Role of exosomes in the pathogenesis, diagnosis, and treatment of central nervous system diseases

Central nervous system (CNS) disorders represent a spectrum of diseases ranging from mild neurological impairment that may have motor, sensory, visual, speech, cognitive manifestations, or a combination thereof, to comatose states and brain death [1]. At present, nearly one in six people worldwide suffer from disorders of the CNS [2]. Common CNS diseases, such as multiple sclerosis, Alzheimer's disease, and Parkinson's disease, also bring huge economy burden to the society [3, 4]. After years of investigation, CNS diseases still remain clinical challenges with limited diagnostic and therapeutic approaches [5]. Under physiological conditions, CNS is protected against potential intruders by a unique microvasculature, the blood–brain-barrier (BBB), which is composed of endothelial cells connected by tight junctions and adherent processes [6]. However, the restrictive nature of the BBB also provides an obstacle for drug delivery to the CNS [7]. Achieving sufficient drug delivery across the BBB into the brain is a key challenge for the treatment of CNS diseases, which sieves out major proportion of therapeutics [7, 8]. To overcome this problem, biologically active cargo carriers such as exosomes and nanoparticles are attracting more attentions.

Exosomes are a large component of the broader class of nanoparticles termed extracellular vehicles (EVs) (9). Small extracellular vesicles containing transferrin receptors were first reported in 1983 by Johnstone and colleagues while culturing reticulocytes [10]. These vesicles were then named as "exosome" by Johnstone in 1987 [11]. With more studies conducted, now exosomes are generally accepted as small cell-derived single-membrane vesicles (30–150 nm) released by almost all types of cells into extracellular space via fusing plasma membrane and multivesicular bodies (MVBs) [12]. Contents embedded in exosomes include nucleic acids, proteins, lipids, amino acids, metabolites, glycoconjugates, cytosolic and cell-membrane proteins [13]. Via releasing these contents to neighboring cells, as a form of paracrine signaling, and/or to distant cells, acting as a type of endocrine signaling, exosomes are able to regulate cell-to-cell communications and multiple autocrine and paracrine cellular phenotypes [14]. Exosomes recently have emerged as the most promising biomarkers for disease diagnosis and targeted drug transporter for disease treatment [15]. Compared with other synthetic drug-delivery vehicles such as liposomes and nanoparticles, exosomes have extensive and unique advantages because of their endogeneity, favorable pharmacokinetic, special immunological properties, and their ability to penetrate physiological barriers [16]. Besides, surface modification of exosomes imparts additional functionality and enable site specific drug delivery of exosomes [17]. Together, these characters make exosomes favorable in CNS diseases diagnosis and treatment.

Recent studies have revealed effective role of exosomes in the diagnosis and treatments of CNS diseases. For example, α-Synuclein in blood exosomes can help distinguish PD from multiple system atrophy [18]. Blood neuro-exosomal synaptic proteins, including GAP43, neurogranin, SNAP25, and synaptotagmin 1, act as effective biomarkers for prediction of AD 5 to 7 years before cognitive impairment [19]. Isolated exosomes could be used alone as a neurorestorative therapy in stroke and neurological injury [20]. The potential of exosomes as biomarkers and therapies for CNS disorders is being actively investigated. In this review, we systematically summarized the advanced research progress of exosomes in several common nervous system diseases. We also propose the opportunities for exosome-based approaches to neuro-restoration and targeted drug delivery in various CNS diseases.

Exosomes are generated through a continuous process that involves double invagination of the plasma membrane and the formation of intracellular multivesicular bodies (MVBs) (Fig. 1) [14]. Biogenesis of exosomes starts with the de novo formation of early-sorting endosomes (ESEs). At start, cell membrane invaginates and forms a cup-shaped structure that contains cell surface proteins and extracellular components, such as soluble proteins, lipids, metabolites, small molecules, and ions [21]. Then ESEs take shape and subsequently either fuse with the endoplasmic reticulum (ER), trans-Golgi network (TGN) or a preexisting ESE. ESEs next mature into late sorting endosomes (LSEs) [22]. The second invagination in LSEs leads to the production of intraluminal vesicles (ILVs), which can further modify the load of future exosomes and allow cytoplasmic components to enter the newly formed ILVs. LSEs are further transformed into multivesicular bodies (MVBs), which can be degraded by fusion with lysosomes or autophagosomes, or they can be fused with plasma membrane to release ILVs as exosomes [23]. Exosomes are released by cells either as a reaction to specific stimuli, or under normal physiological conditions [24]. Released exosomes function as carriers of molecular information to transfer cargo molecules from parent to recipient cells and regulate cell-to-cell communications involving in physiological and pathological processes [25].

Exosomes are parts of those vesicle-producing cells. Quantity and contents of exosomes reflect the state of their cells of origin. Besides, exosome provide a mechanism by which cells can manipulate the molecular composition and function of extracellular matrix (ECM) [26]. In addition, exosomes transmit signals and molecules via a pathway of intercellular vesicle traffic, exerting local paracrine or distal systemic effects [9]. Exosome-mediated responses can be either disease promoting or restraining, depending on the composition and cell state. Furthermore, engineered exosomes can deliver diverse therapeutic payloads, including short interfering RNAs, antisense oligonucleotides, chemotherapeutic agents, and immune modulators [14]. The biological functions of exosomes span a large swath of biology, and we just focus on those aspects related to CNS diseases in this review. Exosomes released by neurons, glia and other cells in CNS contribute to the complex network of interconnected messages that underlie both the physiology and the pathology of this system [27]. Protective mechanisms of exosomes in CNS diseases which we discuss in the following include angiogenesis promotion, immune regulation, inhibiting the apoptosis of neurons and promoting the formation of myelin sheath and axon growth [28].

Exosomes and the biomolecules they carry mediate the communication between cells in CNS which participate in the development and function of CNS [41, 56]. This is of great significance for us to study the onset and progression of CNS diseases and provide potential targets for disease diagnosis and treatments. Exosomes are involved in maintaining normal physiological functions, such as tissue repair, immune surveillance, antigen presentation, and blood coagulation processes [56‐58]. However, the effects of exosomes on the body are not all beneficial, depending on their original cells and the specific molecules they contain. For instance, activated glial cell-derived exosomes carrying amyloid-β peptides and α-synuclein are pathologically linked to Alzheimer’s disease (AD) and Parkinson’s disease (PD), respectively [58]. Exosomes detected in the tumor microenvironment are found to facilitate tumorigenesis by regulating angiogenesis, immunity, and metastasis [59]. In the following text, we summarize and discuss the roles of exosomes in some specific CNS diseases, hoping to provide more information for further study and clinical application.

In this review, we first summarize the potential mechanisms of neuroprotective effects of exosomes. Then we discussed the role and application of exosomes in several CNS diseases. As we discussed above, exosomes participate in the onset and progression of a variety of neurological diseases. These exosome-mediated responses can be either disease promoting or restraining, depending on their contents and the intrinsic properties of diseases. Besides, the properties of exosomes in regulating complex intracellular pathways have advanced their potential as therapeutic targets and biomarkers for early diagnosis of diseases [14]. In addition, exosomes can be engineered to deliver diverse therapeutic payloads to a desired target. Ongoing technological and experimental advances enhance our ability to harness their therapeutic and diagnostic potential. There is an increasing number of clinical trials investigating the clinical application of exosomes. Though at present, there is no finished or published clinical trials regarding the role of exosomes in the treatment of CNS diseases, several undergoing clinical trials and studies using exosomes as biomarkers are worthy of notice. For example, s clinical study performed by researchers from Isfahan University of Medical Sciences aimed to assay the effects of MSC derived exosomes on improving the disability of patients with acute ischemic stroke (ClinicalTrials.gov Identifier: NCT03384433). Another study performed by researchers from China Medical University Hospital explored the role of acupuncture-induced exosomes in the treatment of post-stroke dementia (ClinicalTrials.gov Identifier: NCT05326724). In addition, study conducted by researchers from Neurological Associates of West Los Angeles evaluated the safety and efficacy of exosome together with concurrent transcranial ultrasound in treating patients with refractory, treatment resistant depression, anxiety, and neurodegenerative dementia (ClinicalTrials.gov Identifier: NCT04202770). Researchers from Ruijin Hospital, Shanghai, China evaluated the safety and efficacy of allogenic adipose MSCs derived exosomes in patients with Alzheimer's disease (ClinicalTrials.gov Identifier: NCT04388982). Other studies are also being conducted to explore the possibility of exosomes as new biomarkers of stroke (ClinicalTrials.gov Identifier: NCT05370105), TBI (ClinicalTrials.gov Identifier: NCT04928534), intracerebral hemorrhage (ClinicalTrials.gov Identifier: NCT05035134) and PD (ClinicalTrials.gov Identifier: NCT01860118).

Despite great progress in exosome-based disease-related studies, there are still some unanswered questions that need to be addressed in the future research. For example, the exact content sorting and secretory regulation mechanisms of exosomes remain largely unknown. Besides, to better distinguish exosomes from other extracellular particles, advanced selection and isolation techniques are required for exosome separation and quantification. In addition, exosomal surface modification and therapeutic cargo loading to enable their specific cell-targeted delivery and treatment present new challenges. Most importantly, application of exosomes in the diagnosis and treatment CNS diseases and the underlying regulating mechanisms need more support evidence, especially the biomarkers for exosome isolation, the dosage, measurement standards, and administration routes of exosomes for disease treatment. Side effects, immunogenicity effects and the heterogeneity of exosomes should also be taken into consideration. Despite the existing obstacles, using exosomes as potential biomarkers and therapeutics is attractive in CNS disease, which is worthy of more investigation in the future.