Extracellular vesicles: multiple signaling capabilities and translation into promising therapeutic targets to promote neuronal plasticity

Extracellular vesicles (EVs) are cell-derived, lipid membrane-enclosed vesicles carrying a broad spectrum of biologically active molecules (including proteins, RNAs, and bioactive lipids) which play important roles in intercellular communication. EVs crucially control neuronal energy metabolism under physiological conditions, constrain oxidative stress and brain inflammatory responses, and promote neuronal survival and plasticity upon brain damage. Originating from the right cells, e.g., mesenchymal stromal cells (MSCs), EVs can exhibit striking neurological recovery-promoting activities in various brain disease models (Hermann et al., 2024). In rodent middle cerebral artery occlusion (MCAO) models, for example, intravenously administered MSC-derived EVs enhanced motor coordination recovery similar to parental MSCs by mechanisms involving long-term neuroprotection, neurogenesis, axonal sprouting, remyelination, and synaptic plasticity (Xin et al., 2013; Doeppner et al., 2015). In contrast to pharmacological compounds that target specific signaling pathways, EVs depending on their cellular origin exhibit multiple signaling abilities, enabling them to regulate several disease processes simultaneously in a clinically relevant way (Hermann et al., 2024). In the long term, EVs are expected to surpass and replace many current pharmaceuticals, as their multimodal mechanism of action can synergistically and contextually modify disease outcomes more effectively. The successful clinical translation will decisively depend on the selection of the right targets.

Brain-derived extracellular vesicles are key players in the regulation of neuronal plasticity: Upon brain injury, such as ischemic stroke, neurons, their axons, dendrites, and synapses undergo profound structural and functional plasticity. New neurons are formed by neurogenesis. Following an initial phase of retraction, axons and dendrites of existing neurons in the vicinity and at a distance to brain lesions sprout, enabling functional neuronal network rewiring via the formation and stabilization of synapses. Neuronal plasticity is facilitated by trophic support by astrocytes, oligodendrocytes, and regulatory immune cells. EVs play decisive roles in the signaling between brain cells. In mice and rats exposed to MCAO, MSC-EVs were shown to promote neurogenesis, axonal, dendritic, and synaptic plasticity (Xin et al., 2013; Doeppner et al., 2015).

EVs are centrally involved in the physiological regulation of synaptic plasticity. Thus, the coordinated growth of presynaptic and postsynaptic spines is regulated by activity-dependent EV release from presynaptic neurons. This EV release involves proteins otherwise implicated in synaptic vesicle secretion, such as syntaxin-1A (Hermann et al., 2024). Synaptic EVs were found to carry the Wingless-binding protein Evenness-interrupted/Wntless to its receptor Frizzled-2 at the pre- and postsynaptic membranes, inducing concerted synapse growth (Hermann et al., 2024). The growth factors brain-derived neurotrophic factor and basic fibroblast growth factor control the sorting of EV cargos (Antoniou et al., 2023) and EV release (Kumar et al., 2020), respectively.

Astrocytes regulate neuronal energy metabolism by lactate shuttling, and they also promote neurite growth via trophic influences through EVs. EVs released from astroglial cells were found to spread over long distances along the corticospinal tract to induce axonal growth (Jin et al., 2023). The axonal plasticity-promoting effects of astroglial EVs involve surface contact mechanisms. As such, the growth promotion by astroglial EVs depended on the presence of the oligomannose-mimicking peptide synapsin-I and of hepatocyte cell adhesion molecule (HepaCAM) on EVs and the presence of the synapsin-I-binding protein neural cell adhesion molecule on neurons (Jin et al., 2023; Hermann et al., 2024). HepaCAM possesses immunoglobulin-like extracellular domains that interact with extracellular matrix components. HepaCAM is also called glial cell adhesion molecule.

Neurons possess long axons that require the continuous provision of energy-rich substrates and trophic stimuli enabling their long-term stability. The provision of both depends on axonal transport mechanisms and external support by myelin sheath-forming oligodendroglial EVs. Oligodendroglial EVs were found to stimulate the anterograde and retrograde transport of BDNF-mCherry-carrying vesicles in mouse hippocampal neurites under oxidative stress or nutrient starvation conditions (Frühbeis et al., 2020). EV release was reduced in cultured oligodendrocytes obtained from proteolipid protein (Plp)^–/– and 2′,3′-cyclic-nucleotide-3′-phosphodiesterase (Cnp)^–/– mice exhibiting progressive corticospinal degeneration (Frühbeis et al., 2020). Anterograde and retrograde axonal transport were compromised in Plp^–/– and Cnp^–/– mice (Frühbeis et al., 2020).

The combined evidence of these studies establishes brain-derived EVs as crucial regulators of neuronal plasticity. Neurodegenerative processes may overshadow endogenous plasticity responses, which can be rescued by EVs providing trophic stimuli.

Metabolic disturbances and oxidative stress are prominent extracellular vesicle targets that set the stage for neuronal plasticity: Mitochondrial disturbances and oxidative stress are joint hallmarks of several brain diseases. In response to neuronal injury, mitochondrial metabolism is rapidly regulated to reduce energy consumption. Upon critical cell damage, the mitochondrial balance of Bcl-2 family proteins shifts from anti-apoptotic to pro-apoptotic proteins. Once a critical degree of neuronal damage is reached, this pro-apoptotic shift sets the stage for mitochondrial permeability transition pore formation, mitochondrial cytochrome-c release, reactive oxygen species release, and caspase-9/-3 activation. Energy breakdown, oxidative stress, and apoptotic injury are direct causes of neuronal death and brain infarction in ischemic stroke. In traumatic brain injury, mitochondrial disturbances and oxidative stress contribute to secondary brain damage. EVs potently regulate mitochondrial integrity and function, oxidative stress, and cell death pathways, laying the ground for neuronal survival (Hermann et al., 2024).

The recovery of energy metabolism following injury depends on the ability of the NAD-dependent deacetylase sirtuin-2 (Sirt2), which controls energy metabolism and oxidative stress in mitochondria, and the chaperone heat-shock protein-72, which restores misfolded proteins, to reestablish mitochondrial integrity. In the spinal cords of Sirt2^–/– mice, EVs obtained from wildtype oligodendrocytes were found to transfer Sirt2 to neurons, where they induced mitochondrial adenine nucleotide translocase-1/2 deacetylation, increased intracellular ATP level, and enhanced mitochondrial integrity (Chamberlain et al., 2021). EVs obtained from Sirt2^–/– oligodendrocytes did not exert these actions in Sirt2^–/– mouse spinal cords (Chamberlain et al., 2021). Reduced Sirt2 and heat-shock protein-72 levels were responsible for the progressive corticospinal degeneration in Plp^–/– and Cnp^–/– mice (Frühbeis et al., 2020). EVs of Plp^–/– and Cnp^–/– oligodendrocytes revealed decreased Sirt2 and heat-shock protein-72 levels compared with wildtype oligodendrocyte EVs (Frühbeis et al., 2020). When transferred to nutrient-deprived cortical neurons, EVs of wildtype oligodendrocytes, but not Plp^–/– and Cnp^–/– oligodendrocytes were able to rescue neuronal metabolic state (Frühbeis et al., 2020).

The regulation of axonal energy metabolism by EVs requires the communication of axons with their somas and nuclei. In a mouse model of sciatic nerve injury, macrophages recruited to neuronal lesions by reactive oxygen species were shown to release EVs containing functional NADPH oxidase-2 (Nox2), an NO radical-producing enzyme (Hervera et al., 2018). Nox2⁺ EVs were taken up by injured axons via endocytosis and retrogradely transported to the soma in axonal endosomes (Hervera et al., 2018). The retrograde Nox2 transport involved an importin-β1-dynein-dependent mechanism. In the soma, Nox2 oxidized phosphatase and tensin homolog (Pten), leading to its inactivation, as shown in dorsal root ganglion cell cultures (Hervera et al., 2018). Inactive Pten allowed phosphatidylinositol-3 kinase/Akt signaling to take over control of cell metabolism, resulting in axonal growth.

Excessive oxidative stress acts as a trigger of delayed neuronal degeneration, brain atrophy, and cognitive impairment in rat traumatic brain injury models (Zhang et al., 2021). Astroglial EVs attenuated mitochondrial oxidative stress, neuronal loss, and brain atrophy in traumatic brain injury by activating nuclear factor erythroid-2-related factor-2 (Nrf2)/heme oxygenase-1 signaling and increasing antioxidant superoxide dismutase and catalase activity (Zhang et al., 2021). The neuroprotective effects of astroglial EVs were abrogated in brain-specific Nrf2^–/– mice.

Microglia are sensors of cell injury and neuroinflammation, which may stabilize or destabilize neuronal integrity and plasticity, depending on their polarization. In mice exposed to MCAO, EVs obtained from hypoxically preconditioned M2-like microglia decreased periinfarct brain edema, pro-inflammatory cytokine responses and astrogliosis, increased aquaporin-4 polarization on astrocytic endfeet, reestablished cerebrospinal fluid flow and induced neurological recovery (Xin et al., 2023).

Protective effects of EVs obtained from anti-inflammatory M2-like microglia on dendritic spine integrity, β-amyloid burden, and cognitive function were observed in transgenic 5×FAD mice with Alzheimer’s pathology. M2 microglial EVs were found to contain miR-7670-3p, which reduced endoplasmic reticulum stress by activating transcription factor-6 expression and preserved dendritic spine integrity of cortical and hippocampal neurons (Chen et al., 2023).

Accordingly, EVs from various cell sources control mitochondrial function and oxidative stress in the injured and degenerating brain to promote neuronal survival. By stabilizing mitochondrial function, EVs set the stage for neuronal plasticity.

Mesenchymal stromal cell-extracellular vesicles are a potent tool for stimulating neuronal integrity and plasticity post-injury most likely via anti-inflammatory actions: In a variety of neurological disease models, EVs obtained from different cell sources, including astrocytes, microglia, and neural precursor cells, were shown to exert anti-inflammatory actions that contribute to brain tissue survival and neurological recovery (Hermann et al., 2024). MSC-EVs potently stimulate motor-coordination recovery in MCAO mice by anti-inflammatory actions (Wang et al., 2020). When intravenously administered in the acute stroke phase (immediately post-MCAO), MSC-EVs effectively reduced brain neutrophil, monocyte/macrophage, and lymphocyte infiltrates. Neutrophil depletion by delivery of an antibody against the neutrophil-specific antigen Ly6G mimicked the effects of intravenously administered MSC-EVs on neurological deficits, ischemic injury, brain monocyte/macrophage and lymphocyte counts (Wang et al., 2020). In neutrophil-depleted mice, however, MSC-EVs did not have any additional effect on neurological deficits and ischemic injury, and brain monocyte/ macrophage and lymphocyte infiltrates were not influenced by MSC-EVs. Of note, the role of neutrophils in mediating post-ischemic actions of MSC-EVs was not restricted to the acute stroke phase. When administered in the post-acute stroke phase (starting 24 hours post-MCAO), MSC-EVs were found to promote peri-infarct angiogenesis, but these angiogenic effects of EVs were also abolished by neutrophil depletion (Gregorius et al., 2021). Neutrophils are early brain invaders after MCAO, which exacerbate ischemic damage in the acute stroke phase, but support brain tissue remodeling in the post-acute phase.

The anti-inflammatory effects of MSC-EVs may help evade the challenge of EV blood-brain barrier passage, which is a major obstacle for EV therapies. The brain accumulation of intravenously or intranasally administered MSC-EVs is poor in macaque monkeys (Driedonks et al., 2022), which is due to limited paracellular or transcellular EV transport. Yet, EVs are rapidly taken up within minutes after systemic delivery by leukocytes (specifically by monocytes and neutrophils) (Driedonks et al., 2022). Since leukocytes abundantly enter the injured brain, they are attractive EV targets that may allow modifying brain responses in clinical settings even under conditions of preserved blood-brain barrier integrity.

This paper highlights three major findings. First, brain-derived EVs are key players in the regulation of neuronal plasticity, supporting coordinated synaptic growth, axonal integrity, and neuronal rewiring (Figure 1). In the regulation of neuronal plasticity, neuronal, astroglial, and oligodendroglial EVs are involved. These EVs control neuronal integrity under physiological conditions and promote plasticity in the injured brain. Secondly, mitochondrial disturbances and oxidative stress are joint hallmarks of neuronal injury in a variety of neurological conditions, which may outweigh endogenous plasticity responses and put at risk the brain for irreversible neuronal injury. Of note, mitochondrial disturbances and oxidative stress also represent important EV targets, which may protect the neurons against injury and set the stage for neuronal plasticity (Figure 1). Indeed, a variety of studies in models of brain or spinal cord ischemia, trauma, or neurodegeneration showed that the restitution of mitochondrial function and regulation of oxidative stress by EVs induced neuronal plasticity and neurological recovery. In view of their location inside the brain or spinal cord, neuronal mitochondrial function and neuronal plasticity are difficult to target with systemically (in particular, intravenously) administered EVs. Fortunately and thirdly, intravenously administered EVs potently modulate immune responses in the blood to induce anti-inflammatory responses in the brain that promote neuronal survival and plasticity (Figure 1). These anti-inflammatory activities have been explored most systematically in ischemic stroke models (Wang et al., 2020; Gregorius et al., 2021), in which MSC-EVs enhanced axonal and synaptic plasticity (Xin et al., 2013; Doeppner et al., 2015). This perspective is a strong credo in the metabolic and anti-inflammatory actions of EVs. The tight links between mitochondrial dysfunction, oxidative stress, inflammatory responses, neuronal survival, and plasticity provide a strong framework for EV-based therapeutics.

Figure 1.

Mitochondrial dysfunction, oxidative stress, immune responses, neuronal survival, and plasticity are important targets of EVs.

In response to injury, mitochondrial dysfunction, oxidative stress, immune responses, neuronal survival, and plasticity are closely interconnected. EVs potently control all five targets. Deregulation of neurons at the levels of mitochondrial function, oxidative stress, and immune responses puts at risk the neurons for irreversible damage. Stabilizing the neurons at each of these three levels supports neuronal integrity, survival, and plasticity. From this perspective, the modulation of mitochondrial dysfunction, oxidative stress, and immune responses is particularly promising with respect to neurological recovery. Among the targets shown, immune responses are unique with respect to clinical translation. Immune responses can easily be targeted in peripheral blood by intravenously administered EVs. Intravenously administered EVs were found to inhibit inflammatory leukocyte responses in ischemic stroke models, mediating neuroprotection and neurological recovery. Hereby, EVs may protect the brain even under conditions of preserved blood–brain barrier integrity, in which the brain accumulation of EVs is poor. Created with BioRender.com. EVs: Extracellular vesicles.

This work was supported by the German Research Foundation (grants 514990328, 389030878, 405358801/428817542 (within FOR2879) and 449437943 (within TRR332, project C06), and by German Federal Ministry of Education and Science (3DOS; grant 161L0278B) (to DMH).

DMH and BG hold patents for the application of extracellular vesicles for the treatment of inflammatory conditions (EP2687219A1; US9877989B2). BG is the founding director and DMH advisor of Exosla Ltd. No conflicts of interest exist between Exosla Ltd. and publication of this paper.