRS 1.10 ± 1.50 17.82 ± 6.51 t(87,1)=−15.70 <0.001 **
CIRS-G 6.03 ± 3.96 10.86 ± 4.50 t(87,1)=−5.292 <0.001 **
MoCA 26.20 ± 2.10 25.46 ± 3.01 t(88,1)=1.3 20 0.19
EX-mtDNA status, mean ± SD
Levels (log scale) 3.06 ± 1.38 3.06 ± 0.96 F(88,1)=0.001 0.97
Estimated mtDNA deletion (log scale) 0.64 ± 0.72 1.06 ± 1.04 F(83,1)=4.402 0.039 *
Inflammatory markers, mean ± SD
TNFRI (log scale) 3.00 ± 0.15 3.01 ± 0.23 F(58,1)=.010 0.92
TNFRII (log scale) 3.27 ± 0.17 3.29 ± 0.21 F(58,1)=.120 0.73
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Statistics derived from Chi-square test of independence (χ2), independent samples t-test (t) and Analysis of variance (F).

HC: healthy control; LLD: Late-life Depression; BMI: body mass index; MADRS: The Montgomery-Asberg Depression Rating Scale; CIRS-G: Cumulative Illness Rating Scale-Geriatric; MoCA: Montreal Cognitive Assessment; EX-mtDNA: mtDNA encapsulated within extracellular vesicles; TNFRI: tumor necrosis factor receptor 1; TNFRII: tumor necrosis factor receptor 2.

*

p<0.05

**

p<0.01.

Evaluation of EX-mtDNA levels and deletion rate in individuals with and without LLD

We found no significant difference in the levels of EX-mtDNA when comparing the two groups before and after adjusting for age, sex, tobacco usage and BMI (F(88,1)=0.001, p=0.97; F(79,1)=0.691, p=0.41, respectively) (Figure 1a). No effect of sex or sex*diagnosis interaction was found for EX-mtDNA levels (F(84,1)=0.891, p=0.35; F(84,1)=0.466, p=0.50, respectively). Notably, elevated levels of EX-mtDNA were significantly associated with worse cognitive performance in the whole cohort (rho=−.328, p=0.002) (Figure 2), and this association remained significant after multiple corrections.

Figure 1 -. EX-mtDNA levels and estimated deletion rate in Late-Life Depression (LLD) and healthy control (HC) individuals.

Figure 1 -

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a) Analysis of variance showed that there was no significant difference in the levels of EX-mtDNA between LLD and HC groups, both before and after accounting for covariates. b) The individuals with LLD exhibited a significantly higher estimated deletion rate compared to HC before adjusting for covariates. The results were determined using univariate analysis of variance (two-tailed): *p<0.05, **p<0.01, ns indicates non-significant. The cross symbol indicates the mean value.

Figure 2 – Correlation matrix and scatter plots of associations between EX-mtDNA status (levels and deletion rate) and demographic, clinical outcomes and inflammatory markers.

Figure 2 –

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The results were determined using Pearson or Spearmans correlation test: *p<0.05, **p<0.01. The upper triangle shows correlation coefficients with significance levels, the lower triangle displays scatter plots with regression lines, and the diagonal presents density plots for each variable.

Abbreviations: MADRS: Montgomery-Åsberg Depression Rating Scale, a measure of depression severity. MoCA: Montreal Cognitive Assessment, a screening tool for cognitive impairment. CIRS-G: Cumulative Illness Rating Scale for Geriatrics, assessing comorbidity. TNFRII: Tumor Necrosis Factor Receptor II, a biomarker related to inflammation. TNFRI: Tumor Necrosis Factor Receptor I, another biomarker related to inflammation.

In a secondary analysis, we included age as a covariate to address potential confounding effects on the relationship between EX-mtDNA levels and global cognitive performance. Our findings revealed that the significant association between EX-mtDNA levels and MoCA scores (r=−.270, p=0.01) persisted. No other significant correlations between EX-mtDNA and demographic/clinical variables were found (Figure 2).

The estimation of deletion rate was conducted subsequent to the confirmation of a significant positive correlation between the levels of EX-mtDNA MT-ND2 and EX-mtDNA MT-ND4 (rho=.540, p<0.001), which remained significant after multiple corrections. We observed that the individuals with LLD exhibited greater estimated deletion rate when compared to HC (F(83,1)=4.402, p=0.039) (Figure 1b). After adjusting for covariates, we observed a trend of significant difference between groups (F(74,1)=3.060, p=0.084). There was no effect of sex or sex*diagnosis interaction was found for the estimated deletion rate (F(79,1)=0.333, p=0.57; F(79,1)=0.036, p=0.85, respectively). Correlation analysis conducted with the entire sample revealed a significant negative correlation between the estimated deletion rate and MoCA score (rho=−.335, p= 0.002) (Figure 2), which retained significance after multiple corrections. This underscores a potential impact of the deletion rate on cognitive performance. In a subsequent analysis, we incorporated age as a factor to address potential confounding effects on the relationship between deletion rate and global cognitive performance. Our results indicated that the correlation between the estimated deletion rate and MoCA scores (r=−.268, p=0.014) remained significant. There were no further significant correlations observed between the estimated deletion rate and demographic/clinical variables (Figure 2).

EX-mtDNA and plasma TNFRI and TNFRII levels

The levels of TNFRI and TNFRII did not display significant differences between the groups (F(58,1)=.010, p=0.92, F(58,1)=.120, p=0.73, respectively). A significant positive correlation was observed between the levels of EX-mtDNA and TNFRII (rho=.367, p=0.004; Figure 2), which remained significant after multiple corrections. However, no significant correlation was found with TNFRI (rho=.222, p=0.088; Figure 2). Furthermore, the estimated deletion rate did not show significant correlations with TNFRI (rho=.079, p=0.562) or TNFRII (rho=.073, p=0.595) levels (Figure 2).

Discussion

Our study examined the levels and integrity of EX-mtDNA in LLD and compared them to older healthy controls. We did not observe differences regarding the levels of EX-mtDNA when comparing older adults with depression with older healthy individuals. We further investigated the EX-mtDNA deletion rate and we found that individuals with LLD individuals exhibited higher rates compared to controls. Both the levels of EX-mtDNA and estimated deletion rate were correlated with poorer cognitive performance, but not with demographic characteristics of the sample like age, years of education, and number of medical comorbidities. Furthermore, we observed a significant correlation between EX-mtDNA and plasma TNFRII levels in our sample, while no correlation was observed with deletion rate.

Despite the growing evidence of alterations in extracellular mitochondrial molecule levels in depression, such as circulating cell-free mtDNA (ccf-mtDNA) (Ampo et al., 2022; Gonçalves et al., 2021; Lindqvist et al., 2018), there is a notable gap in studies addressing the potential role of mtDNA engulfed within exosomes. Different forms of extracellular mtDNA can play diverse roles and have various implications, including inducing either pro or anti-inflammatory immune response (Al Amir Dache and Thierry, 2023; West et al., 2015), participating in cell-to-cell communication (Al Amir Dache and Thierry, 2023), and bioenergetics profile restoration (Caicedo et al., 2015; Miliotis et al., 2019). For instance, ccf-mtDNA is thought to originate from damaged or dying cells or by active secretion (Bronkhorst et al., 2022) and can activate innate immunity and inflammation (Mills et al., 2017) being considered mitochondrial damage-associated molecular patterns (DAMPs) (De Gaetano et al., 2021; Galluzzi et al., 2012; Weinberg et al., 2015; West et al., 2015). When mtDNA is packed within exosomes, it is protected from degradation, possibly enhancing its uptake by recipient cells and thereby influencing cellular functions and signaling pathways (Colombo et al., 2014; Gurung et al., 2021; Kalluri and LeBleu, 2020). There are a limited number of studies that have evaluated mtDNA in extracellular vesicles, particularly in exosomes (Sansone et al., 2017; Wang et al., 2022; Wang et al., 2020). Sansone et al. highlighted the presence of whole mitochondrial genomes in exosomes, with a potential role in horizontal transfer inducing resistance to cancer treatment (Sansone et al., 2017). Recently, it has been demonstrated that a higher presence of mtDNA and other mitochondrial components within exomes is influenced by the mitophagy pathway (Wang et al., 2022). Furthermore, it has been found that exosomes released in cultured human neuroblastoma cells and plasma contain extended mtDNA sequences and degraded mtDNA fragments, which could potentially inform the individual’s status of mitochondria function (Wang et al., 2020). Research assessing the quantity of mtDNA in extracellular vesicles has indicated a decrease in the levels of mtDNA associated with these vesicles with aging (Lazo et al., 2021), and an increase has been observed in frail individuals (Byappanahalli et al., 2023). However, in our study, we did not observe differences in the levels of EX-mtDNA when comparing individuals with and without LLD. One important outcome of the alteration in the extracellular mtDNA levels, such as EX-mtDNA, would be the activation of the innate immune response via the activation of TLR (De Gaetano et al., 2021; Tresse et al., 2023). The interaction between TLRs and T cells, particularly the involvement of TNFRII, represents a link between innate and adaptive immunity (Siegmund et al., 2016). TNFRII, activated by TNF, plays a multifaceted role in modulating T cell responses, contributing to the effective recognition and elimination of pathogens (Ye et al., 2018). Torralba et al. have demonstrated that activated T cells release extracellular vesicles, including exosomes, containing genomic and mitochondrial DNA (Torralba et al., 2018). Additionally, the TNF receptors I and II are thought to inhibit mitophagy, leading to altered mitochondrial function, metabolic abnormalities, and elevated cytosolic levels of mtDNA (Willemsen et al., 2021). The above studies suggest that there may be positive feedback regulation loops between EX-mtDNA and the TNFRI and II pathway, indicating their mutual involvement in shaping immune responses. Our finding of a positive correlation between EX-mtDNA and plasma TNFRII levels aligns with the understanding that extracellular mitochondrial DNA can lead to an inflammatory state observed in depression (Behnke et al., 2023; Kageyama et al., 2018).

There is a large gap in our understanding of how damages to mtDNA can further trigger pathological conditions and how its spread can lead to the clinical manifestations (Vaidya et al., 2022). Tresse et al. reported the presence of mtDNA deletions within the genes MT-ND4 and MT-ND5 (subunits of the complex I respiratory chain) in the postmortem brains of individuals diagnosed with Parkinson’s disease (Tresse et al., 2023). Similarly, increased levels of the mtDNA common 4977bp deletion, encompassing the MT-ND4 region, have been reported in post-mortem brain tissue derived from individuals with bipolar disorder (Bodenstein et al., 2019; Kato et al., 1997), Alzheimer’s disease (AD) (Corral-Debrinski et al., 1994; Hamblet and Castora, 1997) and Huntington’s disease (Horton et al., 1995). Controversial findings have also been reported, indicating either no disparities or reduced levels of this deletion in individuals with AD (Blanchard et al., 1993; Lezza et al., 1999).

Building on these observations, earlier studies have shown that the damaged mtDNA is regulated through the process of mitophagy (Palozzi and Hurd, 2023; Wasner et al., 2022), or released either freely or encapsulated within extracellular vesicles such as EXs (De Gaetano et al., 2021). In our study, we assessed the integrity of the mtDNA within EXs and we observed that individuals with LLD presented higher levels of estimated deletion rate when compared to healthy controls. Considering the essential role of the mitochondrial gene MT-ND4 in the proper function of complex I (Kim et al., 2019), vital for energy metabolism, higher levels of deletions may indicate compromised mitochondrial function in LLD individuals. This is in line with studies showing an oxidative stress imbalance and higher DNA oxidation by-products in LLD (Diniz et al., 2018; Vieira et al., 2021). Goetz and colleagues (2021) focused on investigating 14 mitochondrial protein levels within plasma neuron-derived extracellular vesicles, all transcribed by nuclear genes, in individuals with major depressive disorder (MDD) before and after selective serotonin reuptake inhibitor (SSRI) treatment. They observed lower levels of OXPHOS complexes I and III in individuals with MDD compared to controls, regardless of SSRI responsiveness. Among SSRI-responsive participants, complex III protein levels were fully restored post-treatment, while complex I levels remained largely unchanged. This implies that restoring complex I function in individuals with depression may require boosting the levels of proteins encoded by mitochondrial genes associated with complex I, such as MT-ND4. We speculate that elevated levels of mtDNA deletions in individuals with depression may disrupt the functioning of the oxidative phosphorylation system (OXPHOS) complexes function, leading to reduced ATP production and heightened oxidative stress in bodily cells. This phenomenon is particularly notable in high-energy-demanding cells such as those found in the brain (Nissanka and Moraes, 2018).

Increased oxidative stress is additionally linked to greater cognitive decline, serving as one possible mechanism driving neuroprogression and accelerated aging in mood disorders (Diniz et al., 2018; Maurya et al., 2016; Vieira et al., 2021; Vieta et al., 2013; Wolkowitz et al., 2010). Conversely, we reported here that the increased estimated deletion rate of EX-mtDNA was associated with worse cognitive performance. This association suggests that the presence of dysfunctional mitochondria associated with the observed deletion could initiate a cascade of biological events, potentially heightening vulnerability among individuals with LLD to adverse health outcomes, including decreased cognitive performance.

Our results should be viewed in light of some limitations. We incorporated a small number of participants from a specialized geriatric psychiatry treatment center. Additionally, the cross-sectional nature of our study prevents us from establishing any causal relationships between LLD and EX-mtDNA status. Lastly, the complex relationship between peripheral findings and events taking place in the central nervous system precludes a fully mechanistic interpretation of the findings from this study. Nevertheless, the robust characterization of both the LLD group and the comparison individuals, coupled with the noteworthy aspect that all participants were not undergoing antidepressant treatment during the blood collection, are major strengths in our study.

Conclusion

EXs are crucial mediators of intercellular communication, playing a significant role in the pathophysiology of depression. Our findings suggest that levels and integrity of EX-mtDNA could potentially serve as indicators of inflammation status and cognitive outcomes in the context of LLD. Additional research is warranted to investigate how mitochondrial DNA trafficking within exosomes interacts with cellular mechanisms related to mitochondrial quality control and degradation and cellular energy production. Moreover, it is essential to gain a deeper understanding of the association between EX-mtDNA and cognitive function, as well as to investigate its potential utility as a biomarker in clinical settings.

Highlights.

  • Depressed older adults exhibited a significantly higher estimated deletion rate in mitochondrial DNA within exosomes (EX-mtDNA) compared to individuals in the control group.

  • Alterations in the amount and deletions of EX-mtDNA serve as markers of impaired mitochondrial function and may heighten susceptibility to adverse health outcomes.

  • The levels and integrity of EX-mtDNA hold potential as indicators of inflammation status and cognitive outcomes in the context of late-life depression.

Acknowledgements

We are grateful to our LLD patients, to their families and the caregivers, for their willingness and interest in scientific research. We also thank all doctors for their continuous support to the clinical research on LLD at CAMH. We thank Brandon Pierre, Jennifer Petryschuk, Fatima Rownak-Selim, and Madison Bak for technical assistance in the participant recruitment and bloodwork. Dr. Erica Vieira is supported by the CAMH-Discovery Fund. Dr Ana Paula Mendes-Silva’s fellowship was supported by the CAMH WomenMind program at the time of the research was conducted, and she is currently affiliated with the Department of Psychiatry at the University of Saskatchewan. This study was partially funded by NIMH grant from Dr. Breno Diniz (1R01MH115953–01A1). Dr. Yuliya Nikolova is supported by a Koerner New Scientist Award and a Paul Garfinkel Catalyst Award administered by the CAMH Foundation. Dr. Vanessa Gonçalves is supported by Larry and Judy Tanenbaum Foundation. This work was supported by CAMH Discovery Fund Seed Funding Competition.

Footnotes

Declaration of Interest statement

Dr. James L. Kennedy is a member of the Scientific Advisory Board of Myriad Neuroscience (unpaid) and holds several patents relating to pharmacogenetic tests for psychiatric medications. Dr. Tarek K. Rajji has received research support from Brain Canada, Brain and Behavior Research Foundation, BrightFocus Foundation, Canada Foundation for Innovation, Canada Research Chair, Canadian Institutes of Health Research, Centre for Aging and Brain Health Innovation, National Institutes of Health, Ontario Ministry of Health and Long-Term Care, Ontario Ministry of Research and Innovation, and the Weston Brain Institute. Dr. Rajji also received for an investigator-initiated study in-kind equipment support from Newronika, and in-kind research online accounts from Scientific Brain Training Pro, and participated in 2021 and 2022 in an advisory activity for Biogen Canada Inc. Dr. Rajji is also an inventor on the United States Provisional Patent No. 17/396,030 that describes cell-based assays and kits for assessing serum cholinergic receptor activity. Dr. Diniz receives research support from the US National Institute of Health (NIH). All other authors report no conflict of interest related to this study.

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