Cell-free DNA in extracellular vesicles: A candidate biomarker of schizophrenia

Abstract

Schizophrenia (SCZ) is a severe mental disorder with significant impacts on individuals, families, and society. Previous research has indicated that SCZ patients will commonly face substantial impairments in mitochondrial function and oxidative phosphorylation in the brain. Cell-free DNA (cf-DNA), serving as a direct biomarker of apoptosis, offers a valuable vantage point to understand the complex cellular mechanisms underlying SCZ. This review is to explore the role of cf-DNA in the etiology and progression of SCZ and evaluate the potential of cf-DNA within extracellular vesicles (EVs) as a diagnostic biomarker. A review approach was employed to gather and analyze relevant literature on the role of cf-DNA in SCZ, especially focusing on the potential of cf-DNA within EVs as a diagnostic biomarker. This review found that cf-DNA within EVs holds the potential to improve diagnostic methods. It can offer more accurate and sensitive means for detecting SCZ. Moreover, it has the ability to optimize disease management strategies by providing information about the disease state. Also, it can promote the development of pharmacological treatments for SCZ. Integrating cf-DNA analysis into clinical practice can help clinicians utilize cf-DNA levels and its unique characteristics for early and accurate diagnosis. The analysis of cf-DNA, particularly cf-DNA within EVs, has significant potential in the context of SCZ. It can transform our understanding of the disorder, improve diagnostic approaches, optimize disease management, and foster the development of pharmacological treatments.

Key Words: Schizophrenia; Cell-free DNA; Extracellular vesicles; Mitochondrial damage; Inflammation

Core Tip: Schizophrenia (SCZ) is a severe mental disorder characterized by multi-dimensional dysfunction in perception, cognition, emotion, and behavior. Recent research has highlighted the critical role of cell-free DNA (cf-DNA) carried by extracellular vesicles in SCZ. Abnormal expression of cf-DNA may be potentially linked to the pathological progression of SCZ. This review provides the molecular regulatory mechanisms of extracellular vesicle-derived cf-DNA in the pathogenesis and progression of SCZ, emphasizing its potential as a novel diagnostic marker and its promising clinical applications, thereby offering a new avenue for exploring the biological underpinnings of the disease and developing objective diagnostic tools.

INTRODUCTION

Schizophrenia (SCZ) is a prevalent psychiatric disorder, which significantly contributes to the global burden of disease[1]. It is characterized by the presence of symptoms, including hallucinations, delusions, and cognitive impairments[2]. Although the scientific community generally believes that the interaction between environmental and genetic factors contributes to the etiology and pathogenesis of SCZ, the causes of SCZ are complex, and the mechanisms underlying its onset remain unclear to date. Multiple etiological hypotheses have been proposed for SCZ, including the abnormal neurotransmission hypothesis, neurodevelopmental and genetic hypothesis, immune system dysfunction hypothesis, and neuronal apoptosis hypothesis[3].

The majority of factors released by activated microglia in patients with SCZ exhibit neurotoxic effects, potentially contributing to the occurrence of neuronal apoptotic events[4]. Moreover, the available evidence suggests that antipsychotic drugs demonstrate neuroprotective effects through the inhibition of inflammation, prevention of neuronal damage, and reduction of apoptosis[5]. The excitatory imbalance between pyramidal neurons and inhibitory gamma-aminobutyric acid (GABA) interneurons, induced by glutamate during the critical stage of neural development, can lead to a localized surge of glutamic acid, triggering excessive dendritic pruning and activating the apoptosis mechanism in local dendritic cells[6]. Apoptosis has been shown to contribute to 20% to 80% neuronal demise in the central nervous system[7]. Repetitive pruning of dendritic spines, in conjunction with aberrant synaptic plasticity, can give rise to cerebral misconnections and synaptic inefficiency, which underlie the prominent negative symptoms and cognitive deficits observed in individuals with SCZ[8-10]. Therefore, it is postulated that apoptosis plays a pivotal role in the pathogenesis and progression of SCZ.

Despite extensive research, the etiology of SCZ remains incompletely understood, and reliable diagnostic biomarkers are lacking. Conventional diagnostic methods are primarily based on clinical assessments and subjective patient reports due to the absence of robust physiological and biochemical markers[11]. This reliance imposes a substantial burden on clinicians’ expertise, heightening the potential for misdiagnosis or overlooked diagnoses and impeding timely administration of optimal treatment. The development of diagnostic markers has the potential to significantly enhance the establishment of standardized criteria for clinicians, which is imperative for the early identification and diagnosis of mental illnesses. Biomarkers of SCZ can be divided into peripheral and central biomarkers. Changes in immune markers, such as C-reactive protein and interleukin, were found in the blood of SCZ patients[12,13]. The most studied genes in the neuroendocrine system of SCZ are FKBP5 and CRHR1, and their single nucleotide polymorphisms[14,15]. In terms of neuroimaging, there are also some potential biomarkers, such as immune dysregulation, reduced cortical gray matter volume, and N-methyl-d-aspartate receptor hypofunction[16,17]. Zhang et al[18] analyzed the differentially expressed genes in the blood of SCZ patients through bioinformatics, and screened three potential hub genes (CTSS, DOK2 and ENTPD1). Jin et al[19] conducted a bibliometric analysis by systematically searching SCZ and biomarkers. The results show that the number of articles related to SCZ and biomarkers has gradually increased in recent years. Various peripheral proteins have been evaluated in SCZ, including nerve growth factor, brain-derived neurotrophic factor, homocysteine, and vitamin B, etc[17]. In terms of gut microbes, the relative abundance of Christensenellaceae, Enterobacteriaceae, and Victivallaceae increased at the family level compared with healthy controls (HCs). However, the relative abundance of Pasteurellaceae, Turicibacteraceae, Peptostreptococcaceae, Veillonellaceae, and Succinivibrionaceae decreased[20]. The diagnostic markers of SCZ include neurotransmitter-related markers, such as increased serotonin or serotonin 2A receptor activity, and D2 receptor hyperactivity[21,22]. Currently, most studies have been established by in vivo imaging or postmortem brain tissue, and occasionally in cerebrospinal fluid (CSF). CSF proteomics for SCZ involves a lumbar puncture, an invasive procedure with risks such as post-puncture headache, bleeding, and infection. Its proteomic changes are affected by multiple factors, have low specificity, and may occur late in disease progression. Magnetic resonance imaging for SCZ requires expensive equipment, specialized technicians, is time-consuming, and may not be widely available. Functional magnetic resonance imaging measures brain activity by detecting changes in blood oxygenation level-dependent signals. Some patients have contraindications, and measured blood oxygenation level-dependent signals can be affected by various factors, leading to inaccurate results.

Cell-free DNA (cf-DNA) refers to small fragments of DNA that circulate freely in the bloodstream and other biological fluids[23]. This extracellular DNA is released into the circulation from various cells through processes such as apoptosis (programmed cell death), necrosis (cell injury and death), and active secretion by some cells. Cf-DNA can be obtained non-invasively via a simple blood draw, is more acceptable to patients, and is suitable for large-scale screening and repeated monitoring. It directly carries disease-related genetic information, can detect genetic and epigenetic alterations early, and helps identify high-risk individuals for early intervention. Cf-DNA testing is more accessible as it only requires basic laboratory facilities, making it suitable for widespread use. As a molecular biomarker, cf-DNA offers a more direct and specific measure of SCZ-related genetic and epigenetic changes, providing more accurate diagnostic information.

This review aims to provide comprehensive and in-depth exploration of the role of cf-DNA in the etiology and progression of SCZ. Concurrently, it examines the biological mechanisms governing the release and function of extracellular vesicles (EVs) within the SCZ context. Moreover, this review systematically integrates existing studies on cf-DNA, along with the correlation between cf-DNA encapsulated in EVs and SCZ. The overarching objective is to comprehensively evaluate the potential value of cf-DNA in both the diagnosis and prognosis of SCZ.

CF-DNA

Sources and release mechanisms of cf-DNA

The presence of cf-DNA was first observed in human blood cells by Mandel and Metais in 1948[24]. Different subtypes of cf-DNA have been characterized in the circulation, including double-stranded and single-stranded fragments, mitochondrial DNA (mtDNA), extrachromosomal circular DNA, as well as viral and bacterial DNA[25]. Cf-DNA can be categorized into two distinct types: Cf genomic DNA (cf-gDNA), encompassing nuclear DNA derived from the genome within the nucleus; and cf-mtDNA), originating from mitochondria[26]. The sizes of cf-DNA exhibit significant variation, ranging from 80 to 10000 bp. Notably, cf-gDNA typically manifests peaks at 166 bp fragments, while the range for cf-mtDNA is between 20 and 100 bp, contingent upon the mechanisms governing fragmentation and release[27]. Cf-gDNA fragments primarily originate from intracellular and extracellular enzymatic cleavage of DNA[28]. The processes of cell death, such as apoptosis and necroptosis, induced by various internal and external factors, commonly lead to incomplete degradation of intracellular nuclear and mtDNA, contributing to the accumulation of cf-DNA in circulation. Cf-DNA has been detected in multiple body fluids, including blood, urine, and CSF, and is thought to enter the circulation via both active and passive mechanisms[29]. The majority of cf-DNA enters the bloodstream through the secretion of EVs, such as exosomes, microparticles, and apoptotic bodies[30,31]. The cf-DNA primarily comprises linear double-stranded DNA, but it can also exist in the forms of circular DNA and single-stranded DNA[32]. They are present in minute quantities within the healthy population, primarily originating from hematopoietic cells[33]. In individuals without any underlying comorbidities, plasma cf-DNA levels typically remain below 10-30 ng/mL[30]. However, the levels may show a significant increase in individuals with acute or chronic illnesses, often by several orders of magnitude. Levels of cf-mtDNA do not appear to exhibit a direct correlation with cellular demise[34]. However, certain neurodegenerative disorders demonstrate an observed decrease in circulating cf-mtDNA levels[34].

Currently, it is widely recognized that the predominant source of circulating cf-DNA is attributed to active cellular apoptosis, necrosis, NETosis[35], and active release (Figure 1)[36]. The released cf-DNA is capable of interacting with high mobility group protein B1, cathelicidin (leucine leucine-37), mitochondrial transcription factor A, and other related proteins. Pro-apoptotic stimuli, such as inflammation, autoimmunity, and oxidative stress, can induce the accumulation of reactive oxygen species (ROS) within mitochondria and disrupt oxidative metabolic processes, thereby triggering apoptosis in both neuronal and glial cells[37,38]. Therefore, the comprehensive analysis of cf-DNA across diverse conditions, encompassing quantitative variations, size discrepancies, and specific disease distributions, holds immense potential for elucidating the pathogenesis of SCZ and establishing a robust foundation for biomarker research of SCZ.

Figure 1 Sources of cell free DNA. A: Apoptosis; B: Necrosis; C: NETosis; D: Active release.

Cf-DNA and mitochondrial damage

Mitochondria are the primary cellular sites for energy metabolism, where oxidative phosphorylation represents the principal pathway of aerobic metabolism occurring on the mitochondrial membrane to generate adenosine triphosphate[39]. Research suggests that mitochondrial dysfunction, whether resulting from hyperactivity or blockade of the oxidative phosphorylation process, can lead to a substantial production and accumulation of ROS[40]. Additionally, aberrant pro-inflammatory or inflammatory cytokine production, immune system suppression, and glutamine deficiency can collectively contribute to the accumulation of ROS and heightened oxidative stress[41-43]. Moreover, perturbations in the dopamine neurotransmitter may also contribute to the process[44,45]. Excessive oxidative phosphorylation and inflammation in the body can both induce intracellular oxidative stress, leading to upregulation of the antioxidant system and triggering of cell apoptosis[46]. The elevation of intracellular oxidative stress can directly induce DNA and protein damage, thereby initiating the process of apoptosis. Conversely, it can induce the release of cytochrome c and elicit an inflammatory response to facilitate apoptosis[47]. Excessive oxidative stress induces mitochondrial damage, leading to the release of mtDNA[48-50]. Therefore, cf-mtDNA accurately reflects both the extent of oxidative stress and immune homeostasis, while mild neurocognitive impairment is associated with the presence of cf-mtDNA in CSF[26,51].

Cf-DNA and inflammation

The white blood cells in the bloodstream are tightly regulated to execute precise physiological functions, encompassing the mediation of inflammation, eradication of pathogens, and perception of biological signals from the body to elicit appropriate responses[52]. The activity of these white blood cells is tightly regulated to ensure their functional responsiveness while mitigating excessive reactions that may lead to tissue damage. Inflammation is believed to play a pivotal role in the pathogenesis and persistence of psychosis and cognitive impairment observed in individuals with SCZ[53]. In certain SCZ patients, the concomitant occurrence of an acute psychotic episode and low-grade systemic inflammation is observed[54]. Many studies have established a robust correlation between pro-inflammatory cytokines and SCZ[54]. The heightened metabolic activity in the peripheral environment leads to the generation of a substantial quantity of metabolic waste products, such as cf-DNA and RNA. Consequently, there exists a correlation between the inflammatory status and the level of cf-DNA[55].

The transcriptomic, proteomic, and metabolomic profiles of brain tissue in patients with SCZ have revealed significant impairments in mitochondrial function and oxidative phosphorylation[56]. The oxidation of cf-DNA significantly enhances its capacity to traverse cellular membranes[57]. Oxidized cf-DNA fragments that enter the cytoplasm can induce diverse responses, including the activation of signaling pathways associated with cytoplasmic nucleic acid receptors such as Toll-like receptor 9 (TLR9), absent in melanoma 2, cyclic GMP-AMP synthase, and retinoic acid-inducible gene-1-like receptors[58-60].

The interaction between CpG-rich fragments and the TLR9 receptor constitutes one of the mechanisms by which cf-DNA gains entry into cells[61]. The TLR9, a pivotal innate immune receptor in humans, plays a critical role in diverse cellular functions, particularly in orchestrating immune responses[62,63]. The activation of TLR9 triggers downstream signaling pathways dependent on myeloid differentiation primary response protein 88, resulting in the production of type 1 interferons mediated by interferon regulatory factor 3 and proinflammatory cytokines regulated by nuclear factor-kappa B[64,65]. In vitro, Ershova et al[66] employed a cellular model to compare the bioactivity of cf-DNA samples derived from individuals with SCZ and HCs, the cf-DNA was observed to induce augmented transcriptional activation of genes involved in the regulation of cellular proliferation, cytokine production, programmed cell death, autophagy, and mitochondrial biogenesis. However, there was no statistically significant disparity observed in the cellular response to both types of cf-DNA. Given the significantly elevated levels of cf-DNA in the bloodstream of patients compared to HCs, it is plausible that cf-DNA may potentially elicit an up-regulation of proinflammatory cytokines in individuals with SCZ.

Cf-DNA and SCZ

The diverse clinical manifestations of SCZ may be mechanistically explained by the presence of excessive oxidative stress and immune dysregulation, which could potentially underlie its pathophysiology[67,68]. Cf-DNA has been demonstrated to be a constituent of the damage-associated molecular patterns[69]. Cf-DNA can be recognized by pattern recognition receptors, thereby initiating a form of sterile inflammation[70]. It has been established that inflammation plays a pivotal role in the pathogenesis of severe psychiatric disorders, such as SCZ[71], bipolar disorder[72], and depressive disorders[73]. However, the precise mechanisms underlying the inflammatory processes associated with psychiatric disorders remain inadequately understood. Thus, cf-DNA represents a promising avenue for further investigation in this regard.

Circulating cf-DNA primarily derives from apoptotic events and serves as a direct indicator of apoptosis. Thus, investigating cf-DNA allows for a comprehensive assessment of the apoptotic status in patients. Recent studies on SCZ and cf-DNA offer a comprehensive overview of the current research in this field (Table 1). Ershova et al[74] employed PicoGreen fluorescence and flow cytometry to quantify the concentration of circulating cf-DNA in plasma and to assess the flow cytometry fluorescence 1-8-oxo-7,8-dihydro-2'-deoxyguanosine (flow cytometry fluorescence 1-8-oxodG) index in lymphocytes. The results showed no statistically significant difference in cf-DNA concentration between SCZ patients and HCs[74]. However, elevated levels of 8-oxoguanine in DNA (8-oxodG) were observed in the patient group, and a strong correlation was found between cf-DNA concentration and the 8-oxodG index[74]. Jiang et al[75] employed fluorescence correlation spectroscopy to examine the concentration and size of cf-DNA, subsequently verifying the results through real-time quantitative polymerase chain reaction. The findings of the study indicated that the concentration of cf-DNA was approximately twofold higher in SCZ patients than in HCs, and that the fragments of cf-DNA were shorter in length[75]. Ershova et al[76] conducted an examination of plasma cf-DNA concentration, plasma nucleic acid endonuclease activity, and the copy number of ribosomal DNA in SCZ patients and HCs. They found that the plasma cf-DNA concentrations in the SCZ patient group were two-fold higher than those in HCs, while plasma nucleic acid levels were four-fold higher and cf-ribosomal DNA concentrations were three-fold higher. Qi et al[77] reported that the concentration of amyloid precursor protein-cleaving enzyme-1 was elevated in SCZ patients and positively correlated with certain inflammatory biomarkers, including interleukin-1β and interleukin-18. Chen et al[78] employed real-time quantitative polymerase chain reaction analysis to compare cf-DNA concentrations in 174 patients with SCZ and 100 matched HCs and they revealed that SCZ patients exhibited elevated cf-DNA levels in their peripheral blood. Jestkova et al[79] examined the concentration of cf-DNA in patients with paranoid and alcoholic SCZ and found that the concentration of cf-DNA in the plasma of SCZ patients was 2.2-fold higher compared to the HC group, and exhibited a 1.8-fold increase in patients with alcohol-induced psychosis. Nikitina et al[80] analyzed the concentrations of cf-DNA and the levels of 8-hydroxy-2’-deoxyguanosine and histone H2AX phosphorylation in children with SCZ and revealed that cf-DNA concentrations were elevated in the patient group compared to HCs. Most studies have found that the concentration of cf-DNA in the plasma or serum of patients with SCZ is significantly elevated compared to that of HCs[75-83]. However, one study did not demonstrate a statistically significant difference between the two groups[74].

Table 1 The association between cell-free DNA and schizophrenia.

Ref.	Sample source	Sample capacity	Cf-DNA types	Method	Results
Ershova et al[74]	Plasma	SCZ: 83, HCs: 30	Cf-DNA	PG	Cf-DNA no change
Jiang et al[75]	Plasma	SCZ: 65, HCs: 62	Cf-DNA	FCS, RT-PCR	Cf-DNA concentrations increased
Ershova et al[76]	Plasma	SCZ: 100, HCs: 96	Cf-DNA, cf-rDNA	PG, NQH	Cf-DNA concentrations increased; cf-rDNA copy number increased
Qi et al[77]	Serum	SCZ: 164, HCs: 100	Cf-DNA	RT-qPCR	Alu concentration increased
Chen et al[78]	Serum	SCZ: 174, HCs: 100	Cf-DNA	RT-qPCR	Alu concentration increased
Jestkova et al[79]	Plasma	SCZ: 381, HCs: 95	Cf-DNA	PG	Cf-DNA concentrations increased
Nikitina et al[80]	Plasma	SCZ: 38, HCs: 34	Cf-DNA	PG	Cf-DNA concentrations increased; mtDNA-CN decreased
Ershova et al[81]	Plasma	SCZ: 100, HCs: 60	Cf-DNA	RT-qPCR	Cf-DNA concentrations increased
Lubotzky et al[82]	Plasma	SCZ: 29, HCs: 31	Cf-DNA	PG, NGS	Cf-DNA concentrations increased
Li et al[83]	Plasma	SCZ: 191, HCs: 65	Cf-DNA	FCS	Cf-DNA concentrations increased, cf-gDNA increased, cf-mtDNA no change

SCZ: Schizophrenia; cf-DNA: Cell-free DNA; HCs: Healthy controls; PG: PicoGreen fluorescence; FCS: Fluorescence correlation spectroscopy; RT-qPCR: Real-time quantitative polymerase chain reaction; cf-rDNA: Cell-free ribosomal DNA; NQH: Nonradioactive quantitative hybridization; mtDNA-CN: Mitochondrial DNA copy number; NGS: Next-generation sequencing; cf-gDNA: Cell-free genomic DNA; cf-mtDNA: Cell-free mitochondrial DNA.

Some research has been conducted on the association between cf-gDNA and SCZ, and it was found that the SCZ patients demonstrated a significantly elevated concentration of cf-gDNA compared to HCs[77,78,82]. Ouyang et al[84] conducted a quantitative analysis of the plasma cf-mtDNA levels in patients with SCZ and observed no statistically significant difference compared to those in HCs. However, following drug therapy, the levels of cf-mtDNA in patients decreased and demonstrated a positive correlation with the Positive and Negative Syndrome Scale scores, indicating an association between alterations in plasma cf-mtDNA levels and clinical progression[84]. Ershova et al[81] examined the copy number of mtDNA and the concentration of cf-DNA in peripheral blood mononuclear cells. They observed a reduction in copy number of mtDNA and an increase in cf-DNA concentration in SCZ patients compared to HCs[81]. Lubotzky et al[82] investigated plasma concentrations of cf-DNA in SCZ patients and observed elevated levels compared to HCs. Notably, these levels were not associated with psychotropic drug use[82]. Li et al[83] found that plasma cf-DNA concentrations were significantly elevated in SCZ patients, irrespective of disease stage or antipsychotic use. Subsequent studies revealed that cf-gDNA was elevated in individuals with SCZ, whereas cf-mtDNA levels remained unaltered. These findings indicate a strong correlation between cf-DNA and both the etiology and progression of SCZ. The results of Ershova’s study differed from those of other investigators, possibly due to the different detection methods used[81]. Reasons for these different results may be due to the inconsistency of study samples. Therefore, large samples and data from other research institutions are needed for confirmation.

DNA methylation (DNAm) is an epigenetic modification that introduces methyl groups to DNA nucleotides, predominantly cytosines and adenines. The physiological functions of DNAm encompass a broad spectrum of processes, including embryonic development, transposable element inhibition, genomic imprinting, and X chromosome inactivation[85,86]. The processes of DNAm and hydroxyl methylation play a pivotal role in the normal development and functioning of the brain, encompassing neural stem cell proliferation and differentiation, synaptic plasticity, neuronal repair, as well as learning and memory[87]. Considering that the onset of SCZ is influenced by environmental factors, it is hypothesized that DNAm might contribute to the pathogenesis of this disease. Environmental factors, such as prenatal stress, have been demonstrated to induce the upregulation of DNA methyltransferase 1 and DNA methyltransferase 3A expression in GABAergic interneurons, resulting in aberrant promoter methylation and subsequent gene silencing of RELN and GAD1[88]. The reduction in protein expression levels of Reelin and glutamic acid decarboxylase 67 (GAD67), which are encoded by RELN and GAD1, respectively, may result in a decrease in DNAm levels[88]. The reduction of Reelin may contribute to a decrease in the density of dendritic spines, dysfunction of the glutamate ionotropic receptor NMDA type subunit 1 receptor, and impairment of long-term potentiation[89]. The enzymatic action of GAD67 facilitates the conversion of glutamate into GABA, which serves as the principal inhibitory neurotransmitter. The reduction of GAD67 Leads to a decrease in the synaptic release of GABA, thereby compromising the synchronization and inhibitory function of excitatory pyramidal neurons. Consequently, this disruption ultimately gives rise to behavioral and cognitive impairments observed in individuals with SCZ[90]. Luo et al[91] conducted a comparative analysis of methylation profiles in hepatocellular carcinoma, normal tissue samples, and plasma cf-DNA, and developed a screening model based on differential methylation blocks. The findings demonstrated the potential utility of cf-DNA-based DNAm analysis as a non-invasive screening tool for early detection of hepatocellular carcinoma in clinical settings. Therefore, the evaluation of cf-DNAm status in plasma or serum can provide valuable insights into patients’ conditions.

From circulating cf-DNA to EV-encapsulated cf-DNA in SCZ

While circulating cf-DNA has provided valuable insights into SCZ pathology, its clinical utility is limited by rapid degradation in biofluids and lack of cellular origin specificity. These limitations have initiated interest in EV-encapsulated cf-DNA. EV membranes confer protection against nucleases, enhancing biomarker stability. EV surface markers permit cell-type-specific tracing of cf-DNA origins. EV cargo sorting mechanisms may reflect selective packaging of pathological nucleic acids. EV-associated cf-DNA mirrors broader trends in liquid biopsy research, where EVs are increasingly recognized as biological “time capsules” preserving disease-relevant molecular signatures.

EVs

EVs are phospholipid bilayer particles, ranging in diameter from approximately 30 nm to 10 μm. They are commonly categorized into four subtypes: Exosomes, microvesicles, apoptotic bodies, and oncosomes[92,93]. The heterogeneity and functional diversity of EVs primarily arise from their cargo, encompassing nucleic acids, proteins, lipids, cytokines, chemokines, as well as neurotoxic and pathogenic end-stage metabolic products[94,95]. Exosomes, a type of EVs, are membrane-enclosed vesicles actively secreted by cells into the extracellular environment and are found in high abundance in various bodily fluids. Microvesicles are initiated by the translocation of phosphatidylserine from the inner leaflet to the outer leaflet of the plasma membrane, thereby triggering budding and fission processes in the plasma membrane[96]. The dimensions of microvesicles are characterized by a larger diameter ranging from 50 to 2000 nm in contrast to exosomes. Apoptotic bodies are membrane vesicles generated during the apoptotic process through the release of membranous structures from apoptotic cells, exhibiting a diameter ranging from 1000 to 5000 nm[97]. EVs are structures released into the extracellular environment by all cells. They are enclosed by a lipid bilayer and contain components derived from the releasing cells. Exosome biogenesis begins with the formation of multiple intraluminal vesicles within endocytic compartments known as multivesicular bodies[98]. Other types of EVs can be directly shed from the plasma membrane, including ectosomes, microvesicles, microparticles, large oncosomes, and apoptotic bodies. The retrovirus-like particles, resembling retroviral capsids in electron microscope images, are formed through budding from the plasma membrane and exhibit a diameter ranging from 1000 to 5000 nm[99].

EVs are membranous structures that protect their cargo from degradation during extended transportation, while maintaining biological functionality. They represent a novel mechanism of intercellular communication, facilitating the transfer of biologically active molecules such as DNA, proteins, and RNA from donor to recipient cells. This process enables the exchange of genetic information and the reprogramming of recipient cells[100]. EVs can cross the blood-brain barrier (BBB) and enter systemic circulation, with immune cells and T cells in the peripheral environment also generating these vesicles (Figure 2). EVs serve various functions, including early diagnosis, prognostic assessment, efficacy monitoring, and personalized treatment. In individuals with SCZ, disease-specific changes in the brain lead to DNA fragmentation, which is released into peripheral blood via EVs traversing the BBB, resulting in elevated cf-DNA levels.

Figure 2 The sources and application of extracellular vesicles in health and disease. Neurons and immune cells can produce extracellular vesicles (EVs) through mechanisms such as cellular apoptosis, necrosis, NETosis, and active release. The EVs generated by neurons have the capability to traverse the blood-brain barrier and enter the peripheral environment to exert their physiological effects. Furthermore, immune cells and T cells in the peripheral environment also produce EVs, which can enter the brain.

CF-DNA IN EVs AND SCZ

Recently, a plethora of studies have unequivocally demonstrated the immense potential of exosomes as highly promising and robust biomarkers for SCZ[101]. Neuron- or glia-derived EVs may contribute to the onset of SCZ by transporting toxic (or misfolded) proteins and neurotransmitters, resulting in clinical symptoms like cognitive deficits[102]. A total of 25 perturbed metabolites in EVs of SCZ patients, associated with the metabolism of glycerophospholipids, phenylalanine, tyrosine, or tryptophan, have been identified to be linked with SCZ[103]. Patients diagnosed with SCZ exhibited decreased protein expression levels in subunits 1 and 6 of nicotinamide adenine dinucleotide-ubiquinone oxidoreductase, as well as subunit 10 of cytochrome b-c1 oxidase[104]. This finding provides evidence supporting the involvement of astrocyte-related neuroinflammation in the association between dysregulated protein expression and increased ROS.

Tsivion-Visbord et al[105] evaluated the viability of intranasal administration of EVs derived from mesenchymal stem cells (MSCs) as a potential therapeutic approach for ameliorating SCZ-like behaviors in the phencyclidine model of SCZ. The findings demonstrated that intranasal administration of MSC-derived EVs exhibited ameliorative effects on social interactions and disrupted prepulse inhibition in mice treated with phencyclidine[105]. Moreover, it was observed that this treatment preserved the neuronal population in the prefrontal cortex while reducing glutamate levels in CSF[105]. It can be reasonably inferred that the diverse substances present within EVs exert a significant influence on the pathogenesis of SCZ. Cf-DNA, enclosed within EVs generated through apoptotic processes in the brain, gains access to the CSF and is subsequently transported via the circulatory system into the peripheral bloodstream, where it exhibits remarkable stability. Previous studies have demonstrated that the majority of cf-DNA in human plasma is predominantly localized within exosomes[31]. Under normal physiological conditions, macrophages efficiently eliminate apoptotic and necrotic cells[106], consequently, the concentration of cf-DNA in healthy human blood remains exceedingly low. However, in cases of malignant tumors, intense physical activity, or systemic inflammation, macrophages may exhibit impaired clearance of apoptotic and necrotic cells, thereby resulting in the extracellular release of cellular DNA and subsequent entry into the peripheral bloodstream via the circulatory system[107,108]. Emerging research findings have sparked increasing interest in the potential value of EVs in peripheral blood for early disease diagnosis, prognosis assessment, and therapeutic efficacy monitoring. The expression of plasma EVs-cf-DNA is synchronized with that of the brain, accurately reflecting its pathological state[109]. This biomarker exhibits disease specificity, high stability, abundance, and ease of detection. In comparison to alternative diagnostic methods, the analysis of EVs-cf-DNA in blood offers specific data for the diagnosis of SCZ. However, there is a lack of consistency regarding sample type (plasma/serum), sample collection and processing methods, free or cell-surface bound DNA, cf-DNA extraction and quantification techniques, as well as the presentation and interpretation of quantitative cf-DNA results. Therefore, it is imperative to establish a systematic scientific framework with collaborative efforts for conducting large-scale multicenter trials and prospective analyses.

LIMITATIONS

Although this review has certain value, there are some limitations. The research scope is restricted as current studies on cf-DNA in EVs in SCZ may be incomplete, with some small-scale or emerging research overlooked due to regional and team-based variances. The specific molecular mechanisms of EV cf-DNA in SCZ etiology and progression have not been explored in-depth, with the focus mainly on its diagnostic biomarker potential. There is a lack of clinical validation as the assessment of EV cf-DNA as a diagnostic biomarker lacks large-scale, multi-center, large-sample clinical trials to verify its accuracy, specificity, and sensitivity in different populations. Moreover, therapeutic applications are limited. This review mainly aims at improving diagnosis, management, and treatments but does not discuss specific therapeutic uses of EV cf-DNA, merely positioning it as a diagnostic biomarker without in-depth exploration of its role in treatment monitoring and drug development. At the same time, this review only investigates the role of cf-DNA in the etiology and progression of SCZ, assessing the potential of cf-DNA in EVs as a diagnostic biomarker. It cannot rule out the possibility that cf-DNA serves as a biomarker for other psychiatric or neurodegenerative disorders, given that it is a marker of apoptosis. Indeed, there are also some researches indicating that cf-DNA can act as a biomarker for other psychiatric or neurodegenerative disorders.

CONCLUSION

SCZ research has revealed that cf-DNA in EVs offer a promising path for better understanding and managing this complex disorder. Reports consistently show elevated cf-DNA levels in the peripheral blood of SCZ patients, suggesting it as a potential biological marker. These increased levels are linked to underlying pathophysiological mechanisms. Neuroinflammation and cellular apoptosis, both key in SCZ pathogenesis, cause the release of cf-DNA into the bloodstream. Measuring cf-DNA can provide insights into the severity and progression of these processes. The distinct size distribution and concentration variations of cf-DNA in the bloodstream are significant. Different fragment sizes may be associated with specific cellular events in SCZ. This makes cf-DNA valuable for early SCZ diagnosis, which is crucial for timely intervention and improved patient outcomes. EVs provide a non-invasive biomarker detection method. They can be easily obtained from blood and urine and can cross the BBB. Analyzing EVs with encapsulated cf-DNA shows great promise as a blood-based biomarker for early SCZ detection, as it offers information on brain pathological changes. Integrating cf-DNA analysis into clinical practice can revolutionize our understanding of SCZ. Clinicians can use cf-DNA levels and characteristics for accurate early diagnosis and develop personalized treatment strategies. Detection of cf-DNA in EVs may require advanced techniques such as next-generation sequencing or quantitative polymerase chain reaction, which can be costly initially in terms of equipment and reagents. However, as technology advances, costs are decreasing.

Specific future research directions hold great potential to further advance the field, such as the integration of multi-omics approaches. By combining genomics, transcriptomics, proteomics, and metabolomics methods to analyze cf-DNA in EVs, a more comprehensive understanding of the molecular mechanisms underlying SCZ can be achieved. This multi-omics approach may uncover novel biomarkers and pathways that are not detectable through single-omics analysis, leading to more precise diagnosis and treatment. Clinical trials guided by EV-cf-DNA-based therapy are another crucial area of future research. These trials can evaluate the efficacy and safety of treatments tailored according to the characteristics of cf-DNA in EVs. However, large-scale studies are still required to validate these findings and standardize cf-DNA and EV analysis protocols in clinical settings. Overall, the combined analysis of cf-DNA and EVs, along with the exploration of multi-omics approaches and EV-cf-DNA-based clinical trials, holds the key to enhancing patient outcomes and advancing SCZ research.